Sexual dimorphism of the human fetal pelvis exists at the onset of primary ossification.

Human adolescent and adult skeletons exhibit sexual dimorphism in the pelvis. However, the degree of sexual dimorphism of the human pelvis during prenatal development remains unclear. Here, we performed high-resolution magnetic resonance imaging-assisted pelvimetry on 72 human fetuses (males [M]: females [F], 34:38; 21 sites) with crown-rump lengths (CRL) of 50-225 mm (the onset of primary ossification). We used multiple regression analysis to examine sexual dimorphism with CRL as a covariate. Females exhibit significantly smaller pelvic inlet anteroposterior diameters (least squares mean, [F] 8.4 mm vs. [M] 8.8 mm, P = 0.036), larger subpubic angle ([F] 68.1° vs. [M] 64.0°, P = 0.034), and larger distance between the ischial spines relative to the transverse diameters of the greater pelvis than males. Furthermore, the sacral measurements indicate significant sex-CRL interactions. Our study suggests that sexual dimorphism of the human fetal pelvis is already apparent at the onset of primary ossification.

Formation of tendinous intersections in the human fetal rectus abdominis.

Previous studies have poorly described the initial development process of the tendinous intersections of the rectus abdominis muscle (RAM). The present study aimed to observe the formation of tendinous intersections in the RAM during the early fetal period using diffusion tensor imaging (DTI). Fifteen human fetal specimens (crown-rump length [CRL]: 39.5-93.7 mm) were selected. Three-dimensional measurements revealed that Zone-4 (i.e., the zone between the pubic symphysis and the caudal base of the umbilical ring in the RAM) had a smaller width and was thicker than Zone-1 and Zone-2 (i.e., the zones between the costal arch and the cranial base of the umbilical ring) and Zone-3 (i.e., the zone at the umbilical ring). Characteristics of tendinous intersections in the RAM during the early fetal period were assessed according to number, size, type, laterality, and sex. The mean number of tendinous intersections on both sides was 3.1 (range: 2.0-4.0), and 21% of specimens had only two tendinous intersections, which was higher than that reported in previous adult studies. The present data suggest that the formation of tendinous intersections was still in progress in specimens with two tendinous intersections in the RAM and that the third tendinous intersection was formed in Zone-2. Ordinal logistic regression via generalized estimating equations revealed that the odds for a higher type of tendinous intersections in Zone-1 and Zone-2 were significantly higher than those in Zone-4 (adjusted odds ratio: 14.85, 8.84). The odds for the presence of incomplete types (tendinous intersections that could not completely transverse the RAM) in Zone-3 were significantly higher than those in Zone-1 (adjusted odds ratio: 7.4). The odds for missing tendinous intersections in Zone-4 were significantly higher than those in Zone-1 (adjusted odds ratio: 20.5). These zonal differences in the formation of tendinous intersections were consistent with those observed in previous adult studies. In this study, DTI detected tendinous intersections in a sample with a CRL of 45.8 mm (approximately 11 weeks of gestation), which is earlier than that in previous histological findings, indicating that the RAM does not have mature tendinous intersections until the 17th week of gestation. In conclusion, DTI could detect the premature differentiation of tendinous intersection formation. Our data may aid in elucidating the developmental processes of tendinous intersections in the RAM.

Epithelial development of the urinary collecting system in the human embryo.

The urinary collecting system (UCS) consists of organized ducts that collect urine from the nephrons and transport it to the ureter and bladder. Understanding the histogenesis of the UCS is critical. Thirty human embryos between the Carnegie stages (CS) 18 and 23 were selected from the Congenital Anomaly Research Center, Kyoto, Japan. Epithelia of the UCS, ureter, and bladder of each sample were randomly selected. Histological findings of the epithelia were analyzed according to the following criteria: type of epithelium, presence or absence of glycogen, percentage of migrated nuclei, percentage of cells in mitosis, and the surrounding mesenchyme. A thickened epithelium lining a narrow luminal cavity was observed in the pre-expanded pelvic specimens at CS18-CS23. At CS23, after pelvic expansion, the UCS showed a thin epithelium with a large luminal cavity mainly located on the early branches, whereas the epithelium covering the subsequent branches had medium thickness. Histological characteristics differed depending on the UCS part and sample stage. The degree of differentiation was evaluated, revealing that in CS18-CS23 pre-expanded pelvis specimens, the undifferentiated epithelium was found in the zeroth to third/fifth generation, whereas at CS23, after pelvic expansion, a differentiated epithelium covered the UCS zeroth to seventh generation. In a comparison of the urothelial epithelium between the UCS, ureter, and bladder, we found that urinary tract differentiation may be initiated in the bladder, followed by the ureter, UCS zeroth to seventh generations, and finally, UCS eighth to end generations. An understanding of the histogenesis of embryonic stage UCS can aid in the clinical management of congenital urinary tract defects and other diseases.

