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. 2022 Sep 2;242(2):174–190. doi: 10.1111/joa.13760

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

Toru Kanahashi 1,✉, Hirohiko Imai 2, Hiroki Otani 3, Shigehito Yamada 1,4, Akio Yoneyama 5, Tetsuya Takakuwa 1
PMCID: PMC9877484  PMID: 36053545

Abstract

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.

Keywords: diaphragm, diffusion tensor imaging, fetus, morphogenesis, MRI, three‐dimensional

The human diaphragm gradually developed a dome‐like shape at Carnegie stage (CS) 20 or later. At the sternal, costal, and lumbar parts and the area surrounding the esophageal hiatus, the fiber orientation became more distinct with growth at CS19 or later. The thickness and fractional anisotropy (FA) values tended to have increased growth in samples with a CRL ≥46 mm, and the center and the left and right hemidiaphragmatic domes did not display thickening and had lower FA values.

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1. INTRODUCTION

The human diaphragm is a double‐domed musculotendinous sheet located at the inferior‐most aspect of the rib cage. It divides the thoracic and abdominal cavities (Drake et al., 2016). The central tendon is located at the diaphragmatic apex. Myofibers from muscular parts (sternal, costal, and lumbar regions) enter the central tendon. The diaphragm has multiple functions, most principally its critical role in respiration (Mead & Loring, 1982). The diaphragm repeatedly contracts and relaxes, changing the volume of the thoracic cavity and of the lungs to produce inspiration and expiration, respectively.

The embryonic diaphragm comprises four major structural components (Moore et al., 2015): the central tendon, derived from the transverse septum; a dorsal midline portion, derived from the foregut mesentery; two dorsolateral shelves of tissue, derived from the pleuroperitoneal membranes (folds); and peripheral components, derived from the lateral body wall. Several studies have been conducted on diaphragm development in humans (Wells, 1954), rats, and mice (Allan & Greer, 1997; Iritani, 1984; Merrell et al., 2015). The general description of human diaphragm development found in textbooks is as follows (Alamo et al., 2015; Moore et al., 2015). In the 4th week, the embryo possesses a single horseshoe‐shaped body cavity. By the 6th week, the anteriorly located transverse septum becomes the central tendon of the diaphragm. The septum grows dorsally and begins to form a semicircular shelf that separates the heart from the liver. Simultaneously, the posterolateral pleuroperitoneal membranes extend progressively in a ventral direction and fuse with the transverse septum and dorsal mesentery of the esophagus. At the 7th week, the transverse septum fuses laterally with the muscular body walls, completing the membranous diaphragm and division between the thorax and abdomen. At 8–10 weeks of gestation, muscular fibers arising from cervical myotomes grow in the lateral body wall and incorporate the intercostal muscles within the membranous diaphragm (Moore et al., 2015; Restrepo et al., 2008). The internal layer of the body wall then forms the muscle of the peripheral diaphragm. Posterolateral remnants of the pleuroperitoneal membranes form a fibrous trigone in each hemidiaphragm that constitutes most of its muscular portion (Taylor et al., 2009), whereas the median part of the muscular diaphragm forms from the dorsal mesentery of the esophagus. Previous studies have been conducted using classic gross anatomy and histopathological analyses and have particularly focused on the period of fusion of each diaphragmatic segment during the embryonic period. 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.

Magnetic resonance imaging (MRI) techniques based on water diffusion, such as diffusion‐weighted imaging and diffusion tensor imaging (DTI), have been developed (Hagmann et al., 2006; Le Bihan et al., 2001). DTI is affected by the directional movement of water molecules that diffuse differently through the tissue depending on its microstructure, and on the integrity and type of barriers that are present. This gives information about its orientation and anisotropy (Lanzman & Wittsack, 2017). Fractional anisotropy (FA), is the most commonly used method; it measures the degree of diffusion anisotropy within the tissue and ranges between 0 and 1, where 0 indicates isotropic diffusion and 1 indicates anisotropic diffusion (Basser & Pierpaoli, 1996). Fiber tractography is a 3‐D technique used to visually represent structural orientation (Basser et al., 2000). DTI is now widely used in clinical and research fields because of its consistency as a noninvasive tool and its ability to indirectly provide information on the macrostructure of white matter in the central nervous system (Li & Zhang, 2020). Although DTI has also been applied to the analysis of muscles outside the cranial nervous system, most applications have focused on the cardiac muscle. Few analyses have involved other muscles (Mekkaoui et al., 2017; Noseworthy et al., 2010), and, to our knowledge, no evaluation of diaphragmatic morphogenesis has been performed using DTI.

