ENDOMETRIOSIS. a Leuven University Fertility Centre, Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Leuven,

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1 ENDOMETRIOSIS Increased peritoneal and endometrial gene expression of biologically relevant cytokines and growth factors during the menstrual phase in women with endometriosis Cleophas M. Kyama, M.Sc., a,b Lutgart Overbergh, Ph.D., c Sophie Debrock, Ph.D., a Dirk Valckx, c Sarah Vander Perre, a Christel Meuleman, M.D., a Attila Mihalyi, Ph.D., a Jason M. Mwenda, Ph.D., b Chantal Mathieu, M.D., Ph.D., c and Thomas M. D Hooghe, M.D., Ph.D. a,b a Leuven University Fertility Centre, Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Leuven, Belgium; b Division of Reproductive Biology, Institute of Primate Research, Karen, Nairobi, Kenya; and c Laboratory of Experimental Medicine and Endocrinology, University Hospital Gasthuisberg, Leuven, Belgium Objective: To examine differential messenger RNA (mrna) expression of relevant cytokines, metalloproteases, growth and adhesion factors in endometrium and peritoneum from women with endometriosis when compared with women without the disease during menstrual and luteal phases of the cycle. Design: Patients with endometriosis were compared with control patients. Setting: University hospital. Patient(s): A total of 35 patients (20 patients during the luteal phase and 15 patients during the menstrual phase) were selected for this study on the basis of cycle phase and presence or absence of endometriosis. Intervention(s): In this study, endometriosis was laparoscopically and histologically confirmed in 24 women with endometriosis of revised American Society for Reproductive Medicine (ASRM) stage I II (n 12) and revised ASRM stage III IV (n 12), and the presence of a normal pelvis was documented by laparoscopy in 11 control patients. The macroscopically normal peritoneum tissues were collected from lateral wall left or right, near the colon ascendens or descendens. Main Outcome Measure(s): The expression levels were determined as ratios between the target molecules and -actin as housekeeping gene. Result(s): In women with endometriosis, peritoneal mrna levels of matrix metalloproteinase (MMP) 3, transforming growth factor-, interleukin (IL)-6, and intercellular adhesion molecule-1 and endometrial mrna levels of MMP-3, tumor necrosis factor (TNF), and IL-8 were significantly higher during the menstrual phase when compared with luteal phase. During the menstrual phase of the cycle, both endometrial expression of TNF-, IL-8, and MMP-3 mrna levels and peritoneal expression of transforming growth factor-, IL-6, and intercellular adhesion molecule-1 mrna levels were significantly higher in women with endometriosis when compared with controls. Immunohistochemical staining confirmed the presence of TNF- in peritoneum and endometrium in both women with endometriosis and controls. Conclusion(s): Increased endometrial and peritoneal cytokine mrna expression during menstruation may contribute to a pelvic inflammatory microenvironment favoring the development of endometriosis. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Endometrium, endometriosis, cytokines, peritoneum, gene expression, real-time RT-PCR Endometriosis is a multifactorial complex disease process characterized by the ectopic presence of endometrial glands and stroma. Endometriosis can be present as peritoneal disease, endometriotic ovarian cysts, and/or deeply infiltrating rectovaginal endometriosis and is associated with pelvic pain, adhesion formation, and infertility. Endometriosis occurs in 30% 40% of women with infertility and is a progressive disease in 40% 50% of reproductive-aged women (1, 2). Received June 1, 2005; revised and accepted November 18, Supported by grants from the Leuven University Research Council and from the Flemish Fund for Scientific Research, Leuven, Belgium. Presented at the 20th Annual Meeting of the European Society for Human Reproduction and Embryology in Berlin, Germany, June 27 30, Reprint requests: Thomas M. D Hooghe, M.D., Ph.D., Leuven University Fertility Center, Herestraat 49, Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Leuven B-3000, Belgium (FAX: ; thomas.dhooghe@ uz.kuleuven.ac.be) /06/$32.00 Fertility and Sterility Vol. 85, No. 6, June 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 1667

