1 Department of Obstetrics & Gynaecology and 2 Department of Pathology, Academisch Ziekenhuis Maastricht and Maastricht University, Maastricht, The Netherlands
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Abstract |
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Key words: adhesion/amnion/endometriosis/peritoneum/menstruation/model
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Introduction |
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Materials and methods |
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Collection of menstrual effluent
Antegradely shed menstrual effluent was collected with a menstrual cup as described previously (Koks et al., 1997). Volunteers agreed to donate menstrual fluid after having given informed consent. Menstrual effluent was collected during 23 h on the first, second or third day of a regular menstrual cycle. Immediately after collection, the menstrual fluid was delivered to the laboratory in a plastic container.
Preparation of menstrual endometrial tissue
Menstrual effluent samples were centrifugated at 800 g for 6 min. The supernatant was removed and the pellet was resuspended in complete culture medium (CM) consisting of Dulbecco's modified Eagles's medium (DMEM)/Ham's F12 (Life Technologies BV, Breda, The Netherlands) supplemented with 10% fetal calf serum, L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). A Ficoll-Paque gradient was used to remove red blood cells and endometrial tissue was obtained after centrifugating at 1000 g for 6 min. After resuspension, the menstrual endometrial tissue was filtered through a 100 µm nylon filter (Micronic, Lelystad, The Netherlands) and a 30 µm polyamid filter (Stokvis & Smits, IJmuiden, The Netherlands). The endometrial fragments retained on the 100 and 30 µm filters were collected, washed and seeded on either side of the untreated and stripped amnion and on the mesothelial side of peritoneum.
Amnion
Fresh human placentas were obtained at the time of normal term delivery. The amnion was separated from the chorion. Amnion overlying the placenta as well as amnion reflectum were used. The amnion part overlying the placenta has cylindrical epithelial cells which are only loosely connected to the basement membrane and are easy to remove. This part of the amnion was stripped from its epithelial lining and extracellular matrix, as described by Liotta et al. (1980). In short, amnion was washed for 1 h in distilled water and 2 mM N-ethylmaleimide (NEM), extracted for 1 h with 1 M NaCl, 20 mM EDTA, 2 mM NEM and finally treated with 4% deoxycholate for 1 h. After each step the membranes were scraped with a rubber policeman to remove epithelial cells and most of the interstitial stroma.
The amnion reflectum, with cuboidal epithelial cells, was rinsed in phosphate-buffered saline (PBS). For light microscopic studies the untreated and stripped membranes were stored at 4°C in PBS for several days. For SEM and TEM studies the untreated amnion was rinsed, collected in CM and used the same day.
Peritoneum
Small strips of peritoneum (2x3 cm) were collected during abdominal surgery for benign gynaecological conditions. Patients agreed to donate peritoneum after having given informed consent. Since the mesothelial lining is very vulnerable, the tissue was handled with utmost care, immediately stored in culture medium and used the same day.
Adhesion studies
Untreated and stripped amnion were suspended between two sterile stainless steel rings. Isolated menstrual endometrial fragments were layered on either side of the untreated and stripped amnion. Peritoneal strips are too small to suspend between rings, and therefore a sterile stainless steel ring was placed on top of the mesothelial side of the peritoneum and the menstrual endometrium was layered inside the ring. After incubation overnight at 37°C in CM, the membranes were rinsed several times in PBS to remove unattached cells and fragments.
For light microscopy the amniotic membranes were snap-frozen in isopentane embedded in dry ice. All samples were stored in 70°C until analysed. Cryostat sections were cut and stained with hematoxylineosin to study the morphology and adhesion of menstrual tissue to basement membrane, extracellular matrix and epithelium of amnion. Light microscopy on peritoneum was performed on Epon-embedded tissue.
Electron microscopy
After rinsing in PBS, the amnion and peritoneum samples were mounted on a piece of cork and fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4). Part of the amnion and peritoneum samples were processed for SEM and part for TEM. Following fixation, the amnion specimens for SEM were dehydrated in a graded series of alcohols, critical point dried with CO2 and sputter-coated with gold. The samples were examined under a Philips 505 scanning electron microscope (Philips, Eindhoven, The Netherlands). After dehydration in alcohol, the peritoneal samples were placed in acetone overnight to extract fat, since the presence of fat interferes with critical point drying. The other samples were prepared for TEM according to the method described by Luft (1961) and Luft and Wood (1963). In short, the samples were postfixed in 1% osmium tetroxide, dehydrated and embedded in epoxy resin. For the peritonal samples fat was partly extracted overnight in propylene oxide. The Epon blocks were trimmed and sectioned. Sections of 1 µm thickness were stained with toluidine blue for evaluation under a light microscope. Subsequently, ultrathin sections were cut using a Reichert ultramicrotome an a diamond knife. The sections were contrasted with uranyl acetate and lead citrate and examined under a Philips CM10 transmission electron microscope.
