Normal Human Embryos Research Articles

Barren in the Promised Land: Childless Americans and the Pursuit of Happiness

In a current medical news release, French researchers claim that normal human embryos can be obtained by the injection of round spermatids into oocytes. Authors believe that many patients with hitherto untreatable and unexplained azoospermia will be able to benefit from this new technique of assisted fertilization. 1 Thus, the new specialty of infertility continues its rapid advance since the astonishing announcement in 1978 of the birth of Baby Louise as a result of in vitro fertilization by Drs Patrick Steptoe and R. Edwards of England. 2 Judging from the title of Elaine Tyler May's Barren in the Promised Land: Childless Americans and the Pursuit of Happiness , and the fact that her father, Dr Ed Tyler, Jr, was a prominent pioneer and leader in infertility at his Los Angeles Tyler Clinic, where he launched his specialty in the early 1950s, I expected that her book would detail the rapid and

Simultaneous enumeration of chromosomes 13, 18, 21, X, and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy.

A fluorescence in situ hybridization (FISH) protocol to simultaneously enumerate chromosomes 13, 18, 21, X, and Y in interphase cell nuclei for application in preimplantation genetic diagnosis (PGD) of aneuploidy was tested. Strict scoring criteria were developed to minimize recording errors. The protocol used optimized probes for chromosome-specific DNA repeat and single-copy loci and showed a significantly higher efficiency (95%) than previously published protocols. The other purpose of this study was to differentiate between two signals originating from a single split target or from two targets in close proximity. These criteria were based on the FISH results obtained from the analysis of all or most of the cells from 50 chromosomally normal or mosaic human embryos, 20 aneuploid embryos, and five polyploid embryos donated for research. Subsequently, 183 human embryos underwent PGD of aneuploidy in one of their cells. In 64 embryos that were not transferred back to the uterus, the rest of cells were also analyzed, and the previous results were confirmed in 91% of these embryos, with 0% (0/21) of the embryos classified as normal embryos being abnormal and 14% (6/43) of the embryos classified as abnormal being normal. Compared to previous protocols, these criteria minimize the risk of transferring abnormal embryos after PGD analysis.

Truncus arteriosus malformation: a developmental arrest at Carnegie stage 14.

The pathogenesis of truncus arteriosus malformation has been ascribed to deficiency of conotruncal ridges, failure to form a pulmonary conus, absence of the aortopulmonary septum, or hemodynamic factors. To re-examine this issue, we reviewed the morphology of 28 hearts with truncus arteriosus malformation and compared the findings to the sequences of cardiogenesis in 351 normal human embryos of the Carnegie Embryological Collection. All malformed hearts had an absent muscular outflow tract (conal) septum. The truncal valve had four commissures and/or raphes, a fused commissure, in seven cases, three in 20, and uncertain status in one. All but one heart had fibrous continuity between the anterior mitral leaflet and the truncal valve. In embryos, the outflow tract was circular and lined by a layer of cardiac jelly early in Carnegie stage 14 but acquired an elliptical configuration and four cushions, with fusion of the two larger, by stage 16. Semilunar valve leaflets form at the downstream end of the cushions. The more frequent occurrence of three rather than four commissures in the truncal valve associated with absence of a conal septum suggests that the embryonic outflow tract failed to acquire an elliptical shape and cushions that could fuse to subdivide the outflow tract and the semilunar valves anatomically. The presence of truncal valve leaflets shows that three or four cushions did form and the usual presence of mitral-truncal valve fibrous continuity supports otherwise normal outflow tract development. We conclude that a failure or delay of the circular outflow tract of early Carnegie stage 14 to acquire the elliptical configuration needed to induce formation of four cushions, two of which fuse to subdivide the outflow tract and semilunar valve primordia, is the cause of truncus arteriosus malformation.

Histocytochemical and immunohistochemical studies related to the role of glycogen in human developing digestive organs.