Regional differences in the umbilical vein and ductus venosus at different stages of normal human development.

During the fetal period, oxygenated blood from the placenta flows through the umbilical vein (UV), portal sinus, ductus venosus (DV), and inferior vena cava (IVC) to the heart. This venous route varies regionally in many aspects. Herein, we sought to characterize the venous route's morphological features and regional differences during embryonic and early-fetal periods. Twenty-nine specimens were selected for high-resolution digitized imaging; 18 embryos were chosen for histological analysis. The venous route showed a primitive, large, S-shaped curved morphology with regional narrowing and dilation at Carnegie stage (CS) 15. Regional differences in vessel-wall differentiation became apparent from approximately CS20. The vessel wall was poorly developed in most DV parts; local vessel-wall thickness at the inlet was first detected at CS20. The lumen of the venous route changed from a nonuniform shape to a relatively round and uniform morphology after CS21. During the early-fetal period, two large bends were observed around the passage of the umbilical ring and at the inlet of the liver. The length ratio of the extrahepatic UV to the total venous route increased. The sectional area gradually increased during embryonic development, whereas differences in sectional area between the DV, UV, and IVC became more pronounced in the early-fetal period. Furthermore, differences in the sectional area between the narrowest part of the DV and other hepatic veins and the transverse sinus became more pronounced. In summary, the present study described morphological, morphometric, and histological changes in the venous route throughout embryonic and early-fetal development, clarifying regional characteristics.

Three-dimensional imaging analysis of the developmental process of posterior meniscofemoral ligaments in rat embryos.

INTRODUCTION: The posterior meniscofemoral ligament (pMFL) of knee joint is a ligament that runs posterior to the posterior cruciate ligament (PCL) and it is known that the height of the pMFL attachment site causes meniscus avulsion. Therefore, understanding the three-dimensional (3D) structure of the pMFL attachment site is essential to better understand the pathogenesis of meniscus disorders. However, the developmental process of pMFL has not been well investigated. The purpose of this study was to analyze pMFL development in rat knee joints using 3D reconstructed images produced from episcopic fluorescence image capture (EFIC) images and examine its relationship with other knee joint components. METHODS: Knee joints of Wistar rat embryos between embryonic day (E) 16 and E21 were observed with HE stained tissues. Serial EFIC images of the hindlimbs of E17-E21 were respectively captured, from which 3D images were reconstructed and the features of pMFL structure: length and angle, were measured. Besides, the chronological volume changes and the volume ratio of the knee joint components compared to E17 were calculated to identify the differences in growth by components. RESULTS: pMFL was observed from E17 and was attached to the medial femoral condyle and lateral meniscus at all developmental stages, as in mature rats. The lack of marked variation in the attachment site and angle of the pMFL with the developmental stage indicates that the pMFL and surrounding knee joint components developed while maintaining their positional relationship from the onset of development. CONCLUSION: Current results may support to congenital etiology of meniscus disorder.

Pyramidalis muscle formation during human embryonic and early fetal periods.

