Peritoneal fluid concentrations of interleukin-8 in patients with endometriosis depend on the severity of the disorder and are higher in the luteal phase

C. Calhaz-Jorge1,3, A.P. Costa1, M.C. Santos2 and M.L. Palma-Carlos2

1 Human Reproduction Unit, Department of Obstetrics and Gynaecology and 2 Lisbon University Haematology and Immunology Centre, Hospital de Santa Maria, Av. Prof. Egas Moniz, Lisboa, Portugal

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Hospital de Santa Maria, Av. Egas Moniz, 1649-028 Lisboa, Portugal. e-mail: calhazjorge{at}mail.telepac.pt


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Previous evaluations of the relationship between the concentrations of interleukin-8 (IL-8) in the peritoneal fluid and endometriosis led to non-consistent results. Our purpose was to investigate the correlation of the concentrations of IL-8 in the peritoneal fluid with the stage of endometriosis, the presence of red lesions and the phase of the menstrual cycle. METHODS: Ninety-two patients with infertility (n = 87) or undergoing sterilization (n = 5) had peritoneal fluid samples collected at laparoscopy. IL-8 determinations were performed using an enzyme-linked immunosorbent assay. RESULTS: The concentrations of IL-8 in the peritoneal fluid of the 68 women with endometriosis were not significantly different from those of the 24 controls. Patients with moderate/severe stages had IL-8 significantly higher than controls (P = 0.008) and marginally higher than patients with minimal/mild endometriosis (P = 0.053). Concentrations of IL-8 were significantly higher in patients than in controls in the luteal phase. Red lesions were associated with significantly increased levels of peritoneal fluid IL-8 only in the luteal phase. CONCLUSIONS: Our findings reinforce the importance of IL-8 in the pathogenesis of endometriosis.

Key words: endometriosis/IL-8/luteal phase/peritoneal fluid/peritoneal red lesions


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometriosis, defined as the presence of endometrial glands and stroma outside the uterine cavity, is a common gynaecological disorder in women of reproductive age. Sampson’s theory of implantation of endometrial cells and fragments refluxed during the menstrual period (Sampson, 1927Go) is generally accepted. The role of peritoneal fluid cellular population and soluble factors has been extensively studied and they are considered to contribute to the implantation of endometrial cells and progression of the situation. Interleukin-8 (IL-8), one of those factors, is a chemokine that is secreted by several cell types including monocytes (Yoshimura et al., 1987Go), fibroblasts (Larsen et al., 1989Go), mesothelial cells (Topley et al., 1993Go), endometrial cells (Arici et al., 1996aGo) and endometriotic cells (Akoum et al., 2001Go). IL-8 induces chemotaxis on neutrophils, has a strong angiogenic activity (Koch et al., 1992Go) and has the capability of promoting endometrial cell growth (Arici et al., 1998bGo).

After the first report by Ryan et al. (1995Go), a few other studies evaluated the concentrations of IL-8 in the peritoneal fluid of women with endometriosis ( Arici et al., 1996bGo; Rana et al., 1996Go; Gazvani et al., 1998Go; Iwabe et al., 1998Go). Although all of them showed some kind of increment in the IL-8 concentrations, their results are somewhat conflicting. Their different designs, non-coincident criteria in the selection of patients, and/or the heterogeneity of the situation itself may have contributed to this. The existence of different types of peritoneal endometriotic lesions is one factor to take into consideration. In fact, several studies have reported a more important neovascularization around and within endometriotic implants (Nisolle et al., 1993Go; Matsuzaki et al., 1998Go) in red lesions when compared with black and white lesions, strongly supporting the possibility of an increased angiogenic status of red lesions.