To elucidate the role of glycogen in the epithelium of developing digestive organs, we investigated the appearance of glycogen and glycogen phosphorylase (GP) in these organs. We studied 64 externally normal human embryos at Carnegie stages 13-23 (5.1-28.0 mm in crown-rump length, 4-8 weeks of gestation) by histocytochemical staining for glycogen and immunohistochemical staining with antibodies against two isoenzymes of GP: brain-type (BGP) and muscle-brain-type (MBGP) GP. At stage 13, glycogen appeared in the epithelium of the digestive tract and the parenchyma of the pancreas. As development advanced, glycogen granules increased in number and size in these tissues, and they became evenly distributed in the epithelium of the digestive tract as either single particles or aggregates, as deduced by electron microscopy at late embryonic stages. Immunoreactivity specific both for BGP and for MBGP was detected in the digestive tract and the pancreas from stage 13. As development advanced, both BGP- and MBGP-immunoreactive cells increased in number and in immunoreactivity, and the number of MBGP-immunoreactive cells became larger than that of BGP-immunoreactive cells. By contrast, in hepatic cells, which serve as a major storage site for glycogen in adults, glycogen was detected only from stage 20, in smaller amounts, without formation of aggregates, and no immunoreactivity specific for BGP or MBGP was apparent throughout the embryonic stages examined. Thus, in the epithelium of the digestive tract and the parenchyma of the pancreas, but not in hepatic cells, the appearance and localization of GP coincided almost exactly with that of glycogen. These observations suggest that glycogen in the epithelium of the digestive tract and the parenchyma of the pancreas has not only been synthesized but also degraded from an early embryonic period and may, thus, be related to active cellular metabolism that is specific for embryonic development, including proliferation of the epithelium and interactions between epithelium and mesenchyme.

Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities

To determine some of the unresolved questions related to chromosome anomalies in early human embryos, such are the detection of any advanced maternal age effect; the complete assessment of mosaicism, which requires analysis of all cells; and the relationship with embryonic dysmorphism. Fluorescence in situ hybridization has been used in this study to answer these issues. Fluorescence in situ hybridization analysis of human embryos using simultaneously probes for three or five chromosomes. Five hundred twenty-four cleavage-stage human embryos obtained by IVF were analyzed by fluorescence in situ hybridization. Embryos were allocated into three groups according to morphological and developmental characteristics (arrested; slow and/or fragmented; morphologically and developmentally normal). The embryos also were analyzed according to maternal age. Dysmorphic embryos had higher rates of polyploidy and diploid mosaicism. Aneuploidy increased with maternal age in nonarrested embryos. Preimplantation genetic diagnosis successfully detected these abnormalities. This study demonstrates that, in morphologically and developmentally normal human embryos, cleavage-stage aneuploidy significantly increases with maternal age. The results suggest that implantation failure in older women largely could be due to aneuploidy.

The developing human biliary system at the porta hepatis level between 29 days and 8 weeks of gestation: a way to understanding biliary atresia. Part 1.

The developing biliary system in normal human embryos from 29 days to 8 weeks post-fertilization was studied. The primitive extrahepatic bile duct that originates from the embryonic hepatic foregut diverticulum is in contact with the hepatic anlage from the start of organogenesis and remains so throughout the gestational ages examined. The primitive extrahepatic bile duct maintains continuity with the ductal plate from which intrahepatic bile ducts are eventually formed. Contrary to long-held concepts of biliary development, no 'solid stage' of entodermal occlusion of the common bile duct lumen was found at any stage of gestation in the material investigated. Therefore, biliary atresia is not caused by incomplete vacuolization of the 'solid stage'.

Extrahepatic biliary atresia: A first-trimester event? Clues from light microscopy and immunohistochemistry

Biliary atresia is an obliterative disorder of the bile ducts, causing obstructive jaundice in neonates. In this study, the developing biliary system of normal human embryos and fetuses was examined and compared with the resected extrahepatic biliary remnants from 205 cases of biliary atresia. At the porta hepatis level, it was found that the primary biliary ductal plate undergoes a specific sequence of remodelling, resulting in the formation of large tubular bile ducts surrounded by thick mesenchyme, between 11 and 13 weeks postfertilisation. These developing ducts are in luminal continuity with the extrahepatic biliary tree throughout gestation. Contrary to long-held belief, no “solid phase’ was observed in the development of the extrahepatic bile duct. Examination of the biliary remnants in biliary atresia showed that the porta hepatis is encased in fibrous tissue, and a variable pattern of obliteration of the common hepatic and common bile ducts was observed. Anticytokeratin immunostaining showed similarities between the abnormal ductules within the porta hepatis in biliary atresia, and the developing bile ducts in the first trimester. Biliary atresia may be caused by failure of the remodelling process at the hepatic hilum, with persistence of fetal bile ducts poorly supported by mesenchyme. As bile flow increases perinatally, bile leakage from these abnormal ducts may trigger an intense inflammatory reaction, with subsequent obliteration of the biliary tree.

