Pregnancy complications such as gestational diabetes (GDM) and hypertensive disorders of pregnancy (HDP) are frequent and influence not only fetal outcomes but also the maternal cardiac function. GDM and HDP may act as a proxy for increased metabolic and cardiovascular risk later in life. Speckle tracking echocardiography (STE) is a relatively new imaging technique that provides more sensitive assessment than conventional echocardiography of the maternal cardiac function. Recent research suggests that STE can be used during pregnancy and postpartum as a useful method of early detection of subclinical maternal cardiac changes related to pregnancy complications, such as GDM and HDP, and as an indicator for future maternal cardiovascular disorders. The aim of this review was to underline the current value of STE in the follow-up protocol of high-risk pregnant women, as a mean for pre- and postpartum monitoring. A review of the literature was conducted in the PubMed database to select relevant articles regarding the association of STE changes and HDP or GDM in the prenatal and postpartum maternal evaluations. Both GDM and HDP are associated with subtle myocardial changes in shape, size and function; these preclinical cardiac changes, often missed by conventional evaluation, can be detected using STE. Left ventricular global circumferential strain might be an important predictor of maternal cardiovascular disorders and might help to define a high-risk group that requires regular monitoring later in life and timely intervention. Echocardiography is a consecrated diagnostic and follow-up method for the adult population with cardiovascular (CV) risk factors and cardiac disease. In pregnancy, maternal echocardiography is reserved for selected cases, with previous cardiac pathologies or onset of symptoms suggesting heart failure either when assessed by the cardiologist or for research purposes, as observed in the recent literature ,2 A new The aim of this review is to underline the value of newer echocardiography techniques in the follow-up protocol of high-risk pregnancies, as a mean for pre- and post-partum monitoring. Pregnancy complications, such as gestational diabetes (GDM) or hypertensive disorders of pregnancy (HDP), can foreshadow subsequent cardiovascular and metabolic disease in future life . TherefoSpecific and standardized assessment of the cardiac function has been the subject of broad and permanent interest ,6. The aHowever, echocardiography is a widely available, non-invasive, not irradiating technique, suitable for maternal heart monitoring in pregnancy and postpartum. Its main disadvantages are related to image acquisition, which is operator- and acoustic-window dependent and to that the assessment of ventricular function is related to geometric assumptions of the normal heart, with less reliability in some cases .M-Mode echocardiography, a method that measures the mid-left ventricle diameters in the short axis and volumes obtained by cubing the diameters, estimates left ventricular fractional shortening and ejection fraction  Figure . A more Global myocardial performance has been assessed by other echocardiographic methods, derived from Doppler analysis, such as myocardial performance index (Tei index) and tissue Doppler mitral annular systolic velocity . These mAnother approach to assess left cardiac function is three-dimensional echocardiography that eliminates the geometric assumptions and operator dependent measurements . A relatively new tool for assessing left ventricular function through myocardial strain with a high temporal and spatial resolution is speckle-tracking imaging . This meCurrently, speckle tracking echocardiography is the most applicable method in the context of pregnancy related complications and has shown the most promising results in detection of subclinical myocardial dysfunction. Two-dimensional speckle-tracking echocardiography is proposed as a novel technique for objective and quantitative evaluation of global and regional myocardial function. The myocardial deformation strains are obtained by frame-by-frame automatic measurement of the distance between two points of each LV segment during the cardiac cycle. The ST allows quantification of myocardial or more correct endocardial deformation. This technique enables us to calculate the segmental, as well as the global longitudinal, strain and strain rate. It measures myocardial velocities and deformation parameters and has evolved to be the imaging modality of choice to detect subclinical cardiac dysfunction .Subclinical cardiac dysfunction precedes the development of heart failure and other cardiovascular diseases, but often goes undiagnosed by conventional echocardiography. Recognition of subclinical myocardial dysfunction offers clinicians an opportunity for early intervention and prevention of symptomatic cardiovascular disease and novel imaging method, such as STE, might be a useful screening strategy. For example, subclinical LV systolic dysfunction in the general population with diabetes mellitus, detected using LV global longitudinal strain, is associated with an increased incidence of cardiovascular events, defined as a composite of acute coronary syndrome, cerebrovascular stroke, cardiovascular death and hospitalization for heart failure ,23.Speckle tracking echocardiography is an advanced, non-invasive imaging technique that provides fast and accurate assessment of cardiac function, global and regional function of both atrial and ventricular chambers, in-plane translation motion and independently from the angle of insonation . The infIt has been demonstrated over the last 10 years that speckle tracking echocardiography is a reliable method in predicting heart failure. Studies in non-pregnant population showed that there is a specific pattern of changes when using speckle tracking, not only in arterial hypertension, but also following diabetes mellitus . Early sIn comparison with other methods of assessing heart function, the main benefit of SPE is represented by the capacity to evaluate the two-dimensional displacement of cardiac muscle spots during the cardiac cycle and to calculate, in an angle-independent way, heart deformation through the distance variation between the speckles in the analysed segment . TherefoMyocardial deformation during cardiac cycle, contraction and relaxation, is defined by the term strain . It reprThe adult myocardium possesses a complex structure, based on right-handed helical fibers at endocardial layer and left-handed helical fibers in epicardial layer . HarmoniDefined by a positive curve, radial strain shows the radial myocardial deformation corresponding to systolic thickening. This parameter is assessing left ventricular dyssynchrony and, hence, has proved to be one of the most accurate methods to identify potential responders to cardiac resynchronization therapy in patients with end-stage heart failure . CircumfThe rotation degrees of the intermediate myocardial planes gradually change from base to apex and the rotation is null at papillary muscles level. A parasternal short-axis section at basal and apical planes can assess left ventricular twisting value .Gestational diabetes affects one out of seven pregnancies and is associated with adverse perinatal outcomes for both the mother and the fetus. Women whose pregnancies were complicated with GDM have a seven-fold higher incidence of type 2 diabetes later in life and a two-fold higher risk of cardiovascular events during the first 10 years postpartum  MoreoverWe did a search on PubMed with no time restriction for words such as “echocardiography” and “pregnancy” and “gestational diabetes”. We retrieved 354 results out of which only 19 were related to maternal cardiac echocardiography in relation to diabetes. After reading the studies, we found eight studies that used STE and reported subtle differences between cardiac function in pregnant women with and without diabetes. .Increased post-partum cardiovascular risk after GDM might be explained by subclinical cardiac changes which appear during pregnancy in women exposed to high glycemic levels and insulin resistance. Most of the studies reported early changes at STE; the main finding when comparing the pregnancies complicated by GDM to normal population is reduced left ventricular global longitudinal strain (LV-GLS).A recent large prospective longitudinal study  enrolledp < 0.001 for both) and lower left ventricular ejection fraction. Early subclinical systolic functional impairment is easily proven by speckle tracking in fetuses with gestational diabetic mothers; functional changes were more evident in the right ventricle [Gestational diabetes also has an impact on the offspring cardiac function, manifesting early signs of subclinical cardiac dysfunction since intrauterine life. A recent study illustrated that fetuses of mothers with gestational diabetes mellitus possessed more globular hearts with higher right and left ventricular sphericity index  was extensively studied in the context of hypertensive disorders of pregnancy. Left ventricular-GLS represents LV myocardial shortening in the longitudinal axis and is an important index of global LV function. It is an early marker of subclinical alterations of subendocardial longitudinal fibers, which are the first to be affected in ischemic injuries and arterial hypertension . AlthougA recent systematic review regarding STE findings in pregnancies complicated by hypertensive disorders analyzed 16 relevant studies and reported that LV-GLS was decreased in women with any form of hypertensive complication. Additionally, women with early onset or severe form of preeclampsia showed other deformation changes, such as decreased left ventricular global radial and circumferential strain . These pReduced sensitivity of conventional echocardiography in detecting subclinical cardiac changes is supported by the results of a systematic review on women with a history of preeclampsia assessed by conventional echocardiography, which failed to show any difference in left ventricular ejection fraction, isovolumetric relaxation time, or deceleration time. The results suggest that cardiac morbidity associated with preeclampsia is a result of an increased incidence of the cardiovascular risk factors within the population group , rather than the preeclampsia alone. In terms of measurement of global myocardial deformation, speckle tracking imaging is a useful tool to assess maternal cardiac function, showing reduced strain in preeclampsia as compared to normotensive pregnancies .A prospective observational study  includinAnother study published in 2020 by Vasapollo et al.  reportedAssessing both preeclampsia and fetal growth restriction in fetal cardiac remodeling, Youssef et al.  demonstrDuring pregnancies affected by preeclampsia, mild diastolic dysfunction can be detected with standard echocardiographic techniques, but STE can also identify systolic dysfunction . A recenFurthermore, the subtle systolic and diastolic dysfunction observed in late pregnancy, when complicated by gestational diabetes, also persist for at least six months post-partum . Long-teAll these data support the coordinated use of speckle tracking by teams of maternal fetal specialists and cardiologists in patients with pregnancy complications, both pre and postpartum. The desired outcome would be to design tailored monitoring protocols for every clinical situation, in order to foresee possible long-term effects temporarily unmasked during pregnancy .It may well be that speckle tracking is for hypertensive diseases of pregnancy what the oral glucose tolerance test is for gestational diabetes mellitus, both for pregnancy and for the long-term follow-up. The current literature is limited concerning the use of speckle tracking in patients with pregnancy complications such as maternal gestational diabetes, preeclampsia and fetal growth restriction and comparative studies. Focus on a larger number of cases with a continuous postnatal monitoring is needed, especially for complications such as preeclampsia, in order to improve early prediction of frequent maternal complications.