Ovarian steroids are generally accepted as a fundamental factor for the development and maintenance of endometriosis. Endometriotic lesions express sex steroid receptors and can undergo cyclical changes, although not as clearly and regularly as endometrium (Nisolle et al., 1997Go). Peritoneal fluid has concentrations of sexual steroids that are significantly higher than those of serum at the same phase of the cycle (Koninckx et al., 1980Go). The peritoneal fluid concentrations of estrogens and progesterone were shown to increase dramatically following ovulation and to be very much higher in the luteal phase than in the follicular phase (Koninckx et al., 1980Go; Bouckaert et al., 1986Go; De Leon et al., 1986Go). It was reported that in women with endometriosis the post-ovulatory peritoneal fluid progesterone increase could be less pronounced (Barry-Kinsella et al., 1994Go) or even absent (Chew et al., 1987Go) in comparison with controls.

In this study we investigated the correlation of the concentrations of IL-8 in the peritoneal fluid with the stage of the disease, the phase of the menstrual cycle and the presence of red lesions in an important number of women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ninety-two consecutive patients undergoing laparoscopy were included in the study; 87 had infertility and 5 were submitted to tubal ligation. Infertility was defined as a delay in conception of >=12 months. None of the patients had any autoimmune disease, had been on hormonal medication, had experienced signs of pelvic infection, or had undergone hysterosalpingography in the 2 months prior to the surgical procedure. Also, none had been pregnant or breastfeeding in the previous 6 months. The local Ethics Committee approved the study.

Laparoscopies were performed under general anaesthesia and throughout the menstrual cycle except during menstruation. The phase of the cycle was determined by the patient’s last menstrual period and confirmed by histological examination of the endometrium; the visualization of either a corpus luteum or a dominant follicle was also taken into consideration.

Endometriosis was diagnosed and staged by visual inspection of the pelvis according to the American Society of Reproductive Medicine classification. Peritoneal lesions were categorized as red, black and white lesions as proposed in the latest revision of that classification (American Society for Reproductive Medicine, 1997). Endometriosis was found in 68 patients. It was classified as minimal/mild (stages I–II) in 53 cases and moderate/severe (stages III–IV) in 15 women. Red lesions were present in 42 patients, isolated or associated with other types of lesions. In 26 patients no red lesions were identified. Patients with and without red lesions were equally distributed by subgroups with endometriosis I–II and III–IV. The control group consisted of 24 patients with no evidence of endometriosis and no pelvic adhesions. In women with endometriosis, the indication for surgery was infertility (n = 65) and sterilization (n = 3). For those without endometriosis, surgery was performed because of infertility (n = 22) or sterilization (n = 2).

Aspiration of the peritoneal fluid was performed under direct visualization from the posterior cul-de-sac and anterior vesico-uterine fold through a second puncture using a 2 mm metal cannula, before any internal manipulation and with the patient still in the horizontal supine position. Patients with bleeding into the peritoneal cavity from the abdominal puncture sites were excluded. Fluid samples were transferred into a chilled sterile plastic tube and kept on ice until arrival at the laboratory. They were centrifuged at 300 g for 15 min and the supernatant collected, divided into aliquots and stored at –70°C until assayed. The concentrations of IL-8 were measured by enzyme immunoassay using a commercial kit for hIL-8 (Quantikine®; R&D Systems, Abingdon, UK) which is said to have no measurable cross-reactivity with other relevant cytokines. The limit of sensitivity is 10 pg/ml and the intra-assay and inter-assay coefficients of variation were <10%. The assays were performed blind to laparoscopic findings.

Demographic and clinical data of patients and controls were compared by using Student’s t-test with the Welch correction for unequal variances and by Fisher’s exact test. The concentrations of IL-8, which are not normally distributed and are left-censored at the limit of sensitivity of the assay method, were compared with the Mann–Whitney rank sum test. The effects of the stage of the disease and the phase of the menstrual cycle on IL-8 concentrations were tested with an analysis of variance (ANOVA) model that included both variables and an interaction term. The analysis was performed on rank-transformed IL-8 values and the results confirmed on the raw IL-8 values. Two-sided P-values < 0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and controls were comparable in age, duration of infertility and proportion of secondary infertility, and distribution for the phases of menstrual cycle (Table I) including early, mid and late (follicular and luteal) subphases. In total, four patients (all with endometriosis I–II) and one control were evaluated before day 10 of the menstrual cycle.