2 The theory that has gained most supportive evidence for the pathogenesis of endometriosis is Sampson s theory (3)of retrograde menstruation. Retrograde menstruation has been reported in 83% of baboons (4) and in 70% 90% of women with spontaneous endometriosis (5). The existence of endometrial cells in the peritoneal fluid has been reported in 59% 79% of women during menses or during the early follicular phase (6, 7). According to Sampson s hypothesis (3), menstrual debris, refluxed into the peritoneal cavity, contains viable endometrial cells that can implant and develop into endometriotic lesions. However, the Sampson theory does not explain why refluxed endometrial cells survive, implant, and infiltrate in the pelvis of some but not all women. It is generally accepted that aberrant expression of cytokines and inflammation may play a role in the pathogenesis of endometriosis (8, 9), although the precise relationship between increased inflammation and the development of endometriosis is unknown. Increased peritoneal inflammation, as evidenced by increased peritoneal fluid (PF) volume and increased PF concentration of white blood cells and inflammatory cytokines, has been reported in women with endometriosis (10, 11). Several cytokines can stimulate endometrial cell adhesion to peritoneal mesothelial cell monolayers in vitro (12 14), as well as to specific extracellular matrix proteins (15). Interleukin (IL)-8 and tumor necrosis factor (TNF) are known to promote proliferation and adhesion of endometrial cells. A positive correlation has been described between PF concentrations of TNF- (8) and IL-8 (9) and size and number of active lesions and revised American Society for Reproductive Medicine (ASRM) stages of endometriosis. The increased concentration of TNF- reflects enhanced secretory activity of the peritoneal macrophages (16) and also may contribute to decreased expression of endogenous tissue inhibitors of matrix metalloproteinases (MMPs and tissue metalloproteinases) (17), resulting in increased MMP activity. However, increased levels of transforming growth factor (TGF) in PF of women with endometriosis when compared with controls have been reported (18). Interleukin-6 has been suggested to be secreted by endometriotic cells and, in concert with interferon-, may up-regulate soluble intercellular adhesion molecule 1 (sicam-1) production by macrophages in patients with endometriosis (19). In a study performed in baboons (20), it was shown that intrapelvic injection of menstrual endometrium induces endometriosis to a more severe degree when compared with intrapelvic injection of endometrium obtained during luteal phase of the cycle. But it is not known whether proinflammatory cytokines that act in concert with MMPs and are present in eutopic endometrium and peritoneum at the time of menstruation may contribute to development of endometriosis. Therefore, this study was performed to test the hypothesis that the messenger RNA (mrna) for biologically relevant molecules TNF-, IL-8 and -6, TGF-, vascular endothelial growth factor (VEGF), ICAM-1, and MMP-1 and -3 is quantitatively and differentially expressed in menstrual endometrium and menstrual peritoneum from women with endometriosis when compared with women without the disease or when compared with endometrium and peritoneum obtained during the luteal phase of the cycle. MATERIALS AND METHODS Patients and Tissue Collection This study was performed on 60 samples from women; the samples were selected from the tissue bank at the Leuven University Fertility Centre tissue bank, where tissue samples of women undergoing laparoscopies for infertility and pain have been stored since All patients had signed a written informed consent before recruitment, and the study protocol was approved by the institutional ethical and review board of University Hospital Gasthuisberg for the protection of human subjects. Samples from 35 patients (20 patients during the luteal phase and 15 patients during the menstrual phase) were selected for this study on the basis of cycle phase and presence or absence of endometriosis. Paired samples of either endometrium and/or normal peritoneum were available in 25 women (9 controls and 16 with endometriosis). Unpaired samples of either endometrium (n 3) or normal peritoneum (n 7) were available in 10 women (2 controls, 8 with endometriosis; Table 1). In this study, endometriosis was laparoscopically and histologically confirmed in all 24 women with endometriosis of revised ASRM stage I II (n 12) and revised ASRM stage III IV (n 12) (21), and the presence of a normal pelvis was documented by laparoscopy in 11 control patients. Tissue samples of endometrium and of macroscopically normal peritoneum were collected from women with endometriosis (n 40; endometrium [n 18] and peritoneum [n 22]) and from controls (n 20; endometrium [n 10] and peritoneum [n 10]) via endometrial biopsy and laparoscopic biopsy, respectively, snap-frozen in liquid nitrogen, and then stored at 80 C until use (Table 1). The macroscopically normal peritoneum tissues were collected from lateral wall left or right, near the colon ascendens or descendens. The selected samples for immunohistochemistry included endometrium obtained during menstrual (n 10) or luteal phase (n 10) and normal peritoneum obtained during menstrual (n 11) or luteal (n 12) phase (Table 1). Isolation of RNA and complimentary DNA Synthesis Total RNA was extracted from deep-frozen endometrial and peritoneal tissues with the Promega SV RNA Isolation System Z3100 (Promega Corp, Madison, WI). Briefly, RNA concentration was determined spectrophotometrically (Pharmacia, Uppsala, Sweden) by absorption at 260 nm. Target RNA was reverse transcribed by using the Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD). In the first step, 5 M Oligo(dT) 16 (Applied Biosystems, Foster City, CA) was added to 500 ng of total RNA and annealed at 70 C for 10 minutes. Then, 100 U of Superscript II reverse transcriptase was added in the pres Kyama et al. Cytokine mrna levels in women with endometriosis Vol. 85, No. 6, June 2006

3 TABLE 1 Overview of endometrial and peritoneal tissue samples collected during menstrual and luteal phases of the cycle that were used for real-time RT-PCR analysis and immunohistochemistry in women with and without endometriosis. Cycle phase by group Total Controls Endometriosis Endometriosis (ASRM I II) Endometriosis (ASRM III IV) Patients Total Menstrual Luteal Endometrium (n 28) Menstrual 14 5 a (2 a ) 9 5 a (4 a ) c 4 a (4 a ) Luteal 14 4 a 1 b (3 a ) 9 4 a (3 a ) 3 a 2 b (3 a 1 b ) Normal peritoneum (n 32) Menstrual 15 5 a (2 a 2 b ) 10 5 a (4 a ) 4 a 1 b (3 a ) Luteal 17 4 a 1 b (3 a 1 b ) 12 4 a 3 b (4 a ) 3 a 2 b (3 a 1 b ) Note: Samples used in immunohistochemistry are indicated in parentheses. ASRM revised ASRM stages. a Paired tissue samples (endometrial and normal peritoneal biopsies were obtained from same patient). b Unpaired tissue samples (endometrial and normal peritoneal biopsies obtained from different patients). ence of 50 mm Tris-HCL, ph 8.3; 75 mm KCL; 3 mm MgCl 2 ; and 5 mm unlabeled deoxynucleotides and was incubated at 42 C for 80 minutes. Reverse-transcriptionminus controls (i.e., RNA samples that were treated similarly but without addition of Superscript II RT enzyme) were included as negative controls for subsequent polymerase chain reactions (PCRs). Real-Time RT-PCR All amplification of primers and TaqMan probes used (Table 2) were designed with Primer Express software (Applied Biosystems, Foster City, CA). 6-Carboxyfluorescein was used as the reporter dye, and 6-carboxytetramethylrhodamine was used as the quencher dye. Real-time PCR was performed for TNF-, IL-8, IL-6, TGF-, VEGF, ICAM-1, MMP-1, -actin, and MMP-3 as described elsewhere (22). Briefly, realtime PCR amplification was performed in duplicate by using 96-well microtiter plates on the ABI Prism 7700 detection System, which contains a Gene-Amp PCR system 9600 (Applied Biosystems, Foster City, CA). The reactions were performed in a total volume of 25 L, containing 0.5 L of cdna sample, 1 real-time PCR buffer, 0.5 mm deoxynucleotides, 5 mm MgCl 2, 5 pmol each primer, 5 pmol dual-labeled probe, and U of Hot Gold Star (Eurogentec, Liege, Belgium). Reaction conditions were 10 minutes at 94 C, followed by 45 cycles (15 seconds at 94 C and 1 minute at 60 C). To correct for variations linked to differences in amount of input RNA, or for different levels of inhibition during reverse transcription (RT) or PCR, we normalized the expression levels by using -actin as a housekeeping