The generalized use and technical improvements in ultrasonographic screening examinations during pregnancy in recent years have led to a considerable increase in the rate of prenatal diagnosis of diaphragmatic pathologies (Alamo et al., 2015). Congenital diaphragmatic eventration has been reported to result from focal thinning of the diaphragm from defective development of the musculature (Jurcak‐Zaleski et al., 1990). An appropriate understanding of diaphragmatic development will enable a better understanding of congenital pathologies.

Thus, in this study, we aimed to evaluate the morphogenesis of the fetal diaphragm using T1‐weighted (T1W) MR images. We also analyzed the fiber architecture of the developing diaphragm in the human fetus using DTI tractography.

2. METHODS

The ethics committee of Kyoto University Faculty and Graduate School of Medicine approved this study's use of human embryonic and fetal specimens (E986, R0316, and R2224).

2.1. Human fetal specimens

Thirty‐seven human embryos (crown‐rump length [CRL]: 8.0–26 mm, Carnegie stage [CS]: 16–23) and 20 human fetuses (CRL: 34–88 mm, from the early fetal period) were selected for this study (Table 1). These had been stored at the Congenital Anomaly Research Center of Kyoto University (Yamaguchi & Yamada, 2018) and Shimane University. Most specimens were acquired when pregnancy was terminated owing to socioeconomic reasons under the Maternity Protection Law of Japan. The specimens were collected from 1963 to 1995 according to the relevant regulations. Written informed consent was not required by parents at that time. Instead, parents provided verbal informed consent to have the specimens deposited, and each participant's consent was documented in the medical record. Approximately 40,000 fetuses were stored; of these, 20% were well preserved. Aborted fetal specimens were brought to the laboratory, measured, examined, and staged using the criteria proposed by O'Rahilly and Müller (1987).

TABLE 1.

Samples used in the present study

ID Carnegie stage CRL (mm) Images MRI condition a
22,322 16 8 MRI Condition 1
29,261 16 9 MRI/PCX‐CT Condition 1
2578 16 8 PCX‐CT —
22,554 16 9 PCX‐CT —
30,102 16 10 PCX‐CT —
24,578 17 12 MRI Condition 1
22,903 17 11 MRI Condition 1
23,241 17 16 PCX‐CT —
26,702 17 11 PCX‐CT —
29,248 17 11 PCX‐CT —
30,801 17 10 PCX‐CT —
33,315 17 12 PCX‐CT —
30,608 17 12 PCX‐CT —
31,455 17 11 PCX‐CT —
31,870 18 12 MRI/PCX‐CT Condition 1
17,746 18 12 MRI/PCX‐CT Condition 1
19,809 18 15 PCX‐CT —
25,914 18 16 PCX‐CT —
33,785 18 12 PCX‐CT —
24,638 18 12 PCX‐CT —
19,989 19 15 MRI/PCX‐CT Condition 1
26,979 19 16 MRI Condition 1
16,127 19 15 PCX‐CT —
19,605 19 16 PCX‐CT —
23,229 19 18 PCX‐CT —
22,171 20 17 PCX‐CT —
23,116 20 16 MRI/PCX‐CT Condition 1
23,125 20 18 PCX‐CT —
24,742 20 19 PCX‐CT —
24,977 20 19 MRI Condition 1
28,070 20 17 PCX‐CT —
30,377 20 18 PCX‐CT —
32,721 21 21 MRI Condition 1
1047 21 23 MRI Condition 1
27,050 22 22 MRI Condition 1
29,399 22 23 MRI Condition 1
52,578 23 26 MRI Condition 1
F2329 F 34 MRI Condition 2
F2274 F 34 MRI Condition 2
F2249 F 38 MRI Condition 2
F2037 F 40 MRI Condition 2
F2219 F 41 MRI Condition 2
F3254 F 46 MRI Condition 3
F2224 F 46 MRI Condition 3
F2371 F 49 MRI Condition 3
F2424 F 49 MRI Condition 3
F1485 F 50 MRI Condition 3
F2396 F 52 MRI Condition 3
33,087 F 59 MRI Condition 3
F2983 F 67 MRI Condition 3
71,030 F 67 MRI Condition 4
F2133 F 70 MRI Condition 3
F2240 F 71 MRI Condition 3
F2248 F 71 MRI Condition 3
F2373 F 71 MRI Condition 3
F2149 F 74 MRI Condition 4
37,304 F 88 MRI Condition 3
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Abbreviations: CRL, Crown‐rump length; F, fetus; MRI, magnetic resonance imaging; PCX‐CT, phase‐contrast X‐ray computed tomography.

a

MRI conditions (coils and acquisition parameters) are summarized in Table 2.