View this table:
[in this window]
[in a new window]
 
Table I. Patient demographics
 
The concentrations of IL-8 in the peritoneal fluid of women with endometriosis were higher but not significantly different from those of the controls (P = 0.072). In patients with moderate/severe stages, IL-8 was significantly higher than in controls (P = 0.008) and marginally higher than in women with minimal/mild endometriosis (P = 0.053) (Figure 1).



View larger version (10K):
[in this window]
[in a new window]
 
Figure 1. Concentrations of interleukin-8 (IL-8) in the peritoneal fluid of control subjects and women with endometriosis. Women with endometriosis are subdivided in minimal/mild and moderate/severe subgroups. Median concentrations are represented by horizontal bar. Patients with moderate/severe stages of endometriosis had median concentration of IL-8 (31.1 pg/ml) significantly higher than controls (12.7 pg/ml; P = 0.008) and marginally higher than patients with minimal/mild endometriosis (15.6 pg/ml; P = 0.053). The dotted line represents the limit of sensitivity of the assay. *P < 0.01 versus controls; P = 0.053 versus stages I–II.

 
ANOVA showed a statistically significant effect of endometriosis on the concentration of IL-8 (P = 0.008 for ranks, P = 0.03 for raw values), but not of the phase of the menstrual cycle (P = 0.33 for ranks, P = 0.12 for raw data). The interaction term was also statistically significant (P = 0.013 for ranks, P = 0.001 for raw data). Pair-wise comparisons of the groups formed by the combination of levels of the study variables (Table II) help us to understand how these effects act on the IL-8 concentrations. The effect of endometriosis is reflected by the significant increase of median IL-8 concentration in moderate/severe endometriosis over the controls. The interaction of endometriosis by the phase of the menstrual cycle explains the observation of a much larger difference in the luteal phase than in the follicular phase of the menstrual cycle in median IL-8 levels. This resulted in a significant difference between patients as a whole and controls (P = 0.019) and in a larger difference between patients with moderate/severe endometriosis and the other groups.


View this table:
[in this window]
[in a new window]
 
Table II. Peritoneal fluid concentrations of interleukin-8 (IL-8)
 
Patients with peritoneal implants classified as red lesions had concentrations of IL-8 in the peritoneal fluid (median: 16.1 pg/ml; range: 10–662.9) not different from the controls (P = 0.08) and from the patients without red lesions (median: 20.3 pg/ml; range: 10–407.2; P = 0.72). Women with and without red peritoneal lesions did not differ in the proliferative phase or in the luteal phase. However, patients with red lesions evaluated in the luteal phase had higher concentrations of IL-8 in their peritoneal fluid than controls in the luteal phase (P = 0.021). No significant difference was found between the IL-8 concentrations of patients without red lesions in the luteal phase and those of controls in the luteal phase.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peritoneal environment is considered an important contributor for the installation and maintenance of pelvic endometriosis. IL-8 is one of the several soluble factors in the peritoneal fluid whose participation in the pathogenesis of endometriosis has been the subject of investigation. A few previous studies evaluated the peritoneal fluid concentrations of IL-8 in women with endometriosis. The different designs and characteristics and size of the populations may explain their somewhat inconsistent results. In brief, three groups (Ryan et al., 1995Go; Arici et al., 1996bGo; Gazvani et al., 1998Go) studied patients in all stages of endometriosis (18 patients, 9 controls; 45 patients, 28 controls; and 25 patients, 22 controls respectively) whereas all 17 patients evaluated by Rana et al. (1996Go) had endometriosis III–IV. Another group (Iwabe et al., 1998Go) studied 24 patients with red lesions and compared them with 16 controls; no information was given concerning the stage of the disorder. Endometriosis is also a very diversified situation, and the extension of lesions, the different types of lesions (Jansen and Russell, 1986Go) and the variations of the hormonal environment, i.e. the phase of the menstrual cycle, are important variables.