gene (Applied Biosystems, Foster City, CA). All experiments were performed with negative controls. Immunohistochemistry Immunohistochemistry was performed to confirm the presence of TNF- in endometrium and peritoneum. Paraffinembedded biopsies of endometrium and peritoneum were cut in 5- m sections and put on silane-coated glass slides. Tissue sections were preheated for 1 hour at 55 C. The tissue sections were deparaffinized in toluene twice for 5 minutes and rehydrated in ethanol twice for 5 minutes. To quench the endogenous peroxidase activity, sections were blocked for 30 minutes by using an endoperoxidase solution of 0.5% hydrogen peroxide (H 2 O 2 ) and 0.1% sodium azide (NaN 3 )in methanol. After washing in 0.01 M tris(hydroxymethyl)aminomethane buffered saline (TBS), the tissue sections were retrieved in 0.01 M Tris buffer, ph 9, with 0.5 M ethylenediaminetetraacetic acid for 2 hours at 80 C. Tissue sections were rinsed again in 0.01 M TBS, then incubated for 15 minutes at room temperature with 0.2% bovine serum albumin, 0.1% nonfat dried milk, and 0.1% Tween-80 with addition of normal goat serum (1/30) in 0.01 M TBS. The primary antibody was added and incubated overnight at 4 C. A mouse monoclonal antibody raised against human TNF- (1:10 dilution; R and D Systems, Minneapolis, MN) was used at 50 g/ml. Then tissue sections were washed in TBS and blocked again with 0.2% bovine serum albumin, 0.1% nonfat dried milk, and 0.1% Tween-80 with addition of normal goat serum (1/30) for 15 minutes at room temperature. Then a secondary antibody peroxidase-conjugated goat anti-mouse IgG (1/100, 30 minutes; Jackson ImmunoResearch Lab Inc, West Grove, PA) was added, supplemented with normal human serum (1: 25). Peroxidase activity was detected by 3,3=-diaminobenzidine tetrahydrochloride DAB (Sigma Chemical Co., St. Louis, MO), and slides were counterstained with Harris hematoxylin. Fertility and Sterility 1669

4 TABLE 2 Primer and probe sequences for real-time RT-PCR of human cytokines, chemokines, adhesion molecules, and metalloproteinases. Substance Sequence (5= 3=) Amplicon length (bp) Accession number a TNF- FW TCTTCTCGAACCCCGAGTGA 151 NM_ RV CCTCTGATGGCACCACCAG NT_ TAGCCCATGTTGTAGCAAACCCTCAAGCT TGF- FW CAGCAACAATTCCTGGCGATA 136 NM_ RV AAGGCGAAAGCCCTCAATTT NT_ CTGCTGGCACCCAGCGACTCG IL-6 FW CACCGGGAACGAAAGAGAAG 97 NM_ RV CCCAGGGAGAAGGCAACTG NT_ CCTTCTCCACAAGCGCCTTCGGT IL-8 FW TGGCAGCCTTCCTGATTTCT 61 NM_ RV TTAGCACTCCTTGGCAAAACTG NT_ CAGCTCTGTGTGAAGGT ICAM-1 FW CGGCTGACGTGTGCAGTAAT 127 NM_ RV CACCTCGGTCCCTTCTGAGA NT_ CATCTACAGCTTTCCGGCGCCCA VEGF FW ACCAAGGCCAGCACATAGAGA 134 NM_ RV CTTGCGCTTTCGTTTTTGC NT_ CAGCACAACAAATGTGAATGCAGACCAA MMP-1 FW GGGAGATCATCGGGACAACTC 80 NM_ RV CAATACCTGGGCCTGGTTGA NT_ CCTTTTGATGGACCTGGAGGAAATCTTGC MMP3 FW TTCCGCCTGTCTCAAGATGAT RV ATCACAGTTGGCTGGCGTC 129 NM_ CCTCCCCCTGACTCCCCTGAGACC NT_ Note: FW forward primer; RV reverse primer; TaqMan probe, dual-labeled with 5=FAM and 3=TAMRA. a Genbank accession number of cdna and corresponding gene, available at Sections incubated without the primary antibody were included as negative control, whereas breast cancer tissue was used as a positive-control tissue. All tissue sections were examined with a light microscope (DMLB; Leica, Wetzlar, Germany). Statistical Analysis In the real-time RT-PCR part of the study, comparisons were made regarding the endometrial and peritoneal mrna expression of all the above-mentioned cytokines between menstrual and luteal phases of women with and without endometriosis and between women with and without endometriosis during the menstrual phase and the luteal phase. Results are expressed as mean SEM. Statistical analysis was performed by using t tests and analysis of variance. A P value of.05 was considered to be statistically significant. Statistical analysis was performed by using the prism3 software (GraphPad, San Diego, CA). RESULTS Menstrual Phase versus Luteal Phase In women with endometriosis, both the endometrial mrna expression of IL-8, TNF-, and MMP-3 (Fig. 1A, C, and F) 1670 Kyama et al. Cytokine mrna levels in women with endometriosis Vol. 85, No. 6, June 2006