The pyramidalis muscle (PM) is a paired small triangular muscle of the anterior abdominal wall; however, its physiological significance is unclear. Recent studies have failed to detect this muscle during embryonic period. Hence, the present study aimed to determine the time when PM is emerging and reveal its features using high-resolution magnetic resonance imaging. Fourteen embryos between Carnegie stage (CS)18 and CS23 and 59 fetuses (crown-rump length: 39.5-185.0 mm) were selected for this study. The PM was first detected in one of the three samples at CS20. It was detected in five of the seven samples (71.4%) between CS21 and CS23. Forty-eight samples (81.4%) at early fetal period had PMs on both the right and left sides, and 3 (5.1%) had it only on the right side. Eight samples (13.6%) had no PMs. No side-differences or sexual dimorphisms were detected. The PM length was larger than the width in most samples, although the length/width ratio varied among the samples. The PM/rectus abdominis muscle length and PM/umbilicus-pubic symphysis length ratios were almost constant, irrespective of the crown-rump length. The PM was located ventrally inferior to the rectus abdominis and closer to the medial muscle groups of the lower limb than the rectus abdominis. The present study demonstrated that PM formation occurred in the late embryonic period, and that the frequency, side differences, sex dimorphism, and spatial position of the PM in the early fetal period were similar to those in adults.

Bronchial tree of the human embryo: Examination based on a mammalian model.

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors.

Morphogenesis of the pulmonary vein and left atrial appendage in human embryos and early fetuses.

The left atrium wall has several origins, including the body, appendage, septum, atrial-ventricular canal, posterior wall, and venous component. Here, we describe the morphogenesis of left atrium based on high-resolution imaging (phase-contrast X-ray computed tomography and magnetic resonance imaging). Twenty-three human embryos and 19 fetuses were selected for this study. Three-dimensional cardiac images were reconstructed, and the pulmonary veins and left atrium, including the left atrial appendage, were evaluated morphologically and quantitatively. The positions of the pericardial reflections were used as landmarks for the border of the pericardial cavity. The common pulmonary vein was observed in three specimens at Carnegie stages 17-18. The pericardium was detected at the four pulmonary veins (left superior, left inferior, right superior, and right inferior pulmonary veins) at one specimen at Carnegie stage 18 and all larger specimens, except the four samples. Our results suggest that the position of the pericardial reflections was determined at two pulmonary veins (right and left pulmonary vein) and four pulmonary veins almost simultaneously when the dorsal mesocardial connection between the embryo and heart regressed. The magnetic resonance images and reconstructed heart cavity images confirmed that the left atrium folds were present at the junction between the body and venous component. Three-dimensional reconstruction showed that the four pulmonary veins entered the dorsal left atrium tangentially from the lateral to the medial direction. More specifically, the right pulmonary veins entered at a greater angle than the left pulmonary veins. The distance between the superior and inferior pulmonary veins was shorter than that between the left and right pulmonary veins. Three-dimensional reconstruction showed that the venous component increased proportionally with growth. No noticeable differences in discrimination between the right and left parts of the venous component emerged, while the junction between the venous component and body gradually became inconspicuous but was still recognizable by the end of the observed early fetal period. The left superior pulmonary vein had the smallest cross-sectional area and most flattened shape, whereas the other three were similar in area and shape. The left atrial appendage had a large volume in the center and extended to the periphery as a lobe-like structure. The left atrial appendage orifice increased in the area and tended to become flatter with growth. The whole left atrium volume^(1/3) increased almost proportionally with growth, parallel to the whole heart volume. This study provided a three-dimensional and quantitative description of the developmental process of the left atrium, comprising the venous component and left atrial appendage formation, from the late embryonic to the early fetal stages.

Femoral posture during embryonic and early fetal development: An analysis using landmarks on the cartilaginous skeletons of ex vivo human specimens.