Septation of the respiratory and digestive tracts in human embryos: crucial role of the tracheoesophageal sulcus.

Esophageal atresia and tracheoesophageal fistula, common malformations of the respiratory and digestive tracts, are of unsettled pathogenesis. Part of the difficulty in understanding these abnormalities arises from the uncertainties about the normal developmental processes in the region. This study examined the development and fate of the tracheoesophageal septum. Normal human embryos from the Carnegie Embryological Collection and fetuses from the Hopkins Pathology Collection were examined, and reconstructions of selected specimens were made from photomicrographs of serial histologic sections. The results show that the lung bud appears in Carnegie stage 12, rapidly enlarges, and bends caudally, thereby producing a sulcus between the foregut and the respiratory system on its caudal aspect. The cranial aspect of this tracheoesophageal sulcus remains fixed at the levels of the first cervical vertebra throughout subsequent embryonic and fetal development. At the same time the trachea and esophagus elongate to bring those part of the respiratory and digestive systems into their definitive anatomic positions. Examination of the tracheoesophageal sulcus shows that its growth-limiting properties may be explained by its catenoidal configuration. Catenoidal, or saddle-shape, sulci have been shown to have similar regional growth-limiting properties in the embryonic heart. These regions contrast with outwardly convex regions in both the developing heart and lung where growth of the tissues occurs. The observations made here suggest that the origin of the tracheoesophageal malformations must be sought in a configurational abnormality in the area of the developing lung bud in Carnegie stage 12.

Three-dimensional reconstructions of the primary palate region in normal human embryos.

Our knowledge of the precise spatial relationships of human primary palate morphogenesis remains poorly defined. This is due to intrinsic difficulties that exist in the study of the subject matter and a lack of adequate methodologies. We present a novel new method to allow precise three-dimensional (3-D) visualization of developing embryonic structures in previously sectioned embryos. In our study we focus on human primary palate development. Five normal human embryos from the Carnegie collection were used. 3-D reconstructions appear similar to scanning electron micrographs (SEM); however, unlike in SEM studies, the original specimen has been previously sectioned histologically. 3-D reconstruction from serial sections involved 1) histological preparation of specimen, 2) projection onto digitizing board, 3) digitization, 4) automated reassembly, and 5) relay to interactive optical disc recorder. Detailed observations of each reconstruction were then made. Data generated in this manner may also be used in the near future for quantitative morphometrics. Thus, 3-D reconstruction techniques presented in this paper generated precise spatial information on the development of the human primary palate.

Development of the lens in human embryos: a histochemical and ultrastructural study.

The early development of the lens was examined, using 36 externally normal human embryos at Carnegie stages 13-23 (4 to 8 weeks of gestation). Twenty-two embryos were sectioned serially and stained with periodic acid-Schiff and a modified method of PAS. In 14 embryos, not only the differential distribution of glycogen but also the ultrastructural change in the developing lens, with special reference to junctional complexes, were examined electron microscopically. At stage 15, when the lens vesicle was formed, glycogen was observed in the cytoplasm of the lens epithelium, especially in the posterior lens epithelium. From stages 16 to 18, when the posterior lens epithelium was differentiated into the primary lens fibers and elongated toward the anterior lens epithelium, the amount of glycogen increased in the basal cytoplasm of the primary lens fiber, where the intracellular organelles, such as the tubular vesicles, mitochondria and multivesicular bodies, began to aggregate. At stage 20, when the lens cavity was obliterated, glycogen was also present in the anterior lens epithelium. At stage 21, as the formation of the secondary lens fibers proceeded, glycogen was noted in the secondary lens fibers in the equator region. These findings suggest that the distribution of glycogen is associated with the formation of the primary and secondary lens fibers. In addition, we provide additional information that a lot of glycogen is distributed in the region where many intracellular organelles aggregate in the embryonic lens vesicles.