5 FIGURE 1 Women with endometriosis: menstrual phase vs. luteal phase. Endometrial IL-8 (A), TNF- (C), and MMP-3 (F) mrna expression and peritoneal TGF- (B), IL-6 (D), ICAM-1 (E), and MMP-3 (F) mrna expression were significantly higher during the menstrual phase when compared with the luteal phase. Intercellular adhesion molecule-1 mrna (E) and TGF- (B) levels were significantly lower in endometrium when compared with the peritoneum during the menstrual phase and the luteal phase, respectively. Red bars, endometrium; blue bars, peritoneum. *P.05. **P.01. and the peritoneal mrna expression of TGF-, IL-6, ICAM-1, and MMP-3 (Fig. 1B, D, E, and F) were significantly higher during the menstrual phase when compared with the luteal phase of the cycle. In women with endometriosis, the expression levels of ICAM-1 mrna and TGF- mrna also were significantly lower in endometrium than in peritoneum during the menstrual phase (Fig. 1E) and luteal phase (Fig. 1B), respectively. No significant differences were observed for MMP-1 and VEGF mrna in any of the above comparisons (data not shown). In women without endometriosis, peritoneal expression of ICAM-1 mrna was significantly higher during the menstrual phase when compared with the luteal phase (data not Fertility and Sterility 1671

6 shown). There were no significant differences observed for TNF-, IL-8, TGF-, IL-6, MMP-3, MMP-1, and VEGF mrna, either in endometrium or peritoneum, during the menstrual phase when compared with the luteal phase (data not shown). Women With Endometriosis versus Controls During the menstrual phase of the cycle, both endometrial expression of IL-8, TNF-, and MMP-3 mrna (Fig. 2A, C, and F) levels and peritoneal expression of IL-6, TGF-, and ICAM-1 (Figs. 2B, D, and E) mrna levels were significantly higher in women with endometriosis when compared with controls. No significant differences were observed for MMP-1 and VEGF mrna levels in any of the comparisons described (data not shown). During the luteal phase, increased peritoneal expression of MMP-3 and TNF- (Fig. 3B and C) mrna levels but decreased peritoneal expression of ICAM-1 mrna was FIGURE 2 Women with endometriosis vs. controls: menstrual phase. Increased endometrial mrna levels for IL-8 (A), TNF- (C), and MMP-3 (F) and increased peritoneal mrna levels of IL-6 (B), TGF- (D), and ICAM-1 (E) in women with endometriosis when compared with controls. Red bars, endometrium; blue bars, peritoneum. *P Kyama et al. Cytokine mrna levels in women with endometriosis Vol. 85, No. 6, June 2006

7 FIGURE 3 Women with endometriosis vs. controls: luteal phase. Increased peritoneal mrna levels for TNF- and MMP-3 (B, C) and decreased peritoneal mrna levels of ICAM-1 (A) in women with endometriosis when compared with controls. Red bars, endometrium; blue bars, peritoneum. *P.05, **P.01. observed in women with endometriosis when compared with controls (Fig. 3A). No significant differences were observed for TGF-, MMP-1, IL-6, IL-8, and VEGF mrna in any of the above comparisons (data not shown). Immunohistochemistry Immunohistochemical staining confirmed the presence of TNF- in peritoneum and endometrium (Fig. 4). Only a few positive cells were identified in peritoneum (Fig. 4A and B), whereas cell-specific staining was observed in some epithelial and stromal cells in endometrium (Fig. 4C and D). DISCUSSION Real-time RT-PCR has been used previously to quantify and compare mrna levels of estrogen receptor alpha (ER- ) and estrogen receptor beta (ER- ) in ovarian endometriotic cysts and in certain endometriotic lesions (23, 24). However, to the best of our knowledge, we have used for the first time real-time RT-PCR technology to evaluate differential gene expression of cytokines and growth factors in endometrium and peritoneum from women with and without endometriosis during the menstrual and luteal phases. The data from our study suggest for the first time that menstrual endometrium from women with endometriosis has more pronounced inflammatory and proteolytic properties than that of women without the disease. In women with endometriosis, the endometrial mrna expressions of TNF-, IL-8, and MMP-3 were significantly higher during the menstrual phase compared with the luteal phase, whereas such a difference was not present in women without endometriosis. Furthermore, endometrial mrna expression of TNF-, IL-8, and MMP-3 was significantly higher in women with endometriosis when compared with the case in women without endometriosis during the menstrual phase. In contrast, there were no significant differences between the two groups in the luteal phase. Therefore, we hypothesize that only menstrual endometrium expressing increased levels of in- Fertility and Sterility 1673