The pre-axial border medially moves between the fetal and early postnatal periods, and the foot sole can be placed on the ground. Nonetheless, the precise timeline when this posture is achieved remains poorly understood. The hip joint is the most freely movable joint in the lower limbs and largely determines the lower-limb posture. The present study aimed to establish a timeline of lower-limb development using a precise measurement of femoral posture. Magnetic resonance images of 157 human embryonic samples (Carnegie stages [CS] 19-23) and 18 fetal samples (crown rump length: 37.2-225 mm) from the Kyoto Collection were obtained. Three-dimensional coordinates of eight selected landmarks in the lower limbs and pelvis were used to calculate the femoral posture. Hip flexion was approximately 14° at CS19 and gradually increased to approximately 65° at CS23; the flexion angle ranged from 90° to 120° during the fetal period. Hip joint abduction was approximately 78° at CS19 and gradually decreased to approximately 27° at CS23; the average angle was approximately 13° during the fetal period. Lateral rotation was greater than 90° at CS19 and CS21 and decreased to approximately 65° at CS23; the average angle was approximately 43° during the fetal period. During the embryonic period, three posture parameters (namely, flexion, abduction, and lateral rotation of the hip) were linearly correlated with each other, suggesting that the femoral posture at each stage was three-dimensionally constant and exhibited gradual and smooth change according to growth. During the fetal period, these parameters varied among individuals, with no obvious trend. Our study has merits in that lengths and angles were measured on anatomical landmarks of the skeletal system. Our obtained data may contribute to understanding development from anatomical aspects and provide valuable insights for clinical application.

Tentorium cerebelli formation during human embryonic and early fetal development.

The morphologies of the fetal tentorium cerebelli (TC) and brain influence each other during development. This study aimed to analyze and more comprehensively understand the three-dimensional morphogenesis of the TC and fetal brain. We examined magnetic resonance imaging from 64 embryonic and fetal specimens (crown-rump length range, 9.2-225 mm). During the embryonic period, the lateral folds of the TC elongated to traverse the middle part of the midbrain. The TC and falx cerebri appeared separated, and no invaginations at the parieto-occipital region were observed. In the early fetal period, the cerebrum covered approximately half of the midbrain. The separation of the dural limiting layer at the parieto-occipital region widened from the posterior cerebrum to the cranial cerebellum. The lateral folds of the TC were spread between its tip, continuous with the falx cerebri, and its base plane, located between the midbrain and rostral hindbrain. Differences in the TC components' growth directions gradually diminished as the cerebrum covered the midbrain. We observed rotation of the TC at its median section according to its growth, which ceased in the middle fetal period. The brainstem and cerebellum extended inferiorly via differential growth, with the cerebrum covering them superiorly. The morphology of the TC curved to conform to the cerebellar and cerebral surfaces. Our present study suggests that factors affecting TC morphology differ between the early and middle fetal periods. Present data provided a more comprehensive view of TC formation according to developmental stage.

Three-dimensional morphogenesis of the human diaphragm during the late embryonic and early fetal period: Analysis using T1-weighted and diffusion tensor imaging.

A precise understanding of human diaphragm development is essential in fetal medicine. To our knowledge, no previous study has attempted a three-dimensional (3-D) analysis and evaluation of diaphragmatic morphogenesis and development from the embryonic to the early fetal period. This study aimed to evaluate the morphogenesis and fibrous architecture of the developing human diaphragm during the late embryonic and early fetal periods. Fifty-seven human embryos and fetuses (crown-rump length [CRL] = 8-88 mm) preserved at the Congenital Anomaly Research Center of Kyoto University and Shimane University were analyzed. 3-D morphogenesis and fiber orientation of the diaphragm were assessed using phase-contrast X-ray computed tomography, T1-weighted magnetic resonance imaging (T1W MRI), and diffusion tensor imaging (DTI). T1W MR images and DTI scans were obtained using a 7-T MR system. The diaphragm was completely closed at Carnegie stage (CS) 20 and gradually developed a dome-like shape. The diaphragm was already in contact with the heart and liver ventrally in the earliest CS16 specimen observed, and the adrenal glands dorsally at CS19 or later. In the fetal period, the diaphragm contacted the gastric fundus in samples with a CRL ≥41 mm, and the spleen in samples with a CRL ≥70 mm. The relative position of the diaphragm with reference to the vertebrae changed rapidly from CS16 to CS19. The most cranial point of the diaphragm was located between the 4th and 8th thoracic vertebrae, regardless of fetal growth, in samples with a CRL of ≥16 mm. Diaphragmatic thickness was nearly uniform (0.15-0.2 mm) across samples with a CRL of 8-41 mm. The sternal, costal, lumbar parts, and the area surrounding the esophageal hiatus thickened with growth in samples with a CRL of ≥46 mm. The thickness at the center of the diaphragm and the left and right hemidiaphragmatic domes did not increase with growth. Tractography showed that the fiber orientation of the sternal, costal, and lumbar parts became more distinct as growth progressed in CS19 or later. All fibers in the costal and lumbar regions ran toward the left and right hemidiaphragmatic domes, except for those running to the caval opening and esophageal hiatus. The fiber orientation patterns from the right and left crura surrounding the esophageal hiatus were classified into three types. Distinct fiber directions between the costal and sternal and between the costal and lumbar diaphragmatic parts were observable in samples with a CRL of ≥46 mm. Anterior costal and sternal fibers ran toward the center. Fiber tracts around the center and the left and right hemidiaphragmatic domes; between the costal and lumbar orientations; and between the costal and sternal orientations showed a tendency for decreasing fractional anisotropy values with fetal growth and showed less density than other areas. In conclusion, we used 3-D thickness assessment and DTI tractography to identify qualitative changes in the muscular and tendonous regions of the diaphragm during the embryonic and early fetal periods. This study provides information on normal human diaphragm development for the progression of fetal medicine and furthering the understanding of congenital anomalies.