In this study we have shown that the concentrations of IL-8 in the peritoneal fluid were higher in patients with moderate/severe stage of endometriosis. In this respect, our results coincide totally with those of some other authors (Ryan et al., 1995Go; Arici et al., 1996bGo) and differ from those published by Gazvani et al. (1998Go), who reported significantly higher IL-8 concentrations in the peritoneal fluid of their eight patients with stage I endometriosis than in the 17 patients with more severe stages of the condition.

Like all other groups cited above, we selected patients by visual inspection of the pelvis (with histological confirmation of diagnosis only in patients with endometriosis stages III–IV). Although this is the diagnostic tool used by the vast majority of groups working in the field, some possible inaccuracy in the macroscopic identification of individual lesions (Jansen and Russell, 1986Go; Walter et al., 2001Go) cannot be ruled out. The fact that microscopic endometriosis was reported in 6% of random biopsies of apparently normal peritoneum of women without visible endometriotic lesions (Nisolle et al., 1990Go) made the situation even more complex.

Experimental findings suggest a role of IL-8 in the endometrium and endometriosis. In fact, IL-8 was found to be produced in the human endometrium in vivo (Arici et al., 1998bGo) and endometriotic implants may represent a potential source of IL-8 (Akoum et al., 2001Go). In addition, IL-8 receptors type A and B were expressed in the endometrium, mostly in the stroma (Arici, 2002Go) and IL-8 receptor A was also expressed in endometriotic stromal cells (Iwabe et al., 1998Go). It was also shown that IL-8 induced endometrial stromal cell proliferation in a concentration-dependent manner (an effect inhibited by anti-IL-8 antibody) (Arici et al., 1998aGo; Iwabe et al., 1998Go) and may exert a dose-dependent stimulatory effect on the survival of whole endometrium (Gazvani et al., 2002Go). Finally, increasing doses of IL-8 stimulated endometrial stromal cells adhesive capacity to an extracellular matrix (ECM) integrin-receptor, fibronectin (Garcia-Velasco and Arici, 1999Go), whereas the adherence of endometrial cells to ECM resulted in the secretion of higher levels of IL-8 among other factors (Smith et al., 1997Go). Overall, these data support the hypothesis of a relevant role of IL-8 as an autocrine growth factor in the endometrium and suggest the existence of a positive feedback mechanism that may be important in the progression of endometriosis. They may also explain the increased concentrations of IL-8 in the peritoneal fluid of women with most extensive disease, be it a consequence and/or a promoter of the situation.

The role of the increased angiogenic activity of the peritoneal fluid of women with endometriosis (Oosterlynck et al., 1993Go) in the hypervascularization of red lesions has not been established. Although IL-8 is a well-known angiogenic factor, a direct relationship between the IL-8 concentrations in the peritoneal fluid and angiogenic activity was not confirmed using an experimental model (Maas et al., 2001Go). In our population we found a trend towards a higher concentration of IL-8 in peritoneal fluid of patients with red lesions than in controls. Patients with and without red lesions had similar concentrations of the cytokine. It is not possible to compare our results with those of Iwabe et al. (1998Go), which described a significant correlation between the level of IL-8 in the peritoneal fluid and red lesion score. In fact, all their patients had red lesions; they scored them according to a specific score system and no clinical details of their patients were described.

