Load-dependent extracellular matrix organization in atrioventricular heart valves: differences and similarities.

The extracellular matrix of the atrioventricular (AV) valves' leaflets has a key role in the ability of these valves to properly remodel in response to constantly varying physiological loads. While the loading on mitral and tricuspid valves is significantly different, no information is available on how collagen fibers change their orientation in response to these loads. This study delineates the effect of physiological loading on AV valves' leaflets microstructures using Second Harmonic Generation (SHG) microscopy. Fresh natural porcine tricuspid and mitral valves' leaflets (n = 12/valve type) were cut and prepared for the experiments. Histology and immunohistochemistry were performed to compare the microstructural differences between the valves. The specimens were imaged live during the relaxed, loading, and unloading phases using SHG microscopy. The images were analyzed with Fourier decomposition to mathematically seek changes in collagen fiber orientation. Despite the similarities in both AV valves as seen in the histology and immunohistochemistry data, the microstructural arrangement, especially the collagen fiber distribution and orientation in the stress-free condition, were found to be different. Uniaxial loading was dependent on the arrangement of the fibers in their relaxed mode, which led the fibers to reorient in-line with the load throughout the depth of the mitral leaflet but only to reorient in-line with the load in deeper layers of the tricuspid leaflet. Biaxial loading arranged the fibers in between the two principal axes of the stresses independently from their relaxed states. Unlike previous findings, this study concludes that the AV valves' three-dimensional extracellular fiber arrangement is significantly different in their stress-free and uniaxially loaded states; however, fiber rearrangement in response to the biaxial loading remains similar.

Transcatheter treatment of tricuspid regurgitation by caval valve implantation--experimental evaluation of decellularized tissue valves in central venous position.

BACKGROUND: Caval valve implantation has been suggested for transcatheter treatment of severe tricuspid regurgitation (TR). Combining the interventional technique with the promising surgical experience with decellularized valves, we sought to evaluate the functional and structural outcome of decellularized pericardial tissue valves (dTVs) in the low-pressure venous circulation in a chronic model of TR. METHODS AND RESULTS: Sixteen pericardial tissue valves were heterotopically implanted in the inferior and superior vena cava in a sheep model (54-98 kg; median 74.5 kg, n = 8) of severe TR. The devices were assembled using self-expanding nitinol stents and bovine pericardia decellularized by a detergent-based protocol (group dTV; n = 8). Glutaraldehyde-fixed pericardial tissue valves served as control (GaTV, n = 8). After 6 months, device function and structural maturation were analyzed using echocardiographic, histologic, immunohistologic, and electron microscopic approaches. After implantation, cardiac output increased significantly from 3.7 ± 1.1 l/min to 4.8 ± 1.1 l/min (P < 0.05) and competent valve function was verified by angiography. At 6 months, angiographic and echocardiographic evaluation revealed moderate to severe regurgitation in all GaTV. In contrast, five of the eight dTVs functioned well with only minor regurgitation. In these animals, autopsy revealed preserved valve structure with tender leaflets without signs of thrombosis or calcification. Conversely, GaTV showed severe degeneration with large calcification areas. Microscopic and histologic analysis confirmed endothelial repopulation in both valve types. However, additional interstitial reseeding was observed in decellularized valves. CONCLUSIONS: In the venous circulation in severe TR, decellularized valves show superior functional performance compared to Ga-fixed tissue valves. Macroscopic and microscopic analyses suggest preserved structural integrity and advanced endothelial and interstitial repopulation with evidence of less degradation in dTV. © 2014 Wiley Periodicals, Inc.

A detailed mechanical and microstructural analysis of ovine tricuspid valve leaflets.

