Elevated levels of macrophage migration inhibitory factor in the peripheral blood of women with endometriosis

Elevated levels of macrophage migration inhibitory factor in the peripheral blood of women with endometriosis Mathieu Morin, B.Sc.,a Christian Bellehumeur, M.Sc.,a Marie-Josée Therriault, M.Sc.,a Christine Metz, Ph.D.,b Rodolphe Maheux, M.D.,a and Ali Akoum, Ph.D.a a

Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec (CHUQ), Faculté de Médecine, Université Laval, Québec, Québec, Canada; and b North Shore-Long Island Jewish Research Institute, Manhasset, New York

Objective: To evaluate the concentrations of macrophage migration inhibitory factor (MIF) in the peripheral blood of normal women and patients with endometriosis. Design: Retrospective study using ELISA to measure peripheral blood MIF. Setting: Gynecology clinic and human reproduction research laboratory. Patient(s): Thirty-eight normal women and 55 women with endometriosis. Intervention(s): Peripheral blood samples were obtained a few days before laparoscopy. Main Outcome Measure(s): The MIF concentrations in blood serum. Result(s): This current study showed a 364% increase in MIF concentrations in women with endometriosis as compared to normal women. A significant increase was seen in endometriosis stages I–II, but a more marked increase was observed in the more advanced stages of the disease (III–IV). Both fertile and infertile women with endometriosis had higher levels of MIF than normal controls, but the difference was more significant in infertile women with endometriosis. Women with endometriosis with no pelvic pain had higher levels of MIF than normal controls, but a more significant increase in MIF levels was observed in women with endometriosis reporting pelvic pain. Conclusion(s): This study showed a marked increase in MIF concentrations in the peripheral blood of women with endometriosis and a relationship with disease progress, and suggests that MIF may be involved in endometriosis-related pain and infertility. (Fertil Steril威 2005;83:865–72. ©2005 by American Society for Reproductive Medicine.) Key Words: Endometriosis, infertility, MIF, pain, peripheral blood

Endometriosis is a gynecological disorder characterized by the ectopic growth of tissue similar to the endometrium, mainly on the peritoneum and the organs of the pelvic cavity. A growing body of evidence indicates that aberrant immune responses may be involved in the development of endometriosis (1). Immunological factors may affect the exfoliated endometrial tissue’s likelihood to implant and play an important role in the manifestation of endometriosis-related clinical symptoms such as pain, menstrual disorders, and infertility (1).

prevent the elimination of misplaced endometrial cells. An increased number and activation of peritoneal macrophages has also been described (6, 7), but instead of eliminating regurgitated blood and endometrial cells, macrophages appeared to have a deficient phagocytotic function (8, 9) and secrete numerous growth and angiogenic factors that may favor the ectopic growth of endometrial tissue (10 –12).

Locally, an immunoinflammatory process involving leukocyte recruitment and activation is taking place (2). Reduced cytotoxicity of natural killer (NK) cells has been reported (3–5) and it is believed that such defects may

Endometriotic lesions are likely to contribute to the modulation of these immunological reactions as they have been shown to produce immunosuppressive factors, which can stimulate leukocyte recruitment and activation (1–13). Cyclical reflux of menstrual debris in the peritoneal cavity may also occur, thereby exacerbating the inflammatory reaction (14).

Received May 10, 2004; revised and accepted October 8, 2004. Mathieu Morin and Christian Bellehumeur have equally contributed to this work. Supported by grant MOP-37921 to Ali Akoum from The Canadian Institutes for Health Research. A.A. is a “Chercheur-Boursier National” of the “Fonds de la Recherche en Santé du Québec (FRSQ).” Reprint requests: Ali Akoum, Ph.D., Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, 10 rue de l’Espinay, Local D0-711, Québec, Québec, Canada, G1L 3L5 (FAX: 418-525-4481; E-mail: [email protected]).

Alterations in immune functions observed in patients having endometriosis are not restricted to the peritoneal cavity, where the disease is frequently found. Systemic alterations at both humoral and cellular levels have been reported, including elevated levels of antibodies specific to endometrial antigens (15, 16), reduced cytotoxic effects of lymphocytes on autologous endometrial cells, and increased activation of peripheral blood monocytes (17, 18). These cells play a central role in the maintenance of humoral- and cell-