2.2. T1W MRI and DTI

T1W MRI and DTI of the human embryonic and fetal specimens were performed as previously reported (Nishitani et al., 2020). Briefly, MRI scanning was performed using a 7‐T preclinical MR system (BioSpec 70/20 USR; Bruker BioSpin MRI GmbH) at the Medical Research Support Center, Graduate School of Medicine, Kyoto University. T1W MR images and DTI data sets were acquired using a spoiled gradient echo sequence and diffusion‐weighted spin‐echo sequence, respectively. According to the size of the samples, appropriate settings for data acquisition were selected from the four conditions, where different MRI coils and acquisition parameters were prepared (Table 2). The other setting conditions varied in detail according to the size of the samples.

TABLE 2.

List of the MRI coils and acquisition parameters

Condition 1 Condition 2 Condition 3 Condition 4
Samples Embryos (CRL, 8.0–26 mm; CS 16–23) Fetuses (CRL, 34–41 mm) Fetuses (CRL, 46–88 mm) Fetuses (CRL, 67, 74 mm)
MRI coil (inner diameter) Solenoid coil (19 mm) a Quadrature volume coil (35 mm) b Quadrature volume coil (72 mm) b Quadrature volume coil (75 mm) a
T1W MRI DTI T1W MRI DTI T1W MRI DTI T1W MRI DTI
Spatial resolution (isotropic, um) 40 100 54 150 98 294 125 350
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Abbreviations: CRL, Crown‐rump length; DTI, diffusion tensor imaging; MRI, magnetic resonance imaging.

a

Manufacturer: Takashima Seisakusyo Co., Ltd., Tokyo, Japan.

b

Manufacturer: Bruker BioSpin MRI GmbH, Ettlingen, Germany.

2.3. Phase‐contrast X‐ray computed tomography

3‐D phase‐contrast X‐ray computed tomography (PCX‐CT) images of the human embryos were obtained using a radiographic imaging system (BL14‐C, 17.8 keV) from the Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK). The mechanism and conditions used to acquire the PCX‐CT images of the embryos have been described previously (Yoneyama et al., 2011).

2.4. 3‐D reconstruction

3‐D images of the diaphragm were manually reconstructed from T1W MR images using the Amira software (version 5.5.0 Visage Imaging GmbH). The thickness of the diaphragm was visualized using the filter module of the Amira™ software program for surface thickness (the thickness of the diaphragm was visualized on the surface with a color scale). To observe the position of the diaphragm during growth, landmarks were set at the locations indicated below based on the 3‐D images: the most cranial points of the left and right hemidiaphragmatic domes (left and right cranial), the most cranial point in the middle (between the left and right hemidiaphragmatic domes; medial cranial), and the most caudal points of the left and right hemidiaphragms (left and right caudal). The height in the cranial‐caudal direction of the diaphragm (i.e., the height of the diaphragm) was measured using the number of vertebrae as reference.

We defined the center, lumbar, costal, and sternal parts of the diaphragm with reference to adult anatomy as follows (Drake et al., 2016). The center was defined as the area near the anterior center of the diaphragm. The lumbar region was defined as the area from around the site, where the diaphragm attaches to the lumbar spine, and dorsal wall to the center. The costal section was defined as the area from the left and right body walls and ribs to the center. The sternal part was defined as the area from the anterior wall around the xiphoid process of the sternum to the center.

2.5. DTI tractography and diffusion parameters

Fiber tractography based on DTI of the diaphragm was performed using DSI Studio (http://dsistudio.labsolver.org). The tracking area was set based on diaphragm segmentation using custom‐written scripts in MATLAB (R2018a; MathWorks, Inc., Natick, MA) and FSL (https://fsl.fmrib.ox.ac.uk/fsl/). FA was calculated using the DSI Studio software tool. After performing tractography, the fibers of the organs bordering the diaphragm (mainly of the heart, esophagus, aorta, veins, and muscle fibers of the abdomen and lumbar region) were removed from the images.

3. RESULTS

3.1. Initial morphogenesis of the diaphragm

3.1.1. Closure of the diaphragm

The transverse septum was observed, but not the pleuroperitoneal membrane at CS16 (Figure 1a). The membranous structure found between the liver and the heart was determined to be a transverse septum, which was observed with a lower signal than that of the diaphragm and a slightly indistinct limbus. T1W MR and PCX‐CT images were used to confirm the proximity of the pleuroperitoneal canals during the embryonic period (CS16–20, Table 3). The right and left pleuroperitoneal canals were observed in all embryonic samples at CS16 and CS17, but only in one at CS18. One of the six samples at CS18 was found to be closed on the right side only. At CS19, four of the five samples had closed canals on both sides; one sample was not closed on the left. Both right and left canals were closed in all CS20 samples.

FIGURE 1.