Morphometric analysis of secondary palate development in human embryos.

Rapid shelf elevation and contact of the secondary palate and fusion reportedly occur due to a growth-related equilibrium change in the structures within the oro-nasal cavity. This study aimed to quantitatively evaluate complex three-dimensional morphological changes and their effects on rapid movements, such as shelf elevation and contact, and fusion. Morphological changes during secondary palate formation were analyzed using high-resolution digitalized imaging data (phase-contrast X-ray computed tomography and magnetic resonance images) obtained from 22 human embryonic and fetal samples. The three-dimensional images of the oro-nasal structures, including the maxilla, palate, pterygoid hamulus, tongue, Meckel's cartilage, nasal cavity, pharyngeal cavity, and nasal septum, were reconstructed manually. The palatal shelves were not elevated in all the samples at Carnegie stage (CS)21 and CS22 and in three samples at CS23. In contrast, the palatal shelves were elevated but not in contact in one sample at CS23. Further, the palatal shelves were elevated and fused in the remaining four samples at CS23 and all three samples from the early fetal period. For each sample, 70 landmarks were subjected to Procrustes and principal component (PC) analysis. PC-1 accounted for 67.4% of the extracted gross changes before and after shelf elevations. Notably, the PC-1 values of the negative and positive value groups differed significantly. The PC-2 value changed during the phases in which the change in the PC-1 value was unnaturally slow and stopped at CS22 and the first half of CS23. This period, defined as the "approach period", corresponds to the time before dynamic changes occur as the palatal shelves elevate, the tongue and mandibular tip change their position and shape, and secondary palatal shelves contact and fuse. During the "approach period", measurements of PC-2 changes showed that structures on the mandible (Meckel's cartilage and tongue) and maxilla (palate and nasal cavity) did not change positions, albeit both groups of structures appeared to be compressed anterior-posteriorly. However, during and after shelf elevation, measurements of PC-1 changes showed significant changes between maxillary and mandibular structures, particularly positioning of the shelves above the tongue and protrusion of the tongue and mandible. These results suggest an active role for Meckel's cartilage growth in repositioning the tongue to facilitate shelf elevation. The present data representing three distinct phases of secondary palate closure in humans can advance the understanding of morphological growth changes occurring before and after the horizontal positioning of palatal shelves and their fusion to close the secondary palate in humans successfully.

The return process of physiological umbilical herniation in human fetuses: The possible role of the vascular tree and umbilical ring.