The tricuspid valve ensures unidirectional blood flow from the right atrium to the right ventricle. The three tricuspid leaflets operate within a dynamic stress environment of shear, bending, tensile, and compressive forces, which is cyclically repeated nearly three billion times in a lifetime. Ostensibly, the microstructural and mechanical properties of the tricuspid leaflets have mechanobiologically evolved to optimally support their function under those forces. Yet, how the tricuspid leaflet microstructure determines its mechanical properties and whether this relationship differs between the three leaflets is unknown. Here we perform a microstructural and mechanical analysis in matched ovine tricuspid leaflet samples. We found that the microstructure and mechanical properties vary among the three tricuspid leaflets in sheep. Specifically, we found that tricuspid leaflet composition, collagen orientation, and valve cell nuclear morphology are spatially heterogeneous and vary across leaflet type. Furthermore, under biaxial tension, the leaflets' mechanical behaviors exhibited unequal degrees of mechanical anisotropy. Most importantly, we found that the septal leaflet was stiffer in the radial direction and not the circumferential direction as with the other two leaflets. The differences we observed in leaflet microstructure coincide with the varying biaxial mechanics among leaflets. Our results demonstrate the structure-function relationship for each leaflet in the tricuspid valve. We anticipate our results to be vital toward developing more accurate, leaflet-specific tricuspid valve computational models. Furthermore, our results may be clinically important, informing differential surgical treatments of the tricuspid valve leaflets. Finally, the identified structure-function relationships may provide insight into the homeostatic and remodeling potential of valvular cells in altered mechanical environments, such as in diseased or repaired tricuspid valves. STATEMENT OF SIGNIFICANCE: Our work is significant as we investigated the structure-function relationship of ovine tricuspid valve leaflets. This is important as tricuspid valves fail frequently and our current approach to repairing them is suboptimal. Specifically, we related the distribution of structural and cellular elements, such as collagen, glycosaminoglycans, and cell nuclei, to each leaflet's mechanical properties. We found that leaflets have different structures and that their mechanics differ. This may, in the future, inform leaflet-specific treatment strategies and help optimize surgical outcomes.

Acute In Vivo Functional Assessment of a Biodegradable Stentless Elastomeric Tricuspid Valve.

Degradable heart valves based on in situ tissue regeneration have been proposed as potentially durable and non-thrombogenic prosthetic alternatives. We evaluated the acute in vivo function, microstructure, mechanics, and thromboresistance of a stentless biodegradable tissue-engineered heart valve (TEHV) in the tricuspid position. Biomimetic stentless tricuspid valves were fabricated with poly(carbonate urethane)urea (PCUU) by double-component deposition (DCD) processing to mimic native valve mechanics and geometry. Five swine then underwent 24-h TEHV implantation in the tricuspid position. Echocardiography demonstrated good leaflet motion and no prolapse and trace to mild regurgitation in all but one animal. Histology revealed patches of proteinaceous deposits with no cellular uptake. SEM demonstrated retained scaffold microarchitecture with proteinaceous deposits but no platelet aggregation or thrombosis. Explanted PCUU leaflet thickness and mechanical anisotropy were comparable with native tricuspid leaflets. Bioinspired, elastomeric, stentless TEHVs fabricated by DCD were readily implantable and demonstrated good acute function in the tricuspid position.

Three-dimensional geometry of the tricuspid annulus in healthy subjects and in patients with functional tricuspid regurgitation: a real-time, 3-dimensional echocardiographic study.

BACKGROUND: Most rings currently used for tricuspid valve annuloplasty are formed in a single plane, whereas the actual tricuspid annulus (TA) may have a nonplanar or 3-dimensional (3D) structure. The purpose of this study was therefore to investigate the 3D geometry of the TA in healthy subjects and in patients with functional tricuspid regurgitation (TR). METHODS AND RESULTS: This study consisted of 15 healthy subjects and 16 patients with functional TR who had real-time 3D echocardiography. With our customized software, 8 points along the TA were determined with the rotated plane around the axis at 45 degrees intervals. The TA was traced during a cardiac cycle. The distance between diagonals connecting 2 points was measured. The height was defined as the distance from the plane determined by least-squares regression analysis at all 8 points. Both the maximum (7.5+/-2.1 versus 5.6+/-1.0 cm2/m2) and minimum (5.7+/-1.3 versus 3.9+/-0.8 cm2/m2) TA areas in patients with TR were larger than those in healthy subjects (both P<0.01). Healthy subjects had a nonplanar-shaped TA with homogeneous contraction. The posteroseptal portion was the lowest toward the apex from the right atrium, and the anteroseptal portion was the highest. In patients with functional TR, the TA was dilated in the septal to lateral direction, resulting in a more circular shape than in healthy subjects. A similar 3D pattern was observed in patients with TR, but it was more planar than that in healthy subjects. CONCLUSIONS: Real-time 3D echocardiography showed a complicated 3D structure of the TA, which appeared to be different from the "saddle-shaped" mitral annulus, suggesting an annuloplasty for TR different from that for mitral regurgitation.