FIGURE 1

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Growth of the diaphragm during the embryonic period. (a) Representative mid‐sagittal sections from T1‐weighted magnetic resonance images. Asterisks indicate unclosed areas. Yellow arrowheads indicate the transverse septum. Orange arrowheads indicate the diaphragm. Blue circles indicate the bonding sites of the transverse septum/diaphragm to the anterior body wall. Ag, adrenal gland; CS, Carnegie stage; He, heart; Li, liver; Lu, lung; St, stomach. The scale bar indicates 2 mm. (b) Three‐dimensional reconstruction of the diaphragm. The cranial view is indicated. Asterisks indicate unclosed areas. The blue circles indicate the bonding site of the transverse septum/diaphragm to the anterior body wall. AH, aortic hiatus; CO, caval opening; EH, esophageal hiatus. The scale bar indicates 1 mm.

TABLE 3.

Closing of the pleuroperitoneal canals at embryo Carnegie stages (CSs) 16–20

CS Crown‐rump length (mm) n Number of closed samples (%)
Left Right
16 8–10 5 0 (0) a 0 (0)
17 10–16 9 0 (0) 0 (0)
18 12–18 6 0 (0) 1 (17)
19 13–18 5 4 (90) 5 (100)
20 16–19 7 7 (100) 7 (100)
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a

Numbers in parentheses indicate the sample closure percentage (the number of closed samples divided by the total number in each CS).

3.1.2. Hiatus and dome formation

Three hiatuses, the esophageal hiatus, aortic hiatus, and caval opening, were noted at CS18. The right and left hemidiaphragms, which appeared flat at CS19, gradually formed dome shapes at CS20 or at a later stage (Figure 1a,b). The 3‐D MR images could not distinguish the boundaries between the transverse septum, foregut mesentery, and pleuroperitoneal membranes in the periods before and after fusion. A left–right difference in the shape of the diaphragm was observed at CS22 or later. In comparison to the left hemidiaphragm, the right hemidiaphragm was slightly enlarged dorsoventrally.

3.1.3. Influence of adjacent organs

The morphology of the diaphragm is influenced by that of its neighboring organs. 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 (Figure 1a). In addition, the diaphragm was in contact with the gastric fundus in samples with a CRL of ≥41 mm, and the spleen in samples with a CRL of ≥70 mm (Figure 2a). The right and left crura, which are attached to the lumbar vertebrae, developed prominently in samples with a CRL of >50 mm. The right crus was longer than the left (Figure 2b).

FIGURE 2.

FIGURE 2

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Diaphragm at the early fetal period (CRL 71 mm). (a) Sagittal sections of the diaphragm from T1‐weighted magnetic resonance images. Orange arrowheads indicate the diaphragm. The blue circle indicates the bonding site of the diaphragm to the anterior body wall. Ag, adrenal gland; He, heart; In, intestine; Li, liver; Lu, lung; Sp, spleen; St, stomach. The scale bar indicates 5 mm. (b) Three‐dimensional reconstruction of the diaphragm. Cranial and dorsal views. AH, aortic hiatus; CO, caval opening; CRL, crown‐rump length; EH, esophageal hiatus; LC, left crus; RC, right crus. The scale bar indicates 10 mm.

3.1.4. Relative height position in the body

The most cranial point of the transverse septum was positioned between the seventh cervical and first thoracic vertebrae at CS16 (Figure 3a). The diaphragm rapidly descended from CS16 to CS19 (i.e., samples with CRL between 8 and 16 mm). The most cranial point of the diaphragmatic dome was located between the fourth and eighth thoracic vertebrae, regardless of fetal growth, in samples with a CRL of ≥16 mm. The most cranial point of the right hemidiaphragm was higher than that of the left in samples with a CRL between 16 and 71 mm. The most caudal point of the diaphragm tended to descend by one thoracic vertebral level from samples with a CRL of 16 mm to those with a CRL of 34 mm and was located between the 12th thoracic and third lumbar vertebrae, regardless of fetal growth, in samples with a CRL of ≥34 mm. The most cranial or caudal points were not always located at specific positions among different individuals. Figure 3b shows the height of the diaphragm in the craniocaudal direction based on the number of vertebrae; the change in height occurred rapidly during the embryonic period and then remained constant thereafter.

FIGURE 3.

FIGURE 3

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Changes in the position and height of the diaphragm with respect to the vertebrae. (a) Changes in the position of the diaphragm. The most cranial and caudal parts of the hemidiaphragmatic domes were determined using three‐dimensional reconstructed images of the diaphragm from T1‐weighted magnetic resonance images. The gray background indicates the embryonic stage. C, cervical vertebra; L, lumbar vertebra; Th, thoracic vertebra. (b) Changes in the height of the diaphragm. The height in the cranial‐caudal direction of the diaphragm is based on the number of vertebrae. The gray background indicates the embryonic stage.

