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Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model
FERTILITY AND STERILITY威 VOL. 72, NO. 3, SEPTEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model Foo-Hoe Loh, M.R.C.O.G.,* Ariff Bongso, Ph.D.,* Chui-Yee Fong, M.Sc.,* Dow-Rhoon Koh, Ph.D.,† Szu-Hee Lee, Ph.D.,‡ and Hui-Qin Zhao, B.Sc.* National University of Singapore, Singapore
Received January 25, 1999; revised and accepted April 20, 1999. Supported by a grant from the National Medical Research Council of Singapore. Presented at the VI World Congress on Endometriosis, Quebec City, Quebec, Canada, June 30 to July 4, 1998. Reprint requests: Foo-Hoe Loh, M.R.C.O.G., Department of Obstetrics and Gynaecology, National University Hospital, 5, Lower Kent Ridge Road, Singapore 119074 (FAX: 65-779-4753; E-mail: [email protected]). * Department of Obstetrics and Gynaecology, National University of Singapore. † Department of Physiology, National University of Singapore. ‡ Department of Pathology, National University of Singapore. 0015-0282/99/$20.00 PII S0015-0282(99)00292-7
Objective: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women. Design: Controlled study of peritoneal macrophage function. Setting: University hospital. Patient(s): Patients with a normal peritoneal cavity (n ⫽ 15) and with pelvic endometriosis (n ⫽ 20) undergoing laparoscopy. Intervention(s): Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls. Main Outcome Measure(s): Endometrial cellular proliferation measured by 3H-thymidine incorporation. Result(s): Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control). Conclusion(s): Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro. (Fertil Steril威 1999;72:533– 8. ©1999 by American Society for Reproductive Medicine.) Key Words: Peritoneal macrophages, endometriosis, endometrial proliferation, coculture
Retrograde menstruation is widely accepted as one of the causes of endometriosis (1). Although retrograde menstruation is an almost universal occurrence among menstruating women (2, 3), only certain susceptible women develop endometriosis. It has been suggested that a conducive peritoneal environment may be one of the factors that accounts for this differential susceptibility. Studies on the peritoneal environment have focused on both the peritoneal fluid and its cellular components. The effect of peritoneal fluid from women with endometriosis on proliferation of endometrial cells appeared to show conflicting results in three studies (4 – 6).
This may be due to methodologic differences among these studies; or perhaps the peritoneal fluid is the result of many processes within the peritoneal cavity and may not be specific enough to the disease process to demonstrate a clear difference. Macrophages are the predominant cell type in peritoneal fluid and could play an important role in the host response to refluxed menstrual material. It has been demonstrated that peritoneal macrophages from women with endometriosis release growth factors and cytokines, which may enhance endometrial cell growth (7–9). A study using murine macrophages showed that macrophage-conditioned medium 533
enhanced endometrial stromal cell proliferation in vitro (10). The effects of peritoneal macrophages from patients with endometriosis on the progression of endometrial implants have yet to be demonstrated in a more direct way. In this study, we tested the hypothesis that secretory products of peritoneal macrophages from women with endometriosis enhance cellular proliferation of endometrial epithelial and stromal cells, which may promote the progression of endometriosis. We report the use of an in vitro coculture model to demonstrate this effect by coculturing peritoneal macrophages from women with and without endometriosis with endometrial epithelial and stromal cells separately.
MATERIALS AND METHODS This study protocol was approved by our hospital’s ethics committee. All patients gave informed consent to participate in the study.
Patients The subjects in this study were women of reproductive age undergoing laparoscopy. Fifteen women had a normal peritoneal cavity on careful laparoscopic inspection. These included women undergoing laparoscopy for tubal ligation (n ⫽ 10) and normal women who had male factor as the only identified problem in the infertility of the couple or infertility secondary to ovulatory dysfunction undergoing diagnostic laparoscopy for tubal assessment (n ⫽ 5). Their mean (⫾ SD) age was 32.13 ⫾ 6.86 years, and the mean cycle day of the period during which laparoscopy was performed was 22.07 ⫾ 13.76 days. Twenty patients with endometriosis were included in the study. These included patients who had laparoscopy for infertility, pelvic pain, and endometriotic cyst. Using the American Fertility Society (AFS) revised classification of endometriosis (11), we identified 9 patients with minimal or mild disease (mean ⫾ SD AFS score, 4.22 ⫾ 3.93) and 11 patients with moderate to severe disease (mean AFS score, 64 ⫾ 41.55). The mean (⫾SD) age of the patients with endometriosis was 33.30 ⫾ 4.75 years, and the mean cycle day during which laparoscopy was performed was 17.68 ⫾ 5.88 days. The differences between the groups in the mean age and cycle day during which laparoscopy was performed were not statistically significant. Endometrial curettage specimens were obtained from the group of women undergoing laparoscopy who had normal endometrium as demonstrated on hysteroscopy. Carbon dioxide was the distending medium used during hysteroscopy in all patients. Endometrium was obtained during both the proliferative and secretory phases of the menstrual cycle (median cycle day 17; range, day 6 to day 24). The endometrial samples used in the two different coculture systems were not different with respect to the age of the patient (normal, 32.6 ⫾ 3.4 years; endometriosis, 34.2 ⫾ 2.9 years; 534
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P⬎.05) or the cycle day of the period (normal, day 13.5 ⫾ 5.0; endometriosis, day 15.7 ⫾ 4.2; P⬎.05).
Peritoneal Fluid Macrophage Collection and Culture Peritoneal fluid was obtained from patients undergoing laparoscopy as the first step in the procedure before the patient was placed in the Trendelenberg position, taking care to ensure that the collection (mainly from the pouch of Douglas and the uterovesical fold) was as complete as possible. The fluid was placed in a sterile, heparinized polypropylene container and transported on ice to be processed immediately. The fluid was centrifuged and the pellet was resuspended in RPMI 1640 medium (GIBCO, Grand Island, NY). The suspension was laid over 60% Percoll (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 3,000 rpm for 15 minutes at 4°C. The cells were then collected from the medium–Percoll interface. The macrophages were resuspended at a concentration of 106 per milliliter of RPMI 1640 medium, and 0.5 mL was dispensed into a tissue culture insert with a 3-m polycarbonate membrane (Nunclon, Roskilde, Denmark). After 1 hour of incubation at 37°C in 5% carbon dioxide, an adherent layer of macrophages was obtained on the tissue culture insert. The macrophages were further purified by gentle lavage three times with warm RPMI 1640 culture medium. The tissue culture insert was then placed in the culture well for coculture with endometrial epithelial or stromal cells. An aliquot of peritoneal fluid from each fresh specimen was cytospun onto glass slides and stained with May-Gru¨nwald Giemsa for morphologic examination. Cytospun macrophages were stained for nonspecific esterase activity using alpha-naphthyl acetate as substrate (Sigma Diagnostics, St. Louis, MO). Viability of the cells obtained on the tissue culture insert was determined by trypan blue exclusion.
