PMC6875321_01
(a) Photographs of the Fetus acardius amorphus. Fetus acardius amorphus shown as a rounded ovoid mass with a small pedicle symbolizing the umbilical cord. On one surface there exists a colloidal mass, as well as a short pedicle with little hair on it. (b) The Center of Fetus acardius amorphus illustrates an organized bone structure predicted to be the vertebra column, surrounded by yellowish tissue as well as red masses. (c1 and c2) Facitron X-ray analysis of the skeletal system of the Fetus acardius amorphus. The Fetus acardius amorphus shows a complete organized vertebral column, with a rudimentary rib cage and an ovoid bone structure (white arrow) without axial organization of a head, limbs or cranial-caudal poles. (d1 and d2) CISH analysis of interphase cells within paraffin sections of male gonadal tissue using the CEN X/Y Probe. CEN X/Y Probe hybridizes on regular male interphase cells demonstrated by one red (chromosome X) and one green (chromosome Y) signal per nucleus. The Fetus acardius amorphus shows a male XY genotype. Scale bar = 20 mum (D1), scale bar = 5 mum (D2).
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Macroscopic examination of the fetus acardius amorphus demonstrated a mass, 10.5 x 6.5 x 7.5 cm in diameter, and covered by mature skin with sparse, superficial hair on the surface (Figure 1(a)). Furthermore a small cyst-like structure with opaque fluid inside was observed at one end of the mass, covered by intact skin and a pedicle of hair (Figure 1(a)). The center of the mass demonstrated an organized bone column with cartilage surrounded by colloidal tissue and focal red masses (Figures 1(b), 1(c1) and 1(c2)). X-ray analysis of the skeletal system with Facitron showed the presence of a complete vertebral column with the presence of a rudimentary rib cage (Figures 1(c1) and 1(c2)). At one end of the vertebral column, an ovoid bone structure was noted which could represent a rudimentary pelvis (Figures 1(c1) and 1(c2)). However, there was no axial organization of a head, limbs or cranial-caudal poles present (Figures 1(c1) and 1(c2)). Sex identification analysis showed in CISH analysis a male gender with XY karyotype in interphase cells (Figures 1(d1) and 1(d2)).
(a1): Hematoxylin and eosin (HE) staining of the skin of the Fetus acardius amorphus. Fetus acardius amorphus shows differentiated skin tissue, with a stratum corneum, an epidermis, a dermis and a subcutis. Images are shown in 4x magnification. (a2 and a3) Hematoxylin and Eosin (HE) staining of the core mass of the Fetus acardius amorphus. Fetus acardius amorphus shows organized bronchus-like structures with respiratory epithelium, goblet cells and cilia (black arrow) on the surface. Images are shown in 4x magnification (a2) and in 40x magnification (a3). (b1 and b2) Hematoxylin and Eosin (HE) staining of the core mass of the Fetus acardius amorphus. Fetus acardius amorphus shows differentiated bone and cartilage with bone marrow cells like megakaryocytes, erythrocyte precursor cells as well as myelocytes in between. Images are shown in 10x magnification (b1) and in 40x magnification (b2). (b3 and b4) Hematoxylin and Eosin (HE) staining of the core mass of the Fetus acardius amorphus. Fetus acardius amorphus shows differentiated skeletal muscle with long, shapoid myocytes with an ovoid nucleus in the center. Images are shown in 10x magnification (b3) and in 40x magnification (b4). (c1 and c2) Hematoxylin and Eosin (HE) staining of the cyst-like structure in the Fetus acardius amorphus. Fetus acardius amorphus shows differentiated neurons of the hippocampus. Images are shown in 10x magnification (c1) and in 40x magnification (c2). (c3 and c4) Hematoxylin and Eosin (HE) staining of the gonadal glands in the Fetus acardius amorphus. Fetus acardius amorphus shows gonadal glands with well-formed seminiferous tubules showing Ledyig cells between (black star), and spermatogonia (black arrow) and Sertoli cells (white star) inside. Images are shown in 10x magnification (c3) and in 40x magnification (c4).