We did not find any difference between patients (and their subgroups) and controls in the follicular phase. However, a significant interaction between endometriosis and phase of the menstrual cycle was evident in the luteal phase, and patients as a group (and subgroup of patients with moderate/severe endometriosis) had significantly higher concentrations of IL-8 in their peritoneal fluid. Patients with red lesions also showed significantly higher levels of IL-8 than controls in the luteal phase. Our patients and controls were evenly distributed regarding the phases of the cycle and their subdivisions. To minimize conflicting factors, we did not include in this study women with laparoscopy performed during their menstruation. Moreover, the proportion of patients (and their subgroups) and controls evaluated in the early luteal phase were not different. We can therefore exclude a relevant influence of the high IL-8 concentration in the follicular fluid (Büscher et al., 1999Go) released into the peritoneal cavity during ovulation. Gazvani et al. (1998Go) also studied an evenly distributed group of patients regarding the phases of menstrual cycle but found no difference in IL-8 peritoneal fluid concentrations. However, their study group included only 25 patients. Ryan et al. (1995Go) found no difference in IL-8 regarding the phase of the cycle but >70% of their 18 patients were in the follicular phase. All patients of the study by Rana et al. (1996Go) were in the luteal phase and no information about the phase of the cycle was given by the other groups that evaluated IL-8 in the peritoneal fluid (Arici et al., 1996bGo; Iwabe et al., 1998Go).

It is tempting to speculate about the effect of the increased levels of steroids that are present in the peritoneal fluid after ovulation in our findings. Estrogens are universally accepted as a promoter of endometriosis. Besides an autocrine/paracrine action of their high local concentrations in implants, probably due to aromatase increased capacity (Noble et al., 1996Go), it seems logical to assume that high concentrations in the peritoneal fluid may increase the capability to act on peritoneal lesions. In spite of its unquestionable therapeutic action, the role (if any) of progesterone in the pathogenesis of endometriosis is not clear. The effect of progesterone on IL-8 production by endometrial cells of normal women led to conflicting results with an inhibitory effect reported in endometrial explants (Kelly et al., 1994Go) and a stimulatory effect described in stromal cells (Arici et al., 1996aGo). To our knowledge, no similar studies have been performed either in endometrium of women with endometriosis or in endometriotic cells. Although some reports suggest that in patients with endometriosis there is a less important increase of peritoneal fluid progesterone after ovulation than in controls (Barry-Kinsella et al., 1994Go) or no increase at all (Chew et al., 1987Go), we can only conjecture about the relevance of these data in our results since we did not measure steroids in the peritoneal fluid.

In conclusion, in our study the concentrations of IL-8 in the peritoneal fluid were higher in women with the more severe stages of endometriosis and more so in the luteal phase. A significant association with the presence of peritoneal red lesions was also found in the luteal phase. These data reinforce the importance of IL-8 in the pathogenesis of endometriosis and suggest that there may be an association between the peritoneal fluid levels of sexual steroids and those of IL-8.


    Acknowledgements
 
The authors are most grateful to António Gouveia, MD, PhD for his invaluable advice and help in the statistical analysis and the revision of the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Akoum, A., Lawson, C., McColl, S. and Villeneuve, M. (2001) Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol. Hum. Reprod., 9, 859–866.[CrossRef]

American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril., 67, 817–821.[CrossRef][ISI][Medline]

Arici, A. (2002) Local cytokines in endometrial tissue: the role of interleukin-8 in the pathogenesis of endometriosis. Ann. NY Acad. Sci., 955, 101–109.[Abstract/Free Full Text]

Arici, A., MacDonald, P.C. and Casey, M.L. (1996a) Progestin regulation of interleukin-8 mRNA levels and protein synthesis in human endometrial stromal cells. J. Steroid Biochem. Mol. Biol., 58, 71–76.[CrossRef][ISI][Medline]

Arici, A., Tazuke, S.I., Attar, E., Kliman, H.J. and Olive, D.L. (1996b) Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol. Hum. Reprod., 2, 40–45.[Abstract]

Arici, A., Seli, E., Zeyneloglu, H.B., Senturk, L.M., Oral, E. and Olive, D.L. (1998a) Interleukin-8 induces proliferation of endometrial stromal cells: a potential autocrine growth factor. J. Clin. Endocrinol. Metab., 83, 1201–1205.[Abstract/Free Full Text]