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Results |
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Peritoneum
The mesothelial lining of the peritoneum was intact in some areas, showing flat mesothelial cells with a loose to moderate density of microvilli. Many mesothelial cells showed retraction, shrinkage or were absent, with exposure of the underlying basement membrane (Figure 3a). TEM showed that peritoneum consists of a single layer of flattened cells with microvilli, and various intact intracellular structures (Figure 3b,c
). The cells are connected to one another by desmosome-like complexes. The mesothelium rests on a basement membrane, which is separated from the underlying tissue by a loose connective layer containing several strata of collagen fibres, fibroblasts, adipocytes and blood vessels (Figure 4a,b
). Adhesion of endometrial fragments to fresh peritoneum was observed in all specimens (n = 5) and again only where the mesothelial lining was damaged or absent (Figure 4ae,
Table II
). Large areas of damaged peritoneum were seen and at these locations small as well as relatively large endometrial fragments adhered to the submesothelial layer. In one of the peritoneal samples no mesothelial cells could be detected and many menstrual endometrial fragments adhered to the submesothelial lining. Again, spreading of cells was seen (Figure 5a
).
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The morphology of the menstrual tissue showed some variation between the different specimens. In some specimens endometrial cells looked intact (Figure 2b,c) and alive (Figure 6
) whereas in others, cells appeared to be degenerating (Figure 2d
). No difference was observed in the adhesion pattern between material collected during the first, second or third day of the menstrual period.
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Discussion |
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Retrogradely shed menstrual endometrial tissue is difficult to obtain. Therefore we have used antegradely shed menstrual endometrium. In a previous study (Koks et al., 1997), using immunohistochemistry to detect expression of marker proteins such as cytokeratin 18, BW 495, and vimentin as well as cultures on extracellular matrix, we have already shown that the tissue obtained with a menstrual cup is indeed viable menstrual endometrium.
It is not obvious which molecular mechanism(s) are responsible for adhesion to the ECM and basement membrane. Expression of cell adhesion molecules on cyclic endometrium, antegradely and retrogradely shed menstrual endometrium, and endometriotic lesions has been studied and it has been shown that these tissues express integrins, CD44 and cadherins (Behzad et al., 1994; Van der Linden et al., 1994
; Lessey et al., 1996
). Since we did not observe cellcell adhesion, it is unlikely that molecules associated with cellcell adhesion, e.g. the cadherins, are involved. Integrins, playing a major role in cellextracellular matrix adhesion, may be responsible for this binding to the subepithelial structures.
Alternatively injury or inflammation within the peritoneal cavity may generate an outpouring of fibrinogen which forms fibrin clots and causes adherence of different structures. Although this type of adhesion is also possible, strands of fibrin were not seen with electron microscopy. In a murine model it has been shown that development of postsurgical adhesions requires trauma to both contacting peritoneal sites (Haney et al., 1994).
It also remains to be elucidated whether the peritoneal defects seen in this study were already existing in vivo, were caused by tissue handling or by the menstrual tissue itself.
If adhesion occurs at locations which were damaged in vivo, this would imply that efforts to mininize trauma to mesothelium are an effective approach to prevent adhesion and the development of endometriosis. An increasing number of reports suggests that surgical procedures which require mechanical manipulations or CO2 pneumoperitoneum may result in trauma of the mesothelium (Bouvy et al., 1996; Volz et al., 1998
). Since mesothelial injuries take ~7 days to repair (Ryan et al., 1973
) we can conclude that to prevent adherence of menstrual endometrium and hence to reduce the risk of developing endometriosis, elective laparotomies and laparoscopies in women of childbearing age are best avoided around and during menstruation.
Menstrual tissue may be able to damage the peritoneum. In-vitro experiments have demonstrated that activated polymorph mononuclear neutrophils are able to adhere to cultured mesothelial cells which leads to ATP depletion, morphological alterations and ultimately mesothelial cell death (Andreoli et al., 1994). In the rat study by Buck (1973), 45 days after intra-abdominal tumour injection, mesothelial cells changed their shape, becoming cuboidal and exposing the basement membrane between adjoining cells. These observations support the contention that the presence of certain factors or cells in the menstrual effluent may have a detrimental effect on the peritoneal lining.
In summary, we have demonstrated that endometrial tissue obtained from antegradely shed menstrual effluent easily adheres to basement membrane and extracellular matrix. Adhesion to the epithelial side is only seen at places where the epithelium has been damaged and the subepithelial structure is exposed.
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Acknowledgments |
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Notes |
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References |
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Submitted on August 14, 1998; accepted on December 4, 1998.