The human intestine elongates during the early fetal period, herniates into the extraembryonic coelom (EC), and subsequently returns to the abdominal cavity (AC). The process by which the intestinal loop returns to the abdomen remains unclear. This study aimed to document positional changes in the intestinal tract with the superior mesenteric artery (SMA) and branches in 3D to elucidate the intestinal loop return process (transition phase). Serial histological cross-sections from human fetuses (crown-rump length [CRL] range: 30-50 mm) in the herniation (n = 1), transition (n = 7), and return (n = 2) phases were selected from the Blechschmidt Collection. The distribution of the SMA trunk and all intestinal and sister branches entering the intestines was visualized so that positional changes in branches were continuous from the herniation to return phases. Positional changes in SMA branches proceeded in an orderly and structured manner; this is essential for continuous blood supply via the SMA to the intestine during transition and for safe intestinal return. Changes in the SMA distribution proceeded prior to the detection of initiation of intestinal tract return, which might start earlier and last much longer than our consensus (i.e., that the return of the herniated intestine begins when the CRL is approximately 40 mm and ends within a short time). In the cross-section of the umbilical ring in the herniation and transition phases, one proximal limb and one distal limb were observed with SMA intestinal branches, which were fully packed in the umbilical ring. The SMA branches were aligned from inferior to superior along the SMA main trunk. In the herniation phase, the distribution of 3rd-13th branches aligned from proximal inferior medial to distal superior left with a slight spiral in the EC, the tips of which suggested an orderly running course of the small intestine. In the transition phase, SMA branches running across the umbilical ring that fed the small intestine were observed, suggesting that the intestine was uncoiled and ran across the umbilical ring almost vertically. The estimated curvature value supported the phenomenon of uncoiling at the umbilical ring; the value at the umbilical ring was lesser than that in the AC and EC. During the transition phase, the proximal and distal limbs transversely ran side by side in the AC, umbilical ring, limbs on the cranial side, and mesentery on the caudal side. The SMA trunk and its branches ran in parallel, cranially to caudally aligned in the mesentery. This layout of the umbilical ring was maintained during the transition phase. In the return phase, the SMA trunk was gently curved from the upper left to the lower right of the AC; around 12 branches spread with a winding staircase appearance. The intestinal tract reached its definitive position immediately after all tissues crossed the umbilical ring and released any restriction. Each SMA branch and the corresponding region of the intestinal tract form a unit and change their position, though the conformation may change within each unit when running across the umbilical ring. We suggest that the slide-stack model requires revision.

Upper arm posture during human embryonic and fetal development.

The upper extremity posture is characteristic of each Carnegie stage (CS), particularly between CS18 and CS23. Morphogenesis of the shoulder joint complex largely contributes to posture, although the exact position of the shoulder joints has not been described. In the present study, the position of the upper arm was first quantitatively measured, and the contribution of the position of the shoulder girdle, including the scapula and glenohumeral (GH) joint, was then evaluated. Twenty-nine human fetal specimens from the Kyoto Collection were used in this study. The morphogenesis and three-dimensional position of the shoulder girdle and humerus were analyzed using phase-contrast X-ray computed tomography and magnetic resonance imaging. Both abduction and flexion of the upper arm displayed a local maximum at CS20. Abduction gradually decreased until the middle fetal period, which was a prominent feature. Flexion was less than 90° at the local maximum, which was discrepant between appearance and measurement value in our study. The scapular body exhibited a unique position, being oriented internally and in the upward direction, with the glenoid cavity oriented cranially and ventrally. However, this unique scapular position had little effect on the upper arm posture because the angle of the scapula on the thorax was canceled as the angle of the GH joint had changed to a mirror image of that angle. Our present study suggested that measuring the angle of the scapula on the thorax and that of the GH joint using sonography leads to improved staging of the human embryo.

Three-Dimensional Analysis of Human Laryngeal and Tracheobronchial Cartilages during the Late Embryonic and Early Fetal Period.