Spinal cord-notochord relationship in normal human embryos and in a human embryo with double spinal cord.

Spinal cord-notochord relationship was analyzed histologically and immunohistochemically in normal human conceptuses between the 4-8 developmental weeks and in a 8-week embryo with double spinal cord. In the early 4-week embryo, the gradual closure of the neural tube along the cranio-caudal body axis was paralleled by the differentiation of the median hindge point cells at the ventral midline of the tube and by its temporary close association with the notochord. During the 5th-8th developmental weeks, the neuroepithelium differentiating into three distinct layers was accompanied by a solid, ventromedially positioned notochord. In the abnormal 8-week embryo, the additional spinal cord was located ventrolaterally from the vertebral column. Both spinal cords appeared bilaterally asymmetric, with their floor and roof plates irregularly formed. An abnormally enhanced pattern of neuroepithelial differentiation characterized their dorsal parts. Furthermore, additional spinal nerves and ganglia and an abnormal bony structure were associated with the spinal cord positioned outside the vertebral column. The underlying vertebral bodies were misshaped and contained scattered supernumerary groups of notochord cells. Our investigation underlines the importance of the notochord-neural tube relationship in the morphogenesis of the spinal cord. We suggest that the double spinal cord was induced by the split notochord.

Ultrasound evidence for variability in the size and development of normal human embryos before the tenth post-insemination week after assisted reproductive technologies

Differences in human chorionic gonadotrophin (HCG), average chorionic sac diameter, embryo/fetus crown-rump length and biparietal diameter were determined in 107 singleton pregnancies delivered after 34 weeks, whose post-insemination age was precisely known as a result of in-vitro fertilization (n = 28) or gamete intra-Fallopian transfer (n = 79). Crown-rump lengths were interpreted in relation to the developmental stage of the embryo. A 7-fold to 10-fold difference in HCG levels was observed on post-insemination days 13-16. A 2-fold to 3-fold difference occurred in average chorionic sac diameter on days 25-36. A 2-fold difference occurred in crown-rump length on seven of 10 days prior to day 44. Biparietal diameter differed by no more than 42% from day 57-68 and 20% after day 68. Chorionic sac diameter, crown-rump length, and biparietal diameter were also related to newborn weight. The results suggest firstly that marked differences occur in the rate of early human development, secondly, that the differences occur prior to day 27 when observable cardiac activity begins, and thirdly, that the differences are minimized after day 68 when the embryonic period of development is completed.

Supra-neuroectodermal cells and fibers on the primary nasal cavity and in the fourth ventricle of mouse and human embryos: scanning and transmission electron microscopic studies.

Neuroectoderm-derived epithelia of the primary nasal cavity and the fourth ventricular floor and roof were observed by scanning (SEM) and transmission electron microscopy (TEM) and SEM-TEM correlative views in mouse embryos of 9th to 13th days of gestation, and in 38 externally normal human embryos ranging at Carnegie stages from 13 to 18 (about 5 to 7 weeks of gestation). Smooth-surfaced spindle-shaped cells with one or more cytoplasmic processes and cord-like cytoplasmic structures were observed by SEM on the wall of the primary nasal cavity of both species. They had morphological features similar to those of neuronal type 1 supraependymal (SE) cells and SE fibers on the floor and roof of the fourth ventricle in both species. Type 1 SE cells, SE fibers, and corresponding structures in the primary nasal cavity were localized in relation to the underlying developing nerve and vascular systems. Furthermore, their processes and fibers ran roughly parallel to these underlying structures and they penetrated the epithelial layer at the ends, suggesting a connection with underlying structures. From TEM and SEM-TEM correlative observations, SE fibers in the fourth ventricle and cord-like structures in the primary nasal cavity, both with a larger diameter, were deduced as single axon-like processes or bundles of processes. Those fibers and cord-like structures of smaller diameters were interpreted as elongated telophase bridges; both contained parallel packed microtubules and connected distant cells. Since these processes and fibers were generally longer and became fewer at later developmental stages, they appeared to be transient neuronal structures. They may play a development-related role in such morphogenetic cell movements as in the developing nerve and vascular systems in the epithelial and/or subepithelial layers, but not as direct rudiments of adult nerve tissues.

Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.

Timing and sequence of the events in the development of extraocular muscles in staged human embryos: ultrastructural and histochemical study.

The ultrastructure and the appearance of glycogen were studied in the extraocular muscles of 14 externally normal human embryos (Carnegie stages 13-21). At stage 16, myofibrils with an immature Z line and glycogen granules appeared in the cytoplasm of the myoblast. The myoblasts came into cluster at stage 18, and fusion between the myotubes was observed at stage 20. At this stage, an M line appeared in the myofibrils. At stage 21, an A band with a Z line and an H band with an M line were observed, the sarcoplasmic reticulum appeared in the cytoplasm of the muscle fibers and glycogen increased in volume in the cytoplasm. In the previous study, we showed that the muscle-specific isoenzymes, such as creatine kinase, beta-enolase and glycogen phosphorylase, appeared from stage 18 to 20 in the extraocular muscles. The previous findings and the present results suggest that the fusion of the muscle cells occurs in the period when some molecular markers of muscle differentiation are expressed in vivo.

Development of the notochord in normal and malformed human embryos and fetuses.

In view of its possible involvement in early embryogenesis and teratogenesis, the developmental characteristics of the human notochord were studied by light and electron microscopy and immunohistochemistry on 20 human conceptuses (5th-22nd week). At the earliest embryonic stages examined, the notochord is closely related to both the pharyngeal endoderm and the neuroectoderm of the posterior (tail) end of the neural tube. In both regions the interspace is bridged by slender cytoplasmic processes, lined with basal lamina and filled with amorphous extracellular material containing collagen types III and IV and laminin. The notochordal cells express cytokeratin brightly and vimentin weakly. As embryonic age progresses, the notochord gradually separates from the epithelia, becomes the axis of developing spinal column and undergoes progressive cell degeneration and rearrangement within the vertebral bodies. This is associated with extensive production of extracellular material and the first appearance of fibronectin. Intracellularly, the expression of vimentin gradually increases, while that of cytokeratin slightly weakens. Changes in the notochord parallel other developmental events in axial organs. In anencephalic fetuses the course of the notochord is irregular and partly interrupted with segments outside the basichondrocranium in specimens with craniorachischisis.

Ultrastructural Study on the Retinal Pigment Epithelium of Human Embryos, with Special Reference to Quantitative Study on the Development of Melanin Granules

The development of the retinal pigment epithelium (RPE) was studied ultrastructurally, using 13 externally normal human embryos, Carnegie stages ranging from 13 to 23 (4-8 week of gestation). Melanosomes in the peripheral and posterior RPE were classified according to Fitzpatrick et al. The melanosome of phase I is formed from the Golgi complex and parcelled off into small vesicles. The vesicle enlarges and elongates to form an oval organelle with membranous structures in it (phase II melanosome). Subsequently, melanin deposits on the membranous structures of the melanosomes (phase III melanosomes), and the completion of this process produces a uniformly electrondense granule without discernible internal structures (phase IV melanosome). Melanosomes of phases III and IV appeared in the RPE at stage 15. As the embryonic stage advanced, the ratio of phase II melanosomes decreased and that of phase IV melanosomes increased. The number of phase III melanosomes reached a peak in the peripheral and posterior RPE at stages 15 and 18, respectively. After stage 17, the increase in melanosomes and intracellular organelles was more prominent in the posterior than in the peripheral RPE. During stages 13 and 15, gap junctions were present not only in the apical but also basal plasma membranes of the RPE. At stage 20, gap junctions in the basal plasma membrane disappeared except for the transitional areas from the RPE to the neural retina (NR). In addition, gap junctions were observed between NR and RPE only in the peripheral region at stage 20. The morphological and quantitative differences in the peripheral and posterior RPE in the embryonic period are discussed.