Endometrial Cell Culture Endometrium was obtained from uterine curettage specimens of patients undergoing laparoscopy. All of the endometrium was noted to be normal on hysteroscopy and further confirmed on histology. The curettage specimens were placed in Hank’s balanced salt solution (HBSS) containing 1% antibiotic and antimycotic mixture (GIBCO) and transported on ice to the laboratory. The tissue was washed with several changes of fresh HBSS to remove mucus and debris and was gently minced into pieces of less than 1 mm in diameter. The endometrial tissue was then incubated with 0.25% collagenase Type 1A (Sigma) at 37°C in an atmosphere of 5% carbon dioxide in air. At the end of the incubation period, the endometrial stromal cells and glands were mechanically dispersed by repeated aspiration through the Pasteur pipette, and the undigested fragments were removed. The suspension of glands and stromal cells was centrifuged at 200 rpm for 5 minutes. Vol. 72, No. 3, September 1999
The supernatant, containing mainly stromal cells, was filtered through a 40-m nylon cell filter (Falcon, Franklin Lakes, NJ). The final purification of stromal cells was obtained by centrifugation at 2,400 rpm for 15 minutes over 60% Percoll to remove the erythrocytes.
FIGURE 1 Setup of the experiment: coculture with peritoneal macrophages and control.
Dissociation of the glands was achieved with 0.05% trypsin– ethylenediaminetetraacetic acid solution (GIBCO) and repeated aspiration through a Pasteur pipette. Digestion was stopped after the addition of RPMI 1640 medium supplemented with 10% fetal bovine serum. The endometrial glandular cells were separated by centrifugation and resuspension twice in medium. The epithelial and stromal cells were counted using a Neubauer hemocytometer, and the suspension concentration was adjusted to achieve 0.3 ⫻ 106 viable cells per milliliter in complete medium: RPMI 1640 with 10% heat-inactivated fetal bovine serum, 1% glutamine, and antibiotic-antimycotic mixture (GIBCO). The cells were then plated separately in duplicate in multiwell dishes (0.75 mL/well) (Nunclon). Samples of the cell cultures were checked for purity with the immunohistochemical stains cytokeratin (Clone MNF116; Dako, Glostrup, Denmark) and vimentin (Clone V9; Dako). Glandular cells stained positive with antibody to cytokeratins, and stromal cells were cytokeratin negative while staining positive for vimentin using the method described by Sharpe et al. (12). The purity of epithelial cell culture reached a mean of 90%, while that of stromal cells had a mean of 85%. Proliferation of endometrial epithelial and stromal cells was assessed after 24 and 72 hours of coculture with macrophages. We used the technique of 3H-thymidine (1 mCu/ mL; Amersham, Uppsala, Sweden) incorporation into the cells to reflect the rate of DNA synthesis. The radioactivity incorporated into the cells was measured with a scintillation counter (Wallac 1410, liquid scintillation counter; Pharmacia Biotech).
Experimental Design For each set of experiments, the endometrial epithelial and stromal cells were cocultured in duplicate with macrophages placed in cell-well inserts and were compared against separate sets of endometrial epithelial and stromal cells obtained from the same patient but cultured with empty inserts acting as an internal control (Fig. 1). The 3H-thymidine counts of macrophage-cocultured endometrial epithelial and stromal cells were expressed as a ratio of the same set of endometrial cultures with empty inserts. This design controlled for variation in the proliferative potentials of different endometrial samples used. The ratios obtained from the coculture systems of peritoneal macrophages obtained from patients with endometriosis were then compared with those cocultured with macrophages obtained from women without endometriosis. FERTILITY & STERILITY威
Loh. Peritoneal macrophages. Fertil Steril 1999.
Statistical Analysis The differences between the ratios of cellular replication of endometrial epithelial and stromal cells at 24 hours and 72 hours of coculture, respectively, with peritoneal macrophages obtained from women with and without endometriosis over their respective internal controls were tested for statistical significance by nonparametric analysis with the Mann-Whitney U test.
RESULTS Peritoneal Fluid The mean (⫾SD) volume of peritoneal fluid obtained was 9.8 ⫾ 9.18 mL. There was little difference in peritoneal fluid volume between patients with endometriosis and women with a normal peritoneal cavity (10.8 ⫾ 11.05 versus 8.47 ⫾ 5.95 mL; P⬎.05). The mean (⫾SD) peritoneal fluid volume of patients with moderate to severe endometriosis was 13.55 ⫾ 13.99 mL, versus 7.44 ⫾ 4.75 mL for those with minimal and mild disease. This difference was not statistically significant (P⫽.099). There was a difference between women who had fluid aspiration during the first 14 days of their menstrual cycle as compared with after the 14th day of the cycle (6.17 ⫾ 4.69 versus 11.38 ⫾ 10.13 mL). This difference approached statistical significance (P⫽.051).
Macrophages Macrophages harvested from the peritoneal fluid were judged to be ⬎95% pure as evaluated by their characteristic morphology after Giemsa staining and by staining for non535
FIGURE 2
FIGURE 3
Comparison of endometrial epithelial cellular proliferation in coculture systems using peritoneal macrophages from women with endometriosis and women with a normal peritoneal cavity: assessment at 24 and 72 hours of culture. Values are expressed as the mean ratio of proliferative activity of endometrial cells cocultured with peritoneal macrophages over controls, with 95% confidence interval (all P values ⬍.05).
Comparison of endometrial stromal cellular proliferation in coculture systems using peritoneal macrophages from women with endometriosis and women with a normal peritoneal cavity: assessment at 24 and 72 hours of culture. Values are expressed as the mean ratio of proliferative activity of endometrial cells cocultured with peritoneal macrophages over controls, with 95% confidence interval (all P values ⬍.05).