3.2. Differentiation of the diaphragm

3.2.1. Thickness

The 3D changes in the thickness of the diaphragm were visualized on the surface using a color scale predetermined for a range of 0–0.3 mm (Figure 4). The thickness values were validated using T1W MR cross‐sectional images of the diaphragm. The diaphragmatic thickness was nearly uniform (0.15–0.2 mm) from samples with CRL 8 mm (CS16) to those with a CRL of 41 mm. It began to increase around the esophageal hiatus in samples with a CRL ≥46 mm, and this thickening extended to the area around the aortic hiatus (lumbar parts; Figure 4). The remaining lumbar and costal regions increased in thickness in samples with a CRL of ≥50 mm, and the sternal region increased in thickness in samples with a CRL of ≥71 mm. The thickness at the center and at the left and right hemidiaphragmatic domes did not increase with growth. The shape of this nonthickening central region resembled that of the central tendon in adults.

FIGURE 4.

FIGURE 4

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Growth of the diaphragm from the embryonic to the early fetal period. Representative cranial view of the three‐dimensional reconstruction of the diaphragm. The thickness of the diaphragm is determined using surface thickness color mapping. Asterisks indicate unclosed areas. AH, aortic hiatus; CO, caval opening; EH, esophageal hiatus. The scale bar indicates 1, 2, or 5 mm.

3.2.2. DTI tractography

The fiber orientation was obscured, and the FA values were overall low at CS16 (FA: 0.05–0.08). Tractography also did not distinguish boundaries between the transverse septum, foregut mesentery, and pleuroperitoneal membranes in the periods before and after fusion. In the lumbar regions, orientation from the right and left crus surrounding the esophageal hiatus was observed at CS19 (Figure 5a). The FA values were also high in areas of thickening from the esophageal hiatus to the right and left crura. Fiber tracts in thickened areas, namely the lumbar, costal, and sternal region, and around the esophageal hiatus became especially prominent with growth and tended to have relatively higher FA values than the other areas in samples with a CRL of ≥46 mm. However, the center and left and right hemidiaphragmatic domes, where the thickness did not increase, showed lower FA values, fewer fibers, and individual differences in fiber orientation (Figure 5a).

FIGURE 5.

FIGURE 5

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Fiber orientation surrounding the esophageal hiatus on diffusion tensor imaging tractography. (a) The gross (left) and closeup (right) cranial views indicate fiber arrangements surrounding the esophageal hiatus in an embryo (CS19) and fetus (CRL 71 mm). Orange squares are shown in the image on the right. White arrows indicate fiber orientation. Asterisks indicate areas with low fractional anisotropy (FA) values and low density. Fiber tracts are visualized according to the FA code. AH, aortic hiatus; CO, caval opening; CRL, crown‐rump length; EH, esophageal hiatus. The scale bar indicates 2 or 5 mm. (b) Three patterns of fiber arrangements surrounding the esophageal hiatus on diffusion tensor imaging tractography are categorized. Fiber tracts are visualized using FA codes (left) and illustrated for explanation (right). The blue illustration indicates fibers of the right crus. The orange illustration indicates fibers of the left crus. AH, aortic hiatus; EH, esophageal hiatus; L, left; R, right.

3.2.3. Diaphragm surrounding the hiatuses

We observed orientation patterns surrounding the caval opening and esophageal hiatus (Figure 5a,b). First, the fiber orientation surrounding the caval opening was evaluated in 25 of the embryos and fetuses. Ten samples were excluded because the fiber tracts surrounding the caval opening were too short and indistinct. In all 25 samples, the fibers from the right crus and other right lumbar parts ran around the caval opening (Figure 5a). The fibers from the left crus could be faintly distinguished on the left side of the caval opening in eight of the 25 samples.

The fiber orientation patterns surrounding the esophageal hiatus could be observed in 27 of the embryos and fetuses (Figure 5b). The fiber tracts were too short and indistinct for evaluation in six of the embryos (CS16–CS18) and in two of the fetuses, which had (CRL values of 38 and 70 mm). The region of interest (ROI) was placed on each side of the aortic hiatus to selectively detect the fibers. Three patterns were identified. The most common pattern (19 samples) involved fibers running to the right ROI splitting on each side of the esophageal hiatus and fibers running to the left ROI running only on the left side of the esophageal hiatus (type 1). In six samples, the left fibers split on each side of the esophageal hiatus, and the right fibers ran only on the right side of the esophageal hiatus (type 2). In two samples, fibers from both sides split and ran on either side of the esophageal hiatus (type 3).