ABSTRACT IMPACT: This work will standardize necessary image pre-processing for diagnostic and prognostic clinical workflows dependent on quantitative analysis of conventional magnetic resonance imaging. OBJECTIVES/GOALS: Conventional magnetic resonance imaging (MRI) poses challenges for quantitative analysis due to a lack of uniform inter-scanner voxel intensity values. Head and neck cancer (HNC) applications in particular have not been well investigated. This project aims to systematically evaluate voxel intensity standardization (VIS) methods for HNC MRI. METHODS/STUDY POPULATION: We utilize two separate cohorts of HNC patients, where T2-weighted (T2-w) MRI sequences were acquired before beginning radiotherapy for five patients in each cohort. The first cohort corresponds to patients with images taken at various institutions with a variety of non-uniform acquisition scanners and parameters. The second cohort corresponds to patients from a prospective clinical trial with uniformity in both scanner and acquisition parameters. Regions of interest from a variety of healthy tissues assumed to have minimal interpatient variation were manually contoured for each image and used to compare differences between a variety of VIS methods for each cohort. Towards this end, we implement a new metric for cohort intensity distributional overlap to compare region of interest similarity in a given cohort. RESULTS/ANTICIPATED RESULTS: Using a simple and interpretable metric, we have systematically investigated the effects of various commonly implementable VIS methods on T2-w sequences for two independent cohorts of HNC patients based on region of interest intensity similarity. We demonstrate VIS has a substantial effect on T2-w images where non-uniform acquisition parameters and scanners are utilized. Oppositely, it has a modest to minimal impact on T2-w images generated from the same scanner with the same acquisition parameters. Moreover, with a few notable exceptions, there does not seem to be a clear advantage or disadvantage to using one VIS method over another for T2-w images with non-uniform acquisition parameters. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results inform which VIS methods should be favored in HNC MRI and may indicate VIS is not a critical factor to consider in circumstances where similar acquisition parameters can be utilized. Moreover, our results can help guide downstream quantitative imaging tasks that may one day be implemented in clinical workflows.


Athlete’s heart (AH) is the result of morphological and functional cardiac modifications due to long-lasting athletic training. Athletes can develop very marked structural myocardial changes, which may simulate or cover unknown cardiomyopathies. The differential diagnosis between AH and cardiomyopathy is necessary to prevent the risk of catastrophic events, such as sudden cardiac death, but it can be a challenging task. The improvement of the imaging modalities and the introduction of the new technologies in cardiac magnetic resonance (CMR) and cardiac computed tomography (CCT) can allow overcoming this challenge. Therefore, the radiologist, specialized in cardiac imaging, could have a pivotal role in the differential diagnosis between structural adaptative changes observed in the AH and pathological anomalies of cardiomyopathies. In this review, we summarize the main CMR and CCT techniques to evaluate the cardiac morphology, function, and tissue characterization, and we analyze the imaging features of the AH and the key differences with the main cardiomyopathies. Physical exercise is useful to prevent many cardiovascular diseases, limiting several risk factors for heart pathologies and reducing the incidence of fatal ischemic events due to coronary artery disease . AthleteLong-lasting athletic training causes cardiac heart overload and it results in morphological and functional cardiovascular adaptations of cardiac chambers, called athlete’s heart (AH). These adaptive modifications are physiological responses to the hemodynamic demands of increased cardiac output but, frequently, overlap with cardiac diseases and the differential diagnosis between AH and cardiomyopathies is a challenging task ,5,6.The clinical evaluation, the electrocardiogram (ECG), and echocardiography are the first-line exams that should be used to detect cardiovascular abnormalities. However, when the results are not conclusive, the cardiologist’s dilemma is often between causing unnecessary disqualification or risking a preventable catastrophic event .The radiologist, specialized in cardiac imaging, could represent an essential support in this clinical decision. Cardiac magnetic resonance allows accurate and reproducible measurements of cardiac anatomy and function and, above all, it provides tissue characterization, that is pivotal for the differential diagnosis between AH and cardiomyopathy. Likewise, cardiac computed tomography (CCT) provides morphological and functional heart evaluation with low radiation dose using the last generation CT scanner. Moreover, the recent technological advances, such as the introduction of dual energy (DE) technology, enabled to extend the CCT application for the myocardial tissue characterization ,8.In this work, the CMR is a non-radiation imaging modality with high spatial and temporal resolution. CMR is useful to evaluate the morphological cardiac findings when the echocardiography alone is not enough, because CMR has no blind spots and it does not have the same limitations, related to the presence of thoracic wall or pulmonary parenchyma or inaccurate wall thickness evaluation, due to short-axis obliquity. Moreover, CMR allows calculating accurately the global cardiac function and evaluating the regional wall motion abnormalities because it provides full ventricle coverage in all cardiac planes, such as short-axis, four-chambers, and two-chambers. Finally, the main added role of CMR is to characterize the myocardial tissue, using different techniques, such as mapping, extracellular volume images (EVC), and late gadolinium enhancement (LGE) .CMR is the reference standard modality to calculate the left ventricular (LV) and the right ventricular (RV) volume and mass. In clinical routine, standard evaluations include the absolute and indexes values of the LV and RV end-diastolic volume (EDV), end-systolic volume (ESV), mass, and wall thickness. The normal ranges are related to sex and age. Due to the high accuracy and reproducibility, these parameters can be used both for diagnosis and follow-up .CMR is the gold standard modality to quantify the global contractile function. In the clinical routine, the evaluations include the LV and the RV stroke volume (SV), cardiac output (CO), and ejection fraction (EF). Recently, using the volume–time curve, other functional ventricular parameters have been introduced, such as the peak ejection rate, the early peak filling rate (PFRE), the peak filling rate at atrial contraction (PFRA), their ratios (PFRE/PFRA), and the values indexed to the EDV . Moreover through atrial volume–time curve can be calculated atrial EDV and ESV and atrial EF .CMR, with use of fast-cine MR sequences, allows evaluation of the regional myocardial kinetic through the subjective evaluation of the systolic-to-diastolic wall thickness changes. The main pathological wall kinetic abnormalities are the hypokinesia, defined as a decreased wall motion, the akinesia, characterized by absent wall motion, and the dyskinesia, described as uncoordinated wall motion .The most recent parameter, introduced to evaluate the myocardial regional function, is the myocardial strain. It defines the degree of deformation of a fixed myocardial point throughout the cardiac cycle, from the systolic to the diastolic phases . The refMapping is an MR imaging technique that allows the pixel-by-pixel estimation of magnetic relaxation times .T1 mapping technique is mainly used in the characterization changes in myocardial tissue composition in various cardiac pathologies, such as myocardial infarction, myocarditis, sarcoidosis, amyloidosis, and overload pathologies .The acquisition consists of multiple T1-weighted images with different longitudinal relaxation times , following either an inversion or saturation preparation. Then, T1 signal intensities at each pixel can be determined using a dedicated equation and T1 mapping images are generated. The main protocols applied are the modified looklocker inversion recovery (MOLLI) for inversion-recovery-based technique and saturation recovery single-shot acquisition (SASHA) for saturation-based technique. .T1 mapping allows a complete myocardial evaluation and it is particularly useful in diffuse myocardial disease. The main benefit of T1 mapping is to quantify the tissue abnormalities to track myocardial changes over time and to control the disease evolution during pharmacological treatments. The main limitation is that T1 values depend on many factors, such as pulse sequence, constructor, cardiac cycle, and magnetic field, and are influenced by age .T1 mapping values increase weakly with myocardial fibrosis and strongly with amyloid and myocardial edema and fall with fatty infiltration and iron overload ,15.T2 mapping technique is mainly used to identify and quantify myocardial edema in different cardiac diseases including myocarditis, myocardial infarction, sarcoidosis, and amyloidosis .The acquisitions are obtained in a single expiratory apnea, during the same phase of the cardiac cycle, and in successive consecutive heartbeats, every 2 to 4 RR intervals, according to the heart rate. In each slice, three T2-weighted images are obtained with different T2 preparation times, usually at 0 ms, 25 ms, and 55 ms. Then, the images are processed applying a motion correction algorithm to reduce respiratory and cardiac motion artifacts. Finally, the T2 mapping images are generated .T2 mapping has several advantages compared to T2-weighted spin-echo inversion recovery sequences, traditionally used to evaluate myocardial edema, which are often of limited value due to susceptibility or slow-motion artefacts. Moreover, T2 mapping allows the quantification of edema .In the absence of iron overload, T2 mapping values increase only with edema. With iron overload or myocardial hemorrhage T2 mapping values decrease .Myocardial ECV refers to the volume of the myocardium, which is not occupied by cells, and it is a marker of interstitial disease. This parameter allows the quantification of the myocardial extracellular volume, expressed as a percentage of myocardial mass .m/∆Rb), where ∆Rm and ∆Rb represent, respectively, the change in T1 