Development of the notochord in human embryos: Ultrastructural, histochemical, and immunohistochemical studies

In the present study of the notochord, the specimens were 54 externally normal human embryos ranging between Carnegie stages 13 and 23. The following staining procedures were used: periodic acid-Schiff (PAS), modified method of PAS, alcian blue, colloidal iron, and toluidine blue. Routine electron microscopic techniques were used. Immunoreactivity of the notochord to alpha-enolase was also examined. The notochord cells were undifferentiated in stage 13 with few intracellular organelles. The microfibrils and deposition of acid mucopolysaccharides appeared in the notochordal sheath in stage 14. The characteristic relation of mitochondria with rough endoplasmic reticulum was observed. Golgi complexes increased in the perinuclear region in stage 15. The layer of microfibrils in the notochordal sheath initially separated from the notochord in stage 16. Glycogen, mucoprotein, neutral mucopolysaccharides, and glycolipids began to increase in the mesenchymal cells around the notochord, starting at stage 16. Acid mucopolysaccharides increased in the notochordal sheath and in the matrix of the precartilage area around the notochord as this embryonic stage advanced. It was also revealed that the immunoreactivity of the notochord to alpha-enolase remained constant during the embryonic period. The results show that the notochord is transformed from an apparently undifferentiated organ into an organ with secretory activity in stage 14, producing microfibrils and depositing acid mucoplysaccharides in the notochordal sheath. The immunoreactivity of the notochord to alpha- and gamma-enolase isoenzymes and the development of the notochord are discussed. This study was undertaken to provide additional information on the development of tumors of notochordal origin.

Development of the choroid plexus anlage and supraependymal structures in the fourth ventricular roof plate of human embryos: scanning electron microscopic observations.

The developing anlage of the choroid plexus and supraependymal structures in the fourth ventricular roof plates of nine normal human embryos ranging from Carnegie stages 14 to 19 were investigated with scanning electron microscopy. In the human embryos at stage 18, the first semimacroscopic choroidal anlage developed in the form of bilateral evaginations that ran dorsomedially and caudally from the bilateral corners of the rhombencephalon. The anlage became evident with even smaller and parallel ridges in the embryo at stage 19. Embryos at earlier stages exhibited surface membrane modifications such as convexity, microvilli, cilia, and spherical protrusions at the middle one-third of the rhombencephalon, which corresponded to the future choroidal anlage region. Two morphologically different groups of supraependymal cells (SE cells) were elucidated throughout the stages examined. Type 1 SE cells has spindle or tear-drop-like bodies, frequently with one or more long cytoplasmic processes. Type 2 SE cells were globular, with numerous fine pseudopodial processes. Type 1 SE cells were distributed mainly at the future choroidal anlage regions or on the anlage itself and were less frequently located at the rostral end of the roof. We found no general pattern in the distribution of type 2 SE cells. Supraependymal fibers (SE fibers) were seen as fine processes that were distributed similarly to type 1 SE cells and extended transversely for a long distance.

Rotation of the junction of the outflow tract and great arteries in the embryonic human heart.

The factors which give rise to the normal relationship between the great arteries and their respective ventricles are unknown. The developmental anatomy of this region was studied by using frontal, sagittal, or transverse serial histologic sections of 17 normal human embryos of Carnegie stages 15-19 from the Carnegie Embryological Collection. Distances and angles between major anatomic landmarks were determined by using computer reconstructions of the serially sectioned embryos, three-dimensional analytic geometry, and Euclidean distance formulas. The findings show that between stages 15 and 19 there is a marked rotation of the axis of the semilunar valves: frontal 121 degrees counterclockwise, sagittal 196 degrees counterclockwise, and transverse 240 degrees clockwise. Simultaneously the great arteries lengthen at a faster rate than the rest of the heart; and there is also an increase in the caliber and wall thickness of the great arteries. These results suggest that the changing rate of growth between the great arteries and the heart is necessary to align the great arteries, the semilunar valves, and the muscular outflow tract septum appropriately with respect to the interventricular septum. Reductions in the rate of growth of the great arteries relative to the heart could, by causing changes in the rotation of great arteries and outflow tract septum, have a role in the pathogenesis of cardiovascular malformations such as tetralogy of Fallot and transposition of the great arteries.