Variations of the Circle of Willis at the End of the Human Embryonic Period.

Variations of the circle of Willis (CW) influence blood supply to the brain and adjacent structures in adults. We examined the formation of the CW in 20 human embryo samples at the end of the embryonic period using 3-D reconstructions of serial histological sections. The CW was closed in all samples, and did not form in a single plane, but was composed of multiple stair-like planes. The artery acutely curved at the caudal part of the CW, namely, at the inlet of the basilar artery and bifurcation of the P1 segment of the posterior cerebral artery (PCA), reflecting flexure of the mesencephalon and diencephalon at this stage. Variations were observed in 17 of 20 samples-only anterior parts (anterior communicating artery [Acom] and anterior cerebral artery [ACA]) in 10 samples, only posterior parts (posterior communicating artery [Pcom]) in one sample, and both anterior and posterior parts in six samples. Variations included the Acom formed as partially duplicated in three samples, duplicated in four, plexiform in three, and no channel as a result of a single azygos ACA in one. The ACA formed as duplicated in two, median ACA in two, and right hypoplasia in one. The Pcom formed in hypoplasia of either side in six samples. Variations observed in this study are similar to those observed in fetuses, neonates, and adults. The P1 segment of PCA was very large in all samples. The present observations indicate that variations in the CW are present from the initiation of CW formation. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Formation of the Periotic Space During the Early Fetal Period in Humans.

The inner ear is a very complicated structure, composed of a bony labyrinth (otic capsule; OC), membranous labyrinth, with a space between them, named the periotic labyrinth or periotic space. We investigated how periotic tissue fluid spaces covered the membranous labyrinth three-dimensionally, leading to formation of the periotic labyrinth encapsulated in the OC during human fetal development. Digital data sets from magnetic resonance images and phase-contrast X-ray tomography images of 24 inner ear organs from 24 human fetuses from the Kyoto Collection (fetuses in trimesters 1 and 2; crown-rump length: 14.4-197 mm) were analyzed. The membranous labyrinth was morphologically differentiated in samples at the end of the embryonic period (Carnegie stage 23), and had grown linearly to more than eight times in size during the observation period. The periotic space was first detected at the 35-mm samples, around the vestibule and basal turn of the cochlea, which elongated rapidly to the tip of the cochlea and semicircular ducts, successively, and almost covered the membranous labyrinth at the 115-mm CRL stage or later. In those samples, several ossification centers were detected around the space. This article thus demonstrated that formation of the membranous labyrinth, periotic space (labyrinth), and ossification of the OC occurs successively, according to an intricate timetable. Anat Rec, 301:563-570, 2018. © 2018 Wiley Periodicals, Inc.

Three-dimensional models of the segmented human fetal brain generated by magnetic resonance imaging.

Recent advances in imaging technology have enabled us to obtain more detailed images of the human fetus in a nondestructive and noninvasive manner. Through detailed images, elaborate three-dimensional (3D) models of the developing brain can be reconstructed. The segmentation of the developing brain has been determined by serial sections. Therefore, in this study, we attempted to develop a 3D model of the fetal brain using magnetic resonance image (MRI). MR images from 19 specimens (11 embryonic specimens and eight fetal specimens from 5.2 to 225 mm in crown rump length) were used to reconstruct 3D models of regionalized developing brains. From this analysis, we succeeded in registering a maximum of nine landmarks on MR images and reconstructing 19 sequential models of the regionalized developing brain. To confirm the validity of the landmarks, we also compared our results with three serial sections from the Kyoto Collection; the same morphological characteristics were observed on both serial sections and MRI. The morphological minutiae could be found on MR images, and regionalized models of the developing brain could be reconstructed. These results will be useful for clinical diagnosis of living fetuses in utero.

Blechschmidt Collection: Revisiting specimens from a historical collection of serially sectioned human embryos and fetuses using modern imaging techniques.