0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2004.10.039

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mediated immunity and by means of numerous secretory products they can play a significant role in the pathogenesis of endometriosis. Subsequent studies showed that peripheral blood monocytes in women with endometriosis secrete elevated levels of proinflammatory mediators such as interleukin (IL)-1 and displayed the ability to stimulate endometrial cell growth in vitro, whereas the monocytes found in normal fertile women suppress the proliferation of these cells (18, 19). We have recently reported that endometriotic lesions, particularly those that are highly vascularized and considered as the earliest and most active stages of the disease, markedly express the macrophage migration inhibitory factor (MIF) (20). We further showed elevated concentrations of MIF in the peritoneal fluid (PF) of patients with endometriosis (21). Originally described as a T-cell derived cytokine that inhibits the random migration of macrophages, MIF is now recognized to be a multifunctional cytokine that modulates the immune response, cell proliferation, and angiogenesis (22–24). The aim of this study was therefore to evaluate MIF levels in the peripheral blood of women with and without endometriosis. In the following study, we report a marked increase in MIF concentrations in the peripheral blood of women with endometriosis and show that they vary according to the disease stage, pelvic pain, and infertility. MATERIALS AND METHODS Subjects Women were recruited into the study after providing their informed consent for a protocol approved by the SaintFrançois d’Assise hospital ethics board. Subjects with endometriosis (n ⫽ 55) were identified after undergoing a laparoscopy for infertility, pelvic pain, or tubal ligation. These women displayed no other pelvic pathology and had not taken any anti-inflammatory or hormone medication at least 3 months before laparoscopy. The stage of endometriosis was determined according to the revised classification of the American Fertility Society (AFS). Control subjects (n ⫽ 38) were fertile women requesting tubal ligation or reanastomosis and having no visible evidence of endometriosis at laparoscopy. Dating of the menstrual cycle was determined according to the regularity of the cycle, the date of the previous menses, and serum levels of P. The main clinical parameters listed in Table 1 include age, infertility, cycle phase, and stage of endometriosis. Collection and Processing of Blood Samples Blood samples were drawn a few days before laparoscopy. For MIF assays, blood was collected in sterile tubes containing ethylenediaminetetracetic acid (EDTA), immediately centrifuged at 2,000 g for 10 minutes at 4°C, and the serum aliquoted and stored at ⫺80°C until assayed. For hormonal assays, blood was collected in red-top tubes and sent to the biochemistry laboratory for current steroid determination. 866

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TABLE 1 Clinical characteristics of patients at the time of laparoscopy.

Controls Endometriosis Stages I–II Stages III–IV Fertile Infertile Without pain With pain Follicular phase Controlsa Endometriosis Luteal phase Controlsa Endometriosis a

No. of patients

Age (yr) (mean ⴞ SD)

38 55 36 19 21 34 13 42

36.7 ⫾ 6.2 33.6 ⫾ 4.7 33.0 ⫾ 5.2 34.3 ⫾ 4.1 36.0 ⫾ 4.8 31.3 ⫾ 3.7 34.6 ⫾ 5.4 32.7 ⫾ 4.9

19 28

35.2 ⫾ 6.1 32.7 ⫾ 5.2

18 27

37.8 ⫾ 6.1 34.6 ⫾ 4.4

The cycle phase for 1 control subject was unknown.

Morin. Peripheral blood levels of MIF in endometriosis. Fertil Steril 2005.

MIF ELISA The MIF concentration was measured by ELISA based on a previously reported procedure (21). Briefly, this technique uses a captured mouse’s monoclonal antihuman MIF antibody (R&D Systems, Minneapolis, MN), a rabbit’s polyclonal antihuman MIF antibody for detection, a goat’s antirabbit alkaline phosphatase-conjugated IgG (Chemicon International Inc, Temecula, CA), and paranitrophenyl phosphate as substrate (Sigma-Aldrich Canada LTD, Oakville, ON, Canada). The optical density (OD) was measured at 405 nm and MIF concentrations were extrapolated from a standard curve using recombinant human MIF. Samples in the MIF ELISA assay were run in duplicate. The interassay coefficient of variation (CV) was 2.9%, and the intra-assay CV was 3.8%. E2 and P Assays Serum E2 and P were measured by competitive immunoassays based on antibody-coated tubes (commercial kits, CoatA-Count; Diagnostic Products Corp., Los Angeles, CA). The intra-assay coeficients of variation measured at low, medium, and high levels of the standard curves were between 1.8% and 8.0% for all the immunoassays. The interassay coeficients of variation were less than 8.0% for E2 and 10% for P. Statistical Analyses The MIF concentrations found in the sera followed a parametric distribution and were therefore expressed as a mean

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⫾ SEM. An unpaired t test was used for comparing those means and the Bonferroni correction procedure was applied whenever more than two groups were compared using GraphPad Software, Prism 3.0 (GraphPad Software, San Diego, CA). An optimal cutoff value for MIF concentrations was selected using receiver operator curves (ROC). Statistical analysis was performed using Fisher’s exact test. The comparison of the patient’s age between the different groups was performed using an unpaired t test. Differences were considered as statistically significant for two-tailed P values ⬍.05.