8 FIGURE 4 Immunohistochemical staining for TNF- showing cell-specific staining in normal peritoneum from a patient without endometriosis (A) and with endometriosis (B) and in both epithelial (C) and stromal (D) compartments of luteal phase endometrium from two different women with moderate to severe endometriosis. Arrowheads show positive staining of TNF-. All bars 50 m. flammatory cytokines and MMPs like TNF-, IL-8, and MMP-3 is capable of implanting ectopically and of leading to the development of endometriosis. We also hypothesize that the natural progression and the stage of endometriosis could be predicted by the levels of expression of inflammatory cytokines and MMPs in menstrual endometrium. Our hypothesis appears to be biologically plausible. Cytokines such as IL-8 and TNF- are known to promote proliferation and adhesion of endometrial cells and angiogenesis. Tumor necrosis factor modulates the stimulation of other cytokines and chemokines such as IL-8 and Regulated upon Activation, Normal T-Cell Expressed and Secreted (25). The increased mrna levels of TNF- also may contribute to increased MMP-3 mrna expression. Indeed, the increased mrna levels of MMP-3 in menstrual endometrium from women with endometriosis observed in our study may explain the increased invasiveness of endometrial fragments in women with endometriosis (26, 27). To the best of our knowledge, our study is the first report illustrating that normal peritoneum is immunologically more active during the menstrual phase than during the luteal phase and that normal peritoneum is biologically different in women with endometriosis when compared with that of controls during both the menstrual and the luteal phases. During the menstrual phase, peritoneal expression of ICAM-1, TGF-, and IL-6 mrna was up-regulated in women with endometriosis when compared with controls. Furthermore, during the luteal phase, peritoneal expression of TNF- and MMP-3 mrna was up-regulated, and ICAM-1 mrna was down-regulated, in women with dis Kyama et al. Cytokine mrna levels in women with endometriosis Vol. 85, No. 6, June 2006

9 ease when compared with controls. These observations may be very relevant with respect to the pathogenesis of endometriosis, and important roles may be played by TGF-, ICAM-1, and IL-6. Our results suggest that the peritoneal production of TGF- may contribute to the reportedly increased levels of TGF- in PF of women with endometriosis when compared with controls (18). Intercellular adhesion molecule-1 is expressed at the surface of numerous cells, including endometrial cells, endothelial cells, and leukocytes (28), and could be involved in endometrial peritoneal adhesion and in the protection of endometrial cells from natural-killer cell mediated cytotoxicity (19, 29). Interleukin-6 also may be involved in this process because IL-6 can be secreted by endometriotic cells and, in concert with interferon-, may up-regulate sicam-1 production of macrophages from patients with endometriosis (30). We hypothesize that the upregulation of IL-6 gene expression in the peritoneum of women with endometriosis also may promote increased production of sicam-1 by endometriotic lesions. In conclusion, increased endometrial and peritoneal cytokine mrna expression during menstruation in patients with endometriosis may contribute to a pelvic inflammatory microenvironment that favors endometrial peritoneal adhesion and leads to the development of endometriosis. REFERENCES 1. Taylor RN, Lundeen SG, Giudice LC. Emerging role of genomics in endometriosis research. Fertil Steril 2002;78: D Hooghe TM, Hill JA. Endometriosis. In: Berek JS, ed. Novak s gynecology. 13th ed. Philadelphia, PA: Williams and Wilkins, 2002: Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927;14: D Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. Increased incidence and recurrence of retrograde menstruation in baboons with spontaneous endometriosis. Hum Reprod 1996;11: Blumenkrantz MJ, Gallaagher N, Bashore RA, Tenckhoff H. Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstet Gynecol 1981;142: Koninckx PR, De Moor P, Brosens IA. Diagnosis of the luteinized unruptured follicle syndrome by steroid hormone assays on peritoneal fluid. Br J Obstet Gynaecol 1980;87: Kruitwagen RF, Poels LG, Willemsen WN, de Ronde IJ, Jap PH, Rolland R. Endometrial epithelial cells in peritoneal fluid during the early follicular phase. Fertil Steril 1991;55: 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: Harada T, Enatsu A, Mitsunari M, Nagano Y, Ito M, Tsudo T, et al. Role of cytokines in progression of endometriosis. Gynecol Obstet Invest Suppl 1999;47: Halme J, Becker S, Hammond MG, Raj S. Pelvic macrophages in normal and infertile women: the role of patent tubes. Am J Obstet Gynecol 1982;142: Oral E, Olive DL, Arici A. The peritoneal environment in endometriosis. Hum Reprod Update 1997;2: Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, et al. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 2000;85: Witz CA, Monotoya-Rodriguez IA, Schenken RS. Whole explants of peritoneum and endometrium: a novel model of the early endometriosis lesion. Fertil Steril 1999;71: Debrock S, Vander Perre S, Meuleman C, Moerman P, Hill JA, D Hooghe TM. In-vitro adhesion of endometrium to autologous peritoneal membranes: effect of the cycle phase and the stage of endometriosis. Hum Reprod 2002;17: Sillem M, Prifti S, Monga B, Arslic T, Runnebaum B. Integrin-mediated adhesion of uterine endometrial cells from endometriosis patients to extracellular matrix proteins is enhanced by tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1). Eur J Obstet Gynecol Reprod Biol 1999; 87: Bullimore DW. Endometriosis is sustained by tumour necrosis factoralpha. Med Hypotheses 2003;60: Gottschalk C, Malberg K, Arndt M, Schmitt J, Roessner A, Schultze D, et al. Matrix metalloproteinases and TACE play a role in the pathogenesis of endometriosis. Adv Exp Med Biol 2000;477: Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994;83: Somigliana E, Vigano P, Gaffuri B, Guarneri D, Busacca M, Vignali M. Human endometrial stromal cells as a source of soluble intercellular adhesion molecule (ICAM)-1 molecules. Hum Reprod 1996; 11: D Hooghe TM, Bambra CS, Raeymaekers BM, De Jonge I, Lauweryns JM, Koninckx PR. Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus, Papio anubis). Am J Obstet Gynecol 1995;173: American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: Fertil Steril 1997;67: Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C. The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 2003;14: Matsuzaki S, Murakami T, Uehara S, Canis M, Sasano H, Okamura K. Expression of estrogen receptor alpha and beta in peritoneal and ovarian endometriosis. Fertil Steril 2001;75: Matsuzaki S, Uehara S, Murakami T, Fujiwara J, Funato T, Okamura K. Quantitative analysis of estrogen receptor alpha and beta messenger ribonucleic acid levels in normal endometrium and ovarian endometriotic cysts using a real-time reverse transcription-polymerase chain reaction assay. Fertil Steril 2000;74: Altman GB, Gown AM, Luchtel DL, Baker C. RANTES production by cultured primate endometrial epithelial cells. Am J Reprod Immunol 1999;42: Sillem M, Prifti S, Koch A, Neher M, Jauckus J, Runnebaum B. Regulation of matrix metalloproteinases and their inhibitors in uterine endometrial cells of patients with and without endometriosis. Eur J Obstet Gynecol Reprod Biol 2001;95: Osteen KG, Bruner-Tran KL, Keller NR, Eisenberg E. Progesteronemediated endometrial maturation limits matrix metalloproteinase (MMP) expression in an inflammatory-like environment: a regulatory system altered in endometriosis. Ann NY Acad Sci 2002;955: Steff AM, Gagne D, Page M, Hugo P, Gosselin D. Concentration of soluble intercellular adhesion molecule-1 in serum samples from patients with endometriosis collected during the luteal phase of the menstrual cycle. Hum Reprod 2004;19: Vigano P, Gaffuri B, Somigliana E, Busacca M, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule (ICAM)-1 mrna and protein is enhanced in endometriosis versus endometrial stromal cells in culture. Mol Hum Reprod 1998;4: Fukaya T, Sugawara J, Yoshida H, Murakami T, Yajima A. Intercellular adhesion molecule-1 and hepatocyte growth factor in human endometriosis: original investigation and a review of literature. Gynecol Obstet Invest 1999;47:11 6. Fertility and Sterility 1675