Sovering annuloplasty rings: experimental pathology in the sheep model.

INTRODUCTION: A new flexible annuloplasty ring (Sovering, Sorin Biomedica Cardio), both closed and open, has been designed and consists of radiopaque silicone core impregnated with barium sulfate and covered by a knitted polyester fabric coated with Carbofilm. The aim of the study was to test the biological compatibility of the new device in large animals in orthotopic position. METHODS: Ten Sovering rings were implanted in 10 female adult sheep, 7 mitral (3 open, 4 closed) and 3 tricuspid (all open). The size was 23-25 mm in the mitral and 28-30 mm in the tricuspid position, and the time in place varied from 63 to 110 days (mean = 89 +/- 14) and from 58 to 63 days (mean = 61 +/- 3), respectively. The morphological analysis consisted of gross, histological, immunohistochemical and ultrastructural investigations. RESULTS: The prosthetic ring appeared well implanted in the valve atrioventricular (AV) junction, encapsulated by a thin fibrous sheath without any evidence of thrombus deposition, fibrinous lining or exuberant fibrous tissue overgrowth. No adverse inflammatory reaction was observed, but rare lymphocytes, macrophages and foreign body giant cells. At electron microscopy, the fibrous tissue appeared to permeate deeply within the fabric network; reendothelization on the surface was noted and confirmed by immunohistochemistry. Sirius red staining at polarized light revealed a higher content of collagen type III in the mitral than in the tricuspid position. CONCLUSIONS: Sovering annuloplasty rings implanted in the AV valves of adult sheep showed excellent biocompatibility, fibrous encapsulation and reendothelization. The absence of thrombosis and exuberant fibrous tissue reaction supports the effective use of Carbofilm covering in annuloplasty devices.

Systolic right ventricular function assessment by pulsed wave tissue Doppler imaging of the tricuspid annulus.

BACKGROUND: Systolic right ventricular (RV) function is an important predictor in the course of various congenital and acquired heart diseases. So far, tricuspid annular motion velocity in systole as obtained by pulsed wave tissue Doppler imaging (TDI) has rarely been investigated for RV function assessment in a sizeable adult patient population. METHODS: 258 individuals were included in the study. Among them, there were 107 individuals without cardiovascular disease, 71 patients with predominant RV dysfunction, 40 patients with pulmonary artery hypertension, and 40 patients with predominant left ventricular dysfunction. The reference methods for RV systolic function assessment were biplane two-dimensional echocardiography and magnetic resonance imaging (MRI; n = 31) for the calculation of RV ejection fraction (EF). Lateral tricuspid valve annular motion velocities in systole (TVlat, cm/s) were recorded using pulsed wave TDI from the apical 4-chamber view (long axis function). RESULTS: RV EF as determined by biplane echocardiography correlated significantly with respective values as assessed by MRI: RVEFecho = RV EFMRI + 1.6; r2 = 0.569, p <0.0001. Using the best TVlat threshold of 12 cm/s, distinction between the group with RV dysfunction and the other groups was possible with 86% sensitivity and 83% specificity. There was a direct and significant correlation between TVlat and RV ejection fraction (p <0.0001). Using TVlat thresholds of 12 and 9 cm/s, distinction between normal RV EF (>55%), moderately reduced (30-55%) and severely reduced RV EF (<30%) was possible with 84% sensitivity and 81% specificity, respectively with 83% sensitivity and 67% specificity. CONCLUSION: Systolic long axis velocity measurement of the lateral tricuspid annulus is useful and accurate to assess RV systolic function in a broad patient population. Thresholds of 12 and 9 cm/s allow differentiation between normal, moderately reduced and severely reduced RV ejection fraction.

Structural changes in a porcine xenograft after implantation for 105 months.

Morphologic studies including light microscopy and transmission and scanning electron microscopy were performed on a formaldehyde-fixed porcine xenograft prosthesis that functioned well for 8 years and had been implanted for 105 months in the tricuspid valve position. It is the oldest implanted valve studied in this manner. Although all leaflets had some gross deterioration, only one had adherent thrombus. The degenerative changes in the connective tissue from all leaflets were similar to those recently reported in glutaraldehyde-fixed porcine valves implanted for shorter periods. The surfaces of the two nonthrombosed leaflets and small portions of the surface of the thrombosed leaflet were endothelized, and the endothelium appeared to have little or no effect on the degenerative changes in the subjacent connective tissue. Further, the endothelium did not extend over the examined portion of the synthetic anulus of the prosthesis, suggesting that its origin was possibly from cells circulating in the blood.

