Details of the bronchial tree formation remain unknown because of the difficulty in analyzing extremely small embryos. We aimed to elucidate the morphogenesis of the human embryonic bronchial tree using phase-contrast X-ray computed tomography (PXCT) images. The three-dimensional (3D) branching of the bronchial trees was reconstructed using PXCT images in a sample of embryos (between n. The images revealed that branching variants arose during the embryonic period and continued throughout life. All proximal bronchi, except the were formed by a monopodial branching mode. The 3D reconstructions of the embryonic bronchial trees provided novel insights into how bronchial trees are generated in the small embryos.
The bronchial tree of the human lung is composed of conducting and respiratory airways [1]. This organ has a highly ramified structure in the lungs. An understanding of branching morphogenesis is essential for the diagnosis and treatment of congenital anomalies. However, how such complicated branching networks are generated during development remains unclear because of the difficulty in analyzing extremely small embryos. Recently, we provided new insights into the branching tree formation in the human embryonic lung by analyzing 3D reconstructions of the human embryonic bronchial tree [2, 3].
Figure 1: Image processing of the bronchial tree and illustration showing the change of bronchus length with monopodial and dipodial branching. a (i) Transverse section using phase-contrast X-ray computed tomography. Heart (H). (ii) Reconstructed bronchial tree. Scale bar: 1mm. (iii) Centerline was calculated based on reconstruction. b The parent bronchus (PBr) length may shrink with monopodial branching just after generation of child bronchus (CBr) (i), but may not shrink or elongate with dipodial branching (ii).
First, we extended the morphological analysis to the end of the embryonic period [2]. The embryonic period is from the time of fertilization to 8 weeks post-fertilization. It begins with the formation of the body structures, generally described by a standardized system of 23 stages called the Carnegie stages (CS) [4]. Previous morphological studies have demonstrated the general morphogenetic processes of the human bronchial tree during the embryonic period. The primordium of the tree buds extends from the middle of the foregut at approximately CS 12 (at approximately 4 weeks after fertilization) [5]. After that, this pulmonary primordium bud continues to extend and branches out continuously to form the lobar, segmental, and more peripheral branches. The first generation of sub-segmental bronchi is complete at CS19 [6]. However, morphological changes in the trees after CS20 have not been elucidated.
Second, we analyzed how the proximal bronchus of the human lung branched off [3]. Previous studies have proposed two simple branching modes: monopodial and dipodial [7, 8]. With monopodial branching, the child branches extend from the sidewall of the parent branch. With dipodial branching, the tip of the bronchus bifurcates. Previous studies estimated the branching mode based only on visual assessments.
Thus, we aimed to describe the morphogenesis of bronchial trees during the human embryonic period. We reconstructed 3D branching trees using phase-contrast X-ray computed tomography (PXCT) images, observed the morphological changes in the trees in detail, and categorized the branching mode as monopodial and dipodial based on the bronchus length.
A total of 48 embryos between CS15 and CS23 (about 5-8 weeks after fertilization, 8-30 mm crown-rump length) [4] of the Kyoto Collection were selected [9]. Imaging data of all samples were acquired using PXCT. The system was set up at the vertical wiggler beamline (BL-14C). The PXCT imaging data provided a resolution of ≥ 18 μm/pixel [10], which enabled the non-destructive observation of intrabody structures in detail, and the highly sensitive morphometry of the embryos. The structure of the bronchial tree was reconstructed for all samples using Amira software (version 6.2.0; Visage Imaging GmbH, Berlin, Germany) (Fig. 1a). The center of the airway was observed linearly with the centerline module. The coordinates were analyzed using MATLAB v. R2018a (MathWorks, Inc., Natick, MA, USA) to calculate the generation of all branches and branch lengths.
We categorized the branching modes of the lobar, segmental, and subsegmental bronchi. After calculating each bronchus length, we categorized the branching mode of the analyzed bronchi based on whether the parent bronchus was divided after generating the analyzed bronchi (Fig.1b).