Loh. Peritoneal macrophages. Fertil Steril 1999. Loh. Peritoneal macrophages. Fertil Steril 1999.
specific esterase activity. Viability of the macrophages as evaluated by trypan blue uptake was consistently ⬎98%. The mean (⫾SD) total number of macrophages obtained from the study population (n ⫽ 35) was 8.99 ⫾ 7.37 ⫻ 106. There was little difference in the total macrophage number between women with endometriosis and normal women (9.08 versus 8.87 ⫻ 106/mL; P⬎.05). The mean (⫾SD) macrophage density in peritoneal fluid was 1.17 ⫾ 0.76 ⫻ 106/mL. There was little difference in macrophage density of peritoneal fluid from women with endometriosis as compared with normal women (1.05 versus 1.32 ⫻ 106/mL; P⬎.05).
Endometrial Proliferation When Cocultured With Macrophages Endometrial Epithelial Cells Cellular proliferation of endometrial epithelial cells when cocultured with macrophages from women with endometriosis was increased when compared with coculture systems using peritoneal macrophages from normal women after 24 hours of culture (1.56 ⫾ 0.72 versus 1.03 ⫾ 0.19 times control; P⫽.001) and after 72 hours of culture (1.55 ⫾ 0.76 versus 1.10 ⫾ 0.20 times control; P⫽.030) (Fig. 2). Within the group of normal patients, there was no difference between the proliferative activity of epithelial cells cocultured with macrophages from fertile patients undergo536
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ing ligation and those from infertile patients with a normal peritoneal cavity when assessed at 24 hours (1.07 versus 0.95 times control; P⬎.05) and at 72 hours (1.10 versus 1.09 times control; P⬎.05). Within the group of patients with endometriosis, there was no difference between the proliferative activity of epithelial cells cocultured with macrophages from patients with minimal to mild endometriosis and those from patients with moderate to severe endometriosis when assessed at 24 hours (1.72 versus 1.43 times control; P⬎.05) and at 72 hours (1.39 versus 1.68 times control; P⬎.05). Endometrial Stromal Cells Cellular proliferation of endometrial stromal cells when cocultured with macrophages from women with endometriosis was enhanced when compared with coculture systems using peritoneal macrophages from normal women after 24 hours of culture (1.65 ⫾ 0.70 versus 1.17 ⫾ 0.20 times control; P⫽.001) and after 72 hours of culture (1.65 ⫾ 0.55 versus 1.21 ⫾ 0.20 times control; P⫽.011) (Fig. 3). Within the group of normal patients, there was no difference between the proliferative activity of stromal cells cocultured with macrophages from fertile patients undergoing ligation and those from infertile patients with a normal peritoneal cavity when assessed at 24 hours (1.15 versus Vol. 72, No. 3, September 1999
1.20 times control; P⬎.05) and at 72 hours (1.25 versus 1.37 times control; P⬎.05).
trial epithelial and stromal cells significantly by between 50% and 60% above the controls.
Within the group of patients with endometriosis, there was no difference between the proliferative activity of stromal cells cocultured with macrophages from patients with minimal to mild endometriosis and those from patients with moderate to severe endometriosis when assessed at 24 hours (1.60 versus 1.70 times control; P⬎.05) and at 72 hours (1.46 versus 1.80 times control; P⬎.05).
There appeared to be little difference between macrophages obtained from patients with minimal or mild disease and those with moderate or severe disease. However, the AFS staging of endometriosis (11) scores the disease in large part on the amount of adhesions present as opposed to the activity and nature of the lesions. Hence, it is not surprising that severity of the disease is not reflected in the amount of macrophage activity on endometrial cellular proliferation. Most of the patients in the study population had a heterogeneous mix of lesions with red, vesicular, and the classic blue-black lesions. The small numbers involved in this study did not allow any meaningful substratification to analyze differential effects these lesions may have on macrophage activity and endometrial cellular proliferation.
DISCUSSION The macrophage plays an important role in the host defense of the peritoneal cavity. Refluxed menstrual material, containing viable endometrial cells, represents a challenge to this local defense system. Failure or aberration of the normal function of peritoneal macrophages has been implicated in the pathogenesis of endometriosis. Numerous studies have shown some aberration of macrophage function that may help to facilitate the establishment and progression of the implant. In patients with endometriosis, peritoneal macrophages have reduced capacity to destroy endometrial cells and ectopic endometrial cells have increased resistance to macrophage-mediated cytolysis (13). Peritoneal macrophages also have been found in increased numbers (14) and with increased activation (15, 16) and secrete many different forms of growth factors (7–9) in patients with endometriosis, which may contribute to the progression of endometriosis. Macrophage-conditioned culture media from patients with endometriosis demonstrated a mitogenic effect on endometrial carcinoma cell line ECC-1, which may be blocked by antibody to epidermal growth factor receptor (17). These findings show that peritoneal macrophages have the ability to promote endometrial cellular proliferation. However, there has not been a direct demonstration of this effect on endometrial cells, which are the presumed targets of such activity. The present study with the coculture model allowed study of the interaction between macrophages and the endometrium. The results provide evidence of the direct role of macrophages in patients with endometriosis on the pathogenesis of the disease in their ability to promote both endometrial epithelial and stromal cellular proliferation. Peritoneal macrophages from normal women did not significantly increase endometrial epithelial cellular proliferation above that found under control conditions with culture media alone. For stromal cells, coculture with macrophages from normal women raised cellular proliferation marginally by approximately 20% above controls. Coculture systems using macrophages from women with endometriosis were able to stimulate the proliferative activity of both endomeFERTILITY & STERILITY威
The difference between peritoneal macrophages in women with endometriosis and normal women in this study was qualitative rather than quantitative. We did not find any difference in peritoneal fluid volumes or total macrophage numbers and densities between the groups, although there was a trend toward increasing peritoneal fluid volumes with more severe disease. This may be a type 2 error, whereby a difference existed but was not detected by the analysis because of the small sample size and large SD. However, we speculate that this is a true observation, whereby the difference in macrophage function may be more fundamental than a mere reaction to the inflammatory environment in the peritoneal cavity with endometriosis. Indeed, this effect of macrophages on endometrial cells may not be limited to a local peritoneal phenomenon. The results of the study by Braun et al. (18), which looked at monocyte influence on endometrial cell proliferation, suggested that there may be a systemic aberration of monocytemacrophage function. The fact that physical contact was not required for the effects of macrophages on endometrial cellular proliferation suggests that soluble factors are responsible for the observed effects. The choice of material is important if the correct conclusions are to be drawn from this study. In this study, we assumed that the endometrial cell is equivalent to the desquamated menstrual material deposited during reflux of menstruation. Matthews et al. (19) found that eutopic and ectopic endometrial cells were morphologically and biochemically similar in culture. However, other studies have suggested that endometriotic lesions may be unique and different from the uterine endometrium, both in their biochemical expression (20) and their physical invasiveness (21). There may also be concern that endometrial cells from patients with endometriosis may have inherently greater proliferative potential than endometrial cells from patients without endometriosis (22), but this has not been a consistent 537
finding (23). We attempted to address this concern in the design of the study by expressing the results as a ratio of the endometrial proliferation cocultured with macrophages over the cellular proliferation of endometrium from the same patient cultured with an empty tissue culture well insert as control. A value ⬎1 would thus express any additional mitogenic effect of the macrophages for the endometrial cells over the effect of culture media alone. This design should remove differences in proliferative potential of individual endometrial tissues in different phases of the menstrual cycle. When the data were grouped according to the phase of the menstrual cycle from which the endometrium was derived, there was no difference in the value of the ratios obtained. Most experiments in our study used autologous endometrium. The study by Braun et al. (18) suggested that there may be a difference in the endometrial responsiveness when cultured in combination with heterologous monocytes. Further studies are needed to elucidate the different contributions to the disease process of macrophage function and inherent endometrial responsiveness to signals within the peritoneal cavity. The coculture model provides an ideal vehicle to test this issue. Before cellular proliferation, it is essential that the refluxed cells are able to adhere to the peritoneum and establish a viable blood supply. These processes are dependent on the expression of cell adhesion molecules and the induction of angiogenesis by substances such as vascular endothelial growth factor. Macrophages may play a role in these two key processes as well. Macrophages have been found to express vascular endothelial growth factor (24 –26) and cell adhesion molecules (27, 28). Macrophages represent but one of the many players in the drama of endometriosis played out in the peritoneal cavity. The pathogenesis of endometriosis remains an enigma, but as more and more bits of information are revealed, the peritoneal macrophage may prove to be a key player in the final analysis.