Along with the Carnegie Collection in the United States and the Kyoto Collection in Japan, the Blechschmidt Collection (Georg-August-University of Göttingen, Germany) is a major historical human embryo and fetus collection. These collections are of enormous value to human embryology; however, due to the nature of the historical histological specimens, some stains are fading in color, and some glass slides are deteriorating over time. To protect these specimens against such degradation and ensure their future usefulness, we tried to apply modern image scanning and computational reconstruction. Samples of histological specimens of the Blechschmidt Collection were digitized into images using commercial flatbed scanners with a resolution of 4800 pixels per inch. Two specimens were reconstructed into three-dimensional (3D) images by using modern techniques to vertically stack two-dimensional images of the slices into 3D blocks. The larger specimen of crown-rump length (CRL) 64.0 mm, a series of very large histological sections in human embryology, was reconstructed clearly, with its central nervous system segmented before stacking. The smaller specimen of CRL 17.5 mm was also reconstructed into 3D images. The outer surface of the embryo was intact, and its development was classified according to the widely used Carnegie stages (CSs). The CS of the specimen was identified as the later half of CS 20. The invaluable Blechschmidt Collection can be revisited for further research with modern techniques such as digital image scanning and computational 3D reconstruction.

Cartilage formation in the pelvic skeleton during the embryonic and early-fetal period.

The pelvic skeleton is formed via endochondral ossification. However, it is not known how the normal cartilage is formed before ossification occurs. Furthermore, the overall timeline of cartilage formation and the morphology of the cartilage in the pelvis are unclear. In this study, cartilage formation in the pelvic skeletons of 25 human fetuses (crown-rump length [CRL] = 11.9-75.0 mm) was observed using phase-contrast computed tomography and 7T magnetic resonance imaging. The chondrification center of the ilium, ischium, and pubis first appeared simultaneously at Carnegie stage (CS) 18, was located around the acetabulum, and grew radially in the later stage. The iliac crest formed at CS20 while the iliac body's central part remained chondrified. The iliac body formed a discoid at CS22. The growth rate was greater in the ilium than in the sacrum-coccyx, pubis, and ischium. Connection and articulation formed in a limited period, while the sacroiliac joint formed at CS21. The articulation of the pubic symphysis, connection of the articular column in the sacrum, and Y-shape connection of the three parts of the hip bones to the acetabulum were observed at CS23; the connection of the ischium and pubic ramus was observed at the early-fetal stage. Furthermore, the degree of connection at the center of the sacrum varied among samples. Most of the pelvimetry data showed a high correlation with CRL. The transverse and antero-posterior lengths of the pelvic inlet of the lesser pelvis varied among samples (R2 = 0.11). The subpubic angle also varied (65-90°) and was not correlated with CRL (R2 = 0.22). Moreover, cartilaginous structure formation appeared to influence bone structure. This study provides valuable information regarding the morphogenesis of the pelvic structure.

Positional Changes of the Ocular Organs During Craniofacial Development.

The present study aimed to describe the positional changes of the ocular organs during craniofacial development; moreover, we examined the relationships among the ocular organs and other internal structures. To do this, we traced the positions of the ocular organs in 56 human early fetal samples at different stages of development using high-resolution magnetic resonance imaging and phase-contrast X-ray computed tomography. The eyes were located on the lateral side in the ventral view at Carnegie stage (CS) 16, and then changed their positions medially during development. The eyes remained in the neurocranium until CS17. However, the eyes changed their positions medially and caudally in the viscerocranium after CS18. The positional relationship between the eyes and pituitary gland changed in the lateral view as development progressed. Specifically, they were close to each other at CS17, but moved apart during the later stages of development. These positional changes were also demonstrated quantitatively with morphometric analyses. Based on the present data, the positional changes of the eyes can be categorized into phases, as follows: Phase 1, dramatic positional changes (early fetal period until CS23); and Phase 2, mild positional changes (stabilized; early fetal period after CS23). Notably, all absolute lengths measured in the present study linearly increased as the crown-rump length increased irrespective of the phase, while features of the measured angles and ratios differentially changed in Phases 1 and 2. The present data may help improve our understanding of both the normal and abnormal development of the ocular organs and craniofacial area. Anat Rec, 300:2107-2114, 2017. © 2017 Wiley Periodicals, Inc.