Figure 2: 3D reconstructions enable novel analyses of the human embryonic bronchial tree. a Representative reconstructions of the bronchial tree. Scale bar: 1mm. b (left) Generation of end-branching (rainbow color) at CS18, CS21, and CS23. The colored circle indicates generations of each end-branch when the lobar bronchus was defined as 0th branch. Color bar indicates the corresponding colors. (right) Change of generation in each lobe by Carnegie stage during CS18 and CS23. LIL, left inferior lobe; LSL, left superior lobe; RIL, right inferior lobe; RML, right middle lobe; RSL, right superior lobe. c Branching mode of the right superior and middle lobar bronchi. The length changes of the right proximal bronchi are shown. Compared with the right primary bronchial bud (RPBB) length before branching, temRMB length and total length of right main bronchus (RMB) and intermediate bronchus (IB) were shorter. RIB, right inferior bronchus; temRMB, temporary RMB branch from the tracheal bifurcation to the base of the right middle lobe.
Three-dimensionally reconstructed bronchial trees revealed a timeline of morphogenesis during the embryonic period (Fig. 2a). The right superior lobar bronchus was formed after the generation of both the right middle and left superior lobar bronchi. The distribution of the end-branch generation among the five lobes was significantly different (Fig. 2b). The median branching generation value in the right middle lobe was significantly lower than that in the other four lobes. Variations found between CS20 and CS23 were all described in the human adult lung, indicating that variation in the bronchial tree may arise during the embryonic period and continue throughout life.
All lobar bronchi were formed with monopodial branching (Fig. 2c). Twenty-five bifurcations were analyzed to categorize the branching mode of the segmental and subsegmental bronchi. Of these, 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.
High-resolution imaging data of human embryonic specimens using PXCT enabled the reconstruction of the three-dimensional bronchial tree, revealing morphogenetic changes during the human embryonic period. Our novel understanding of bronchial tree development will provide a crucial resource for elucidating congenital anomalies.
Fujii, T. Takakuwa (Kyoto Univ.)
REFERENCES
Standring, S, Gray’s Anatomy (Elsevier. 2015)
Fujii, T. Muranaka, J. Matsubayashi, S. Yamada, A. Yoneyama and T. Takakuwa, J. Anat.237 (2020).
Fujii, T. Muranaka, J. Matsubayashi, S. Yamada, A. Yoneyama and T. Takakuwa, PLoS ONE16, 1 (2021)
O’Rahilly and F. Müller, Developmental stages in human embryos: including a revision of Streeter’s” horizons” and a survey of the Carnegie Collection, (Carnegie Institution of Washington Publishing. 1987).
Streeter, G.L., Contributions to Embryology Carnegie Institution of Washington, 31 (1945)
Wells, L. and Boyden, E. American Journal of Anatomy, 95 (1954)
Palmer, D. M. Ohio Journal of Science, 36 (1936).
Metzger RJ, Klein OD, Martin GR and Krasnow MA, Nature. 453, 7196 (2008).
Nishimura, K. Takano, T. Tanimura and M. Yasuda, Teratology, 1 (1968).
Yoneyama, S. Yamada and T. Takeda, “Fine Biomedical Imaging Using X-Ray Phase-Sensitive Technique” in Advanced Biomedical Engineering, edited by G. D. Gargiulo and A. McEwan, 107-128. InTech Publishinng 2011.
The cause of spontaneous abortion of normal conceptuses remains unknown in most embryos because of the difficulty of diagnosing too small embryos. We aimed to reveal latent liver abnormalities using novel phasecontrast radiographic computed tomography (PXCT). Embryos with liver volumes ≥ 2 SD above or below the mean for the stage of development were screened from 1156 MR images from the Kyoto Collection. Selected embryos were further analyzed by using PXCT. Liver abnormality was detected in 9 embryos by our protocol. Most of such liver abnormality embryos do not survive, as liver function becomes essential.
The cause of spontaneous abortion of normal conceptuses remains unknown in most embryos [1] because of the difficulty of obtaining appropriate embryo materials as well as diagnosing internal abnormalities in very small embryos. The crown-rump length of an embryo is ranging 23 to 32 mm during the embryonic period by Carnegie stage (CS) 23 (about 56–60 days after fertilization) [2], by which stage all primordia of the organs are already provided.
Figure 1: Protocol for detecting latent abnormal embryos that may result in spontaneous abortion.
Many factors are involved in spontaneous abortion, not only the embryonic factor itself but also maternal health and the intrauterine environment affected by unspecified modulators. Such complicated factors distract attention from the laborious inspection of embryos, especially when embryos are externally normal.