Besides, a cross of muscle fibers of the right or left crus, before it divides into two bundles that surround the esophagus, were observed in each of the three types. In right crus of type 1, the left bundle was placed superiorly to the right bundle in 14 of 19 samples (Figure 6a). In the remaining five cases, the left and right bundles could not be separated in detail. In left crus of type 2, the left bundle was superior to the right bundle in four of six samples (Figure 6b). In the remaining two cases, the left and right bundles could not be separated in detail. In one sample of type 3, the left bundle was superior to the right bundle even with the right or left crus. In another sample, the left bundle was superior to the right bundle with left crus. However, the left and right bundles could not be separated with right crus. The left and right bundles could not be separated due to increased short fibers and artifacts at the anterior and cranial end of the hiatus where the two bundles of the right crus meet the left crus.

FIGURE 6.

FIGURE 6

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The right and left bundle arrangements of the right or left crus that surround the esophagus. (a) The gross (left) and closeup (right) medial views indicate right and left bundle arrangements of the right crus surrounding the esophageal hiatus in the fetus (CRL 71 mm). Orange squares are shown in the image on the right. Fiber tracts are visualized using fractional anisotropy (FA) codes. CRL, crown‐rump length; EH, esophageal hiatus. (b) The gross (left) and closeup (right) medial views indicate right and left bundle arrangements of the left crus surrounding the esophageal hiatus in the fetus (CRL 46 mm). Orange squares are shown in the image on the right. Fiber tracts are visualized using FA codes. EH, esophageal hiatus.

3.2.4. Diaphragm at the lumbar region

Thicker areas were observed in the other lumbar regions. However, we did not identify any regular patterns regarding these locations (Figure 7a). A greater distinction of fiber directions began to be observable between the costal and lumbar parts of the diaphragm among samples with a CRL of ≥46 mm (Figure 7b). All fibers in the costal and lumbar region ran toward the left and right hemidiaphragmatic domes, except for those running to the caval opening and esophageal hiatus. Right and left triangular areas, with reduced FA values and lower density, were observed on tractography between the costal and lumbar fiber orientations in samples with a CRL of ≥67 mm (indicated by * in Figure 7b). The triangles were in the region where the 11th or 12th rib was close to the diaphragm. Eight samples, with a CRL between 67 and 88 mm, were examined for the presence or the absence of left and right triangles. Bilateral triangles were evident in five of the eight samples. Two had triangles on the right side only, and a single sample had an isolated triangle on the left side.

FIGURE 7.

FIGURE 7

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The lumbar region of the diaphragm in the early fetal period. (a) The dorsal view of the reconstructed diaphragm according to the growth. The thickness of the diaphragm is indicated using surface thickness color mapping. AH, aortic hiatus; CRL, crown‐rump length; LC, left crus; RC, right crus. The scale bar indicates 5 mm. (b) Dorsal view of the reconstructed diaphragm with the rib and vertebrae (left), diffusion tensor imaging tractography (right), and closer views (lower) in a fetus (CRL 71 mm). Orange circles indicate the areas where the diaphragm meets the ribs. Fiber tracts are visualized using fractional anisotropy (FA) codes. White arrows indicate the fiber orientation. Asterisks indicate areas with low FA values and low density. The scale bar indicates 5 mm.

3.2.5. Diaphragm at the costal and sternal regions

In the costal diaphragm, areas of increased thickness tended to expand from the ventral region to other lateral parts in samples with a CRL of ≥50 mm. Aspects of the seventh to 12th ribs were observed to be close to the diaphragm and partly in contact with the diaphragm (Figure 8a). The intercostal region tended to be thicker than areas close to the ribs. On tractography, the tract ran straight from each rib attachment site or intercostal space to the left and right hemidiaphragmatic domes; no clear differences were observed between thick and thin areas.

FIGURE 8.

FIGURE 8

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The costal (a) and sternum (b) regions of the diaphragm in the early fetal period. (a) The right lateral view of the reconstructed diaphragm with/without the thoracic cage (left and right), and diffusion tensor imaging tractography (lower) in a fetus (CRL 71 mm). Orange circles indicate the areas where the diaphragm meets the ribs. Fiber tracts are visualized with fractional anisotropy (FA) codes. The scale bar indicates 5 mm. (b) The ventral views of the reconstructed diaphragm with/without the thoracic cage (left and right) and diffusion tensor imaging tractography (lower) in a fetus (CRL 88 mm). Fiber tracts are visualized using FA codes. White arrows indicate the fiber orientation. Asterisks indicate areas with low FA values and low density. CRL, crown‐rump length; CP, costal part; SP, sternal part. The scale bar indicates 5 mm.