5. 6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16. 17.
18. 19. 20.
21. 22. 23. 24. 25.
Acknowledgment: The authors thank Associate Professor Samuel S.W. Tay for his help with the immunohistochemical analysis of the endometrial cell cultures.
26.
References 1. Sampson JA. The development of the implantation theory for the origin of peritoneal endometriosis. Am J Obstet Gynecol 1940;40:549 –57. 2. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64:151– 4. 3. Kruitwagen RFPM, Poels LG, Willemsen WNP, Jap PH, Thomas CM, Rolland R. Endometrial epithelial cells in peritoneal fluid during early follicular phase. Fertil Steril 1991;55:297–303. 4. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis
538
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27.
28.
patients on endometrial stromal cell proliferation in vitro. Obstet Gynecol 1990;76:792–7. Koutsilieris M, Allaire-Michaud L, Fortier M, Lemay A. Mitogen(s) for endometrial like cells can be detected in human peritoneal fluid. Fertil Steril 1991;56:888 –93. Overton CE, Fernandez-Shaw S, Hicks B, Barlow DH, Starkey P. In vitro culture of endometrial stromal and gland cells as a model for endometriosis: the effect of peritoneal fluid on proliferation. Fertil Steril 1997;67:51– 6. Halme J, White C, Kauma S, Estes J, Haskill S. Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. J Clin Endocrinol Metab 1988;66:1044 –9. Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 1996;65:925–30. Leslie CC, Musson RA, Henson PM. Production of growth factor activity for fibroblasts by human monocyte-derived macrophages. J Leukoc Biol 1984;36:143–59. Olive DL, Montoya I, Riehl RM, Schenken RS. Macrophage-conditioned media enhance endometrial stromal cell proliferation in vitro. Am J Obstet Gynecol 1991;164:953– 8. The American Fertility Society. Revised American Fertility Society classification of endometriosis: 1985. Fertil Steril 1985;43:351–2. Sharpe KL, Zimmer RL, Khan RS, Penney LL. Proliferative and morphogenic changes induced by the co-culture of rat uterine and peritoneal cells: a cell culture model for endometriosis. Fertil Steril 1992;58:1220 –9. Braun DP, Gebel H, Rana N, Dmowski WP. Cytolysis of eutopic and ectopic endometrial cells by peripheral blood monocytes and peritoneal macrophages in women with endometriosis. Fertil Steril 1998;69:1103– 8. Olive DL, Weinberg JB, Haney AF. Peritoneal macrophages and infertility: the association between cell number and pelvic pathology. Fertil Steril 1985;44:772–7. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7. Samejima T, Masuzaki H, Ishimaru T, Yamabe T. Activity of peritoneal macrophages in endometriosis. Asia-Oceania J Obstet Gynaecol 1989;15:175– 81. Zhang RJ, Wild RA, Medders D, Gunupudi SR. Effects of peritoneal macrophages from patients with endometriosis on the proliferation of endometrial carcinoma cell line ECC-1. Am J Obstet Gynecol 1991; 165:1842– 6. Braun DP, Muriana A, Gebel H, Rotman C, Rana N, Dmowski WP. Monocyte mediated enhancement of endometrial cell proliferation in women with endometriosis. Fertil Steril 1994;61:78 – 84. Matthews CJ, Redfern CPF, Hirst BH, Thomas EJ. Characterization of human purified epithelial and stromal cells from endometrium and endometriosis in tissue culture. Fertil Steril 1992;57:990 –7. Sharpe KL, Zimmer RL, Griffin WT, Penny LL. Polypeptides synthesized and released by human endometriosis differ from those of the uterine endometrium in cell and tissue explant culture. Fertil Steril 1993;60:839 –51. Gaetje R, Kotzian S, Herrmann G, Baumann R, Starzinski-Powitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995;346:1463– 4. Wingfield M, Macpherson A, Healy DL, Rogers PAW. Cell proliferation is increased in the endometrium of women with endometriosis. Fertil Steril 1995;64:340 – 6. Jurgensen A, Mettler L, Volkov NI, Parwaresch R. Proliferative activity of the endometrium throughout the menstrual cycle in infertile women with and without endometriosis. Fertil Steril 1996;66:369 –75. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:410 –22. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 1998;153:587–98. Kauma S, Clark MR, White C, Halme J. Production of fibronectin by peritoneal macrophages and concentration of fibronectin in peritoneal fluid from patients with or without endometriosis. Obstet Gynecol 1988;72:13– 8. Dou Q, Williams RS, Chegini N. Inhibition of transforming growth factor-beta 1 alters the growth, anchor dependent cell aggregation and integrin mRNA expression in human pro-monocytes: implications for endometriosis and peritoneal adhesion formation. Mol Hum Reprod 1997;3:383–91.