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Histology analysis of the core mass showed differentiated skin tissue with all layers including epidermis, dermis and subcutis (Figure 2(a1)) as well as bronchus-like structures with respiratory epithelium with apical cilia and mucous-producing cells on top (Figures 2(a2) and 2(a3)). Furthermore, the core mass also demonstrated bone and cartilage tissue, as well as bone marrow with megakaryocytes, erythrocyte precursor cells as well as myelocytes in between (Figures 2(b1) and 2(b2)). Differentiated muscle cells with shapoid myocytes harboring an ovoid nucleus in the center were present as well (Figures 2(b3) and 2(b4)). The cyst-like structure showed brain tissue with hippocampal neurons (Figures 2(c1) and 2(c2)). Additionally parts of the urogenital tract were detected with male gonadal structures with testicles consisting of seminiferous tubules surrounded by a thin smooth muscle layer (Figures 2(c3) and 2(c4)). Inside the seminiferous tubules, spermatogonia as well as Sertoli cells were present, Leydig cells were embedded between the semiferous tubules, respectively (Figures 2(c3) and 2(c4)).
Hematoxylin and Eosin (HE) staining and immunohistochemistry staining for villin, mucin 2 and calretinin in the intestine of the Fetus acardius amorphus. Villin is stained in red (ALEXA 568), mucin 2 in green (ALEXA 488) and nuclei in blue (Hoechst). Fetus acardius shows differentiated small (a) and large (b) intestine with positive apical villin staining (red) marking the intestinal brush border in cells of the crypt and villi and mucin 2 (green) positive Goblet cells in crypts and villi. Ganglion cells of the Auerbach and Meissner Plexus were also present shown by positive Calretinin staining. Images are shown in 10x and in 20x magnifications.
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Moreover, within the focal red masses parts of the gastrointestinal tract with small as well as large intestine were identified (Figures 3(a) and 3(b)). The intestine was organized in a crypt villous axis, and enterocytes showed an apical brush border strongly positive for the brush border protein villin in both, crypts and villi (Figure 3). Goblet cells were identified with positive mucin 2 stains (Figure 3). Ganglion cells positive for Calretinin were detected within the Meissner and Auerbach Plexus (Figure 3).
(a) PAS staining and immunohistochemistry staining for villin of fetal renal tissue. In immunohistochemistry, villin is stained in red (ALEXA 568) and nuclei in blue (Hoechst). Fetus acardius shows differentiated kidney with cortex and medulla, but lack of proximal tubules as shown by negative villin staining. Additionally, distal tubules of the fetus acardius amorphus show mild nephrocalcinosis (white star). PT (proximal tubule), G (glomerulus). Images are shown in 10x, 20x and 40x magnifications. (b) Hematoxylin and Eosin (HE) staining and immunohistochemistry staining for MAC 387 staining in fetal renal tissue. MAC 387 positive cells were detected in the interstitial tissue of the kidney in the Fetus acardius amorphus, those cells are marked with a black arrow in the Hematoxylin and Eosin (HE) staining labeling erythrocyte precursor cells. MAC 387 is stained in red (ALEXA 568), nuclei stained with in blue (Hoechst). Images are shown in 20x magnification. (c) CD31 immunohistochemistry staining in fetal renal tissue. CD31 (brown) is staining vessels in the renal cortex and medulla, nuclei are stained with hematoxylin in blue. Increased number of CD31 positive vessels is detected, in particular in the renal cortex of the fetus acardius amorphus.Images are shown in 10x magnification. (d) Smooth muscle actin (SMA) staining in fetal renal tissue. Increased number of SMA positive vessels in particular the cortex of the fetus acardius amorphus. SMA is stained in green, nuclei stained with Hoechst in blue. Glomeruli are marked with a white star, tubules with white arrows. Images are shown in 10x magnification.
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Intriguingly, the most curious finding of this case of Fetus acardius amorphus was the abnormalities within the renal tissue (Figure 4). Fetus acardius amorphus demonstrated differentiated glomeruli, but a complete lack of proximal tubules, confirmed by PAS staining as well as villin immunohistochemistry (Figure 4(a)). Findings of the fetus acardius amorphus were compared to a fetus without any evidence of abnormalities (control) and a fetus with bilateral renal hypoplasia (disease control). In both control cases, proximal tubules were present (Figure 4(a)). In addition, within the medulla, clusters of erythrocyte precursor cells were detected, usually just found in the spleen and liver as a sign for extramedullary hematopoiesis (Figure 4(b)). Immunofluorescence of those clusters of cells showed positivity for the myeloid marker MAC 387, identifying these cells as erythrocyte precursor cells (Figure 4(b)). Stainings of vessels with CD31 showed particularly in the renal cortex of the Fetus acardius amorphus increased presence of CD31 positive vessels, mainly within the interlobular area (Figure4(c)). Interestingly, those vessels were strongly positive in immunofluorescence stains for smooth muscle actin (Figure 4(d)). Those SMA positive vessels also seemed to have a higher degree of dilation, when compared to both controls (Figure 4(d)).