Nerve fibres containing neuropeptide Y in the atrioventricular valves of Japanese monkey and rat; a light and electron microscopic study.

Dense distribution of varicose fibres containing neuropeptide Y-like immunoreactivity (NPY-LI) was found in the atrioventricular valves of the Japanese monkey, and moderately in the rat. The immunoelectron microscopy using immunogolds resulted in the localization of NPY-LI within the dense-cored vesicles which existed with the small clear vesicles in the unmyelinated axons near the endocardium. These NPY-LI-containing fibres may participate in regulation of vasomotor role or other functions of the atrioventricular valves.

BIOSA bileaflet pericardial valve: experimental study in sheep model.

BACKGROUND: BIOSA is a single sheet, two-cuspal-shaped ("bileaflet") glutaraldehyde fixed bovine pericardial valve prosthesis (BP), designed to minimize stress and prevent mechanical failure. We tested this device in the adult sheep model. METHODS: Seven BIOSA and two Baxter Carpentier-Edwards (Perimount) pericardial BPs, 25 mm in size, were implanted in the tricuspid position of adult sheep. A mid term BIOSA explant (81 days) died of infective endocarditis. The remaining BPs were divided in three early explants (0-3 days: two BIOSA and one Perimount) and five late explants (162-189 days: four BIOSA and one Perimount). Protocol of the study included gross examination, mammography X-ray (score 0-4), histology, scanning (SEM) and transmission (TEM) electron microscopy and atomic absorption spectroscopy. RESULTS: (a) Early explants: The mesothelium was detached, collagen-elastic fibers and pericardiocytes of the fibrosa showed optimal preservation. Calcium content in BIOSA BPs was 6.22 mg/g dry weight (mean) versus 7.75 mg/g of the Perimount BP. (b) Late explants: At naked eye all BPs showed regular cusp pliability and coaptation, without tears, perforations, fibrous pannus or calcific deposits. X-ray was either negative (three BPs) or exhibited score 1 (two BPs). Microscopic features were excellent both in terms of collagen-elastic fibers preservation and absence of inflammation. The calcium content was 4.95 mg/g dry weight (mean) in BIOSA BPs versus 5.29 mg/g in the Perimount BP. CONCLUSIONS: Tissue characteristics of BIOSA BPs were optimal, without difference with Perimount BPs and in this animal model no case of structural valve deterioration occurred in the long term.

Prevalence of cardiac valve abnormalities in afebrile injection drug users.

OBJECTIVES: To determine the prevalence of occult valvular pathology in afebrile injection drug users (IDUs) compared with an afebrile, non-IDU population. To characterize the type of valvular pathology present in light of current recommendations regarding periprocedural antibiotic prophylaxis against endocarditis. METHODS: This was a comparative, cross-sectional study involving a convenience sample of 98 patients with a history of injection drug use, and 99 non-IDU patients presenting to a large urban ED. Patients were excluded if they had one or more of the following: a history of cardiac valve abnormality, a history of endocarditis, fever on presentation, or a cardiac murmur. Data were collected that included demographics, medical history, and details of injection drug use. Transthoracic echocardiography (echo) was performed on each subject in the ED, and read in a blinded fashion by a single board-certified cardiologist. RESULTS: Among 98 IDUs and 99 non-IDUs, 12% of the IDUs had aortic valve thickening, compared with 5.1% of the non-IDUs (99% CI for difference of 7.8% = -3.0% to 18.6%). Forty-four percent of the IDUs had mitral valve thickening, compared with 25% of the non-IDUs (99% CI for difference of 18.3% = 0.9% to 35.7%). Eleven percent of the IDUs vs 1% of the non-IDUs had tricuspid valve thickening (99% CI for difference of 10.4% = 1.6% to 19.2%). No patient had pulmonic valve thickening. Six percent of the IDUs vs 0% of the non-IDUs had mitral annulus thickening (99% CI for difference of 6.3% = -0.1% to 12.8%). Twelve percent of the IDUs vs 3% of the non-IDUs had mitral chordae thickening (99% CI for difference of 9.5% = -0.4% to 19.4%). Tricuspid chordae thickening was recorded in 2% of the IDUs vs 0% of the non-IDUs (99% CI for difference of 2.1% = -1.7% to 6.0%). Most important, the prevalence of valvular regurgitation was small, and evenly distributed in the two groups. No valvular vegetations were seen. CONCLUSION: Both non-IDUs and IDUs have occult valvular pathology. There is an increased prevalence in IDUs of tricuspid and mitral valve thickening. The prevalence of valvular regurgitation, a reported indication for periprocedural antibiotic prophylaxis, was small and the affected valves were not statistically different between the two groups. These findings question the selected routine use of antibiotic prophylaxis in IDU patients undergoing invasive procedures.