relaxation time in the myocardial muscle and in the blood before and after contrast administration. The delay acquisition is usually performed 10′ after contrast agent injection. In this way, it is possible to obtain an estimation of extracellular matrix, the space within the myocardium, without intracellular and intravascular compartments [The ECV evaluation is based on the distribution of the contrast agent in the myocardium and its calculation exploits the characteristics of the paramagnetic contrast agent, which is intravascular, extracellular, and interstitial. Considering the variation of the T1 relaxation time of the myocardium and the blood before and after contrast administration and using the hematocrit (Hct) value, the ECV estimation is obtained: ECV = (1 − Hct) ×  ,27.The main CCT advantages compared with CMR are the feasibility and short examination time. Therefore, CCT is particularly useful in patients with relative and absolute contraindications for CMR or in the case of claustrophobic patients, which may be unable to undergo the examination ,28.CCT allows the evaluation of the main cardiac morphological parameters, such as volume, mass, and wall thickness. The values demonstrated excellent reproducibility and a very good linear relationship compared to those obtained with CMR, even if the normal CCT values are significantly lower than those obtained with CMR . The knoMoreover, CCT provides global and regional functional analysis. Many prior studies validated, via comparisons with CMR, the global functional parameters, such as SV, CO, and EF, and the evaluation of regional functional findings, such as hypokinesia, akinesia, and dyskinesia .Morphological and functional findings are comprehensively assessed using a retrospective ECG-gated helical scanning protocol, which is characterized by continuous data acquisition throughout some consecutive cardiac cycles. This retrospective technique allows the evaluation of any cardiac phase and it is particularly useful in patients with high or irregular heart rhythm and, even if it is associated with relatively high radiation doses, it allows with the same acquisition the evaluation of the coronary artery disease ,29.Myocardial ECV provides a remodeling value of the cardiac tissues and offers a measure of the proportion of the extracellular space within the total mass of the myocardium. ECV is a reproducible index and it demonstrated good agreement with the same value calculated with CMR. Indeed, iodine-based contrast medium has similar pharmacokinetic characteristics compared to the paramagnetic contrast agent ,30.m/ΔHUb), where ΔHU LVm and ΔHUb represent the Hounsfield Unit difference between the pre-contrast and the post-contrast phase, respectively, in the myocardium and in the blood. The post-contrast phase is usually performed 5–7 min after contrast injection [ECV is calculated with the formula: ECV = (1 − Htc) × , usually a low (from 70 to 90 kV) and a high (from 140 to 150 kV) voltage. This acquisition allows generation of monoenergetic images with a high contrast-to-noise ratio to detect LIE. Different DE technologies can be used, such as the rapid switching of X-ray tube potential, the multilayer detector, or dual X-ray tubes. The total volume of the iodine contrast medium depends on the contrast concentration and on the patient’s weight, and usually, the range is from 1.2 mL/kg to 1.5 mL/kg ,32.AH is characterized by all the cardiac adaptations related to physical training. These cardiac changes are due to systemic arterial resistance reduction and volume overload. Therefore, the morphological modifications are the increased cardiac volumes and the myocardial hypertrophy. The main functional adaptation is an augmented stroke volume with a usually normal or slightly reduced EF at rest, showing a good contractile reserve ,2,6.Intense prolonged training can induce cardiac changes that overlap with cardiomyopathy and occasionally athletes may die suddenly during training or competition. For this reason, it is essential to distinguish athletic cardiac remodeling and cardiomyopathies ,6.Hypertrophic cardiomyopathy (HCM) is a non-ischemic cardiac disease, characterized by a wall thickness ≥ 15 mm at ED, in at least one LV myocardial segment, or a ratio between septal thickness and inferior wall at mid-ventricular level >1.3, without pressure overload or infiltration. LV wall thickness ≥ 30 mm is associated with SCD .The evidence of pathological ECG changes, particularly abnormal T wave inversion, without echocardiogram anomalies represent the main indication for the CMR or CCT examinations. CMR or CCT can identify the apical HCM and can precisely measure the extension and the pattern of LV wall thickness, especially in segments less reliably visualized by echocardiography when the acoustic window is poor. Furthermore, they can provide information about myocardial fibrosis and they can reveal features, such as papillary muscle hypertrophy, basal-apical muscle bundle, and clefts ,35.Athletes who train regularly may have physiological LV hypertrophy, which can be difficult to differentiate from HCM ,10.2/mL, and an increased number of papillary muscles, that are usually displaced and hypertrophic (> 11 mm). Other typical secondary signs of HCM are myocardial crypts, para-septal muscle bundle, and abnormal apical trabeculation. Moreover, AH hypertrophy regresses after a rest detraining period. Indeed, a reduction greater than 2 mm, in the absence of exercise over a three-month period, supports the diagnosis of AH, while an unchanged hypertrophy suggests the diagnosis of HCM [Sports with a high-static component cause a concentric hypertrophy with an increased ventricle mass without chamber dilation. Sports with high-dynamic components determine an eccentric hypertrophy with an increase in ventricle mass and size. In AH, LV hypertrophy is usually symmetrical. The main morphological findings that support the diagnosis of HCM are the presence of an asymmetric apical or septal hypertrophy, a value of maximum diastolic thickness/minimum diastolic thickness ≥ 1.3, a value of LV diastolic wall-to-volume ratio ≥ 0.15 mm/ms of HCM ,10.In AH, global and regional functions are preserved. HCM, due to structural alterations such as fibrosis and the disarray of myocardial fibers, is usually characterized by a diastolic dysfunction, with normal or supranormal SV and EF. Finally, strains and strain rate can be mildly reduced in HCM, instead, in AH, they are preserved ,20.Mapping and ECV can be used to differentiate between AH and HCM. Indeed, in AH, hypertrophy is due to a percentage increase in the cellular component and a relative reduction of the extracellular space, on the contrary in HCM, the hypertrophic component is due to the presence of myocardial fibrosis, which causes a relative higher extracellular component and a percentage reduction of the cellular space. Hence, T1 mapping and ECV values are reduced in AH and increased in HCM. T1 value > 1217 ms and ECV value > 22.5% have the greatest sensitivity and specificity for the diagnosis of HCM ,9,10. ThLGE is normally absent in AH and when present it appears as a mesocardial stria located at the anterior and/or posterior interventricular junctions and at the insertion point of the trabeculae on the ventricular wall . The maiAlthough the detection of LGE favors the diagnosis of HCM, the absence does not exclude the possibility of HCM. For these reasons, considering the actual clinical evidence, even if current guidelines recommend the only fibrosis evaluation, a comprehensive CMR tissue characterization that comprises mapping, ECV and LGE should be performed in every athlete with suspected HCM ,9.Particularly when CMR is contraindicated, the evaluation with CCT could be considered. CCT provides anatomical information about HCM based on key morphological characteristics , LVEF and the extent of myocardial fibrosis. As for the CMR, mean ECV values with HCM are significantly higher than in AH ,9.Dilated cardiomyopathy (DCM) is a non-ischemic heart muscle disease. It is defined as dilation of both ventricles, exceeding normal value of 112%, with EF lower than 45%, in the absence of coronary artery disease or increased load conditions. Occasionally, it is also associated with hyper-trabeculation of both ventricles ,38.CMR and CCT are particularly useful to evaluate the volumes and the EF in subjects with poor acoustic echocardiographic windows and to characterize the tissue composition ,38.The AH overlaps features of DCM . About hDetraining is a common test used to differential diagnosis between AH and DCM. Indeed, despite the limited data, in most athletes, there is a reduction in the cardiac dilation after 3 months from the cessation of training. However, adherence to detraining is poor and the identification of the AH using non-invasive imaging remains preferable .Considering functional findings, AH has a slightly reduced ejection fraction , especially in sports with high-dynamic activity. In this case, the use of stress CMR-imaging may be helpful to correctly differentiate AH from DCM. Indeed, while athletes can significantly increase EF during exercise, patients with DCM less so. A cutoff value of 11.2% for the increase in LVEF from rest to peak exercise has a sensitivity of 93% and a specificity of 90% to differentiate AH from DCM. However, the role of stress CMR-imaging is not validated, and the best threshold of contractile reserve has not been well defined .Mapping and ECV are useful techniques to differentiate AH and DCM. T1 mapping, T2 mapping, and ECV values are lower in athletes than in DCM, and T1 mapping seems to be the most accurate parameter to differentiate these two conditions .LGE is present in about 30–50% of the patients with DCM. The most characteristic pattern is a linear LGE, located in the mesocardial layer, at the infero-lateral wall or at the interventricular septum .In the diagnostic workup of DCM, CCT is recommended to exclude coronary artery disease with low pretest probability. In addition, as a one-stop-shop exam, CCT can evaluate LV global systolic dysfunction and calculate LV size, wall thickness, and LIE. Moreover, CCT allows demonstrating expansion of ECV in patients with DCM compared with healthy subjects ,43.Left ventricular non-compaction (LVNC) cardiomyopathy is a myocardial phenotype that overlaps with hypertrophic and dilated cardiomyopathies and is characterized by prominent LV trabeculation .Athletes, especially in high-dynamic sports, showed an increased prevalence in LV trabeculation and it is more common in African/Afro-Caribbean compared with Caucasian athletes. For this reason, the