Dynamics of gyrification in the human cerebral cortex during development.

This study quantitatively characterized cortical gyrus folding over human neocortical development by calculating the gyrification index (GI) in 22 human fetal specimens from 16 to 40 weeks with magnetic resonance imaging data. GI values remained constant at approximately 1.0 until the fetal specimens reached 500 g body weight and 200 mm crown-rump length (CRL), respectively, and then increased in correlation with the body weight and CRL. The rostrocaudal GI distribution in the cerebral cortex revealed a correspondence of GI peaks with indentations of early-generated primary sulci at 21 weeks of gestation and more frequently increased GI values in the parieto-occipital region than in the fronto-temporal region at 31 and 40 weeks of gestation. These results provide a quantitative reference set for gyrification in normal human cortical development, which may help reveal the mechanism of neurodevelopmental disorders.

Morphogenesis of the middle ear ossicles and spatial relationships with the external and inner ears during the embryonic period.

We describe the three-dimensional morphogenesis of the middle ear ossicles (MEOs) according to Carnegie stage (CS) in human embryos. Seventeen samples including 33 MEOs from CS18 to 23 were selected from the Kyoto Collection. The primordia of the MEOs and related structures were histologically observed and three-dimensionally reconstructed from digital images. The timing of chondrogenesis was variable among structures. The stapes was recognizable as a vague condensation of the mesenchymal cells in all samples from CS18, whereas the malleus and incus were recognizable at CS19. Chondrogenesis of all MEOs was evident in all samples after CS21. The chondrocranium was recognizable in all samples by CS18, and the perichondrium border of the auricular cartilage and otic capsule was distinct in all samples at CS23. At CS19, the MEOs were positioned in the anterior to posterior direction, following the order malleus, incus, stapes, which adjusted gradually during development. The MEOs connected in all samples after CS22. The stapes was located close to the vestibular part of the inner ear, although the basal part was not differentiated into the "footplate" form, even at CS23. The handles of the malleus were close to the tubotympanic recess at CS23, but were distant from the external auditory meatus. Determining the timeline of the formation of MEOs and connection of the external and inner ears can be informative for understanding hearing loss caused by failure of this connection. These data may provide a useful standard for morphogenesis, and will contribute to distinguishing between normal and abnormal MEO development. Anat Rec, 299:1325-1337, 2016. © 2016 Wiley Periodicals, Inc.

Formation of the circle of Willis during human embryonic development.

The circle of Willis (CW) is a circulatory anastomosis that supplies blood to the brain and adjacent structures. We examined the timing of formation of CW in 20 Japanese human embryo samples by using 3-dimensional reconstruction of serial histological sections. The CW was closed in 1 (n = 6), 2 (n = 8), 2 (n = 3) and 2 (n = 3) samples at Carnegie stages 20, 21, 22, and 23, respectively. The CW was unclosed in 13 samples (unclosed at ACOM alone, 6 samples; ACOM and bilateral P1, 4; left PCOM and right P1, 1; right PCOM and right P1, 1; ACOM and left PCOM, 1). It was difficult to predict whether the circle would close during further development, as such variations frequently exist in adults.

The Digestive Tract and Derived Primordia Differentiate by Following a Precise Timeline in Human Embryos Between Carnegie Stages 11 and 13.