Laryngeal and tracheobronchial cartilages are present as unique U-shaped forms around the respiratory tract and contribute to the formation of rigid structures required for the airway. Certain discrepancies still exist concerning cartilage formation in humans. To visualize the accurate timeline of cartilage formation, tracheobronchial and laryngeal cartilages were 3D reconstructed based on serial tissue sections during the embryonic period (Carnegie stage [CS] 18-23) and early fetal period (crown rump length [CRL] = 35-45 mm). The developmental phases of the cartilage were estimated by histological studies, which were performed on the reconstructed tissue sections. The hyoid greater horns were recognizable at CS18 (phase 2). Fusion of 2 chondrification centers in the mid-sagittal region was observed at CS19 in the hyoid bone, at CS20 in the cricoid cartilage, and in the specimen with CRL 39 mm in the thyroid cartilage. Phase 3 differentiation was observed at the median part of the hyoid body at CS19, which was the earliest among all other laryngeal and tracheobronchial cartilages. Most of the laryngeal cartilages were in phase 3 differentiation at CS22 and in phase 4 differentiation at CS23. The U-shaped tracheobronchial cartilages with phase 2 differentiation covered the entire extrapulmonary region at CS20. Phase 3 differentiation started on the median section and propagates laterally at CS21. The tracheobronchial cartilages may form simultaneously during the embryonic period at CS22-23 and early fetal periods, similar to adults in number and distribution. The spatial propagation of the tracheal cartilage differentiation provided in the present study indicates that cartilage differentiation may have propagated differently on phase 2 and phase 3. This study demonstrates a comprehensible timeline of cartilage formation. Such detailed information of the timeline of cartilage formation would be useful to improve our understanding of the development and pathophysiology of congenital airway anomalies.

Early development of the cortical layers in the human brain.

The cortical plate (CP) first appears at seven postconceptional weeks (pcw), when it splits the preexisting preplate into two layers, the marginal zone and the presubplate (pSP). Although three-dimensional (3D) analysis using fetal magnetic resonance imaging and two-dimensional tissue observations have been reported, there have been no studies analyzing the early development of the layer structure corresponding to the pSP stage in 3D. Here, we reconstructed 3-D models of the brain with a focus on the cortical layers in pSP stage. To achieve this, we digitized serial tissue sections of embryos between CS20 and CS23 from the Kyoto Collection (n = 7, approximately 7-8.5 pcw), and specimens at early fetal phase from the Blechschmidt Collection (n = 2, approximately 9.5-12 pcw, crown rump length [CRL] 39 and 64 mm). We observed tissue sections and 3D images and performed quantitative analysis of the thickness, surface area, and volume. Because the boundary between pSP and the intermediate zone (IZ) could not be distinguished in hematoxylin and eosin-stained sections, the two layers were analyzed together as a single layer in this study. The histology of the layers was observed from CS21 and became distinct at CS22. Subsequently, we observed the 3-D models; pSP-IZ was present in a midlateral region of the cerebral wall at CS21, and an expansion centered around this region was observed after CS22. We observed it over the entire cerebral hemisphere at early fetal phase (CRL 39 mm). The thickness of pSP-IZ was visible in 3D and was greater in the midlateral region. At the end of the pSP stage (CRL 64 mm), the thick region expanded to lateral, superior, and posterior regions around the primordium of the insula. While, the region near the basal ganglia was not included in the thickest 10% of the pSP-IZ area. Middle cerebral artery was found in the midlateral region of the cerebral wall, near the area where pSP-IZ was observed. Feature of layer structure growth was revealed by quantitative assessment as thickness, surface area, and volume. The maximum thickness value of pSP-IZ and CP increased significantly according to CRL, whereas the median value increased slightly. The layer structure appeared to grow and spread thin, rather than thickening during early development, which is characteristic during pSP stages. The surface area of the cerebral total tissue, CP, and pSP-IZ increased in proportion to the square of CRL. The surface area of CP and pSP-IZ approached that of the total tissue at the end of the pSP stage. Volume of each layer increased in proportion to the cube of CRL. pSP-IZ and CP constituted over 50% of the total tissue in volume at the end of the pSP stages. We could visualize the growth of pSP-IZ in 3D and quantify it during pSP stage. Our approach allowed us to observe the process of rapid expansion of pSP-IZ from the midlateral regions of the cerebral wall, which subsequently becomes the insula.

A 3D analysis of growth trajectory and integration during early human prenatal facial growth.

Significant shape changes in the human facial skeleton occur in the early prenatal period, and understanding this process is critical for studying a myriad of congenital facial anomalies. However, quantifying and visualizing human fetal facial growth has been challenging. Here, we applied quantitative geometric morphometrics (GM) to high-resolution magnetic resonance images of human embryo and fetuses, to comprehensively analyze facial growth. We utilized non-linear growth estimation and GM methods to assess integrated epigenetic growth between masticatory muscles and associated bones. Our results show that the growth trajectory of the human face in the early prenatal period follows a curved line with three flexion points. Significant antero-posterior development occurs early, resulting in a shift from a mandibular prognathic to relatively orthognathic appearance, followed by expansion in the lateral direction. Furthermore, during this time, the development of the zygoma and the mandibular ramus is closely integrated with the masseter muscle.