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Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model Foo-Hoe Loh, M.R.C.O.G.,* Ariff Bongso, Ph.D.,* Chui-Yee Fong, M.Sc.,* Dow-Rhoon Koh, Ph.D.,† Szu-Hee Lee, Ph.D.,‡ and Hui-Qin Zhao, B.Sc.* National University of Singapore, Singapore
Received January 25, 1999; revised and accepted April 20, 1999. Supported by a grant from the National Medical Research Council of Singapore. Presented at the VI World Congress on Endometriosis, Quebec City, Quebec, Canada, June 30 to July 4, 1998. Reprint requests: Foo-Hoe Loh, M.R.C.O.G., Department of Obstetrics and Gynaecology, National University Hospital, 5, Lower Kent Ridge Road, Singapore 119074 (FAX: 65-779-4753; E-mail: [email protected]). * Department of Obstetrics and Gynaecology, National University of Singapore. † Department of Physiology, National University of Singapore. ‡ Department of Pathology, National University of Singapore. 0015-0282/99/$20.00 PII S0015-0282(99)00292-7
Objective: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women. Design: Controlled study of peritoneal macrophage function. Setting: University hospital. Patient(s): Patients with a normal peritoneal cavity (n ⫽ 15) and with pelvic endometriosis (n ⫽ 20) undergoing laparoscopy. Intervention(s): Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls. Main Outcome Measure(s): Endometrial cellular proliferation measured by 3H-thymidine incorporation. Result(s): Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control). Conclusion(s): Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro. (Fertil Steril威 1999;72:533– 8. ©1999 by American Society for Reproductive Medicine.) Key Words: Peritoneal macrophages, endometriosis, endometrial proliferation, coculture
Retrograde menstruation is widely accepted as one of the causes of endometriosis (1). Although retrograde menstruation is an almost universal occurrence among menstruating women (2, 3), only certain susceptible women develop endometriosis. It has been suggested that a conducive peritoneal environment may be one of the factors that accounts for this differential susceptibility. Studies on the peritoneal environment have focused on both the peritoneal fluid and its cellular components. The effect of peritoneal fluid from women with endometriosis on proliferation of endometrial cells appeared to show conflicting results in three studies (4 – 6).
This may be due to methodologic differences among these studies; or perhaps the peritoneal fluid is the result of many processes within the peritoneal cavity and may not be specific enough to the disease process to demonstrate a clear difference. Macrophages are the predominant cell type in peritoneal fluid and could play an important role in the host response to refluxed menstrual material. It has been demonstrated that peritoneal macrophages from women with endometriosis release growth factors and cytokines, which may enhance endometrial cell growth (7–9). A study using murine macrophages showed that macrophage-conditioned medium 533
enhanced endometrial stromal cell proliferation in vitro (10). The effects of peritoneal macrophages from patients with endometriosis on the progression of endometrial implants have yet to be demonstrated in a more direct way. In this study, we tested the hypothesis that secretory products of peritoneal macrophages from women with endometriosis enhance cellular proliferation of endometrial epithelial and stromal cells, which may promote the progression of endometriosis. We report the use of an in vitro coculture model to demonstrate this effect by coculturing peritoneal macrophages from women with and without endometriosis with endometrial epithelial and stromal cells separately.
MATERIALS AND METHODS This study protocol was approved by our hospital’s ethics committee. All patients gave informed consent to participate in the study.
Patients The subjects in this study were women of reproductive age undergoing laparoscopy. Fifteen women had a normal peritoneal cavity on careful laparoscopic inspection. These included women undergoing laparoscopy for tubal ligation (n ⫽ 10) and normal women who had male factor as the only identified problem in the infertility of the couple or infertility secondary to ovulatory dysfunction undergoing diagnostic laparoscopy for tubal assessment (n ⫽ 5). Their mean (⫾ SD) age was 32.13 ⫾ 6.86 years, and the mean cycle day of the period during which laparoscopy was performed was 22.07 ⫾ 13.76 days. Twenty patients with endometriosis were included in the study. These included patients who had laparoscopy for infertility, pelvic pain, and endometriotic cyst. Using the American Fertility Society (AFS) revised classification of endometriosis (11), we identified 9 patients with minimal or mild disease (mean ⫾ SD AFS score, 4.22 ⫾ 3.93) and 11 patients with moderate to severe disease (mean AFS score, 64 ⫾ 41.55). The mean (⫾SD) age of the patients with endometriosis was 33.30 ⫾ 4.75 years, and the mean cycle day during which laparoscopy was performed was 17.68 ⫾ 5.88 days. The differences between the groups in the mean age and cycle day during which laparoscopy was performed were not statistically significant. Endometrial curettage specimens were obtained from the group of women undergoing laparoscopy who had normal endometrium as demonstrated on hysteroscopy. Carbon dioxide was the distending medium used during hysteroscopy in all patients. Endometrium was obtained during both the proliferative and secretory phases of the menstrual cycle (median cycle day 17; range, day 6 to day 24). The endometrial samples used in the two different coculture systems were not different with respect to the age of the patient (normal, 32.6 ⫾ 3.4 years; endometriosis, 34.2 ⫾ 2.9 years; 534
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P⬎.05) or the cycle day of the period (normal, day 13.5 ⫾ 5.0; endometriosis, day 15.7 ⫾ 4.2; P⬎.05).
Peritoneal Fluid Macrophage Collection and Culture Peritoneal fluid was obtained from patients undergoing laparoscopy as the first step in the procedure before the patient was placed in the Trendelenberg position, taking care to ensure that the collection (mainly from the pouch of Douglas and the uterovesical fold) was as complete as possible. The fluid was placed in a sterile, heparinized polypropylene container and transported on ice to be processed immediately. The fluid was centrifuged and the pellet was resuspended in RPMI 1640 medium (GIBCO, Grand Island, NY). The suspension was laid over 60% Percoll (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 3,000 rpm for 15 minutes at 4°C. The cells were then collected from the medium–Percoll interface. The macrophages were resuspended at a concentration of 106 per milliliter of RPMI 1640 medium, and 0.5 mL was dispensed into a tissue culture insert with a 3-m polycarbonate membrane (Nunclon, Roskilde, Denmark). After 1 hour of incubation at 37°C in 5% carbon dioxide, an adherent layer of macrophages was obtained on the tissue culture insert. The macrophages were further purified by gentle lavage three times with warm RPMI 1640 culture medium. The tissue culture insert was then placed in the culture well for coculture with endometrial epithelial or stromal cells. An aliquot of peritoneal fluid from each fresh specimen was cytospun onto glass slides and stained with May-Gru¨nwald Giemsa for morphologic examination. Cytospun macrophages were stained for nonspecific esterase activity using alpha-naphthyl acetate as substrate (Sigma Diagnostics, St. Louis, MO). Viability of the cells obtained on the tissue culture insert was determined by trypan blue exclusion.