differential diagnosis between AH and LVCN can be a challenging task and it is usually over-diagnosed in athletes, particularly black athletes ,4.CMR and CCT are particularly useful to detect the exact extension of hyper-trabeculation and to differentiate the physiological hyper-trabeculation in AH and the pathological LVNC, usually associated with marked repolarization abnormalities and low EF ,4.The main finding for the differential diagnosis between hyper-trabeculation in AH and LVNC is the morphological one, which can be evaluated using different criteria. Petersen’s criteria indicate the diagnosis of non-compaction with a ratio between non-compacted (NC) myocardium and compacted (C) myocardium > 2.3, evaluated in ED, and in four-chambers plane, in at least two consecutive segments. Usually, the most frequent cardiac segments involved are the mid-apical ones of the LV inferior-lateral wall and septum. Subsequently, to improve the sensitivity and diagnostic specificity, Jacquier’s criteria were introduced, which support the diagnosis of non-compact myocardium with a ratio between NC/C mass > 20%, evaluated in ED, and in short-axis plane. Finally, Captur’s criteria suppose the diagnosis of non-compaction considers the complexity of the myocardial NC distribution based on mathematical model and fractal analysis. However, none of the criteria demonstrated adequate diagnostic accuracy and with a low pretest probability of LVNC, these values could generate false positive diagnoses ,8.Considering functional findings, LVNC, although often has slightly reduced EF, usually shows strain and strain rates reduction. This observation implies that strain myocardial alteration occurs early in the course of the disease and underlines the utility of deformation CMR-imaging for evaluating LV function compared to LVEF alone ,4,8,44.T1 mapping values are higher in LVNC patients compared with normal controls, even in the absence of LGE, suggesting that T1 mapping can detect myocardial fibrosis in an early stage .In LVNC, LGE has usually a linear pattern localized in the mesocardial layer and does not closely correlate with the NC areas. It is interesting to underline that LGE is the only independent predictor of systolic LV impairment and it is an independent parameter of poor outcome. On the contrary, the thickness of LV trabeculation does not have a significant prognostic role ,42.CCT allows the detection of trabeculations along the LV wall, the precise measurement between NC and C thickness ratio, and the diagnosis of the subendocardial areas of LIE. Additional cardiac CT findings may include LV systolic dysfunction evaluation with low radiation dose protocol ,42,43,44Arrhythmogenic cardiomyopathy (AC) is a non-ischemic heart disease characterized by the mutation of the desmosomes, the intercellular junctions between myocardial cells. The cascade of the pathophysiological process begins with the rupture of defective desmosomes followed by apoptosis or necrosis of myocardial cells and subsequent replacement by fibro-adipose tissue. It is necessary to underline that fibro-fatty replacement does not occur homogeneously at the cardiac muscle. The disease first affects the subepicardial layer and then becomes transmural. For this reason, there are several variants of AC. The two main forms of AC are arrhythmogenic right ventricular cardiomyopathy (ARVC), the most frequent, and the atypical form, left dominant (LD) AC, which caused a high percentage of AC-related deaths ,45.CMR is the most suitable imaging modality to study the cardiac chambers because, compared to echocardiography, it has a higher sensibility in the identification of anatomical, functional, and tissue pathological changes, especially in early AC ,47.ARVC is characterized by the replacement of the RV myocardium with fibro-adipose tissue, resulting in RV dilation, dysfunction, and arrhythmia .Especially at an early stage, the cardiac aspects of ARVC can overlap AH, making ARVC a challenging diagnosis in athletes. However, the differential diagnosis between physiological or pathological modification of RV is very important, because ARCV is related to up to 20% of sudden cardiac death in athletes .2  or ≥100 mL/m2  or RVEF ≤ 40% and, and in the minor criteria, with RVEDV from ≥100 to <110 mL/m2  or ≥90 to <100 mL/m2  or RV ejection fraction from >40% to ≤45% [In 2010, the criteria for the ARCV diagnosis were changed, adding the quantitative CMR-parameters. These criteria include akinesia or dyskinesia or regional dyssynchronous contraction of RV associated, in the major criteria, with RVEDV ≥ 110 mL/m to ≤45% ,48.The diagnostic criteria of ARVC in CMR are based on a combination of regional and global kinetic anomalies and anatomical alterations. However, there could be several problems in the distinction between AH and ARVC. Firstly, athletes may exhibit mild to moderate RV dilation because the thin RV wall and the propensity for a volume overload adaptation explain the earlier anatomical modifications of the RV compared to LV. Indeed, prolonged and intense physical exercise, such as occurs endurance sports, often causes moderate RV dilation. Conversely, intense and short-term efforts are associated with mild or without RV dilation. Other elements that need to be considered in evaluating the RV dilation are the athlete’s age and the number of years of training, because older athletes with more years of training show a greater degree of RV dilation. Secondly, the misdiagnosis of ARVC in athletes can occur frequently because the assessment of regional functional impairment of the RV wall is subjective and depends on the operator’s personal experience. Finally, athletes may also exhibit a mild RVEF decrease ,12,48.Fortunately, there are some significant differences between AH and ARVC. The first difference is that in AH, unlike ARVD, the dilation of the RV is associated with a concomitant LV volume increment, due to a global and symmetrical adaptation of the heart induced by training. For this reason, a ratio of RVEDV/LVEDV < 1.2 has been proposed to distinguish between physiological remodeling and ARVC. The second difference is that AH does not show the regional contractile RV abnormalities and has normal strain and strain rate, differently ARVC may have strain reduction. The third difference concerns the RVEF. In athletes, although there may be a mild reduction in RVEF, rarely this parameter is < 45%, as occurs in patients with ARVD ,49.Tissue characterization is not included in the CMR diagnostic criteria for ARVC. However, it may represent an important element of differential diagnosis because the presence of LGE in the free wall of the RV with morphological and functional abnormalities can be useful to support the diagnosis of ARVD. However, some possible difficulties in assessing the LGE need to be considered. The first pitfall concerns the identification. Indeed, the diagnosis of LGE in the thin RV wall may be problematic. However, notable advances in MR scanners and rapid pulse sequence updates have improved this possibility. The second pitfall is about the interpretation. Indeed, although the LV does not show dilation and dysfunction, CMR reveals a subepicardial LGE stria at the infero-lateral LV wall, up to 70% of patients with ARVD. The underlying disease process of ARVC affects the entire myocardium and left myocardial LGE can be demonstrated in ARVC patients, even in the absence of LV regional function abnormalities. For the differential diagnosis, it is essential to remember that LGE, in AH, is usually mesocardial at anterior and/or posterior interventricular junctions ,49.Finally, in AH, the exercise-induced remodeling of the RV is a dynamic process and can decrease or increase in relation to the training load. Conversely, in ARVC, RV dilation is irreversible ,49.LDAC is characterized by mild LV dilation without RV involvement, and the fibro-fatty replacement initially concerns the subepicardial layer of the LV infero-lateral wall ,51.In athletes, LDAC causes ventricular arrhythmias during exercise and the probability of detection with the ECG and echocardiography is low. For this reason, the identification of myocardial fibrosis replacement may be useful ,51.The occurrence of exercise-induced repetitive premature ventricular beats with a right bundle-branch block may merit the cardiac evaluation with the use of CMR to rule out the subepicardial scar in the infero-lateral LV wall, which is the imaging hallmark of LDAC and may represent the only abnormal cardiac finding suggesting the differential diagnosis with AH ,51.Infiltrative cardiomyopathy (IC) is a non-ischemic cardiomyopathy that includes a variety of myocardial disorders, resulting from the deposition of abnormal substances within extracellular and intracellular cardiac spaces. The main IC disorders are cardiac amyloidosis, sarcoidosis, and hemochromatosis and cause a gradual wall thickening, dysfunction up to heart failure .The AH is characterized by a slight thickening of the LV myocardium and differential diagnosis with IC is required. Morphologically, IC is characterized by myocardial thickness, normal ventricular size, and atrial dilation. IC causes diastolic dysfunction, with reduction of CMR-strain values .Furthermore, mapping, ECV, and LGE are crucial for the differential diagnosis. T1 mapping and ECV values are higher in cardiac amyloidosis and sarcoidosis than in AH. Differently, in hemochromatosis, T1 values are lower due to the iron deposition. LGE in cardiac amyloidosis typically has a diffuse circumferential endomyocardial distribution. Unlike in cardiac sarcoidosis, LGE has a nonspecific distribution, but usually involves the interventricular septum or the LV lateral wall and papillary muscles with a patchy mesocardial layer pattern and relative sparing of the subendocardial layer .AH is associated with structural cardiac changes due to intense and prolonged exercise. These modifications frequently simulate those observed in cardiomyopathies that represent one of the leading causes of sudden cardiac death in athletes.Correct differentiation between physiological AH changes and cardiomyopathies requires a comprehensive knowledge of the main imaging features and an in-depth consciousness that the cardiac adaptations are related to age, sex, ethnic characteristics, and, above all, intensity and duration of the training exercise.In this context, the radiologist, specialized in cardiac imaging, can have a pivotal role in differentiating between AH and cardiomyopathies, as CMR and CCT are particularly useful when there are nonspecific or inconclusive findings by electrocardiography or echocardiography.