A thickened area surrounding the region of the xiphoid process of the sternum was observed in samples with a CRL of ≥71 mm (Figure 8b). The difference in fiber directions between the costal and the sternal parts of the diaphragm became clear among samples with a CRL of ≥46 mm. Anterior costal and sternal fibers ran toward the center. The border area between the costal and the sternal fibers included fewer fibers and lower FA values (indicated by * in Figure 8b).

4. DISCUSSION

In this study, we characterized human diaphragm formation during the late embryonic and early fetal periods (37–40 days to 12 weeks postfertilization) (O'Rahilly & Müller, 2001a). We used a novel approach based on thickness variation and DTI tractography. Based on the characteristics identified here, we classified the morphogenesis of the diaphragm into three stages.

The first formative stage was from CS16–CS20 in the embryonic period, when the diaphragm was in the process of closing. It subsequently domed, creating the prototypical diaphragm shape. Wells (1954) stated that the closure of the pleuroperitoneal canals begins in Streeter's group 17, ends for many individuals in group 20, and there is complete closure for all by group 21. Another study reported that the pleuroperitoneal canal closes once the CRL reaches 21 mm (Botha, 1959). We described the closure time based on embryonic staging (CS), and the timing did not contradict previously published findings. The relative position of the diaphragm descends rapidly toward the caudal direction during this period. The change in position from cervical to thoracic was consistent with that previously reported by Müller and O'Rahilly (1986). In addition, we observed that the height of the diaphragm, according to the number of vertebrae, was almost the same as in adults among samples with a CRL of ≥34 mm.

The second formative stage was from CS20 in the embryonic period to a CRL of 46 mm in the early fetal period. Over the course of this period, the thickness of the diaphragm remained constant and no obvious change in thickness was observed. However, DTI tractography showed that tracts at the sternal, costal, and lumbar parts gradually became more distinct with growth at CS19 or later. The sternal, costal, and lumbar parts are said to be the area where myoblasts develop and myofibers differentiate in samples with a CRL of ≥18 mm (Wells, 1954). The CS20 samples used in this analysis had a CRL of approximately 18 mm, implying that tractography exhibited muscle fiber differentiation. Several studies have reported different arrangements of the right and left crura in the formation of the esophageal hiatus (Collis et al., 1954; Costa & Pires‐Neto, 2004; Listerud & Harkins, 1958). The patterns observed in this study were identical to those previously reported. In the most common adult pattern, the right crus divides into two bundles that encircle the esophageal hiatus; the left crus combines with the left branch of the right crus. We observed this pattern in the majority of the samples (type 1; 70%). Similarly, for cross patterns of the two bundles of the right crus, the most common pattern in the present study coincided with the most common adults' pattern (Costa & Pires‐Neto, 2004, Listerud & Harkins, 1958). However, the observed proportions of all types differed from those in previous studies (Collis et al., 1954; Costa & Pires‐Neto, 2004). This difference may be attributed not only to differences in the number of samples and assessment methods but also to differences between adults and fetuses as the subjects of observation. However, this result suggested that the arrangement of the right and left crura in the formation of the esophageal hiatus had been already determined during the early developmental period of the diaphragm.

The third formative stage was from samples with a CRL of 46 mm to those with a CRL of 88 mm, in which the sternal, costal, lumbar parts, and the area surrounding the esophageal hiatus thickened. With DTI tractography, the fiber orientation became more distinct with growth. In addition, the FA values of the sternal, costal, and lumbar parts tended to increase. Previous studies on the human fetal heart have also reported a relationship between histological maturation and increasing anisotropy (Mekkaoui et al., 2013; Pervolaraki et al., 2013). Several studies using animal models have reported that FA can reflect the directional coherence of cardiomyocyte orientations and cellular shape changes (Chen et al., 2005; Garrido et al., 1994; Pierpaoli & Basser, 1996; Wu & Wu, 2009). Fiber orientations in the costal, sternal, and lumbar regions were the same as those observed in adults (Drake et al., 2016). Our data suggest that increased FA and thickness are associated with muscle development. However, further research is required to clarify this relationship. In samples with a CRL of ≥46 mm, the distinct development of the diaphragm may be attributed to the completed fusion of the sternal bands (O'Rahilly & Müller, 2001b), the onset of rib and vertebral ossification to stabilize the trunk (Noback & Robertson, 1951), or further development of existing diaphragmatic muscle fibers that can be captured based on the thickness or with DTI tractography.

