1 Harris Birthright Research Centre For Fetal Medicine, King's College School of Medicine and Dentistry, Denmark Hill, London, UK, 2 Institute of Anatomy II, University of Freiburg, Albertstrasse 17, D-79104 Freiburg and 3 Department of Obstetrics and Gynaecology, University of Kiel, Kiel, Germany
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Abstract |
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Key words: lymphatic vessels/nuchal translucency/5'nucleotidase/trisomies 21, 18 and 13/Turner syndrome
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Introduction |
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In previous studies investigating extracellular matrix molecules in the skin of trisomic fetuses with increased nuchal translucency, we found alterations in a number of structural proteins, i.e. collagen type VI, laminin and collagen type IV (Brand-Saberi et al., 1994a, b
; von Kaisenberg et al., 1998a
, b
). It seems that the mechanism leading to the increase of fluid in the skin in Turner syndrome is completely different from fetuses with trisomies.
A microscopic study examining the lymphatic vessels in the skin of spontaneously aborted fetuses with cervical cystic hygromas reported that in non-Turner fetuses, there were numerous dilated lymphatic vessels, whereas in Turner syndrome, there were very few such vessels. In the skin of normal fetuses with no cystic hygromas, lymphatic vessels were evenly distributed (Chitayat et al., 1989).
The aim of this study was to investigate the distribution of lymphatic vessels in nuchal skin tissue from fetuses with Turner syndrome compared to fetuses with trisomies 21, 18 and 13, that also had increased nuchal translucency and chromosomally normal controls. The distribution of vessels was examined by immunohistochemistry using a monoclonal antibody, PTN63, against 5'nucleotidase and an anti-laminin antibody. The enzyme 5'nucleotidase has its highest activity in lymphatic vessels, but it is also present in high endothelial venules of lymphoid tissues (Turner et al., 1987). Laminin is a major component of basement membranes and therefore highlights large lymphatics and both large and small blood vessels. Smaller lymphatics and lymph capillaries are devoid of basement membranes and are therefore not stained using anti-laminin.
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Materials and methods |
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Immunohistochemistry
Tissue samples were sectioned serially at 20 µm using a cryocut 2000 (Leica) and collected onto chromealumgelatin coated slides. Sections were air-dried, treated with bovine serum albumin (BSA) in phosphate buffered saline (PBS) to absorb unspecific antibody-binding, rinsed in PBS and incubated with the first antibody (see below) for 60 min at room temperature in a humid chamber.
PTN63 and anti-laminin were used as first antibodies. PTN63, a monoclonal antibody from mouse raised against the PaTu 8902 cell line established from a human pancreatic adenocarcinoma (Flocke et al., 1992), was used at a dilution of 1:4. Anti-laminin polyclonal antibodies, obtained from Sigma (Munich, Germany), were used at a dilution of 1:200 to stain the basement membranes of major vessels and blood capillaries. After rinsing in PBS, the second antibody was applied for 60 min. For the detection of PTN63, a goat-anti-mouse antibody coupled with Cy3 fluorochrome (Dianova, Hamburg, Germany) yielding a red signal was used. Anti-laminin antibodies were detected by a goat anti-rabbit antibody coupled with fluorescein (Dianova) yielding a green signal. Double-labelling was performed with both primary and secondary antibodies applied to the same sections in a sequence. Sections were covered with mowiol (embedding medium; Hoechst, Frankfurt, Germany) and coverslips. They were viewed and micrographs were taken using an epifluorescence microscope (Carl Zeiss, Oberkochen, Germany), and TMY-400 black-and-white films or colour films.
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Results |
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Discussion |
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These findings are compatible with those from the microscopic studies of Chitayat (Chitayat et al., 1989) who reported lymphatic hypoplasia in the skin of Turner fetuses. Further support for lymphatic hypoplasia in Turner syndrome has been provided by studies investigating women with ovarian dysgenesis as a result of a 45XO karyotype; in these cases, lymphangiography revealed hypoplastic lymphatic vessels in the lower limbs, pelvis and retroperitoneal space (Vittay et al., 1980
).
The enzyme 5'nucleotidase has been shown to be involved in the phenomena of cell adhesion and migration on laminin and fibronectin (Risse et al., 1989; Stochaj et al., 1989
, 1990
). It is tempting to speculate that changes in the distribution of these matrix molecules in Turner syndrome may account for alterations of the distribution of the lymphatic vessels or vessels in general. Since 5' nucleotidase interacts with both laminin and fibronectin and laminin is unchanged between normal and Turner fetuses, it would be interesting to investigate if the distribution of fibronectin is altered in Turner syndrome. If true this may account for decreased migration of lymphangioblasts into the upper dermis resulting in lymphatic vessel hypoplasia.
In summary, we conclude that an aberrant distribution of lymphatic vessels in the upper dermis of the nuchal skin in Turner fetuses may account for the increase in nuchal translucency due to a failure to drain interstitial fluid in this area and due to a congestion of large diameter vessels at the dermis/subcutis junction. These dilated vessels may account for the `cystic' appearance of nuchal hygroma which is typical of Turner syndrome. Since no animal model for Turner syndrome is available at present, it seems unlikely that it will be possible to perform perfusion studies to investigate further the physiology of lymphatic transport in the near future.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 18, 1998; accepted on November 24, 1998.