Proactive aggression (PA) is a planned and unprovoked form of aggression that is most often enacted for personal gain or in anticipation of a reward. Frequently described as “cold-blooded” or goal oriented, PA is thought to be associated with low autonomic arousal. With this view in mind, we performed a scoping review of the biological correlates of PA and identified 74 relevant articles. Physiological findings indicated a robust association between PA and reduced resting heart rate, and to a lesser extent a relationship between PA and decreased heart rate and skin conductance reactivity, perhaps indicating dampened sympathetic function. The twin literature identified PA as a heritable trait, but little evidence implicates specific genes in the pathogenesis of PA. Neuroimaging studies of PA pinpoint impaired amygdala function in the assessment and conditioning of aversive stimuli, which may influence the establishment of behavioral patterns. Nodes of the default mode network were identified as possible neural correlates of PA, suggesting that altered function of this network may be involved in the genesis of PA. Given the overlap of PA with reactive aggression and the overall behavioral complexity of PA, it is clear that multiple endophenotypes of PA exist. This comprehensive review surveys the most salient neurobiologically informed research on PA. Aggression is defined as the noxious behavior of one individual directed at another person or object, in which verbal or physical force is used to injure, coerce, or express anger ,2. WhileThe bimodal distinction between reactive (RA) and proactive aggression (PA) is one of the most widely utilized aggression classification schemes ,5,6. RA Aggression research to date has largely focused on reactive types of aggression and most studies that evaluate PA do so in tandem with RA. To the best of our knowledge, no review to date has evaluated the biological underpinnings of PA. In an attempt to address this gap, we conducted a scoping review of the extant literature on the biological correlates of PA in humans. The aims of this review were the following: (1) to algorithmically identify all studies in the extant literature that explicitly link PA to some aspect of neurobiology, including brain structure and function; (2) to group the identified articles according to area of study and critically review and contrast research findings; and (3) to identify significant gaps in the literature to guide future research.There are several validated tools used to quantify PA. In general, the available methods can be classified according to the following three categories: (1) survey-based assessments, (2) laboratory-based paradigms, and (3) using historical behaviors as a proxy for PA. Survey-based evaluations are relatively inexpensive, simple to administer, and are the most commonly used, followed by aggression paradigms and historical behaviors. However, recognizing the corresponding strengths and limitations of the various tools is critical when selecting the most appropriate methodology.Dodge and Coie  were theAggression paradigms are laboratory-based measures that employ simulated situations to elicit quantifiable aggressive behavior in the subject. Typically, the subject is instructed to compete against a fictitious opponent and is incentivized with points or a monetary reward. The subject may aggress the opponent by interfering with, stealing from, or by administering punishments , where PA is measured as the intensity and number of aggressions the subject directs at the opponent in the absence of provocation. Commonly used paradigms include the Taylor Aggression Paradigm (TAP)  and the Lastly, PA may be inferred by using past behavior as a proxy. Data are most often obtained from offending records, which allows subjects to be categorized as having engaged in PA or not. This approach theoretically measures organic PA that is not influenced by data collection methods. However, it is difficult to label aggressive behavior as purely proactive in nature, especially when data are gleaned only from records. This methodology also assumes that past behavior is predictive of current and future behavior. Past aggression remains the most consistent and stable predictor of future aggression . PA scorTo inform our review, the following keywords, in various combinations, were entered into MEDLINE/PubMed, PsychINFO, and EMBASE databases on May 2020: proactive, premeditated, predatory, planned, unprovoked, offensive, instrumental, or cold-blooded aggression; AND genetic, twin, neuroimaging, neurotransmitter, physiological, or hormone. We restricted our search to articles published in English and excluded animal studies. We scanned systematic reviews and meta-analyses to locate additional articles that were not captured by our search terms.Our search identified a total of 6113 articles . Of the The psychophysiology of aggression measures the biological expression of mental processes in an attempt to identify differences that may be characteristic of aggressive behavior. The so-called “fight or flight” response is controlled by the sympathetic and parasympathetic branches of the autonomic nervous system (ANS) in order to regulate critical life function . The symThe parasympathetic nervous system (PNS) controls the conservation of energy and functions to return the body to equilibrium. Heart rate variability (HRV) and respiratory sinus arrhythmia (RSA) are two parameters commonly used to assess PNS activity. HRV is the beat-to-beat variation in the heart and RSA refers to transient increases and decreases in HR that correspond to inspirations and expirations due to the influence of the vagus nerve on the sinoatrial node of the heart ,117,118.It has been suggested that autonomic under-arousal may underlie PA . Two posXu and colleagues  studied In an attempt to disentangle the autonomic differences between childhood RA and PA, Scarpa and coauthors  evaluaten = 585). PA was negatively correlated with RHR in males, which may have been influenced by higher mean PA scores for males than females. The researchers hypothesized that low RHR is driven by temperament, whereas HRR is related to life experiences, such as early physical abuse or chronic social rejection. Relatedly, others have reported negative associations between PA and RHR and PA and HRR among adolescent boys )Several limitations of this review should be addressed. Notably, our review did not include animal studies. Although PA can be measured in rodent models , animal In conclusion, we report that the neurobiological correlates of PA represent a diverse area of study that goes far beyond the “cold blooded” characterization. The majority of PA findings published to date are based on community samples; thus, bridging the gap between “normal” and pathological PA remains an important research priority. As our review revealed that multiple PA endophenotypes likely exist, future research should focus on defining the corresponding genetic and neurological predictors in an attempt to further disentangle PA as a complex behavior. Furthermore, elucidation of the specific neural structures, networks, and implicated neurotransmitters in PA could only enhance the translation of findings to treat PA with more precision, utilizing behavioral and pharmacological methods.


In addition, they were older at repair (p = 0.002) but underwent earlier pulmonary valve replacement (PVR) . On multivariate Cox regression analysis, adjusted for age at first repair, LV dysfunction remained significantly more associated with DS than del22q11.2 and non-syndromic patients . There were only four episodes of non-sustained ventricular tachycardia in our cohort. Conclusions: Among the cMRI parameters commonly taken into consideration in rToF patients, LV dysfunction seemed to be the only one affected by the presence of a genetic syndrome. The percentage of patients performing PVR appears to be similar in both populations, although syndromic patients were older at repair and younger at PVR. Finally, the number of arrhythmic events in rToF patients seems to be low and unaffected by chromosomal abnormalities.Background: The impact of genetic syndromes on cardiac magnetic resonance imaging (cMRI) parameters, particularly on right and/or left ventricular dysfunction, associated with clinical parameters following the repair of Tetralogy of Fallot (rToF) is not well known. Therefore, this study aimed to assess the differences in clinical, surgical, and cMRI data in syndromic and non-syndromic rToF patients. Methods: All syndromic rToF patients undergoing a cMRI without general anesthesia between 2010 and 2020 who were able to match with non-syndromic ones for birth date, sex, type of surgery, timing of cMRI, and BSA were selected. Demographic, clinical, surgical, MRI, ECG, and Holter ECG data were collected. Results: A total of one hundred and eight rToF patients equally subdivided into syndromic and non-syndromic, aged 18.7 ± 7.3 years, were studied. Del22q11.2 and Down syndrome (DS) were the most frequent syndromes . Regarding the cMRI parameters considered, left ventricular (LV) dysfunction (LVEF < 50%) was more frequently found in syndromic patients ( Tetralogy of Fallot (ToF), the most common cyanotic congenital heart disease (CHD), can be associated with various genetic syndromes ,2. The mTherefore, in addition to the different perioperative outcomes, syndromic patients with ToF could have a worse long-term outcome when compared to non-syndromic ones ,9,10,11.This would mean that despite similar clinical characteristics, syndromic patients could have greater right ventricular (RV) dilation, which could lead to worse RV or left ventricular (LV) dysfunction, worse exercise capacity, and more frequent ventricular arrhythmic events. Consequently, they could require considerable attention and shorter time interval between each follow-up visit and be submitted to pulmonary valve replacement (PVR) more frequently than non-syndromic patients. This may be even more true in DS, as proposed by Al-Biltagi et al. ,14, who Despite the interesting and useful nature of this topic, little is known about the impact of the most common syndromes on the long-term follow-up after rToF ,17,18,19Therefore, this study aims to compare two groups of rToF patients (syndromic vs. non-syndromic) with similar clinical and surgical characteristics to evaluate the impact of the different associated syndromes on cardiac magnetic resonance imaging (cMRI) data, with particular attention to the ejection fraction of both ventricles, QRS duration and adverse cardiac events. In addition, we sought to highlight the main differences between the two most represented syndromes in rToF: DS and del22q11.2. given that these are the most frequent syndromes associated with ToF.2; RV end-systolic volume indexed by BSA (RVESVi) ≥ 80 mL/m2; RV ejection fraction (RVEF) ≤ 47%; pulmonary regurgitation fraction (PRF) ≥ 40%; LV ejection fraction (LVEF) ≤ 55%; moderate or greater tricuspid regurgitation; RV outflow tract obstruction with RV systolic pressure ≥ 0.7 systemic  and compared with normal values published by Kawel-Boehm et al. [The imaging data collected were analyzed offline on a separate workstation using a cardiac post-processing software . LV and RV volumes were measured via manual segmentation of the endocardial border of both ventricles on short-axis cine images at end-diastole and end-systole, which was subsequently calculated using the method of discs . Papillam et al. . In eachm et al. ,28. A seIn addition to RV pressure ≥ 45 mmHg in echocardiography, the presence of two or more of the following cMRI criteria was required to establish the diagnosis of pulmonary artery/RV outflow tract stenosis: (1) flow velocity across the RV outflow tract or a branch pulmonary artery ≥ 3 m/sec, (2) abnormal pulmonary artery size, (3) blood flow maldistribution . RV dysfunction was taken into consideration in patients with a RVEF ≤ 47%, and LV dysfunction in the presence of a LVEF ≤ 50%. p-value < 0.1 at the univariate analysis into the multivariable logistic regression models. Afterward, the backward stepwise regression method was used to select the best fit. Subsequently, variables that did not show a statistically significant effect on the prevalence of LV dysfunction (p ≥ 0.05) were removed from the multivariable models. Associations between the investigated variables and the likelihood of LV dysfunction over the follow-up period were estimated using hazard ratios (HR) and their 95% confidence intervals (95% CI). The Cox proportional hazards model was applied to calculate each clinical variable’s adjusted relative hazards of outcome events. The forward stepwise regression model based on the Akaike information criterion was used to select the multivariable analysis model. Only variables with a p-value < 0.1 in the univariate Cox regression analysis were subsequently introduced into the multivariable Cox regression model. Only p values < 0.05 were considered statistically significant. All tests were two-tailed, and the analyses were performed using computer software packages .The Shapiro–Wilk test was used to assess all continuous variables for normality and for the examination of their histograms. As appropriate, data are presented as percentages and frequencies, mean ± standard deviation, or median and 25–75th percentile . Unadjusted differences were compared using the Wilcoxon Sum Rank Test or t-test, as appropriate. Either Fisher’s exact or the Chi-Square test were used to compare dissimilarities in categorical variables, as appropriate. In order to identify predictors of LV dysfunction, univariate and multivariate logistic regression models were used. We then introduced the variables that had a A total of 108 rToF patients were selected from our cohort. Fifty-four syndromic patients were matched with 54 non-syndromic ones. All characteristics of each group are reported in n = 5), VACTERL (n = 3), Goldenhar (n = 2), Williams (n = 2), Kleefstra (n = 1) and subterminal del 10p15.1 + dup 10p14 (n = 1). The latter showed hypoplastic pulmonary branches. The comorbidities found in our syndromic population were: 4 obesity, 1 hypertension and 8 thyroid dysfunctions  and 17 (31.5%) patients, respectively. In the remaining 14 patients (25.9%), the following syndromes were detected: Noonan (unctions . Thyroidunctions .p = 0.002) than non-syndromic ones. On the contrary, they were slightly younger at PVR , although the percentage of PVR was similar in the two groups. Regarding the MRI parameters, we observed a major presence of LV dysfunction in the syndromic group compared to the non-syndromic one , besides the lower values of LVEDVi  (p = 0.031) and higher PRF  (p = 0.030). There was a low incidence of adverse cardiac events during the study period (only four episodes of NS-VT). Finally, DS remained an independent prognostic factor for LV dysfunction, compared to del22q11.2 and non-syndromic patients with an HR of 5.245  during a median follow-up of 14.5 years (QR 12–23)  . When co= 0.021) . SurprisR 12–23)  on multiOur study, comprising syndromic rToF patients, showed that, among the cMRI parameters usually evaluated in this cardiac disease, LV dysfunction seemed to be the only one significantly affected by the presence of a genetic syndrome. In addition, our study has documented that Down and DiGeorge syndromes, in the absence of extracardiac features, do not appear to have worse outcomes at follow-up in terms of fatal arrhythmic events, sudden death, and PVR rate. These data agree with Blais et al., who document similar long-term survival in 960 ToF patients for both the “classic” non-syndromic ToF group and “classic” ToF with genetic conditions  when evap = 0.053). Blais et al. documented that the presence of genetic conditions could be unassociated with the number of cardiovascular interventions in rToF, except for 22q11 deletions in subjects with ToF and pulmonary atresia [In our cohort, the incidence of PVR was similar in both groups, but syndromic ToF patients were significantly younger at the time of the procedure , as already shown by Michielon et al. ,11. NeveWe did not find a significant difference in the QRS duration between syndromic and non-syndromic patients. On the contrary, DS patients showed shorter QRS duration compared with del22q11 patients. This could be due to the different types of surgery performed in the two groups . Finally, the incidence of arrhythmic events in our overall population was very low, and therefore, although the incidence of arrhythmic events seems not to be affected by the syndrome, we could not draw any conclusions on this matter.This study presents some limitations. Firstly, estimating the differences in transannular patch sizes in our cohort was not possible. Similarly, predicting the extent to which these patches may impact ventricular function and the degree of RV dilation, in addition to the well-known pulmonary insufficiency, is also quite challenging. Moreover, we were unable to correlate ventricular dysfunction with the possible presence and extent of delayed enhancement because this parameter was not taken into consideration, since it had not been evaluated in all patients. Finally, the small number of patients in our cohort may have limited the statistical power of the analysis by potentially failing to identify other significant associations.Our study demonstrated that syndromic ToF patients could have a higher incidence of LV dysfunction than non-syndromic patients, independently from the surgical technique, stage, and age at repair evaluated by cMRI 15 years after repair. However, the incidence of adverse events in the follow-up period examined seems not to have been affected by the presence of a genetic syndrome in the absence of additional extracardiac anomalies.


Heart development is topographically complex and requires visualization to understand its progression. No comprehensive 3-dimensional primer of human cardiac development is currently available. We prepared detailed reconstructions of 12 hearts between 3.5 and 8 weeks post fertilization, using Amira® 3D-reconstruction and Cinema4D®-remodeling software. The models were visualized as calibrated interactive 3D-PDFs. We describe the developmental appearance and subsequent remodeling of 70 different structures incrementally, using sequential segmental analysis. Pictorial timelines of structures highlight age-dependent events, while graphs visualize growth and spiraling of the wall of the heart tube. The basic cardiac layout is established between 3.5 and 4.5 weeks. Septation at the venous pole is completed at 6 weeks. Between 5.5 and 6.5 weeks, as the outflow tract becomes incorporated in the ventricles, the spiraling course of its subaortic and subpulmonary channels is transferred to the intrapericardial arterial trunks. The remodeling of the interventricular foramen is complete at 7 weeks. Hikspoors et al. generate a pictorial account of human embryonic heart development between 3.5 and 8 weeks. This resource provides detailed, interactive 3D anatomical models and quantifies the developmental milestones of key structures in cardiac function. Other examples are Blechschmidt’s models and drawings of human embryos4, and van Mierop’s images of the developing heart5, which were redrawn by Netter6. All these successful approaches have in common that medical artists collaborated with embryologists who had artistic capacities themselves. Because the methods used to make the models were labor-intensive, existing illustrations were often modified rather than new versions being created. An example, documented in detail7, is the frequently cited treatise of Kramer on the septation of the outflow tract8. Such serial modifications, however, tend to propagate concepts rather than observations, and need to be assessed with caution.Embryology is a visual discipline. Many aspects of embryonic development are topographically complex, such that three-dimensional (3D) models are exceedingly helpful in fully understanding temporal events. Examples of often used or cited classical models are Born’s “Plattenmodellen”, and Ziegler’s freehand models of embryos, which were studied and described by His9. This atlas, however, does not address the development of the heart in any detail. Both qualitative10 and quantitative11 tabulations are available for the development of the human heart. Since nomenclature in the developing heart is notoriously variable, a combination of text and illustrations is necessary to provide an understandable account. In this respect, the description of human cardiac development based on magnetic resonance or fluorescent episcopic microscopy is instructive12. The spatial resolution and differential staining properties of these techniques, however, are still limited. To our knowledge, no comprehensive primer of cardiac development is presently available that is based on first-hand segmentation of structures of interest identified in histological sections. Our study has visualized such development in human embryos between 3.5 and 8 weeks of development, extending from Carnegie stages 9 through 23. We describe each of the stages, and the features distinguishing them from the previous stage. The evidence can be inspected in the corresponding interactive 3D-pdfs . By CS9, Hensen’s node, which localizes gastrulation, is found at the caudal end of the columns of somitomeres.The heart becomes morphologically identifiable when ~26 days have passed since fertilization15. At CS8, midway through gastrulation, bilateral heart fields, also known as cardiogenic plates, form craniolaterally in the embryonic mesoderm16. The cranial part of these morphologically still indistinct precursors forms the “first heart field”, and differentiates to form cardiomyocytes during or shortly after the formation of the first somite. The remaining, more caudally located and still proliferating precursors, which form the “second heart field”, start to differentiate at the transition of CS9 into CS1020. Cardiomyocyte contractions arise in the lateral regions of the cardiac crescents of mouse embryos as soon as sarcomeric assembly and Ca2+ transients can be demonstrated22. Visible contractions develop in mouse embryos when 3 somites have formed23, while the first cardiac contractions in human embryos can be visualized with ultrasound at days 28 after conception24. The first heart field can be visualized in mice by the expression of transcription factors Tbx526 and Hcn428, and the second heart field by the expression of the transcription factor Isl129. Fate-mapping studies in mice have shown that the first heart field contributes virtually all cardiomyocytes of the embryonic left ventricle, three-quarters of those in the atrioventricular canal, and half of those making up the atriums and right ventricle. The more slowly evolving second heart field, on the other hand, contributes virtually all cardiomyocytes of the outflow tract, half of the atria and right ventricles, and one-quarter of the atrioventricular canal30. The systemic venous sinus is a separate part of the second heart field that does not express the early cardiogenic transcription factor Nkx2-5, but does express Tbx1832. Based on molecular mouse data, we infer that the center of the heart develops first, and that the upstream venous and downstream arterial components are added successively.The heart is located at the cranial margin of the embryo33 of embryos with ~5 somites, show that the early heart is, like that in mouse embryos34, bilaterally symmetrical. It consists of two tiny vascular networks that course in front of the foregut, with only few vessels connecting them across the midline. Both vascular networks are surrounded by paired, but partially merged swellings of acellular cardiac jelly. The jelly is enclosed, in turn, within an unpaired, bilaterally symmetrical pericardial cavity with a myogenic visceral wall15. The distribution of the jelly, which is produced by the endoderm and the visceral pericardial wall35, reflects the location of the boundaries of the differentiating left ventricular myocardium. The initially non-luminal endocardial tubes of the cardiac vascular network gradually canalize, but at first contain only few erythrocytes. At the venous pole, the heart tubes are continuous with an extensive venous plexus on the periphery of the endoderm. At the arterial pole, near the buccopharyngeal membrane, the heart tubes pass the pharynx laterally to join the paired dorsal aortas. In front of the heart, the primordium of the transverse septum forms as a shelf of thick mesoderm between the endoderm and the pericardial cavity . The single channel has the embryonic left ventricle as its caudal, and the embryonic right ventricle as its cranial component. The umbilical vein, which occupies the boundary of embryonic disk and amnion, and the vitelline plexus, which is situated on the yolk sac, merge at the level of the 4th somite to form the hepatocardiac channels37. These