1 Leuven University Fertility Center and 2 Department of Pathology, UZ Gasthuisberg, KU Leuven, Belgium
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Abstract |
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Key words: adhesion/endometriosis/endometrium/peritoneum/tissue culture
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Introduction |
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The interaction between endometrium and peritoneum in vitro has been studied in various models. Effects of cytokines on adhesion of endometrial cells to monolayered mesothelial cells have been reported (Zhang et al., 1993; Wild et al., 1994
). Some investigators have promoted the amniotic membrane as an in-vitro model for peritoneum and concluded that mesothelium acts as a barrier to the attachment of ectopic endometrium, suggesting that peritoneal damage is required for the adhesion of endometrial fragments (Van der Linden et al., 1996
; Groothuis et al., 1998
). This finding was refuted by others who reported that endometrium can attach to the intact mesothelial surface of the peritoneum, suggesting that peritoneal damage is not required before endometrial implantation (Witz et al., 1999
, 2001
).
To the best of our knowledge, no data are available on how endometrialperitoneal adhesion is affected by the phase of the menstrual cycle, or by the presence or absence of endometriosis. This may be important, since data in baboons indicate that the in-vivo intrapelvic implantation potential of menstrual endometrium is higher than the implantation potential of endometrium derived from either the follicular or luteal phase (DHooghe et al., 1995). Furthermore, it is possible that endometriosis occurs only in women with a high degree of endometrialperitoneal adhesion. Therefore, this study was designed to test the hypothesis that, firstly, in-vitro endometrialperitoneal adhesion is more likely to occur using tissue collected during the menstrual phase of the cycle than during the follicular or luteal phase of the cycle, and secondly, in-vitro endometrialperitoneal adhesion more frequently occurs from samples collected from women with endometriosis than from women with a normal pelvis.
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Materials and methods |
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A diagnostic laparoscopy for investigation of pain and/or infertility was performed in 67 reproductive age women (range 1944 years). Endometriosis was found in 49 women and 18 women had a normal pelvis. The 49 patients with endometriosis had rAFS stage I (n = 26), rAFS stage II (n = 11), rAFS stage III (n = 6) and rAFS stage IV (n = 6) endometriosis, according to the revised system of the American Society of Reproductive Medicine, 1996 (American Society for Reproductive Medicine, 1997). Laparoscopies were performed during the luteal phase of the menstrual cycle in 32 women, during the follicular phase in 15 women and during menses in 17 women (Table I). Laparoscopy was also performed in three women with amenorrhoea (Table I
). Two of these three amenorrhoeic women had endometriosis and had amenorrhoea induced with GnRH agonists for severe disease (rAFS stage IV). The third patient with amenorrhoea did not have endometriosis but a diagnosis of the polycystic ovarian syndrome and was not receiving hormonal therapy. During laparoscopy, biopsies from parietal peritoneum and endometrium were obtained from each patient. A circular peritoneal biopsy of
2 cm2 was surgically excised from the left suprapelvic parietal peritoneum (above the rectosigmoid) by CO2 laser (15 Watt superpulse, Sharplan 40 C; Laser Industries, Tel Aviv, Israel). During the laser excision, the biopsy was not touched at all, except for the laser beam directed at the edge of the biopsy, to prevent macroscopic or microscopic peritoneal damage. At the end of the excision, the biopsy was carefully held and removed with a grasping forceps at the margin (not in the centre) of the biopsy, again to prevent peritoneal damage by surgical manipulation. During removal from the excision site, the biopsy underwent a spontaneous folding process and was slowly and gently extracted through a 5 mm small suprapelvic trocar. The average thickness of a peritoneal biopsy was
2 mm (including subperitoneal fat). Endometrial samples were obtained by curettage. A portion of the biopsy was fixed for histological confirmation of the menstrual phase.
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Histology
After fixation, the endometrialperitoneal explant was cut into three or four pieces that were embedded together in the same paraffin block. This was done to limit the number of sections needed to obtain a full picture of the endometrialperitoneal adhesion process. Serial sections (5 µm) of the paraffin blocks were taken and mounted on 3-aminopropyltriethoxysilane-coated slides (Sigma Chemical Co., Bornem, Belgium). Morphology and adhesion of the endometrial fragments to the peritoneum was evaluated at periodic intervals following haematoxylin and eosin stain. Adhesion was assumed when uninterrupted contact between the epithelial side of the peritoneal biopsy and endometrial fragments was observed at light microscope level. Endometrial biopsies were histologically evaluated for dating the menstrual cycle phase (Noyes et al., 1950). All slides were reviewed by a senior expert in the histopathology of endometrium and endometriosis.