The center and the left and right hemidiaphragmatic domes of the diaphragm were not thickened, and the FA values decreased in samples with a CRL of ≥46 mm. Tracts in the area were relatively short with low density and the shape resembled the central tendon of an adult (Drake et al., 2016). Wells (1954) described the presence of a primitive central tendon in samples with a CRL of 31 mm, and this has a distinct structure in samples with a CRL of 67 mm. Although there may be a slight time lag for the FA values to reflect tendon development, we observed continuous changes in the formation of the central tendons and three muscular parts in samples with a CRL of ≥30 mm. In addition, the early fetal diaphragm structure was similar to that observed in adults.

In samples with a CRL of ≥67 mm, triangles of decreased FA were observed bilaterally between the costal and lumbar fiber orientations. These triangular sites correspond to the locations of the lumbocostal triangles (Kardon et al., 2017; Wells, 1954). Wells (1954) also noted interindividual differences in lumbocostal triangle formation in adults. The undefined location of thickening we observed might be related to individual differences in the formation of the lumbocostal triangles or delayed fibrous development. This should be confirmed in a fetus at a later stage of development.

The embryology of congenital diaphragmatic hernia remains controversial; however, the development of Bochdalek hernia is widely believed to result from delayed or inhibited closure of the pleuroperitoneal canals (Brown et al., 2011). The sternocostal triangle is also the site of congenital Morgagni hernias (Alamo et al., 2015). In samples with a CRL of ≥46 mm, DTI tractography indicated that the number of tracts around the closed area of the pleuroperitoneal canal decreased, and the border area between the costal and sternal fibers (i.e., the sternocostal triangle) had fewer fibers and lower FA values than those of other areas. Individual differences were observed in the thickness surrounding the above area. We provided information on the normal development of sites where congenital diaphragmatic hernias are most likely to occur; however, further morphogenesis needs to be clarified in more detail in the future by additional comparative studies including cases of hernia.

This study has several limitations. First, the samples used were fixed in a medium containing formaldehyde and stored in a sample tube for a long period. Therefore, deformity and shrinkage due to fixation and preservation should be considered. Second, it was difficult to identify the part of the membranous structure of the diaphragm, possibly because of image resolution limitations. As we could not clearly distinguish between the ligaments of the diaphragmatic lumbar region and the areas where muscles and ligaments of the other lumbar muscles are located, the morphogenesis of the lumbocostal arch could not be adequately discussed. Finally, our T1W MRI and DTI methods could not clearly discern the differences between muscular and collagenous fibers. We treated the constituent fibers of the diaphragm collectively. However, MRI and CT images are more suitable than histological sections for detecting continuity and 3‐D changes in membranous structures. Therefore, we believe that this study accurately presents morphological changes in the fetal diaphragm. Moreover, DTI tractography is a unique method that utilizes the diffusion of water molecules and helps elucidate the 3‐D microstructure of tissues that cannot be recognized on TIW MRI and PCX‐CT. Combining DTI tractography with conventional histological methods and morphogenetic analysis using MRI, CT, and other imaging techniques has the potential to greatly advance our understanding of fetal myofiber and membrane structure development as well as the diaphragm itself in the future.

In conclusion, this study presented information collected using T1W MRI on the morphological development of the diaphragm until fusion was complete according to embryonic staging. We used 3‐D thickness assessment and DTI tractography to identify qualitative changes in the muscular and tendon sites that comprise 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 could aid in furthering the understanding of congenital anomalies in the future.

AUTHOR CONTRIBUTIONS

Toru Kanahashi performed data analysis/interpretation and drafting of the manuscript. Hirohiko Imai carried out data analysis/interpretation and acquisition of data. Hiroki Otani contributed to the acquisition of data and critical revision of the manuscript. Shigehito Yamada and Akio Yoneyama contributed to the acquisition of data. Tetsuya Takakuwa contributed to the concept/design and critical revision of the manuscript.

FUNDING INFORMATION

This study was supported by the Japan Society for Promotion of Science (grant nos. 20K22736 and 21K07772) and ISHIZUE 2021 of the Kyoto University Research Development Program.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

STATEMENT OF ETHICS

The ethics committee of the Kyoto University Faculty and Graduate School of Medicine approved this study, which used human embryos and fetal specimens (E986, R0316, and R2224).

ACKNOWLEDGMENTS

The authors thank Dr. Chiharu Endo at the Congenital Anomaly Research Center and Dr. Akihiro Matsumoto at Shimane University for technical assistance in handling human embryos and fetuses. We would like to thank Editage (www.editage.com) for English language editing.

Kanahashi, T. , Imai, H. , Otani, H. , Yamada, S. , Yoneyama, A. & Takakuwa, T. (2023) Three‐dimensional morphogenesis of the human diaphragm during the late embryonic and early fetal period: Analysis using T1‐weighted and diffusion tensor imaging. Journal of Anatomy, 242, 174–190. Available from: 10.1111/joa.13760

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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