Endometrial Cell Culture Endometrium was obtained from uterine curettage specimens of patients undergoing laparoscopy. All of the endometrium was noted to be normal on hysteroscopy and further confirmed on histology. The curettage specimens were placed in Hank’s balanced salt solution (HBSS) containing 1% antibiotic and antimycotic mixture (GIBCO) and transported on ice to the laboratory. The tissue was washed with several changes of fresh HBSS to remove mucus and debris and was gently minced into pieces of less than 1 mm in diameter. The endometrial tissue was then incubated with 0.25% collagenase Type 1A (Sigma) at 37°C in an atmosphere of 5% carbon dioxide in air. At the end of the incubation period, the endometrial stromal cells and glands were mechanically dispersed by repeated aspiration through the Pasteur pipette, and the undigested fragments were removed. The suspension of glands and stromal cells was centrifuged at 200 rpm for 5 minutes. Vol. 72, No. 3, September 1999
The supernatant, containing mainly stromal cells, was filtered through a 40-m nylon cell filter (Falcon, Franklin Lakes, NJ). The final purification of stromal cells was obtained by centrifugation at 2,400 rpm for 15 minutes over 60% Percoll to remove the erythrocytes.
FIGURE 1 Setup of the experiment: coculture with peritoneal macrophages and control.
Dissociation of the glands was achieved with 0.05% trypsin– ethylenediaminetetraacetic acid solution (GIBCO) and repeated aspiration through a Pasteur pipette. Digestion was stopped after the addition of RPMI 1640 medium supplemented with 10% fetal bovine serum. The endometrial glandular cells were separated by centrifugation and resuspension twice in medium. The epithelial and stromal cells were counted using a Neubauer hemocytometer, and the suspension concentration was adjusted to achieve 0.3 ⫻ 106 viable cells per milliliter in complete medium: RPMI 1640 with 10% heat-inactivated fetal bovine serum, 1% glutamine, and antibiotic-antimycotic mixture (GIBCO). The cells were then plated separately in duplicate in multiwell dishes (0.75 mL/well) (Nunclon). Samples of the cell cultures were checked for purity with the immunohistochemical stains cytokeratin (Clone MNF116; Dako, Glostrup, Denmark) and vimentin (Clone V9; Dako). Glandular cells stained positive with antibody to cytokeratins, and stromal cells were cytokeratin negative while staining positive for vimentin using the method described by Sharpe et al. (12). The purity of epithelial cell culture reached a mean of 90%, while that of stromal cells had a mean of 85%. Proliferation of endometrial epithelial and stromal cells was assessed after 24 and 72 hours of coculture with macrophages. We used the technique of 3H-thymidine (1 mCu/ mL; Amersham, Uppsala, Sweden) incorporation into the cells to reflect the rate of DNA synthesis. The radioactivity incorporated into the cells was measured with a scintillation counter (Wallac 1410, liquid scintillation counter; Pharmacia Biotech).
Experimental Design For each set of experiments, the endometrial epithelial and stromal cells were cocultured in duplicate with macrophages placed in cell-well inserts and were compared against separate sets of endometrial epithelial and stromal cells obtained from the same patient but cultured with empty inserts acting as an internal control (Fig. 1). The 3H-thymidine counts of macrophage-cocultured endometrial epithelial and stromal cells were expressed as a ratio of the same set of endometrial cultures with empty inserts. This design controlled for variation in the proliferative potentials of different endometrial samples used. The ratios obtained from the coculture systems of peritoneal macrophages obtained from patients with endometriosis were then compared with those cocultured with macrophages obtained from women without endometriosis. FERTILITY & STERILITY威
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Statistical Analysis The differences between the ratios of cellular replication of endometrial epithelial and stromal cells at 24 hours and 72 hours of coculture, respectively, with peritoneal macrophages obtained from women with and without endometriosis over their respective internal controls were tested for statistical significance by nonparametric analysis with the Mann-Whitney U test.
RESULTS Peritoneal Fluid The mean (⫾SD) volume of peritoneal fluid obtained was 9.8 ⫾ 9.18 mL. There was little difference in peritoneal fluid volume between patients with endometriosis and women with a normal peritoneal cavity (10.8 ⫾ 11.05 versus 8.47 ⫾ 5.95 mL; P⬎.05). The mean (⫾SD) peritoneal fluid volume of patients with moderate to severe endometriosis was 13.55 ⫾ 13.99 mL, versus 7.44 ⫾ 4.75 mL for those with minimal and mild disease. This difference was not statistically significant (P⫽.099). There was a difference between women who had fluid aspiration during the first 14 days of their menstrual cycle as compared with after the 14th day of the cycle (6.17 ⫾ 4.69 versus 11.38 ⫾ 10.13 mL). This difference approached statistical significance (P⫽.051).
Macrophages Macrophages harvested from the peritoneal fluid were judged to be ⬎95% pure as evaluated by their characteristic morphology after Giemsa staining and by staining for non535
FIGURE 2
FIGURE 3
Comparison of endometrial epithelial cellular proliferation in coculture systems using peritoneal macrophages from women with endometriosis and women with a normal peritoneal cavity: assessment at 24 and 72 hours of culture. Values are expressed as the mean ratio of proliferative activity of endometrial cells cocultured with peritoneal macrophages over controls, with 95% confidence interval (all P values ⬍.05).
Comparison of endometrial stromal cellular proliferation in coculture systems using peritoneal macrophages from women with endometriosis and women with a normal peritoneal cavity: assessment at 24 and 72 hours of culture. Values are expressed as the mean ratio of proliferative activity of endometrial cells cocultured with peritoneal macrophages over controls, with 95% confidence interval (all P values ⬍.05).
Loh. Peritoneal macrophages. Fertil Steril 1999. Loh. Peritoneal macrophages. Fertil Steril 1999.
specific esterase activity. Viability of the macrophages as evaluated by trypan blue uptake was consistently ⬎98%. The mean (⫾SD) total number of macrophages obtained from the study population (n ⫽ 35) was 8.99 ⫾ 7.37 ⫻ 106. There was little difference in the total macrophage number between women with endometriosis and normal women (9.08 versus 8.87 ⫻ 106/mL; P⬎.05). The mean (⫾SD) macrophage density in peritoneal fluid was 1.17 ⫾ 0.76 ⫻ 106/mL. There was little difference in macrophage density of peritoneal fluid from women with endometriosis as compared with normal women (1.05 versus 1.32 ⫻ 106/mL; P⬎.05).