FIGURE 1 Distribution of migration inhibitory factor (MIF) concentrations in the peripheral blood of normal controls (n ⫽ 38) and patients with endometriosis (n ⫽ 55) according to the stage of the disease. The horizontal lines represent the mean for each set of data. The dotted line represents the cutoff value (0.57 ng/mL).

RESULTS The distribution of MIF concentrations in normal and endometriosis women (stages I–II and III–IV) is shown in Figure 1. Statistical analysis of the data revealed that MIF concentrations were markedly higher in patients with endometriosis as compared with normal controls, increasing by 364% (P⬍.0001). A significant increase was seen in endometriosis stages I–II (P⬍.01), but a more marked increase was observed in the more advanced stages of the disease (III–IV) (P⬍.0001). Morin. Peripheral blood levels of MIF in endometriosis. Fertil Steril 2005.

Considering that endometriotic lesions are influenced by cyclic changes in ovarian sex steroids, we examined whether peripheral blood MIF concentrations varied according to the phase of the menstrual cycle. Our data

showed no difference in MIF concentrations between the follicular and the luteal phases within the control or the

TABLE 2 Peripheral blood concentrations of migration inhibitory factor (MIF).

Controls Endometriosis Stages I–II Stages III–IV Fertile Infertile Without pain With pain Follicular phase Controlsc Endometriosis Luteal phase Controlsc Endometriosis

No. of patients

Peripheral blood concentration of MIFa (ng/mL)

P valueb

38 55 36 19 21 34 13 42

0.4529 ⫾ 0.08196 1.9680 ⫾ 0.3395 1.3580 ⫾ 0.2690 3.1234 ⫾ 0.7880 1.4770 ⫾ 0.3901 2.2710 ⫾ 0.4908 1.6130 ⫾ 0.6045 2.080 ⫾ 0.4053

⬍.0001 .0030 ⬍.0001 .0032 .0006 .0064 .0006

19 28

0.4234 ⫾ 0.1092 1.5550 ⫾ 0.4216

.0288

18 27

0.5092 ⫾ 0.1290 2.4200 ⫾ 0.5278

.0030

Values are means ⫾ SEM. b Versus controls with t test and Bonferroni correction. c The cycle phase for 1 control subject was unknown. a

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FIGURE 2 Distribution of migration inhibitory factor (MIF) concentrations in the peripheral blood of normal controls (n ⫽ 38) and patients with endometriosis (n ⫽ 55) according to the menstrual cycle phase. The horizontal lines represent the mean for each set of data. The dotted line represents the cutoff value (0.57 ng/mL).

secretory (P⬍.01) than in the follicular phase (P⬍.05). Graphic distribution of MIF concentrations, based on the menstrual cycle phase, is shown in Figure 2. Estradiol and P levels showed no significant difference between women with and without endometriosis. Moreover, no statistically significant correlation (Pearson’s correlation coefficient) between MIF concentrations and E2 or P levels in normal controls or women with endometriosis was noted. The MIF concentrations were then analyzed according to the infertility status of endometriosis patients and the presence or absence of pelvic pain. Both fertile and infertile women with endometriosis had higher levels of MIF than normal controls (P⬍.01, and P⬍.001, respectively), but the difference was more significant in infertile women with endometriosis (Table 2).

Morin. Peripheral blood levels of MIF in endometriosis. Fertil Steril 2005.

endometriosis group (Table 2). However, the increased concentration of MIF in women with endometriosis as compared to normal controls was more significant in the

FIGURE 3 Distribution of migration inhibitory factor (MIF) concentrations in the peripheral blood of normal controls (n ⫽ 38) and patients with endometriosis (n ⫽ 55) according to their fertility status. The horizontal lines represent the mean for each set of data. The dotted line represents the cutoff value (0.57 ng/mL).