More than 40,000 embryo and fetal specimens have been collected in the Kyoto Collection since 1961 for revealing the mechanism of congenital anomalies [3]. An MRI database of approximately 1200 well-preserved human embryos, diagnosed as externally normal, was acquired in 2000–2005 (the Kyoto embryo visualizing project) [4] to observe a number of normal human embryos. The database has proven to be useful for research purposes and as a teaching material [5].
Although the MRI database includes only externally normal embryo specimens, whether the internal organs were also normal cannot be guaranteed. Considering that the cumulative intrauterine mortality rate in normal conceptuses was estimated at 18% [1], the MRI database includes embryos that have potential abnormalities that would have led to spontaneous abortion. Thus, detailed observation of the internal organs of embryo specimens from the database may provide clues to spontaneous abortion in the embryonic and fetal periods. In this connection, we aimed to determine the latent abnormalities that may cause spontaneous abortion by using the MRI database and novel phase-contrast radiographic computed tomography (PXCT).
The MRI database was 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 selected. Embryos with potentially abnormal livers were further analyzed by using PXCT. The PXCT data provide a resolution of ≥ 18 μm/pixel, which enabled highly sensitive measurement, approximately > 1000 times more sensitive than the conventional radiographic method using absorption contrast [6].
Liver abnormality was detected in 9 embryos after the procedure of our protocol (Fig. 1), which consisted of hepatic agenesis (2 embryos), hepatic hypogenesis (4), liver lobe defect (1), involvement of the liver to the thoracic cavity by diaphragm herniation (1), and other (1). Three embryos had only liver abnormalities and 6 exhibited complications in other organs. The prevalence of liver malformations in CS18 and CS21 in the intrauterine population of externally normal embryos is approximately 1.7%. Most of such liver abnormality embryos do not survive, as liver function becomes essential during development [7].
Figure 2: Representative embryos with liver abnormality (liver agenesis). a External view of the embryo at CS21, which shows no obvious external abnormality. b Phase-contrast X-ray computed tomography (PXCT) transverse sections at the level of Th4. No liver (Li) was detected in the plane sections, while the stomach (St) was observed in the midsagittal transverse sections at Th4. The diaphragm is indicated by arrow heads. Lung (Lu). c Left anterior oblique view of the three-dimensional (3-D) PXCT reconstruction of the embryo using Amira software (Visage Imaging, Berlin, Germany), demonstrating the locations of all intrathoracic, retroperitoneal, and intra-abdominal organs. The findings of note were as follows: agenesis of the liver; the stomach was deviated ventrally and cranially; the pancreas (Pa) was deviated ventrally; and the right mesonephros (Ms) and genital ridge (Ge) were absent. Adrenal gland (Ad), metanephros (Mt), and heart (He).
A representative embryo with liver agenesis is shown in Fig. 2. The size and gestational age were within the normal range for the embryo at CS 21. Obvious damage to, or anomalies of, the external forms were not present (Fig. 2a). The liver at CS21 usually occupies a large space in the abdominal cavity, which has a smooth surface due to the contact between the cranial surface and the diaphragm, and between the ventral surface and the abdominal wall [7]. In the present embryos, the liver was not detected in any of the serial plane sections (Fig. 2b). The locations of all intrathoracic, retroperitoneal, and intraabdominal organs were reconstructed in three dimensions (Fig. 2c). The absence of the liver had affected the locations of the other internal organs, especially the stomach, duodenum, and pancreas. The stomach was observed on the midsagittal line in the Th4 transverse sections, indicating that the stomach had deviated cranially and ventrally. The diaphragm was apparent in these sections.
The present study demonstrates that PXCT may be considered a powerful tool for visualization of internal structures of embryos, and for the detection of novel abnormalities during the embryonic period, without the need for histological analysis. The noninvasive and nondestructive properties of the technique are important for analysis of scarce specimens, such as human
embryos. The present study is the first step toward elucidating the latent abnormalities that result in spontaneous abortion in externally normal embryos [8].