Morphology and morphometry of the human early foetal brain: A three-dimensional analysis.

Morphometric analyses in the early foetal phase (9-13 postconceptional week) are critical for evaluating normal brain growth. In this study, we assessed sequential morphological and morphometric changes in the foetal brain during this period using high-resolution T1-weighted magnetic resonance imaging (MRI) scans from 21 samples preserved at Kyoto University. MRI sectional views (coronal, mid-sagittal, and horizontal sections) and 3D reconstructions of the whole brain revealed sequential changes in its external morphology and internal structures. The cerebrum's gross external view, lateral ventricle and choroid plexus, cerebral wall, basal ganglia and thalamus, and corpus callosum were assessed. The development of the cerebral cortex, white matter microstructure, and basal ganglia can be well-characterized using MRI scans. The insula became apparent and deeply impressed as brain growth progressed. A thick, densely packed cellular ventricular/subventricular zone and ganglionic eminence became apparent at high signal intensity. We detected the emergence of important landmarks which may be candidates in the subdivision processes during the early foetal period; the corpus callosum was first detected in the sample with crown-rump length (CRL) 62 mm. A primary sulcus on the medial part of the cortex (cingulate sulcus) was observed in the sample with CRL 114 mm. In the cerebellum, the hemispheres, posterolateral fissure, union of the cerebellar halves, and definition of the vermis were observed in the sample with CRL 43.5 mm, alongside the appearance of a primary fissure in the sample with CRL 56 mm and the prepyramidal fissure in the sample with CRL 75 mm. The volumetric, linear, and angle measurements revealed the comprehensive and regional development, growth, and differentiation of brain structures during the early foetal phase. The early foetal period was neither morphologically nor morphometrically uniform. The cerebral proportion (length/height) and the angle of cerebrum to the standard line at the lateral view of the cerebrum, which may reflect the growth and C-shape formation of the cerebrum, may be a candidate for subdividing the early foetal period. Future precise analyses must establish a staging system for the brain during the early foetal period. This study provides insights into brain structure, allowing for a correlation with functional maturation and facilitating the early detection of brain damage and abnormal development.

Bronchial tree of the human embryo: Categorization of the branching mode as monopodial and dipodial.

Some human organs are composed of bifurcated structures. Two simple branching modes-monopodial and dipodial-have been proposed. With monopodial branching, child branches extend from the sidewall of the parent branch. With dipodial branching, the tip of the bronchus bifurcates. However, the branching modes of the human bronchial tree have not been elucidated precisely. A total of 48 samples between Carnegie stage (CS) 15 and CS23 belonging to the Kyoto Collection were used to acquire imaging data with phase-contrast X-ray computed tomography. Bronchial trees of all samples were three-dimensionally reconstructed from the image data. We analyzed the lobar bronchus, segmental bronchus, and subsegmental bronchus. After calculating each bronchus length, we categorized the branching mode of the analyzed bronchi based on whether the parent bronchus was divided after generation of the analyzed bronchi. All lobar bronchi were formed with monopodial branching. Twenty-five bifurcations were analyzed to categorize the branching mode of the segmental and subsegmental bronchi; 22 bifurcations were categorized as monopodial branching, two bifurcations were not categorized as any branching pattern, and the only lingular bronchus that bifurcated from the left superior lobar bronchus was categorized as dipodial branching. The left superior lobar bronchus did not shorten during the period from CS17 or CS18, when the child branch was generated, to CS23. All analyzed bronchi that could be categorized, except for one, were categorized as monopodial branching. The branching modes of the lobar bronchus and segmental bronchus were similar in the mouse lung and human lung; however, the modes of the subsegmental bronchi were different. Furthermore, remodeling, such as shrinkage of the bronchus, was not observed during the analysis period. Our three-dimensional reconstructions allowed precise calculation of the bronchus length, thereby improving the knowledge of branching morphogenesis in the human embryonic lung.