The precise mechanisms through which the digestive tract develops during the somite stage remain undefined. In this study, we examined the morphology and precise timeline of differentiation of digestive tract-derived primordia in human somite-stage embryos. We selected 37 human embryos at Carnegie Stage (CS) 11-CS13 (28-33 days after fertilization) and three-dimensionally analyzed the morphology and positioning of the digestive tract and derived primordia in all samples, using images reconstructed from histological serial sections. The digestive tract was initially formed by a narrowing of the yolk sac, and then several derived primordia such as the pharynx, lung, stomach, liver, and dorsal pancreas primordia differentiated during CS12 (21-29 somites) and CS13 (≥ 30 somites). The differentiation of four pairs of pharyngeal pouches was complete in all CS13 embryos. The respiratory primordium was recognized in ≥ 26-somite embryos and it flattened and then branched at CS13. The trachea formed and then elongated in ≥ 35-somite embryos. The stomach adopted a spindle shape in all ≥ 34-somite embryos, and the liver bud was recognized in ≥ 27-somite embryos. The dorsal pancreas appeared as definitive buddings in all but three CS13 embryos, and around these buddings, the small intestine bent in ≥ 33-somite embryos. In ≥ 35-somite embryos, the small intestine rotated around the cranial-caudal axis and had begun to form a primitive intestinal loop, which led to umbilical herniation. These data indicate that the digestive tract and derived primordia differentiate by following a precise timeline and exhibit limited individual variations.

Morphometric human embryonic brain features according to developmental stage.

OBJECTIVES: The present study investigated linear, area, and volume measurements of human brain samples according to Carnegie stages (CS) in an attempt to select suitable morphometric features that reflect embryonic development. METHODS: Using magnetic resonance imaging, we measured seven linear segments, three separate areas, and three regional volumes in 101 samples between CS13 and 23. Brain volume was determined via manual segmentation of the magnetic resonance image, whereby a formula was generated to estimate the volume of each linear measurement. RESULTS: All parameters correlated with crown-rump length. Bitemporal length and mesencephalic height increased linearly according to the CS, and a high correlation between bitemporal length and both whole-brain (r = 0.98) and prosencephalon (r = 0.99) volumes was found when brain cavity volume was excluded. CONCLUSION: Morphometric data related to human embryonic stages are valuable for correcting and comparing sonographic data. The present approach may contribute to improvements in prenatal diagnostics by enabling the selection of more suitable measurements during early embryonic stages. © 2016 John Wiley & Sons, Ltd.

Intestinal Rotation and Physiological Umbilical Herniation During the Embryonic Period.

Drastic changes occur during the formation of the intestinal loop (IL), including elongation, physiological umbilical herniation (PUH), and midgut rotation. Fifty-four sets of magnetic resonance images of embryos between Carnegie stage (CS) 14 and CS 23 were used to reconstruct embryonic digestive tract in three dimensions in the Amira program. Elongation, PUH, and rotation were quantified in relation to the proximal part of the superior mesenteric artery (SMA), designated as the origin. Up to CS 16, IL rotation was initially observed as a slight deviation of the duodenum and colorectum from the median plane. The PUH was noticeable after CS 17. At CS 18, the IL showed a hairpin-like structure, with the SMA running parallel to the straight part and the cecum located to the left. After CS 19, the IL began to form a complex structure as a result of the rapid growth of the small intestinal portion. By CS 20, the IL starting point had moved from the right cranial region to an area caudal to the origin, though elongation of the duodenum was not conspicuous-this was a change of almost 180° in position. The end of the IL remained in roughly the same place, to the left of and caudal to the origin. Notably, the IL rotated around the origin only during earlier stages and gradually moved away, running transversely after CS 19. The movements of the IL may be explained as the result of differential growth, suggesting that IL rotation is passive.

A Novel Strategy to Reveal the Latent Abnormalities in Human Embryonic Stages from a Large Embryo Collection.

The cause of spontaneous abortion of normal conceptuses remains unknown in most cases. The study was aimed to reveal the latent abnormalities by using a large collection of embryo images from a magnetic resonance imaging (MRI) database and novel phase-contrast radiographic computed tomography (PXCT). MRI from 1,156 embryos between Carnegie stage (CS) 14 and CS23 from the Kyoto Collection were screened by using the volume of the liver as the target organ. Embryos with liver volumes ≥2 SD above or below the mean for the stage of development were screened and examined precisely on MRI. Embryos with potentially abnormal livers were further analyzed by using PXCT. Liver abnormality was detected in all 7 embryos in the extra-small liver group and in 2 of 8 embryos in the extra-large liver group. The abnormalities in the extra-small liver group consisted of hepatic agenesis (2 embryos), hepatic hypogenesis (4), and liver lobe defect (1). Among the 7 extra-small liver group, 2 had only liver abnormalities and 5 exhibited complications in other organs. Of the 2 embryos in the extra-large liver group, one had only a single liver abnormality and the other had a morphologically abnormal liver with complications in other organs. Most of such liver abnormality cases are not survive, as liver function becomes essential. The prevalence of liver malformations in CS18 and CS21 in the intrauterine population of externally normal embryos is approximately 1.7%. The present study is the first step toward the elucidation of the latent abnormalities resulting in spontaneous abortion in externally normal embryos.