REFERENCES
[1] K. Shiota, Congenital Anomalies 31, 67 (1991). [2] R. O’Rahilly and F. Müller, Developmental stages in human embryos: including a revision of Streeter’s” horizons” and a survey of the Carnegie Collection, (Carnegie Institution of Washington Publishing. 1987). [3] H. Nishimura, K. Takano, T. Tanimura and M. Yasuda, Teratology 1, 281 (1968). [4] K. Shiota, S. Yamada, T. Nakatsu-Komatsu, C. Uwabe, K. Kose, Y . Matsuda, T . Haishi, S. Mizuta and T . Matsuda, Am J Med Genet A 143A, 3121 (2007). [5] T. Takakuwa, Antat Rec (2017), in press. [6] A. Yoneyama, S. Yamada and T. Takeda, Fine Biomedical Imaging Using X-Ray Phase-Sensitive Technique, eds. G. D. Gargiulo and A. McEwan, Advanced Biomedical Engineering 107 (2011).
[7] A. Hirose, T. Nakashima, S. Yamada, C. Uwabe, K. Kose and T. Takakuwa, Anat Rec 295, 51 (2012).
[8] T. Kanahashi, S. Yamada, M. Tanaka, A. Hirose, C. Uwabe, K. Kose, A. Yoneyama, T. Takeda and T. Takakuwa, Anat Rec 299, 8 (2016).
Herniation into the extraembryonic coelom and return to the abdominal coelom
Background
PUH occurred independent of the liver.
Drastic changes occur during the development of the intestinal loop (IL), including physiological umbilical herniation (PUH) and its return. The present study was designed to analyze such developments three-dimensionally during human embryonic and early fetal period.
Materials and Methods
The software AMIRA was used to analyze the 3D digitalized data (high-resolution MRI, phase-contrast X-ray CT) obtained from the Kyoto Collection.
Results and Discussion
Based on the results of our analysis, the following time line and main features of IL formation were revealed:
Herniate phase (Carnegie stage (CS)14-CS23, Crown-rump length (CRL) < 35 mm): IL rotation was initially observed as a slight deviation of the duodenum and colorectum from the median plane up to CS16. The PUH was noticeable after CS16. The IL displayed a hairpin-like structure, with the superior mesenteric artery (SMA) running parallel to the straight part and the cecum located to the left at CS18. The IL rotated around the SMA only during the early stages (until CS19). The IL gradually moved away, running transversely after CS19. Embryos with liver malformation showed PUH, which indicated that PUH occurred independent of liver volume.
Transition phase (CRL = 37, 41, and 43 mm): Intestinal return began from proximal to distal part in samples with CRL of 37 mm. The cecum returned before the distal end of the small intestine (ileum) in samples with CRLs of 41 and 43 mm.
Return phase: The cecum immediately reached its final position in the right lower quadrant of the abdomen (the adult position). The anti-clockwise “en-bloc rotation” described by descent and fixation of the cecum in the abdominal cavity may not exist. A rapid increase in the space available for the intestine in the abdominal coelom that exceeded the intestinal volume in the extraembryonic coelom was observed. The height of the umbilical ring increased in a stepwise manner between the transition and return phases and its height in the return phase was comparable to or higher than that of the hernia tip during the herniation phase. We speculated that the space is generated to accommodate the herniated portion of the intestine, similar to the intestine wrapping into the abdominal coelom as the height of the umbilical ring increases.
Conclusion
The data obtained in the present study demonstrate the precise timeline of IL formations, which indicate several points of discrepancy in the results of previous studies.
References
Nagata A, Hatta S, Imai H, Yamada S, Takakuwa T. Position of the cecum in the extraembryonic and abdominal coelom in the early fetal period. Congenit Anom 2019, in press.
Nagata A, Hatta S, Ji X, Ishikawa A, Sakamoto R, Yamada S, Imai H, Matsuda T, Takakuwa T. Return of the intestinal loop to the abdominal coelom after physiological umbilical herniation in the early fetal period. J Anat, 2019, 234, 456-464.doi: 10.1111/joa.12940.
Kanahashi T, Yamada S, Yoneyama A, Takakuwa T. Relationship Between Physiological Umbilical Herniation and Liver Morphogenesis During the Human Embryonic Period: A Morphological and Morphometric Study. Anat Rec 2019 in press.
