Photon Factory Activity Report 2013 #31 (2014) B

BL-14C/2013G514

Two cases of liver agenesis detected in externally normal human embryos:
A novel abnormality resulting in abortion during the embryonic period
外表正常ヒト胚子にみられた肝臓無形成の２例；流産に陥ると考えられる新たな異常例の報告
Tetsuya T akakuwa and Tohoru Kanahashi
Kyoto University, Kyoto 606-8507, Japan
1 Introduction
Abortion is the termination of pregnancy by theremoval or expulsion of a fetus or embryo from the uterus,
prior to viability. An abortion that occurs spontaneously
is usually called a miscarriage. Most spontaneous
embryonic abortions occur during the first 3 weeks [1]. It
is generally thought that 15–20% of verified pregnancies
that have survived the first 4 weeks post-ovulation arelost through spontaneous abortion. A high incidence of
spontaneous abortion of fetuses with neural tube defects,
cleft lip, and cleft palate has been noted amongmalformed embryos, and many other externalmalformations may be involved in abortions [1,2].
However, the cause of abortion of normal conceptusesremains unknown, mainly due to the following issues: thedifficulty in obtaining appropriate sample materials, andthe small size of the embryos that makes analyses to findthe internal abnormality difficult. The crown-rump lengthof an embryo is a maximum of 30 mm during theembryonic period by Carnegie stage (CS) 23 (about 56-60
days after fertilization) [3].
In the present report, two cases of liver agenesis weredetected using phase-contrast X-ray
computed
tomography (PCT), and described in detail [4]. This novel
abnormality could seriously disrupt embryonicdevelopment, and, as a result, may have led to early
miscarriage. The causes of intrauterine death in externallynormal embryos have not previously been elucidated, andthe present report is a first step in that direction.
2 Experiment (Materials & Methods)
Images of three embryos at CS 21 were acquired using PCT
imaging. The embryos were collected and stored at theCase 1(ID 28153)

Case 2(ID 31874)

Control(ID 25858)

Fig. 1:
External view of
the three embryos
analyzed in thepresent study.
Congenital Anomaly Research Center of Kyoto
University [5]. All three embryos were obtained byinduced abortion (Table 1). The sizes and gestational ages

Table 1. Clinical features of the embryos and their parents
Case 1Case 2Control(ID 28153)(ID 31874)(ID 25858)

2121

21

[mm]

17.8

19.5

19.7

3

422.1

409.6

565.5

06/20/1970

12/05/1972

05/04/1970

Carnegie stage
Crown-rump length
Volume of whole embryo[mm ]Date of surgery
Days after ovulation[days]

534154

Age of father/mother[years]

36/30

32/35

- /42

InducedInducedInduced

Method of abortion

Photon Factory Activity Report 2013 #31 (2014) B

Case 1 (28153)

Control (25858)

LuLuLuLuLi

St

Th9Th4Lu

Lu

Es

Mt

Mt

Ad

Ad

Du

Ad

St

Li

Du

In
In

L1

Th6

Figure 2: Phase-contrast X-ray computed tomography(PCT) transverse sections from Case 1, and a normal
control for comparison. The sectional levels (at Th4,Th6, Th9, and L1) are indicated in each panel.
No liver (Li) was detected in the serial plane sections,while the stomach (St) was observed in the midsagittaltransverse sections at Th4. The diaphragm (△) was notedin these sections. The duodenum (Du) was located on theright ventral side of the abdomen, while the pancreas (▼)
had deviated to the ventral side in the Th6 transverse
section. Umbilical vein and ductus venosus was notshown in this Figure.
Lung (Lu), esophagus (Es), adrenal gland (Ad),intestine (In), metanephros (Mt), liver (Li), portal veins(▲), and hepatic veins (▲).

Photon Factory Activity Report 2013 #31 (2014) B

Case 1

HeLu
St
Ad
Ms

PaGe

were within the normal range. No clinical finding that
may have caused an abnormality (i.e. alcohol, tobacco,drugs, etc) was observed. Obvious damage to, or
anomalies of the external forms were not present (Fig. 1).
The embryo body axes were maintained in the originalform, i.e. not artificially deformed during fixation andpreservation. Therefore, the samples were judged to besuitable for internal structural analysis.
The three-dimensional (3-D) PCT images of thehuman 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.
This technique enables a highly sensitive measurement,
approximately > 1000 times more sensitive than theconventional radiographic method using absorption
contrast. The mechanism and conditions used to acquire
the PCT images of the embryos have been described
elsewhere [4]. The 3-D images included images of theintrathoracic, retroperitoneal, and intra-abdominal organs,reconstructed using Amira software (ver. 5.4.5, Visage
Imaging, Berlin, Germany).