Mechanical properties and ultrastructure of normal human tricuspid valve chordae tendineae.

The mechanical properties and ultrastructure of normal human tricuspid valve chordae tendineae were examined. Mechanical studies showed that these chordae exhibited less extensibility than normal mitral valve chordae of comparable size. The non-linear stress-strain curve of the tricuspid chordae, however, can be attributed to their microstructure. Under scanning electron microscopy these chordae were found to consist of fairly straight collagen bundles that were made up of networks of collagen fibrils. Transmission electron microscopical studies showed that the distribution of collagen fibril size was bimodal with an average fibril diameter of 441 A (S.E. = 2A) while the average fibril density was 198.4 (S.E. = 8.4) per 10(-8) cm2. These data data from the transmission electron micrographs were found to be different from those already reported for the mitral valve chordae. Thus, fine structural differences exist between chordae from both the mitral and tricuspid valves. These differences in fine structure could be due to the fact that these valves are under different in vivo mechanical stresses and they may account for the less extensible nature exhibited by the tricuspid chordae tendineae.

Surface structure of the human cardiac valve. A comparative study of normal and diseased valves.

This article reports the surface structure of the endothelial cells covering the cardiac valves and their changes in chronic inactive rheumatic valvulitis and bacterial endocarditis. The endocardial surface of the normal cardiac valve was covered by a layer of endothelial cells, each of which was recognized because of the presence of centrally located nuclear bulge and marginal folds at the cell boundaries. In the cardiac valves affected by chronic rheumatic valvulitis, the endothelial layer denuded extensively, even in the regions where only slight thickening was noticed in the intravalvular tissues. As compared to the inflow side of the valve, denudation of the endothelial layer was more conspicuous on the outflow side. There was no morphological difference between the two hemodynamically different conditions, i.e. stenosis and regurgitation. In the cardiac valves affected by bacterial endocarditis, in contrast, the destruction of the endothelial layer was preferentially observed along the closing margins of the valve, and in the region of calcification and vegetation. These findings are compatible to the interpretation that differences in morphological changes of the endothelial layer in these two acquired valvular diseases may reflect the differences of their pathogeneses. The present study thus suggest an importance of endothelial cell in elucidating the pathogenesis of acquired valvular diseases.

The inferior right atrial isthmus: further architectural insights for current and coming ablation technologies.

BACKGROUND: Although linear ablation of the right atrial isthmus in patients with isthmus-dependent atrial flutter can be highly successful, recurrences and complications occur in some patients. Our study provides further morphological details for a better understanding of the structure of the isthmus. METHODS AND RESULTS: We examined the isthmic area in 30 heart specimens by dissection, histology, and scanning electron microscopy. This area was bordered anteriorly by the hinge of the tricuspid valve and posteriorly by the orifice of the inferior caval vein. With the heart in attitudinal orientation, we identified and measured the lengths of three levels of isthmus: paraseptal (24 +/- 4 mm), central (19 +/- 4 mm), and inferolateral (30 +/- 3 mm). Comparing the three levels, the central isthmus had the thinnest muscular wall and the paraseptal isthmus the thickest wall. At all three levels, the anterior part was consistently muscular whereas the posterior part was composed of mainly fibro-fatty tissue in 63% of hearts. The right coronary artery was less than 4 mm from the endocardial surface of the inferolateral isthmus in 47% of hearts. Inferior extensions of the atrioventricular node were present in the paraseptal isthmus in 10% of hearts, at 1-3 mm from the endocardial surface. CONCLUSIONS: The thinner wall and shorter length of the central isthmus together with its distance from the right coronary artery, and nonassociation with the atrioventricular node or its arterial supply, should make it the preferred site for linear radiofrequency ablation.