Ueda Y, Yamada S, Uwabe C, Kose K, Takakuwa T, Intestinal rotation and physiological umbilical herniation during the embryonic period, Anatomical Record 299, 197-206, 2016, DOI: 10.1002/ar.23296
To obtain 3D digital data on early human development, we used some micro-imaging modalities such as magnetic resonance microscopy (MRM), episcopic fluorescence image capture (EFIC), and phase-contrast X-ray computed tomography (PXCT).
2.1 Magnetic resonance microscopy (MRM)
MR microscopy is a very powerful tool for 3D measurements of human embryos, because chemically fixed human embryos contain large quantities of mobile or NMR visible protons, which are major components of the formalin preservation fluid. MR imaging is nondestructive and does not need sectioning of embryonic tissues. Using MRM equipped with superconducting magnets ranging from 1.0T to 7.0T, embryos from the Kyoto Collections were imaged (Haishi et al., 2001, Matsuda et al., 2007, Matsuda et al., 2003, Yamada et al., 2010). The first super-parallel MR microscope was implemented on a 1.5 T clinical MRI scanner operated at University Hospital of Tsukuba University.
Kyoto Human Embryo Visualization Project (BIRD project); this is a collaborative research with Prof. Katsumi Kose of Tsukuba University and has been funded by the “Bioinformatics R & D (BIRD)” Project of the Japan Science and Technology Agency (2005-2010). Kyoto and Tsukuba Universities began a project in 1999 to acquire 3D MR microscopic images of thousands of human embryos using a super-parallel MR microscope operated at 2.35T (Shiota et al., 1991; Matsuda et al., 2003, 2007; Yamada et al., 2006). We were successful in acquiring high-resolution sectional images and in identifying the detailed structures of major organs. A database of developing human embryo images was established for future biomedical research (http://bird.cac.med.kyoto-u.ac.jp). This database will be useful not only in the study of human embryology but also for future gene mapping studies in human development.
Kose and his coworkers (Matsuda et al., 2003) developed a four-channel MR microscope with four unshielded gradient coil probes. The four-channel array probe was developed for a 2.35 T superconducting magnet. Each gradient coil probe consists of a rigid aluminum square frame [18 cm (W) × 20 cm (H) × 28 cm (D)] and slide planes made of thin brass plates (0.3 mm thick) acting as an RF shield. The RF coil units are exchangeable, and the diameter can be optimized for a given sample size. In the project, 18 mm diameter four-turn solenoid coils were used for the 100 MHz signal excitation and detection frequency. The pulse sequence was a T1-weighted 3D gradient echo sequence (TR = 100 ms, TE = 8 ms). Embryo specimens were imaged in test tubes (ID = 13.5 mm) filled with 4% formaldehyde solution. Introduction of a superconducting magnet (2.35 T) to MR microscopy significantly improved the quality of the MR images. The resolution was equivalent to that of low-magnification histological sections. The resolution now approaches 80 µm, and it is possible to identify various embryonic structures, such as the brain (and cerebral cortex), eyes, inner ears, pituitary gland, bronchi, lungs, stomach and intestines, kidneys, gonads, liver and spleen in embryos that are less than 30 mm in length. While introduction of a super-parallel MR microscope enabled imaging of 4-8 specimens simultaneously and significantly reduced the time required (Matsuda et al., 2003).
MRI (7T); MR images were acquired using a 7T MR system (BioSpec 70/20 USR; Bruker Biospin MRI GmbH; Ettlingen, Germany) with a 35-mm-diameter 1H quadrature transmit-receive volume coil (T9988; Bruker Biospin MRI GmbH). The 3D T1-weighted images were acquired using a fast, low-angle shot pulse sequence with the following parameters: repetition time, 30 ms; echo time, 4.037–6.177 ms; flip angle, 40°; field of view, 22.5 × 15.0 × 15.0–42.0 × 28.0 × 28.0 μm3; matrix size, 636 × 424 × 424–768 × 512 × 512; spatial resolution, 35.4–54.7 μm3.
It is feasible to apply MR microscopic imaging to fast and efficient screening of embryos in genetic and teratological studies by identification of internal visceral anomalies quickly, efficiently. MRI is nondestructive and does not need sectioning of specimens, which not only saves time and labor, but also allows precious specimens to remain intact.