Mt

Fig.3:Left anterior oblique viewof the three-dimensional
PCT reconstruction of Case
1, demonstrating thelocations of all intrathoracic, retroperitoneal,and intraabdominal organs.
The findings of note were as
follows: agenesis of the liver;
the stomach (St) wasdeviated ventrally andcranially; the pancreas (Pa)was deviated ventrally; andthe right mesonephros (Ms)and genital ridge (Ge) wereabsent.
Lung (Lu), adrenal gland(Ad), metanephros (Mt), andheart (He).
The scale bar in thereconstructed image indicates
1 mm.

3 Results and Discussion
The liver at CS21 usually occupies a large space in theabdominal cavity, which has a smooth surface due to thecontact between the cranial surface and the diaphragm,
and between the ventral surface and the abdominal wall[6]. The PCT image resolution was high enough toobserve not only the morphological structure of theinternal organs but also the internal structure of the liver.
A PCT plane section showed the main structures ofthe vascular systems, such as the umbilical vein, ductus
venosus, hepatic veins, portal vein, and their branches, ina control case (Figure 2). In the two abnormal cases, theliver was not detected in any of the serial plane sections.
In Case 1 (ID 28153), the stomach was observed on themidsagittal line in the Th4 transverse sections, indicatingthat the stomach had deviated cranially and ventrally. The
diaphragm was apparent in these sections. The duodenum
was located in the right ventral part of the abdomen, andthe pancreas deviated ventrally in the Th6 transverse
section. Similar abnormalities were detected in Case 2(ID 31874), which also had no detectable liver (data notshown.

Photon Factory Activity Report 2013 #31 (2014) B

Case 1 (28153)

Control (25858)

StDuDuPa

Fig.4;The 3-D reconstructed images (anterior view) of thestomach (St), duodenum (Du), and pancreas (Pa),demonstrating

The locations of all intrathoracic, retroperitoneal, andintra-abdominal organs were reconstructed in threedimensions, as shown in Figure 3. The liver, whichusually occupied a large space, was not detectable at all.
The right genital ridge and mesonephros were not present.
The absence of the liver in Case 1 had affected thelocations of the other internal organs, especially thestomach, duodenum, and pancreas (Figure 4). The
stomach was rotated. The pyloric antrum was straight,
and ran vertically in the right ventral quadrant. Notably,
the relative positions of the cardia and pylorus were notaffected. The pancreas originated from the duodenum,and its tail had deviated ventrally. Similar complications
were detected in Case 2 (data not shown).
The liver plays an important role in the development offunctional organs [6,7]. The liver becomes ahematopoietic organ after 6 weeks, and, during the fetal
period, begins to metabolize biochemical materials
important for development, such as albumin, bile,glycogen, and fetus-specific proteins [8]. Therefore, liveragenesis may be lethal to development.
The present study demonstrates that PCT may beconsidered a powerful tool for visualization of internal
structures of embryos, and for detection of novel
abnormalities during the embryonic period, without theneed for histological analysis. The non-invasive and nondestructive properties of the technique are important foranalysis of scarce specimens, such as human embryos.
The application of the present method will contribute toadvancing the field of fetal diagnosis and developmental
anatomy.

StPa

their anatomical relationships in Case 1 and in anormal control.
The scale bar in the reconstructed image indicates 1 mm.

References[1] O'rahilly R, Müller F. Human embryology &teratology. 92 (2001)[2] Shiota K. Congenital Anomalies 31, 67 (1991)[3] O'Rahilly R, Müller F. Developmental stages inhuman embryos: including a revision of Streeter's
horizons and a survey of the Carnegie Collection, (1987)[4] Yoneyama A, Yamada S, Takeda T. Advanced
Biomedical Engineering, 107(2011)[5] Nishimura H, et al. Teratology 1,281(1968)[6] Hirose A, et al. Anat Rec 295, 51(2012)[7] Lemaigre FP. Gastroenterology 137, 62(2009)[8] Tavian M, Hallais M, Péault B. Development 126,793(1999)
This study was approved by the Committee of MedicalEthics of Kyoto University Graduate School of Medicine,
Kyoto, Japan (E986).
* tez@hs.med.kyoto-u.ac.jp