channels, in turn, join the systemic venous inflows to the heart. At this stage, both arms of the cardiac inflow tract are transversely oriented vessels, merging in the midline  are indicative of continuing differentiation. The beginning of ballooning of the right atrium, and the leftward transfer of the atrioventricular canal permit the recognition of laterality . This laterality involves differences in both lineage31 and phenotypic properties50 of the right and left atriums. The apical part of the embryonic left ventricle is also beginning to balloon at the outer curvature of the loop. The outflow tract still bifurcates just ventral to the pharynx into the ventral aortas, which continue dorsally on either side of the pharynx to join both dorsal aortas. The dorsal aortas extend caudally to the dorsal wall of the cloaca and have not yet started to fuse. Near the caudal part of the junction of the midgut and stalk of the yolk sac, an arterial plexus with multiple roots in the ventral wall of the dorsal aorta begins to extend ventrally towards the yolk sac. This plexus represents the earliest stage in the development of the celiacomesenteric trunk. On the left side, one arterial vessel has a markedly bigger diameter than all other components of the arterial network. Slightly more caudal, yet another plexus with multiple roots in the dorsal aortas extends along the cranial wall of the cloaca towards the connecting stalk. This plexus represents the earliest stage in the development of the umbilical arteries. The configuration is remarkably similar to that reported by Tandler in a human embryo with 14 somites51.The expansion and the medial fusion of the myocardial walls of the atriums . At the venous and arterial poles, the heart retains its midline connections with the pharyngeal mesenchyme through the remaining parts of the dorsal mesocardium.The appearance of the transverse pericardial sinus in CS10 embryos marks the beginning of cardiac looping. The accompanying rightward tilt of the arterial pole, and the leftward tilt of the embryonic left ventricle, are further overt and early signs of asymmetry in these young embryonic hearts Fig. . In mice13. The reconstructed specimen is shown in Supplemental Fig. 60. A still blind mid-pharyngeal strand, which will eventually canalize to form the common pulmonary vein, can be recognized penetrating the dorsal mesocardium between the arms of the inflow tract . This helical configuration can be shown by the expression pattern of the left-sided marker Pitx2c73, by lineage tracing74 and by the course of the endocardial ridges formed in the outflow tract75. The spiraling direction is clockwise when looking in downstream direction. Cells from the left side of the caudal second heart field end up cranially (superiorly) in the left atrium and atrioventricular canal, whereas those from the right side end up caudally (inferiorly)49. Similarly, the parietal and septal ridges spiral from the right and left side of the proximal myocardial outflow tract to the left and right side of the distal myocardial outflow tract, respectively. The walls of other structures that form loops, such as the intestines76, follow strikingly similar courses. During this phase of looping, the elongating muscular outflow tract forms an acute bend between its transversely oriented proximal part, which is also known as the “conus”, and its ventrodorsally oriented distal part, also known as the “truncus”8. The pronounced “bayonet”77 or “dog-leg” bend78 between these parts marks the junction. This bend may be a critical structural element for effective valveless pumping in these early hearts79. The presence of the bend permits the outflow tract to be described as having proximal and middle parts, which are myocardial, with the non-myocardial distal part being added when the arterial trunks begin to form in CS15 embryos , and continue cranially into the carotid arteries.By this stage, it is possible to recognize the first two pharyngeal arches, along with their accompanying arteries. The vascular space within the ventral pharyngeal mesenchyme that connects the outflow tract with the arteries of the pharyngeal arches is known as the aortic sac. In mice, the endothelium of the first two pharyngeal arches shares its lineage with that of the dorsal and ventral aortas, but differs from that of the subsequent pharyngeal arch arteries13, is considered “phylotypic”. This is because morphologic features and profiles of gene expression are most similar among vertebrate embryos at this stage14. The reconstructed specimen is shown in Supplemental Fig. 81. The heart, within its pericardial cavity, remains surrounded by the transverse septum, the pharynx, and the forebrain. Due to the rapid growth of the brain and foregut between CS9 and CS14, the transverse septum gradually changes in orientation from frontal at CS9 to near-transverse at CS11 . In mice, the myocardial walls of the horns differ from those of the atrial chambers and the pulmonary vein in developing from an Nkx2-5-negative, Tbx18-positive lineage82. By this stage, furthermore, the systemic venous sinus drains exclusively into the right side of the atrial chambers through the right-sided sinuatrial junction .Myocardium has appeared on the epicardial side of the asymmetrically expanding systemic venous sinus, permitting the definition of the sinus horns . The pulmonary vein, which passes between the atrial extensions of the atrioventricular cushions and through the dorsal mesocardium, has now canalized so as to connect with the venous plexuses developing ventral to the lung buds. The rightward margin of the dorsal mesocardium . Cell multiplication at its base and a circumferential growth pattern92 bring about a sharp boundary between the left and right sides of the septum93. Its crest forms the caudal margin of the interventricular foramen, with the inner curvature forming the cranial margin . Meanwhile, endocardial cells that undergo epitheliomesenchymal transformation also populate the endocardial jelly120. The initially more numerous75 neural crest cells are necessary for correct positioning of the ridges, and for patterning of the arterial valvar leaflets119. The feature, therefore, that distinguishes these ridges from the endocardial cushions of the atrioventricular canal is the presence of neural crest cells. For this reason, we describe the outflow entities as ridges, rather than cushions. The prongs within the ridges take a clockwise-spiraling course when observed in the downstream direction, occupying septal and parietal locations at their junction with the developing right ventricle .Cells derived from the neural crest, which surround the arteries of the pharyngeal arches, begin to indent the dorsal wall of the aortic sac. They form a protrusion between its cranial portion, which connects to the arteries of the 3rd and 4th pharyngeal arches, and its caudal portion, which connects to the arteries of the 6th pharyngeal arch. The neural crest cells extend ventrally, having embraced the aortic sac bilaterally, and from there invade the endocardial jelly of the outflow tract as prongs of dense mesenchyme122. These cells of the second wave are dorsally continuous with, and probably originate from a phenotypically similar mass of pharyngeal mesenchyme surrounding the trachea124. This “club” of mesenchyme forms during CS13, and remains an identifiable entity during CS14 and CS15  confined to a small subsection of the middle portion of the outflow tract near the dog-leg bend , suffered from venous congestion. To decide whether or not this condition was atypical, we surveyed the Blechschmidt collection of human embryos. Supplemental Fig. 130. The left hepatocardiac channel was still discernable in all four CS14 embryos, and in one of the six CS15 embryos of the Blechschmidt collection. The latter embryo did not suffer from venous congestion. The configuration of the atrial chambers and their topographic relation with the pulmonary vein are similar to those in the previous stage63. The breakdown of the dorsal portion of the primary atrial septum has created, however, a secondary atrial foramen. In the Blechschmidt collection, we found two CS15 embryos who had not yet formed a secondary foramen and two others who had, like the reconstructed specimen, both primary and secondary foramens. Accordingly, McBride et al.11 reported that ~50% of the CS15 embryos in the Carnegie collection had developed a secondary foramen. We found no notable changes in the architecture of the ventricles. The GlN-positive interventricular ring is still a planar structure131, but a widening of the crest of the muscular ventricular septum identifies the developing branching component of the atrioventricular conduction axis132.At this stage, ~36 days have passed since fertilization133. By this stage, the arteries of the left and right 6th pharyngeal arches have each given rise to a pulmonary artery, which extends caudally within the pharyngeal mesenchyme along the trachea. The most pronounced developmental changes are to be seen in the arrangement of the middle portion of the outflow tract and the aortic sac. Continued axial growth within the myocardial part of the outflow tract . In consequence, the aortic sac acquires a dextrocranial systemic component, which connects the subaortic part of the outflow tract with the arteries of the 3rd and 4th pharyngeal arches, and a sinistrocaudal pulmonary component, which connects the subpulmonary part of the outflow tract with the arteries of the 6th arches .This stage is reached at ~38 days after conceptionges Fig. . With co139). Subsequently, the cranial portion of the interventricular foramen will evolve into the channel between the left ventricle and the subaortic outlet . This increasingly spiraling course of the intrapericardial arterial trunks corresponds in time with the unwinding of the spiraling course of the muscular outflow tract and its endocardial ridges142 . Accordingly, a wide secondary foramen has formed due to the breakdown of the dorsal portion of the primary atrial septum in these hearts. The site of fusion of the atrioventricular cushions in the atrium is reinforced on its right side by the dorsal mesenchymal protrusion, or vestibular spine. Myocardium surrounds the stem of the pulmonary vein, which from this stage onwards begins to expand radially, suggesting an increase in blood flow . In overall architecture, nonetheless, it undergoes an impressive change in appearance. Its length decreases abruptly to <50% of its original length in the ~2 days separating CS16 from CS17 . Much of the effective shortening can be attributed to it changing from a long tubular configuration at CS16 to a shorter figure-of-eight configuration at CS17 , and the addition of new cardiomyocytes from the second heart field has ceased, the fishmouth has lost its characteristic appearance. The non-myocardial cells that continue to populate the distal outflow tract, now interpose between the distal margin of the myocardial walls and the pericardial reflection. Consequently, the myocardial boundary moves away from the pericardial reflection.Since the myocardium of the outflow tract hardly proliferates . The myocardializing area is continuous on its medial side with the crest of the muscular ventricular septum and on its lateral side with the parietal wall of the myocardial outflow tract166. This myocardialization occurs in mammals173 and birds153. It progresses from proximal to distal, in other words, opposite to the direction of septation. In mice, myocardialization of the dorsal mesenchymal protrusion and the fused proximal ridges of the outflow tract both seem to be associated with apoptotic activity in the underlying mesenchymal layer174.The wedge shape of the myocardial component of the outflow tract, with its blunt side over the outer curvature, and the sharp edge in the inner curvature, becomes more pronounced. The septal and parietal endocardial ridges have now fused across their entire length Figs. , and 9b.150. The position of the arterial valves was previously put at the dog-leg bend175. In reality, the arterial roots, containing the semilunar valvar leaflets, have significant length, and derive from the entire middle portion of the outflow tract. Their proximal boundary corresponds roughly with the dog-leg bend, which is present between the two parts of the myocardial outflow tract until CS16. At CS18, this boundary is marked by the transition of the thin layer of myocardium that surrounds the