Phase-contrast X-ray computed tomography (PXCT) is a relatively newer technique of imaging. In this imaging, the X-rays are used as electric waves which has the information of amplitude and phase. When an X-ray passes through a sample, their amplitude is decreased and phase is shifted. Conventional X-ray imaging (radiography) is based on absorption-contrast (i.e. amplitude imaging) and represents the mass-density distribution of X-ray inside the sample. The sensitivity is not sufficient to perform detailed observations of samples consisting of light elements such as biological soft tissues such as embryos without contrast agent or high X-ray dosages. In contrast, PXCT uses the phase-shift, occurring when X-rays pass through samples (Momose and Fukuda 1995). The sensitivity of the phase shift for light elements such as hydrogen, carbon, nitrogen, and oxygen is about 1000 times larger than that of absorption (Momose and Fukuda, 1995). For phase-shift detection, it is essential to convert the phase shift into the change in X-ray intensity. 2D and 3D observations of various biomedical samples have been performed using synchrotron radiation. The devices using this principle have been developed (Becker and Bonse, 1974, Yoneyama et al., 2004). The 3-D PXCT images of the human embryos were acquired using a radiographic imaging system (BL14-C, 17.8 keV) from Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK, Tsukuba, Japan). The data provide a resolution of 18 μm/pixel or better by imaging systems with a two-crystal X-ray interferometer
Hideo Nishimura, Prof. in the Department of Anatomy at Kyoto University School of Medicine, instigated a collection of human conceptuses in 1961. Induced abortions were then legal in Japan under the Maternity Protection Law of Japan. Therefore, pregnancies were terminated for social reasons during the first trimester in a great majority of cases. The Congenital Anomaly Research Center was founded at Kyoto University, School of medicine in 1975, when the number of specimens reached over 36,000. The embryo collection comprises over 45,000 specimens nowadays, which represents the largest human embryo collection in the world.
Over 70% of the embryos were collected in the 1960s and 25.5% in the 1970s. The less than 5% of the specimens joined KC between 1980 and 1999 (Kameda et al, 2011). The specimens were collected from a total of 22 prefectures in six different districts; Kansai, Tokai, Hokuriku, Kanto, Chugoku/Shikoku with aid of approximately 1400 obstetrician.
1.2. How were so many samples collected?
The majority of the specimens were obtained after termination of pregnancy by dilatation or curettage during the first trimester for socioeconomic reasons. Other specimens resulted from spontaneous or threatened abortions. Dilatation and curettage (therapeutic abortions) given to healthy women enabled acquisition of undamaged, intact embryos. Approximately 20% of the specimens are undamaged, well-preserved embryos.
When the aborted materials were brought to our laboratory, the embryos were measured, staged, and examined for gross external abnormalities and presence of intrauterine death under a dissecting microscope. The developmental stage of the embryos (Carnegie stage: CS) was determined according to the criteria proposed by O’Rahilly and Müller (1987). Most of the specimens are between the CS13 and CS23, which is the critical period of teratogenesis. The specimens were fixed in 4% formaldehyde or Bouin solution and stored in 4% formaldehyde.
1.3. Feature of Kyoto Collections
Since the attending obstetricians were not involved in examining the aborted materials, Embryos were collected in a random manner. The collection of embryos was not biased by their outcome (e.g., normal or abnormal, live or dead), thus, the embryo collection is considered representative of the unbiased total intrauterine population in Japan (Nishimura, 1974, 1975, Shiota 1991) Using this representative embryo population, it was reported that the incidence of malformations in embryos were more frequent than that in infants (Nishimura et al., 1968), and that embryos with severe malformations were prone to spontaneous abortion at high rates (Shiota, 1991).
2) KC comprises embryo specimens with a large variety of external malformations as reported by previously published studies (Nishimura et al. 1968; Matsunaga and Shiota 1977; Yamada et al. 2004). Approximately 7.8% of the embryos exhibit external anomalies and 92.2% are without anomalies, which provide a unique opportunity to study the early stages of abnormal morphogenesis. The three most common anomalies were nuchal bleb, holoprosencephaly and spina bifida. Holoprosencephaly is encountered much more frequently (1/250 or more) in the unselected early human embryonic population (Matsunaga and Shiota, 1977).