Endometrial Proliferation When Cocultured With Macrophages Endometrial Epithelial Cells Cellular proliferation of endometrial epithelial cells when cocultured with macrophages from women with endometriosis was increased when compared with coculture systems using peritoneal macrophages from normal women after 24 hours of culture (1.56 ⫾ 0.72 versus 1.03 ⫾ 0.19 times control; P⫽.001) and after 72 hours of culture (1.55 ⫾ 0.76 versus 1.10 ⫾ 0.20 times control; P⫽.030) (Fig. 2). Within the group of normal patients, there was no difference between the proliferative activity of epithelial cells cocultured with macrophages from fertile patients undergo536
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ing ligation and those from infertile patients with a normal peritoneal cavity when assessed at 24 hours (1.07 versus 0.95 times control; P⬎.05) and at 72 hours (1.10 versus 1.09 times control; P⬎.05). Within the group of patients with endometriosis, there was no difference between the proliferative activity of epithelial cells cocultured with macrophages from patients with minimal to mild endometriosis and those from patients with moderate to severe endometriosis when assessed at 24 hours (1.72 versus 1.43 times control; P⬎.05) and at 72 hours (1.39 versus 1.68 times control; P⬎.05). Endometrial Stromal Cells Cellular proliferation of endometrial stromal cells when cocultured with macrophages from women with endometriosis was enhanced when compared with coculture systems using peritoneal macrophages from normal women after 24 hours of culture (1.65 ⫾ 0.70 versus 1.17 ⫾ 0.20 times control; P⫽.001) and after 72 hours of culture (1.65 ⫾ 0.55 versus 1.21 ⫾ 0.20 times control; P⫽.011) (Fig. 3). Within the group of normal patients, there was no difference between the proliferative activity of stromal cells cocultured with macrophages from fertile patients undergoing ligation and those from infertile patients with a normal peritoneal cavity when assessed at 24 hours (1.15 versus Vol. 72, No. 3, September 1999
1.20 times control; P⬎.05) and at 72 hours (1.25 versus 1.37 times control; P⬎.05).
trial epithelial and stromal cells significantly by between 50% and 60% above the controls.
Within the group of patients with endometriosis, there was no difference between the proliferative activity of stromal cells cocultured with macrophages from patients with minimal to mild endometriosis and those from patients with moderate to severe endometriosis when assessed at 24 hours (1.60 versus 1.70 times control; P⬎.05) and at 72 hours (1.46 versus 1.80 times control; P⬎.05).
There appeared to be little difference between macrophages obtained from patients with minimal or mild disease and those with moderate or severe disease. However, the AFS staging of endometriosis (11) scores the disease in large part on the amount of adhesions present as opposed to the activity and nature of the lesions. Hence, it is not surprising that severity of the disease is not reflected in the amount of macrophage activity on endometrial cellular proliferation. Most of the patients in the study population had a heterogeneous mix of lesions with red, vesicular, and the classic blue-black lesions. The small numbers involved in this study did not allow any meaningful substratification to analyze differential effects these lesions may have on macrophage activity and endometrial cellular proliferation.
DISCUSSION The macrophage plays an important role in the host defense of the peritoneal cavity. Refluxed menstrual material, containing viable endometrial cells, represents a challenge to this local defense system. Failure or aberration of the normal function of peritoneal macrophages has been implicated in the pathogenesis of endometriosis. Numerous studies have shown some aberration of macrophage function that may help to facilitate the establishment and progression of the implant. In patients with endometriosis, peritoneal macrophages have reduced capacity to destroy endometrial cells and ectopic endometrial cells have increased resistance to macrophage-mediated cytolysis (13). Peritoneal macrophages also have been found in increased numbers (14) and with increased activation (15, 16) and secrete many different forms of growth factors (7–9) in patients with endometriosis, which may contribute to the progression of endometriosis. Macrophage-conditioned culture media from patients with endometriosis demonstrated a mitogenic effect on endometrial carcinoma cell line ECC-1, which may be blocked by antibody to epidermal growth factor receptor (17). These findings show that peritoneal macrophages have the ability to promote endometrial cellular proliferation. However, there has not been a direct demonstration of this effect on endometrial cells, which are the presumed targets of such activity. The present study with the coculture model allowed study of the interaction between macrophages and the endometrium. The results provide evidence of the direct role of macrophages in patients with endometriosis on the pathogenesis of the disease in their ability to promote both endometrial epithelial and stromal cellular proliferation. Peritoneal macrophages from normal women did not significantly increase endometrial epithelial cellular proliferation above that found under control conditions with culture media alone. For stromal cells, coculture with macrophages from normal women raised cellular proliferation marginally by approximately 20% above controls. Coculture systems using macrophages from women with endometriosis were able to stimulate the proliferative activity of both endomeFERTILITY & STERILITY威
The difference between peritoneal macrophages in women with endometriosis and normal women in this study was qualitative rather than quantitative. We did not find any difference in peritoneal fluid volumes or total macrophage numbers and densities between the groups, although there was a trend toward increasing peritoneal fluid volumes with more severe disease. This may be a type 2 error, whereby a difference existed but was not detected by the analysis because of the small sample size and large SD. However, we speculate that this is a true observation, whereby the difference in macrophage function may be more fundamental than a mere reaction to the inflammatory environment in the peritoneal cavity with endometriosis. Indeed, this effect of macrophages on endometrial cells may not be limited to a local peritoneal phenomenon. The results of the study by Braun et al. (18), which looked at monocyte influence on endometrial cell proliferation, suggested that there may be a systemic aberration of monocytemacrophage function. The fact that physical contact was not required for the effects of macrophages on endometrial cellular proliferation suggests that soluble factors are responsible for the observed effects. The choice of material is important if the correct conclusions are to be drawn from this study. In this study, we assumed that the endometrial cell is equivalent to the desquamated menstrual material deposited during reflux of menstruation. Matthews et al. (19) found that eutopic and ectopic endometrial cells were morphologically and biochemically similar in culture. However, other studies have suggested that endometriotic lesions may be unique and different from the uterine endometrium, both in their biochemical expression (20) and their physical invasiveness (21). There may also be concern that endometrial cells from patients with endometriosis may have inherently greater proliferative potential than endometrial cells from patients without endometriosis (22), but this has not been a consistent 537
finding (23). We attempted to address this concern in the design of the study by expressing the results as a ratio of the endometrial proliferation cocultured with macrophages over the cellular proliferation of endometrium from the same patient cultured with an empty tissue culture well insert as control. A value ⬎1 would thus express any additional mitogenic effect of the macrophages for the endometrial cells over the effect of culture media alone. This design should remove differences in proliferative potential of individual endometrial tissues in different phases of the menstrual cycle. When the data were grouped according to the phase of the menstrual cycle from which the endometrium was derived, there was no difference in the value of the ratios obtained. Most experiments in our study used autologous endometrium. The study by Braun et al. (18) suggested that there may be a difference in the endometrial responsiveness when cultured in combination with heterologous monocytes. Further studies are needed to elucidate the different contributions to the disease process of macrophage function and inherent endometrial responsiveness to signals within the peritoneal cavity. The coculture model provides an ideal vehicle to test this issue. Before cellular proliferation, it is essential that the refluxed cells are able to adhere to the peritoneum and establish a viable blood supply. These processes are dependent on the expression of cell adhesion molecules and the induction of angiogenesis by substances such as vascular endothelial growth factor. Macrophages may play a role in these two key processes as well. Macrophages have been found to express vascular endothelial growth factor (24 –26) and cell adhesion molecules (27, 28). Macrophages represent but one of the many players in the drama of endometriosis played out in the peritoneal cavity. The pathogenesis of endometriosis remains an enigma, but as more and more bits of information are revealed, the peritoneal macrophage may prove to be a key player in the final analysis.