Correlation of external ear auricle formation with staging of human embryos.

The formation of auricles in human embryos was evaluated between Carnegie stage (CS)19 and CS23, and the findings were correlated across the stages. The auricle was categorized into 11 steps according to Streeter's criteria with modifications. Mesenchyme cell condensation was observed at Step 7, and two layers of cartilage consisting of the auricle were recognized at Step 11. The representative steps at each CS shifted from Step 3 to Step 11 during CS16 and CS23, although several steps overlapped between adjacent CSs. These results indicate that observations of the auricle between CS19 and CS23 may be utilized for determining embryo staging as convincing supportive evidence of external features reflecting the internal histological structure, although other findings should also be taken into account.

Morphogenesis of the inner ear at different stages of normal human development.

This study examined the external morphology and morphometry of the human embryonic inner ear membranous labyrinth and documented its three-dimensional position in the developing embryo using phase-contrast X-ray computed tomography and magnetic resonance imaging. A total of 27 samples between Carnegie stage (CS) 17 and the postembryonic phase during trimester 1 (approximately 6-10 weeks after fertilization) were included. The otic vesicle elongated along the dorso-ventral axis and differentiated into the end lymphatic appendage and cochlear duct (CD) at CS 17. The spiral course of the CD began at CS18, with anterior and posterior semicircular ducts (SDs) forming prominent circles with a common crus. The spiral course of the CD comprised more than two turns at the postembryonic phase, at which time the height of the CD was evident. A linear increase was observed in the length of anterior, posterior, and lateral SDs, in that order, and the length of the CD increased exponentially over the course of development. Bending in the medial direction was observed between the cochlear and vestibular parts from the latero-caudal view, with the angle decreasing during development. The position of the inner ear was stable throughout the period of observation on the lateral to ventral side of the rhombencephalon, caudal to the pontine flexure, and adjacent to the auditory ganglia. The plane of the lateral semicircular canal was approximately 8.0°-14.6° with respect to the cranial caudal (z-)axis, indicating that the orientation of the inner ear changes during growth to adulthood.

Magnetic resonance microscopy of chemically fixed human embryos at high spatial resolution.

We acquired magnetic resonance (MR) microscopic images of chemically fixed human embryos of Carnegie stages 16 to 22 with a large image matrix (256 × 256 × 512) using an MR microscope that we developed with a 9.4-tesla vertical wide-bore superconducting magnet and a dual-channel receiver system to extend the dynamic range of the MR signal. The images showed clear anatomical structures at spatial resolutions of (40 µm)(3) to (60 µm)(3). We concluded that the experimental technique we developed will aid construction of the next anatomical database of the collection of chemically fixed human embryos.

Morphological features and length measurements of fetal lateral ventricles at 16-25 weeks of gestation by magnetic resonance imaging.

Normal growth of the lateral ventricles (LVs) was characterized three-dimensionally using magnetic resonance imaging (MRI) data from 16 human fetuses at 16-25 weeks of gestation. The LV was differentiated into four primary regions, the anterior horn, central parts, posterior horn, and inferior horn, at 16 weeks of gestation. The LV changed shape mainly by elongation and narrowing, which corresponded to the external and internal growth of the surrounding cerebrum. Six length parameters measured in the LV correlated with biparietal diameter by simple regression analysis (R(2) range, 0.56-0.93), which may be valuable for establishing a standardized prenatal protocol to assess fetal well-being and development across intrauterine periods. No correlation was found between biparietal diameter and LV volume (R(2) = 0.13).