Arici, A., Seli, E., Senturk, L.M., Gutierrez, L.S., Oral, E. and Taylor, H.S. (1998b) Interleukin-8 in the human endometrium. J. Clin. Endocrinol. Metab., 83, 1783–1787.[Abstract/Free Full Text]

Barry-Kinsella, C., Sharma, S.C., Cottell, E. and Harrison, R.F. (1994) Mid to late luteal phase steroids in minimal stage endometriosis and unexplained infertility. Eur. J. Obstet. Gynecol. Reprod. Biol., 54, 113–118.[ISI][Medline]

Bouckaert, P.X., Evers, J.L., Doesburg, W.H., Schellekens, L.A. and Rolland, R. (1986) Patterns of changes in glycoproteins, polypeptides, and steroids in the peritoneal fluid of women during the periovulatory phase of the menstrual cycle. J. Clin. Endocrinol. Metab., 62, 293–299.[Abstract]

Büscher, U., Chen, F.C.K., Kentenich, H. and Schmiady, H. (1999) Cytokines in the follicular fluid of stimulated and non-stimulated human ovaries; is ovulation a suppressed inflammatory reaction? Hum. Reprod., 14, 162–166.[Abstract/Free Full Text]

Chew, P.C., Loganath, A., Gunasegaram, R., Peh, K.L., Kottegoda, S.R. and Ratnam, S.S. (1987) A comparative study of peritoneal fluid concentration of oestradiol-17 beta, progesterone, and testosterone in controls and patients with endometriosis. Ann. Acad. Med. Singapore, 16, 344–346.[Medline]

De Leon, F.D., Vijayakumar, R., Brown, M., Rao, C.V., Yussman, M.A. and Schultz, G. (1986) Peritoneal fluid volume, estrogens, progesterone, prostaglandin, and epidermal growth factor concentrations in patients with and without endometriosis. Obstet. Gynecol., 68, 189–194.[Abstract]

Garcia-Velasco, J.A. and Arici, A. (1999) Interleukin-8 stimulates the adhesion of endometrial stromal cells to fibronectin. Fertil. Steril., 72, 336–340.[CrossRef][ISI][Medline]

Gazvani, M.R., Christmas, S., Quenby, S., Kirwan, J., Johnson, P.M. and Kingsland, C.R. (1998) Peritoneal fluid concentrations of interleukin-8 in women with endometriosis: relationship to stage of the disease. Hum. Reprod., 13, 1957–1961.[Abstract]

Gazvani, R., Smith, L. and Fowler, P.A. (2002) Effect of interleukin-8 (IL-8), anti-IL-8, and IL-12 on endometrial cell survival in combined endometrial gland and stromal cell cultures derived from women with and without endometriosis. Fertil. Steril., 77, 62–67.[CrossRef][ISI][Medline]

Iwabe, T., Harada, T., Tsudo, T., Tanikawa, M., Onohara, Y. and Terakawa, N. (1998) Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil. Steril., 69, 924–930.[CrossRef][ISI][Medline]

Jansen, R.P. and Russell, P. (1986) Nonpigmentated endometriosis: clinical, laparoscopic, and pathologic definition. Am. J. Obstet. Gynecol., 155, 1154–1159.[ISI][Medline]

Kelly, R.W., Illingworth, P., Baldie, G., Leask, R., Brouwer, S. and Calder, A.A. (1994) Progesterone control of interleukin-8 production in endometrium and chorio-decidual cells underline the role of neutrophil in menstruation and parturition. Hum. Reprod., 9, 253–258.[Abstract]

Koch, A.E., Polverini, P.J., Kunkel, S.L., Harlow, L.A., DiPietro, L.A., Elner, V.M., Elner, S.G. and Strieter, R.M. (1992) Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science, 258, 1798–1801.[ISI][Medline]

Koninckx, P., Heyns, W., Verhoeven, G., Van Baelen, H., Lissens, W.D., De Moor, P. and Brosens, I.A. (1980) Biochemical characterization of peritoneal fluid in women during the menstrual cycle. J. Clin. Endocrinol. Metab., 51, 1239–1244.[Abstract]