The atrioventricular valves of the mouse. I. A scanning electron microscope study.

This paper reports a scanning electron microscope study of the morphology of the atrioventricular (AV) valves in the mouse. The leaflet tissue of the 2 AV valves consists of a continuous veil that shows no commissures or clefts. In all instances, the chordae that arise from the papillary system merge with the free border of the leaflet tissue. No distinct terminations of chordae were observed on the ventricular face of the valves. The leaflet tissue of the right AV valve can be divided into parietal and septal components on the basis of the insertion into the ventricular wall and of the papillary system. While the septal component is similar in shape, location and tension apparatus to the septal tricuspid leaflet in man, the parietal component appears to correspond to the anterior and posterior human leaflets. This segment of the valve is served by 3 papillary muscles that arise from the septal wall. The right AV valve is not a tricuspid structure from the morphological standpoint, but appears to function as such because of the particular attachment of the papillary muscles. The leaflet tissue of the mitral valve is served by 2 papillary muscles, anterior and posterior, which consist of muscular trabeculae extending from the heart apex to the base of the valve. These muscles remain associated with the ventricular wall. The leaflet tissue attaches directly to these papillary muscles, which give rise to a very small number of slender chordae. There are thus several important differences between the AV valves of the mouse and man.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural quantification of collagen in human chordae tendineae.

OBJECTIVE: To determine collagen fibril diameter distributions in the chordae tendineae and to see whether there are any differences between right and left sides of the human heart. DESIGN: Collagen fibril diameters and the percentage volume occupied by collagen fibrils (as opposed to ground substance) were determined by means of a planimeter with a digitising tablet from electron micrographs printed at a magnification of 136; 000. MATERIAL: Human chordae tendineae were obtained at postmortem examination from seven subjects aged 50-75 years. MEASUREMENTS AND MAIN RESULTS: Histograms of collagen fibril diameter distributions showed a sharp, unimodal distribution. The mean collagen fibril diameters associated with the tricuspid and mitral valves were 41.1 nm and 40.5 nm respectively. The percentage volume occupied by collagen was about 39% for each valve. There were no significant differences for any of these values between the right and left sides of the heart. CONCLUSIONS: The greater pressures present on the left side of the heart are not reflected in any difference in collagen fibril diameters within the chordae tendineae compared with the right side.

Formation of the tricuspid valve in the human heart.

BACKGROUND: Some of the problems concerning the origin of the inlet component of the definitive right ventricle were resolved in a previous study in which we showed it to be derived exclusively from the embryonic right ventricle. Questions remain, however, concerning the relative contributions of endocardial cushion tissue and myocardium to the definitive valvar apparatus guarding the right atrioventricular orifice and the origin of the valvar leaflets. METHODS AND RESULTS: The formation of the tricuspid valve was studied by scanning electron microscopic and immunohistochemical techniques. Concurrent with the development of the right atrioventricular connection, a myocardial ridge forms at the boundary between the atrioventricular canal and the embryonic right ventricle. It grows to become a myocardial gully that funnels atrial blood beneath the lesser curvature of the initial heart tube toward the middle of the right ventricle. Fenestrations in the floor of the gully create an additional inferior opening in the funnel, transforming its initial anterior rim into the septomarginal trabeculation. The septum formed by the fusion of the endocardial ridges of the outflow tract becomes myocardialized in its inferior portion to form, in part, the outlet septum and, in part, the supraventricular crest. The smooth atrial surface of the tricuspid valvar leaflets develops from endocardial cushion tissue. The leaflets become freely movable, however, only after delamination of the tension apparatus within the myocardium. The inferior and septal leaflets derive from the gully and the ventricular septum, their delamination being a single, continuous process. The antero-superior leaflet forms by delamination from the developing supraventricular crest. CONCLUSIONS: The leaflets of the tricuspid valve develop equally from the endocardial cushion tissues and the myocardium. The myocardium contributing to the valve comes from two sources, the tricuspid gully complex and the developing supraventricular crest. These findings facilitate the understanding of several congenital malformations.