Human Reproduction, Vol. 14, No. 3, 816-822,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Adhesion of shed menstrual tissue in an in-vitro model using amnion and peritoneum: a light and electron microscopic study
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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We have investigated the adhesion of endometrial tissue isolated from antegradely shed menstrual effluent to amnion and peritoneum. This endometrial tissue was cultured overnight on either side of intact and stripped amnion and on the mesothelial side of peritoneum. Light and electron microscopy were applied to evaluate adhesion. With light microscopy adhesion of endometrial fragments to stripped membranes was observed in nine out of 12 specimens and in 12 out of 13 specimens when layered on the extracellular matrix side of amnion. Adhesion when layered on the epithelial side was seen in only four out of 13 specimens. However, when using scanning electron microscopy adhesion of menstrual endometrial tissue could be visualized in all samples. Numerous adhering fragments were seen when layered on the extracellular matrix side of untreated amnion. On several occasions not only adhesion but also spreading of cells was observed. When layered on the epithelial side of untreated amnion or peritoneum, adhesion was exclusively seen at locations where the epithelium was damaged or absent. These findings were confirmed by transmission electron microscopy. These observations indicate that endometrial tissue isolated from antegradely shed menstrual effluent preferentially adheres to subepithelial structures of amnion and peritoneum. The lack of adhesion to epithelial cells suggests that an intact mesothelial lining prevents adhesion of menstrual endometrial tissue.
Key words: adhesion/amnion/endometriosis/peritoneum/menstruation/model
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The reflux implantation theory postulates that endometriosis is a consequence of the reflux of viable endometrial tissue through the Fallopian tubes during menstruation, with subsequent implantation and growth on the peritoneum (Sampson, 1940
). Retrograde menstruation is a common physiological event in menstruating women with patent tubes, as demonstrated by the presence and viability of endometrial tissue in peritoneal fluid (Koninckx et al., 1980; Kruitwagen et al., 1991). The mechanism responsible for the transformation of refluxed menstrual tissue into endometriotic lesions remains to be elucidated. It is tempting to assume that endometrial tissue adheres to the peritoneal lining, invades the extracellular matrix and develops into an endometriotic lesion (Spuijbroek et al., 1992). Endometrial fragments in the peritoneal fluid and mesothelial cells express adhesion molecules that are possibly involved in cell–cell, and cell–extracellular matrix interactions (Jonjic et al., 1992; Van der Linden et al., 1994; Witz et al., 1998). A myriad of factors in the peritoneal fluid, such as matrix metalloproteinases, steroids, growth factors and cytokines may also be involved in this process. The peritoneum is very fragile and tissue handling easily damages the mesothelial lining. Various in-vitro models have been used to study adhesion of endometrial cells and fragments to the peritoneal lining (Sharpe et al., 1992; Zhang et al., 1993; Wild et al., 1994; Van der Linden et al., 1996, 1998; Groothuis et al., 1998). Amnion, the innermost layer of the fetal membranes, strongly resembles the peritoneum, is less fragile, and has been used in previous studies (Van der Linden et al., 1996, 1998; Groothuis et al., 1998). Amnion has served as a model for basement membrane after stripping of its epithelial lining (Liotta et al., 1980). In these studies proliferative and secretory endometrial tissues, mechanically obtained, were used to study adhesion. Ideally, retrogradely shed menstrual endometrium should be used but this is hard to obtain in sufficient amounts. As second best we have used endometrial tissue isolated from antegradely shed menstrual effluent. In the present study we have investigated the adhesion of endometrial fragments isolated from menstrual effluent to untreated and stripped amnion and human peritoneum. Adhesion of menstrual tissue was evaluated using light, scanning electron (SEM) and transmission electron microscopy (TEM). |
Materials and methods |
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Institutional Review Board approval was obtained for this study.
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 2–3 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 hematoxylin–eosin 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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The results of the adhesion studies are presented in Tables I and II
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Light microscopy on cryostat sections
When layered on stripped amnion, adhesion of menstrual endometrial fragments was observed in nine out of 12 specimens (Figure 1a
, Table I
), and when layered on the matrix side of untreated amnion in 12 out of 13 specimens (Figure 1b, Table I). In four out of 13 specimens layered on the epithelial side of untreated amnion, adhesion of menstrual endometrial tissue was observed (Figure 1c, Table II). Adhesion occurred only at locations where the epithelial lining was damaged.
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Scanning and transmission electron microscopy
Amnion
As observed with SEM and TEM the amnion appeared to have a mostly intact epithelial lining with some isolated degenerating epithelial cells. Figure 2a
shows an amnion epithelium with part of the epithelial lining stripped off, exposing the basement membrane.
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Adhesion of menstrual endometrial tissue to the matrix side was observed at multiple locations in all samples, and some spreading of cells occurred (Figure 2b,c,
Table I). When seeded on the epithelial side of untreated amnion, adhesion was only seen at spots were the epithelial lining was damaged or absent, exposing the submesothelial layer (Figure 2d,e, Table II). Only small areas of the epithelial lining of amnion were damaged and accordingly only sporadic adherence of small fragments, consisting of a few cells, was observed.
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 4a–e, 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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Red blood cells and vaginal epithelial cells were present in nearly all samples, but these cells were easy to distinguish from endometrial tissue fragments (Figure 4c
).
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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study clearly demonstrates that antegradely shed menstrual tissue easily adheres to the ECM and basement membrane of amnion and peritoneum but not to an intact epithelial lining. This conclusion is based on several observations. There was no difference in adhesion to either side of the stripped membrane. Numerous fragments adhered to the ECM side of untreated amnion. Adhesion to the epithelial lining of untreated amnion and peritoneum was observed only at locations were the lining was not intact. The difference in rate of adhesion when layered on the epithelial side of amnion and processed for light microscopy (4/13 samples) versus scanning electron microscopy (8/8 samples) can be explained by the fact that a relatively large surface area can be examined by SEM in contrast to the limited surface area of the 5 µm cryostat cross-ections. The observations and conclusions from the present study are consistent with our previous studies (Van der Linden et al., 1996
, 1998; Groothuis et al., 1998). These functional studies were all performed with proliferative and secretory endometrial tissue and not with menstrual tissue. In addition endometrium that has been manipulated mechanically and enzymatically differs substantially from retrogradely shed endometrium. After collagenase digestion of endometrial fragments, the cells lose cell adhesion molecule expression and their capacity to adhere to stripped amniotic membranes (Van der Linden et al., 1998).
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 cell–cell adhesion, it is unlikely that molecules associated with cell–cell adhesion, e.g. the cadherins, are involved. Integrins, playing a major role in cell–extracellular 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), 4–5 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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We thank Liesbeth Bouchet, Paul Bomans and Rein van Gool for their expert technical support.
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Notes |
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3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Academisch Ziekenhuis Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
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Submitted on August 14, 1998; accepted on December 4, 1998.
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