Larsen, C.G., Anderson, A.O., Oppenheim, J.J. and Matsushima, K. (1989) Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumor necrosis factor. Immunology, 68, 31–36.[ISI][Medline]

Maas, J.W.M., Calhaz-Jorge, C., Riet, G., Dunselman, G.A.J., Goeij, A.F.P.M. and Struijker-Boudier, H.A.J. (2001) Tumor necrosis factor á but not interleukin-1â or interleukin-8 concentrations correlate with angiogenic activity of peritoneal fluid from patients with minimal to mild endometriosis. Fertil. Steril., 75, 180–185.[CrossRef][ISI][Medline]

Matsuzaki, S., Canis, M., Darcha, C., Dechelotte, P., Pouly, J.L. and Bruhat, M.A. (1998) Angiogenesis in endometriosis. Gynecol. Obstet. Invest., 46, 111–115.[CrossRef][ISI][Medline]

Nisolle, M., Paindaveine, B., Bourdon, A., Berlière, M., Casanas-Roux, F. and Donnez, J. (1990) Histologic study of peritoneal endometriosis in infertile women. Fertil. Steril., 53, 984–988.[ISI][Medline]

Nisolle, M., Casanas-Roux, F., Anaf, V., Mine, J.M. and Donnez, J. (1993) Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil. Steril., 59, 681–684.[ISI][Medline]

Nisolle, M., Casanas-Roux, F. and Donnez, J. (1997) Immunohistochemical analysis of proliferative activity and steroid receptor expression in peritoneal and ovarian endometriosis. Fertil. Steril., 68, 912–919.[CrossRef][ISI][Medline]

Noble, L.S., Simpson, E.R., Johns, A. and Bulun, S.E. (1996) Aromatase expression in endometriosis. J. Clin. Endocrinol. Metab., 81, 174–179.[Abstract]

Oosterlynck, D.J., Meuleman, C., Sobis, H., Vandeputte, M. and Koninckx, P.R. (1993) Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil. Steril., 59, 778–782.[ISI][Medline]

Rana, N., Braun, D.P., House, R., Gebel, H., Rotman, C. and Dmowski, W.P. (1996) Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil. Steril., 65, 925–930.[ISI][Medline]

Ryan, I.P., Tseng, J.F., Schriock, E.D., Khorram, O., Landers, D.V. and Taylor, R.N. (1995) Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil. Steril., 63, 929–932.[ISI][Medline]

Sampson, J.A. (1927) Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am. J. Obstet. Gynecol., 14, 422–469.

Smith, R.E., Hogaboam, C.M., Strieter, R.M., Lukacs, N.W. and Kunkel, S.L. (1997) Cell-to-cell and cell-to-matrix interactions mediate chemokine expression: an important component of the inflammatory lesion. J. Leukoc. Biol., 62, 612–619.[Abstract]

Topley, N., Brown, Z., Jorres, A., Westwick, J., Davies, M., Coles, G.A. and Williams, J.D. (1993) Human peritoneal mesothelial cells synthesize interleukin-8. Synergistic induction by interleukin-1 beta and tumor necrosis factor-alpha. Am. J. Pathol., 142, 1876–1886.[Abstract]

Walter, A.J., Hentz, J.G., Magtibay, P.M., Cornella, J.L. and Magrina, J.F. (2001) Endometriosis: correlation between histologic and visual findings at laparoscopy. Am. J. Obstet. Gynecol., 184, 1407–1413.[CrossRef][ISI][Medline]

Yoshimura, T.K., Matsushima, K., Oppenheim, J.J. and Leonard, E.J. (1987) Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin-1 (IL-1). J. Immunol., 139, 788–793.[Abstract/Free Full Text]

Submitted on May 23, 2002; resubmitted on November 13, 2002; accepted on November 21, 2002.