middle outflow tract into the much thicker myocardium of the proximal outflow tract. Their distal boundary, the sinutubular junction, corresponds with the distal end of the endocardial ridges . It is against this fold, which is incorrectly known as the secondary atrial “septum”, that the primary septum will eventually rest to close the oval foramen.At this last Carnegie stage, ~56 days have elapsed since fertilization190. Fragmentation of the myocardial floor of the tricuspid gully gives atrial blood access to the right ventricular cavity via conduits that pass the septomarginal trabeculation cranially (pre-existing) and caudally (newly formed)139. In both ventricles, the papillary muscles begin to form by consolidation of aggregating trabeculations, with the compaction starting at the valve leaflets and moving in the direction of the compact ventricular walls. Epicardially derived cells have begun to induce insulation within the atrioventricular junctions, and have populated the lateral cushions191. During CS21 and CS22, the whorl of neural crest cells in the ridges of the proximal outflow tract all but disappears, while myocardialization continues. As already explained, the so-called tendon of the conus, a cord-like band between the aortic and pulmonary roots172, is an inconsistent distal remnant of the whorl. The still long subaortic outlet now passes between the developing mitral valve, the muscular ventricular septum, and the muscularized septum in the proximal outflow tract. The subpulmonary outlet passes between the muscularized septum in the proximal outflow tract and the free right ventricular wall.By now, the leaflets of the atrioventricular valves are forming, although tendinous cords have yet to develop. The leaflets, furthermore, still contain myocardium on their ventricular surface156, and transforms the transitory outlet septum into the smooth medial wall of the right ventricle, the dorsocranial part of which becomes the “free-standing” muscular subpulmonary infundibulum171. The attribution of the muscular septum of the outflow tract as a mostly right ventricular structure131 can be best appreciated if the ventricular cavities, muscular septum, and (sub-)aortic and (sub-)pulmonary channels are observed from the left . During CS9, the first heart field produces the cardiomyocytes of the embryonic left ventricle and a fraction of those in the atrioventricular canal, atriums, and right ventricle. Ongoing addition of cardiomyocytes from the second heart field to the venous and arterial poles of the first heart field, and differentiation into cardiac compartments, is therefore necessary to form the definitive heart. During CS10, the embryonic right ventricle arises by the addition of cardiomyocytes from the cranial second heart field, followed by the proximal myocardial portion of the outflow tract during CS11 and the distal myocardial portion during CS12. Meanwhile, the caudal second heart field produces the cardiomyocytes of both atriums during late CS10 and early CS11, while those of the venous sinus are added during CS12. The pharyngeal arch arteries are successively added between CS12 and CS14, while the neural crest cells invade the endocardial ridges of the outflow tract during CS14. Endocardial cushions and ridges form during CS14 to allow for separate blood flows. The non-myocardial distal portion of the outflow tract, from which the arterial trunks form, begins to appear at CS15135.Supplemental Table 87. The non-trabeculated distal infundibulum  part of the right ventricle, on the other hand, originates from the incorporation of the smooth-walled proximal outflow tract starting at CS17193. The central structures in the heart, such as the atrioventricular canal, inner heart curvature, and muscular outflow tract, which temporarily retain their relatively undifferentiated status as remnants of the primary heart tube, subsequently contribute to the internal remodeling that is necessary to achieve septation112. The process of septation achieves separate pulmonary and systemic circulations, and is completed by the closure of the middle portion of the interventricular foramen at CS20. It is not known, to our knowledge, whether the completion of septation is associated with a new functional capacity. Since the timing corresponds with the transition of the yolk sac to the (hemo-)chorial placenta194, we submit that the enhanced pumping efficiency or capacity is a determining factor.This “peripheral growth” model of heart development ends when the building plan of the heart has been established. We consider the early ventricles “embryological”, because they still stand to receive substantial additional cell contributions. The free wall of the left ventricle receives an extensive contribution of cardiomyocytes from the adjacent part of the atrioventricular canal starting at CS13184 embryos have developed 30–36 somites195, which makes them comparable to Theiler stage 16 mouse embryos196. This stage is considered “phylotypic” because the basic body plan of vertebrate embryos has been established at this stage and gene-expression profiles between model species of vertebrate groups are most similar14. Subsequent to this very conserved stage, CS14 is characterized by the appearance of an array of new features, such as the venous valves, the primary atrial septum, a patent pulmonary vein, the muscular ventricular septum, the ridges in the outflow tract, the cardiac neural crest, the non-myocardial walls of the distal outflow tract, the beginning transformation of the aortic sac into the arterial trunks, and the appearance of the last and special pair of pharyngeal arch arteries113. The transition of CS13 to CS14 does not, of course, proceed abruptly, but should be considered as a change in developmental pace.The description of the respective developmental stages required an unexpected difference in the number of words needed to delineate stage-specific differences. On average, 500–800 words sufficed to describe the incremental changes in heart development for most stages, but the description of CS12, CS14 and CS(17 + 18) required double that number. The text of CS12 exceeds the average length of a section because it includes the description of the looping of the heart tube. Human CS13 or “Horizon XIII”197. Perhaps to prepare for extrauterine survival marsupials or for perfusion of the (hemo-)chorial placenta in eutherian mammals, the heart extensively remodels during CS17 and CS18. It is at these stages that fusion occurs between the superior and inferior atrioventricular cushions, and between the septal and parietal ridges of the outflow tract. During the same period, the interventricular foramen remodels to accommodate unimpeded systemic and pulmonary blood flows, the mesenchymal components of the atrial septum and the outlet septum myocardialize to buttress the structures to which they contribute, the coronary arteries form to nourish the newly formed compact wall of the ventricles, the muscular outflow tract remodels with the incorporation of its proximal part into the ventricles, and a start is made with the development of valves. It is closure of the middle part of the interventricular foramen by the formation of the membranous septum at CS20 that completes septation.Marsupial embryos at CS18 have advanced sufficiently to survive outside the womb even though, for instance, their interventricular foramen has yet to close194. The subsequent fetal phase varies markedly in length between species. It can, in the case of marsupials, even be nonexistent, with development proceeding in a pouch, which is outside the womb.Based on the features described in the previous paragraphs, we hypothesize that embryonic heart development includes an early phase, during which its basic building plan is laid down, in accordance with the peripheral growth model. There is then a later phase during which the heart remodels to cope with the requirements of postnatal or placental circulation in marsupial and eutherian mammals, respectively196. Instead, it is expressed in embryological days (“E” or “ED”), which refers to the time since midnight before demonstrating the presence of a vaginal plug in the morning. The method is prone to variation, as evidenced by the frequently reported inter- or even intra-litter variation of half a day or more199, which is the equivalent of one Carnegie stage  in which the daily development of the mouse embryo is approximately fourfold faster than that of the human embryo, and a second phase (ED13.5-ED16.5) in which daily development of the mouse embryo exceeds that of the human embryo ~13-fold198. Unfortunately, early heart development is at the very beginning of this comparison. To validate Otis and Brent’s comparison for that early period, we staged our reconstructed early human hearts according to Le Garrec’s system for early mouse hearts52. Supplemental Fig. 198, and that early heart development in mice and humans proceeds at the same rate as subsequent development. Apparently, rapid overall development and insufficiently careful staging of mouse embryos are to blame for the perception that early heart development in the mouse differs from that in humans.The mouse embryo is the most frequently used animal model for  studies of heart development. Accordingly, mouse data is also the main reference for mechanistic extrapolation in this study. Almost invariably, murine development is not based on a staging system, such as that of TheilerWe have taken great care to calibrate the scale cubes that we added to each of the reconstructions. The scale cubes, therefore, permit comparisons of structures between stages in real size. They can be used to settle arguments of the effects of differential growth on shape. Especially in cardiac structures with components that differ greatly in growth rate, such as the respective parts of the outflow tract, proportional comparisons are useful. The pictorial timelines revealed, for instance, that the absence of growth in the myocardial outflow tract was compensated for by growth of the intrapericardial arterial trunks, in particular the aortic trunk Fig. . These c142, becoming transferred to the intrapericardial arterial trunks by the obliquely growing aortopulmonary septum , the helical course of the wall was clockwise in direction when following the bloodstream  has four or five somites, which places it in the least advanced CS10 group, but its cardiovascular development is least developed in the entire series of early hearts described by Davishttps://www.ehd.org/virtual-human-embryo/about.php, http://virtualhumanembryo.lsuhsc.edu/DREM/OnlineDisks.html). These embryos are part of the Carnegie collection, Washington D.C., USA. We reconstructed 12 hearts from human embryos obtained between ~26 and ~56 days of development subsequent to fertilization  project  was used to generate 3D reconstructions. Preliminary alignment of consecutive sections was performed automatically with the least-squares method, followed by further manual alignment. The definitive alignment also accounted for curvatures in the sagittal and transverse planes of the body axis of the embryo. Existing images of the embryo before sectioning or age-matched embryos that were imaged with magnetic resonance were used as the template for proper alignment . Delineation of heart structures was performed manually, using the immunohistochemically stained sections described elsewhere205 as guides.Processing of the digital images, and calculation of voxel size, were performed as described previouslyhttp://www.adobe.com) for the generation of 3D-interactive PDF files . The accuracy of the remodeling process was safeguarded by simultaneous visualization in Cinema 4D of the original output from Amira and the remodeled Cinema model. Subsequently, the Cinema 3D-model was exported via “wrl export” to Adobe’s portable device format (PDF) reader version 9 (For all goniometric measurements, the structures of interest were aligned using long craniocaudal structures, such as the dorsal aorta or neural tube (Supplemental Fig. 211. We use cranial-caudal, dorsal-ventral, and left-right to describe topographical relations, with the cervical and upper thoracic vertebral column being dorsal. The terms proximal and distal refer to positions relative to the center of the heart.The descriptions of the embryonic hearts follow the sequential segmental approachFurther information on research design is available in the Peer Review FileSupplementary InformationReporting Summary