Immunohistochemistry
Immunohistochemical staining was performed on 5 µm tissue sections to identify adhering cells using monoclonal antibodies directed against cytokeratin, endometrial stromal cell marker CD10 (Toki et al., 2002), histiocyte/macrophage marker CD68 (Kp1; Dako, Glostrup, Denmark) and leukocyte marker CD-45 (Dako). It is well known that mesothelial and endometrial epithelial cells, but not endometrial stromal cells, stain positively for cytokeratin.
Staining for cytokeratin included digestion with pepsin (Sigma) in HCl and incubation with a mixture of H2O2 and NaN3 in methanol (30 min), followed by local treatment with a 2% bovine serum albumin solution (15 min). Slides were then incubated with a mouse anti-cytokeratin (1/400, Clone MNF 116; Dako) antibody for 2 h at room temperature. The secondary antibody was peroxidase-conjugated goat anti-mouse IgG (1/100, 30 min; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), supplemented with normal human serum (1/25). Peroxidase activity was detected using 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) and slides were counterstained with Harris haematoxylin.
For immunohistochemical staining with CD10, slides were dewaxed, rehydrated and boiled in 0.01 mol/l of citrate buffer (pH 6.0) for 30 min in a microwave oven. After endogenous peroxidase activity had been blocked, slides were incubated with CD10 (1/20; Novacastra, Newcastle-upon-Tyne, UK) for 30 min at room temperature. The sections were incubated with anti-Mouse EnVision/HRP (undiluted, 30 min; Dako). The slides were finally stained with DAB and counterstained with Harris haematoxylin.
Statistical analysis
Statistical analysis to detect significant differences between groups was performed with 2-tests. P < 0.05 was accepted as significant.
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Results |
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Discussion |
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Several in-vivo and in-vitro models have been developed to study the potential for endometrialperitoneal interactions. The baboon has been proposed as an in-vivo culture model for the study of endometrialperitoneal implantation (DHooghe et al., 1995). Nude mice were used in one study (Nisolle et al., 2000
) to observe transplantation of human endometrium. In vitro, potential interactions have been studied using amnion membranes (Van der Linden et al., 1996
; Groothuis et al., 1998
) or biopsies of human peritoneum (Groothuis et al., 1999
; Witz et al., 1999
, 2001
), but conclusions are controversial. One group suggested that an intact mesothelium may prevent endometrial fragments from adhering to the peritoneum (Van der Linden et al., 1996
; Groothuis et al., 1998
). Electron microscopy supported this hypothesis that the mesothelial lining acts as a barrier for adhesion (Groothuis et al., 1999
). Using endometrial tissue isolated from antegradely shed menstrual effluent, this group confirmed their previous results that adhesion was exclusively seen at locations where the epithelium was damaged or absent (Koks et al., 1999
). In contrast, another group (Witz et al., 1999
, 2001
) reported that endometrium can attach rapidly to the intact mesothelial surface of peritoneum.
In the present study, we used mechanically fragmented endometrium in an in-vitro model to determine whether endometrium could adhere to autologous peritoneal surfaces. In our experiments, the area of endometrium adherent to peritoneum consisted mainly of cells morphologically and immunohistochemically identified as stromal cells, suggesting that stromal cells may play an important role in in-vivo attachment. Our results are in accordance with those of other investigators (Witz et al., 1999), although they also reported that in addition to stromal cells, endometrial epithelium can also attach to mesothelium (Witz et al., 2001
). Experiments in nude mice have also demonstrated that stromal cells are involved in the attachment process and glandular cells in the growth of endometriotic lesions (Nisolle et al., 2000
).
Our results indicated that a high degree of endometrialperitoneal adhesion (80100%) occurred after culturing the explants for 48 h. Unfortunately, only one experiment was performed with an incubation period of 24 h, so we cannot exclude more rapid adhesion. Others have shown that endometrial adhesion occurs within 1 h and that transmesothelial invasion occurs within 18 h (Witz et al., 2001).