Morphogenesis of the spleen during the human embryonic period.

We aimed to observe morphological changes in the spleen from the emergence of the primordium to the end of the embryonic period using histological serial sections of 228 samples. Between Carnegie stages (CSs) 14 and 17, the spleen was usually recognized as a bulge in the dorsal mesogastrium (DM), and after CS 20, the spleen became apparent. Intrasplenic folds were observed later. A high-density area was first recognized in 6 of the 58 cases at CS 16 and in all cases examined after CS 18. The spleen was recognized neither as a bulge nor as a high-density area at CS 13. The mesothelium was pseudostratified until CS 16 and was replaced with high columnar cells and then with low columnar cells. The basement membrane was obvious after CS 17. The mesenchymal cells differentiated from cells in the DM, and sinus formation started at CS 20. Hematopoietic cells were detected after CS 18. The vessels were observed at CS 14 in the DM. Hilus formation was observed after CS 20. The parallel entries of the arteries and veins were observed at CS 23. The rate of increase in spleen length in relation to that of stomach length along the cranial-caudal direction was 0.51 ± 0.11, which remained constant during CSs 19 and 23, indicating that their growths were similar. These data may help to better understand the development of normal human embryos and to detect abnormal embryos in the early stages of development.

Morphogenesis of lateral choroid plexus during human embryonic period.

The morphological and histological changes of the choroid plexus (CP) during Carnegie stage (CS) 18 and CS23 were presented, based on magnetic resonance imaging data and histological serial section of human embryos from the Kyoto Collection of Human Embryos. The primordium of the CP was initially detected as a small lump at CS19 that grew caudally, so that the CP became crescent shaped. It developed in all directions after CS21, as the dorsal and frontal growth also became prominent. The CP formed a number of undulating surfaces at CS20, irregular bulges at CS21, and then three large clusters with two deep fissures on the caudal surface at CS23. The mean volume of the CP was 0.282±0.141 mm(3) at CS19; it reached 16.8±8.77 mm(3) at CS23. Additionally, the histology was different depending on the regions of the CP at all stages after CS20. The epithelium and angioblasts in the center of the stroma were proliferated in the proximal region, whereas the epithelium was differentiated and lobulated in the distal region where the blood vascular system was organized. The histological differentiation was mapped on the CP reconstructed from histological serial sections. The data suggested the correlation between morphological information obtained from magnetic resonance data sets and distribution of the differentiation. With the help of morphological analysis and histological findings, we have been able to categorize each CP into specific stages. These findings will be useful in clinical evaluation of development during the embryonic period.

Morphology and morphometry of fetal liver at 16-26 weeks of gestation by magnetic resonance imaging: Comparison with embryonic liver at Carnegie stage 23.

AIM: Normal liver growth was described morphologically and morphometrically using magnetic resonance imaging (MRI) data of human fetuses, and compared with embryonic liver to establish a normal reference chart for clinical use. METHODS: MRI images from 21 fetuses at 16-26 weeks of gestation and eight embryos at Carnegie stage (CS)23 were investigated in the present study. Using the image data, the morphology of the liver as well as its adjacent organs was extracted and reconstructed three-dimensionally. Morphometry of fetal liver growth was performed using simple regression analysis. RESULTS: The fundamental morphology was similar in all cases of the fetal livers examined. The liver tended to grow along the transversal axis. The four lobes were clearly recognizable in the fetal liver but not in the embryonic liver. The length of the liver along the three axes, liver volume and four lobes correlated with the bodyweight (BW). The morphogenesis of the fetal liver on the dorsal and caudal sides was affected by the growth of the abdominal organs, such as the stomach, duodenum and spleen, and retroperitoneal organs, such as the right adrenal gland and right kidney. The main blood vessels such as inferior vena cava, portal vein and umbilical vein made a groove on the surface of the liver. Morphology of the fetal liver was different from that of the embryonic liver at CS23. CONCLUSION: The present data will be useful for evaluating the development of the fetal liver and the adjacent organs that affect its morphology.