5. 6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16. 17.
18. 19. 20.
21. 22. 23. 24. 25.
Acknowledgment: The authors thank Associate Professor Samuel S.W. Tay for his help with the immunohistochemical analysis of the endometrial cell cultures.
26.
References 1. Sampson JA. The development of the implantation theory for the origin of peritoneal endometriosis. Am J Obstet Gynecol 1940;40:549 –57. 2. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64:151– 4. 3. Kruitwagen RFPM, Poels LG, Willemsen WNP, Jap PH, Thomas CM, Rolland R. Endometrial epithelial cells in peritoneal fluid during early follicular phase. Fertil Steril 1991;55:297–303. 4. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis
538
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27.
28.
patients on endometrial stromal cell proliferation in vitro. Obstet Gynecol 1990;76:792–7. Koutsilieris M, Allaire-Michaud L, Fortier M, Lemay A. Mitogen(s) for endometrial like cells can be detected in human peritoneal fluid. Fertil Steril 1991;56:888 –93. Overton CE, Fernandez-Shaw S, Hicks B, Barlow DH, Starkey P. In vitro culture of endometrial stromal and gland cells as a model for endometriosis: the effect of peritoneal fluid on proliferation. Fertil Steril 1997;67:51– 6. Halme J, White C, Kauma S, Estes J, Haskill S. Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. J Clin Endocrinol Metab 1988;66:1044 –9. Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 1996;65:925–30. Leslie CC, Musson RA, Henson PM. Production of growth factor activity for fibroblasts by human monocyte-derived macrophages. J Leukoc Biol 1984;36:143–59. Olive DL, Montoya I, Riehl RM, Schenken RS. Macrophage-conditioned media enhance endometrial stromal cell proliferation in vitro. Am J Obstet Gynecol 1991;164:953– 8. The American Fertility Society. Revised American Fertility Society classification of endometriosis: 1985. Fertil Steril 1985;43:351–2. Sharpe KL, Zimmer RL, Khan RS, Penney LL. Proliferative and morphogenic changes induced by the co-culture of rat uterine and peritoneal cells: a cell culture model for endometriosis. Fertil Steril 1992;58:1220 –9. Braun DP, Gebel H, Rana N, Dmowski WP. Cytolysis of eutopic and ectopic endometrial cells by peripheral blood monocytes and peritoneal macrophages in women with endometriosis. Fertil Steril 1998;69:1103– 8. Olive DL, Weinberg JB, Haney AF. Peritoneal macrophages and infertility: the association between cell number and pelvic pathology. Fertil Steril 1985;44:772–7. Halme J, Becker S, Hammond MG, Raj MHG, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983;145:333–7. Samejima T, Masuzaki H, Ishimaru T, Yamabe T. Activity of peritoneal macrophages in endometriosis. Asia-Oceania J Obstet Gynaecol 1989;15:175– 81. Zhang RJ, Wild RA, Medders D, Gunupudi SR. Effects of peritoneal macrophages from patients with endometriosis on the proliferation of endometrial carcinoma cell line ECC-1. Am J Obstet Gynecol 1991; 165:1842– 6. Braun DP, Muriana A, Gebel H, Rotman C, Rana N, Dmowski WP. Monocyte mediated enhancement of endometrial cell proliferation in women with endometriosis. Fertil Steril 1994;61:78 – 84. Matthews CJ, Redfern CPF, Hirst BH, Thomas EJ. Characterization of human purified epithelial and stromal cells from endometrium and endometriosis in tissue culture. Fertil Steril 1992;57:990 –7. Sharpe KL, Zimmer RL, Griffin WT, Penny LL. Polypeptides synthesized and released by human endometriosis differ from those of the uterine endometrium in cell and tissue explant culture. Fertil Steril 1993;60:839 –51. Gaetje R, Kotzian S, Herrmann G, Baumann R, Starzinski-Powitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995;346:1463– 4. Wingfield M, Macpherson A, Healy DL, Rogers PAW. Cell proliferation is increased in the endometrium of women with endometriosis. Fertil Steril 1995;64:340 – 6. Jurgensen A, Mettler L, Volkov NI, Parwaresch R. Proliferative activity of the endometrium throughout the menstrual cycle in infertile women with and without endometriosis. Fertil Steril 1996;66:369 –75. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:410 –22. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482–9. Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 1998;153:587–98. Kauma S, Clark MR, White C, Halme J. Production of fibronectin by peritoneal macrophages and concentration of fibronectin in peritoneal fluid from patients with or without endometriosis. Obstet Gynecol 1988;72:13– 8. Dou Q, Williams RS, Chegini N. Inhibition of transforming growth factor-beta 1 alters the growth, anchor dependent cell aggregation and integrin mRNA expression in human pro-monocytes: implications for endometriosis and peritoneal adhesion formation. Mol Hum Reprod 1997;3:383–91.
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