Endometrialperitoneal adhesion was observed at several sites on the peritoneal surface. The number of adhering fragments was not counted since the amount of added endometrium was not quantified before the experiment. Furthermore, overestimation of the number of adhesion sites would have been unavoidable since three or four tissue explants were embedded in the same paraffin block.
Our histological results suggest that a dynamic process occurs at the endometrialperitoneal interface. Invasion of stromal cells into the peritoneum occurred rapidly with the formation of a multicellular layer under the mesothelium. Below the area of attachment of endometrial fragments, no identifiable mesothelium was present, and at the margins, a re-epithelialization process rapidly integrated the implant into the peritoneal layer. Others (Witz et al., 1999) have also observed that intact mesothelium occurs up to the point of attachment. The re-epithelialization process by the mesothelial epithelium is continued into the glandular epithelium of the endometrial implant. Under this epithelialized layer, the endometrial stroma was replaced by a mixture of fine fibrillar and necrotic debris. Finally, in interstitial peritoneal tissue, well-structured collagen fibres disintegrated as a result of elastoid degeneration. Interestingly, these histological observations were made in several experiments using endometrium from different phases of the menstrual cycle, regardless of the presence or absence of endometriosis and the stage of endometriosis.
A prerequisite for the success of adhesion and structural changes in the implant is the presence of viable stromal cells in the endometrial biopsy. Sometimes, necrotic tissue was observed in the endometrial biopsy and/or in the peritoneum. This could prevent the re-epithelialization and the integration of the implant. Probably, necrotic tissue is also responsible for a negative result of the adhesion process and may explain why adhesion was not observed in 100% of our assays.
In our study, peritoneal tissues were collected with the use of a CO2 laser instead of a scalpel. The advantage of laser dissection is that it is not necessary to damage the peritoneum during excision. Technically, the laser technique allows the surgical excision of the biopsy without touching the biopsy. Only at the end of the biopsy can the tissue be grasped with a forceps at the margin and gently extracted from the abdomen. In contrast, the usage of scissors and forceps, and/or electrocoagulation, during peritoneal biopsy involves more manipulation of, and more damage to, the peritoneal surface.
The effect and the presence of the stage of endometriosis on the adhesion process between autologous endometrium and peritoneum has never been studied in detail. Most investigators only included endometrium from women without endometriosis who were undergoing surgery for benign conditions (Groothuis et al., 1999; Witz et al., 1999
, 2001
). In our study, adhesion assays were performed with endometrium from patients with and without endometriosis including women with minimal, mild, moderate and severe endometriosis. No correlation was observed between endometrialperitoneal adhesion and the presence or stage of endometriosis. It is possible that the lack of statistically significant results regarding endometrialperitoneal adhesion in vitro between patients with and without endometriosis can be explained by lack of power. Indeed, although our study contained considerably more patients (n = 67) than the number of subjects (n = 315) studied in previous reports (Groothuis et al., 1999
; Witz et al., 1999
, 2001
), power analysis revealed that at least 27 patients with endometriosis and 27 patients without endometriosis would be needed (ß = 0.8 and
< 0.05) to test the hypothesis that luteal endometrialperitoneal adhesion in vitro occurs significantly more frequently (80%) in women with endometriosis than in women without endometriosis (40%). However, the observation during the menstrual and follicular phase that adhesion occurred in 100% of all patients without endometriosis directly undermined our hypothesis and prompted us to stop the study at an earlier stage.
The effect of the different phases of the cycle on the adhesion process has never been reported. Other investigators have used proliferative (Groothuis et al., 1998, 1999
; Witz et al., 1999
, 2001
) secretory (Groothuis et al., 1998
; Witz et al., 1999
, 2001
) or menstrual endometrium (Van der Linden et al., 1996
; Nisolle et al., 2000
), but did not compare their results with respect to adhesion capacity to peritoneum. Our results indicated that endometrium obtained during the menstrual, luteal or follicular phase had similar potential to implant on autologous peritoneum suggesting that the adhesion of endometrium onto peritoneum is a universal process, independent of the cycle phase. In-vitro models, however, are limited by their descriptive and qualitative nature. A quantitative adhesion assay is needed to study in detail the interaction between peritoneum and endometrium. Using this in-vitro model, the in-vivo situation in women is mimicked, but we cannot exclude several shortcomings in comparison with the in-vivo situation. Firstly, endometrial tissue and cells obtained after a transcervical uterine biopsy are probably different from endometrial tissue and cells present in the peritoneal fluid in vivo. Secondly, the collection, handling and culture of peritoneum may alter the propensity of peritoneum to adhere to endometrium. Therefore, short-term in-vivo co-culture of menstrual endometrium, peritoneal fluid and peritoneum is needed and being developed in the baboon (DHooghe, 1997
) to study the early endometrialperitoneal interactions in a clinically and biologically meaningful way. Further studies are also needed to investigate the precise mechanisms involved in endometrialperitoneal implantation events.