3) Well-preserved samples were stored and some of them were selected to be sectioned serially; a total of 500 normal embryos and 500 abnormal embryos were stored as complete serial sections, including HPE embryos. All histological sections comprised in the library is plan to be digitalized. The project is currently focusing on serial sections of normal embryos. Parts of the digitized serial sections are accessible from our website (http://atlas.cac.med. kyoto-u.ac.jp).
4) Most specimens were collected along with detailed clinical and epidemiological information on the associated pregnancies and the mothers. These data were accumulated in the formats of paper sheets and punch cards. All data gathered in KC were turn into dizitalized textual and imaging data recently (Kameda et al 2011).
1.4 Ethics and legal point of view
The embryo specimens of KC were collected and stored legally and ethically in Japan in the first place.
After World War II, a lot of laws were established in Japan to reconstruct the country. The Eugenic Protection Law was one of such laws, and established in 1948. Revision of the law in 1952 made artificial abortion in early periods of pregnancy legally available by the articles as same as present Maternal Protection Law. Prof. Nishimura explained in his book about the rationale of this human embryo collection by “Japanese Eugenic Protection Law in 1952 (Nishimura 1975). The legal basis for artificial abortion in found in the 1st item of article 14 in Chapter III (Protection of the mother’s life and health) of the law as cited as follows (Nishimura 1966); ‘The physician designated by the Medical Association, which is a corporate juridical body established in the prefectural district as a unit, may exercise artificial interruption of pregnancy, at his discretion, on the person, who falls under any of the following items with consent of the person in question and the spouse: First item: A mother, whose health may be affected seriously by continuation of pregnancy or by delivery from the physical view point or economically’ (Nishimura 1966). Thus, in Japan, a lot of operations of artificial abortion have been performed for the reasons of economical reason. After the operation, embryo and placenta (or decidua) were taken out from maternal body and small embryos before 12 weeks of gestation and placenta were usually disposed as hospital waste. Prof. Nishimura planned to use the human embryo and placenta disposed as hospital waste for the research to elucidate the nature of embryonic morphology. He and his colleagues solicited the cooperation of the obstetricians who explained to their clients the significance of medical research and the contributions the parents could make by leaving the embryos with them. In most cases the mothers acceded to the requests of the obstetricians in whom they had trust. In addition, we regarded ourselves as an extension of the obstetrician’s clinic and could accept the specimens in good faith. The embryos were transferred to our laboratory with their some information such as date of operation, gestational ages, etc., but had no identifying information and cannot be linked to the parents or any relatives. Thus, the operations of artificial abortion could only be performed under the reasons based on the law, with the consent of mother and/or the father. The scientist or obstetrician could force mothers neither to take operation and nor to give the embryo to the scientist or obstetrician.
Therefore, the embryos have been obtained ethically. It is not appropriate to treat the Kyoto Collection and victims of Holocaust. Based on these backgrounds, our research have been approved by IRB of Kyoto University (The Committee of Medical Ethics of Kyoto University Graduate School of Medicine, Kyoto: E986, G377).
Three-dimensional (3D) analysis of the human embryonic and early-fetal period has been performed using digitized datasets obtained from the Kyoto Collection, in which the digital datasets play a primary role in research. Datasets include magnetic resonance imaging (MRI) acquired with 1.5 T, 2.35 T, and 7 T magnet systems, phase-contrast X-ray computed tomography (CT), and digitized histological serial sections. Large, high-resolution datasets covering a broad range of developmental periods obtained with various methods of acquisition are key elements for the studies. The digital data have gross merits that enabled us to develop various analysis. Digital data analysis accelerated the speed of morphological observations using precise and improved methods by providing a suitable plane for a morphometric analysis from staged human embryos. Morphometric data are useful for quantitatively evaluating and demonstrating the features of development and for screening abnormal samples, which may be suggestive in the pathogenesis of congenital malformations. Morphometric data are also valuable for comparing sonographic data in a process known as “sonoembryology.” The 3D coordinates of anatomical landmarks may be useful tools for analyzing the positional change of interesting landmarks and their relationships during development. Several dynamic events could be explained by differential growth using 3D coordinates. Moreover, 3D coordinates can be utilized in mathematical analysis as well as statistical analysis. The 3D analysis in our study may serve to provide accurate morphologic data, including the dynamics of embryonic structures related to developmental stages, which is required for insights into the dynamic and complex processes occurring during organogenesis