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Acknowledgements |
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Notes |
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3 To whom correspondence should be addressed at: Leuven University Fertility Center, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: sophie.debrock{at}uz.kuleuven.ac.be
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References |
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DHooghe, T.M. (1997) Clinical relevance of the baboon as a model for the study of endometriosis. Fertil. Steril., 68, 613625.[ISI][Medline]
DHooghe, T.M., Bambra, C.S., Raeymaekers, B.M., De Jonge, I., Lauweryns, J.M. and Koninckx, P.R. (1995) Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus, Papio anubis). Am. J. Obstet. Gynecol., 173, 125134.[ISI][Medline]
Groothuis, P.G., Koks, C.A., de Goeij, A.F., Dunselman, G.A., Arends, J.W. and Evers, J.L. (1998) Adhesion of human endometrium to the epithelial lining and extracellular matrix of amnion in vitro: an electron microscopic study. Hum. Reprod., 13, 22752281.[Abstract]
Groothuis, P.G., Koks, C.A., de Goeij, A.F., Dunselman, G.A., Arends, J.W. and Evers, J.L. (1999) Adhesion of human endometrial fragments to peritoneum in vitro. Fertil. Steril., 71, 11191124.[ISI][Medline]
Halme, J., Hammond, M.G., Hulka, J.F., Raj, S.G. and Talbert, L.M. (1984) Retrograde menstruation in healthy women and in patients with endometriosis. Obstet. Gynecol., 64, 151154.[Abstract]
Koks, C.A., Groothuis, P.G., Dunselman, G.A., de Goeij, A.F. and Evers, J.L. (1999) Adhesion of shed menstrual tissue in an in-vitro model using manion and peritoneum: a light and electron microscopic study. Hum. Reprod., 14, 816822.
Nisolle, M., Casanas-Roux, F. and Donnez, J. (2000) Early-stage endometriosis: adhesion and growth of human menstrual endometrium in nude mice. Fertil. Steril., 74, 306312.[ISI][Medline]
Noyes, R.W., Hertig, A.R. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 325.[ISI][Medline]
Sampson, J. (1927) Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am. J. Obstet. Gynecol., 14, 422469.
Toki, T., Shimizu, M., Takagi, Y., Ashida, T. and Konishi, I. (2002) CD10 is a marker for normal and neoplastic endometrial stromal cells. Int. J. Gynecol. Pathol., 21, 4147.[ISI][Medline]
Van der Linden, P.J., de Goeij, A.F., Dunselman, G.A., Erkens, H.W. and Evers, J.L. (1996) Endometrial cell adhesion in an in vitro model using intact amniotic membranes. Fertil. Steril., 5, 7680.
Wild, R.A., Zhang, R. and Medders, D. (1994) Whole endometrial fragments form characteristics of in vivo endometriosis in a mesothelial cell co-culture system: an in vitro model of the histogenesis of endometriosis. J. Soc. Gynecol. Invent., 1, 165168.
Witz, C.A., Montoya-Rodriguez, A.I. and Schenken, R.S. (1999) Whole peritoneal explants: a novel model of the early endometriosis lesion. Fertil. Steril., 71, 5660.[ISI][Medline]
Witz, C.A., Thomas, M.R., Montoya-Rodriguez, A.I., Nair, A.S., Centonze, V.E. and Schenken, R.S. (2001) Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium. Fertil. Steril., 75, 385390.[ISI][Medline]
Zhang, R., Wild, R.A. and Ojago, J.M. (1993) Effect of tumor necrosis factor- on adhesion of human endometrial stromal cells to peritoneal mesothelial cells: an in vitro system. Fertil. Steril., 59, 11961201.[ISI][Medline]
Submitted on February 1, 2002; resubmitted on May 14, 2002; accepted on June 12, 2002.