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Endometriosis women with no pelvic pain had higher levels of MIF than normal controls (P⬍.01), but a more significant increase in MIF levels was observed in women with endometriosis reporting pelvic pain (P⬍.001) (Table 2). On the other hand, only three patients with endometriosis were infertile and had no pelvic pain, thereby limiting the assessment of data based on the infertility status and pelvic pain simultaneously and therefore reducing statistical power. Nevertheless, all endometriosis groups (fertile and infertile women with and without pain) had significantly higher MIF concentrations than the control group (P⬍.05), but the highest MIF levels were found in women with pelvic pain and infertility (P⬍.01). Graphic distribution of MIF concentrations based on endometriosis-related infertility and pelvic pain are shown in Figures 3 and 4, respectively. No significant difference in E2 or P concentrations was found between normal controls and women with endometriosis, neither in the follicular nor in the luteal phase of the menstrual cycle (Table 3). In addition, E2 and P levels did not significantly vary according to the fertility status of endometriosis patients or the presence or absence of pelvic pain. The optimal cutoff value for MIF concentrations was 0.57 ng/mL. Twenty-five of 38 control subjects (66%) had MIF concentrations ⬍057 ng/mL, whereas 36 of 55 patients with endometriosis (66%) had MIF concentrations ⬎0.57 ng/mL (P⬍.01). The positive and negative predictive values for this cutoff were 0.57 and 0.74, respectively (Table 4). DISCUSSION In the present study, we have shown that women with endometriosis had higher circulating levels of MIF as compared to normal women having a normal gynecological status at laparoscopy. A significant increase was observed in earlier endometriosis stages (revised classification of the AFS stages I–II), but the most significant elevation of MIF levels was found at the advanced stages of the disease (revised classification of the AFS stages III–IV). This indi-

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biological properties of MIF are consistent with the nature of endometriosis, a disease characterized by an abnormal growth of endometrial cells outside the uterus and a chronic immunoinflammatory reaction, which is frequently observed at the local and systemic levels (1, 13, 25).

FIGURE 4 Distribution of migration inhibitory factor (MIF) concentrations in the peripheral blood of normal controls (n ⫽ 38) and patients with endometriosis (n ⫽ 55) according to the absence or presence of pelvic pain. The horizontal lines represent the mean for each set of data. The dotted line represents the cutoff value (0.57 ng/mL).

Thus, recent data showed an important role for MIF in tumor growth-related angiogenesis in in vivo and in vitro autocrine regulation of endothelial cell proliferation (26, 27). This is consistent with our recent findings, which identified MIF as an important mediator of endothelial cell proliferation released by ectopic endometrial cells (28), showed peritoneal MIF involvement in endothelial cell proliferation, and revealed a marked elevation of MIF concentrations in the PF of patients with endometriosis as well as in active and highly vascularized endometriotic lesions (20, 21). The MIF has also been shown to override the immunosuppressive effects of glucocorticoids on immune cells (29) and to activate T lymphocytes, monocytes, and macrophages (30, 31).

Morin. Peripheral blood levels of MIF in endometriosis. Fertil Steril 2005.

cates a close relationship with the severity of endometriosis and suggests a role for MIF in the progression of the disease. These findings may have an interesting pathophysiologic relevance. First described as a product of activated T lymphocytes that inhibits the random migration of cultured macrophages, recent findings indicate that MIF is rather a multifunctional cytokine endowed with potent mitogenic, immunomodulatory, and proinflammatory properties and show the involvement of this factor in neoplastic and immunoinflammatory disorders (20 –24). These newly discovered

In endometriosis, there is an increased number of activated macrophages in the PF and within endometriotic implants (6, 7, 11, 32). Peripheral blood monocytes of women having endometriosis demonstrate an increased activation as well, and a higher cytotoxicity to culture cell lines as compared to those of normal fertile controls (33, 34). Therefore, our data make possible MIF involvement in the activation of monocytes/macrophages both at the local and the systemic levels. At the present time, it is not clearly determined how monocytes could be involved in the pathogenesis of endometriosis. We do know, however, that they secrete numerous growth factors that can promote endometrial cell proliferation and cytokines believed to play a role in the pathogenesis of the disease and the manifestation of its clinical symptoms (18, 19). Interestingly, monocytes from patients with endometriosis have been shown to stimulate endometrial cell proliferation in vitro, whereas those of normal fertile women suppress the

TABLE 3 E2 and P levels in the serum of patients.

Follicular phase Controls Endometriosis subjects Luteal phase Controls Endometriosis subjects a b

No. of patients

E2 (pmol/L)a,b

No. of patients

P (nmol/L)a,b

18 27

214.2222 ⫾ 27.2032 356.4074 ⫾ 67.0949

19 28

3.3000 ⫾ 0.8231 2.2407 ⫾ 0.4910

18 27

500.3889 ⫾ 171.7706 302.5555 ⫾ 34.1878

18 27

18.3889 ⫾ 2.6337 23.4148 ⫾ 3.6491

Values are means ⫾ SEM. E2 levels were not available for 2 control and 1 endometriosis women, and P levels were not available for 1 control woman.

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eration, immunoglobulin production, and monocyte/macrophage activation (30, 44).

TABLE 4 Distribution of patients according to serum migration inhibitory factor (MIF) cutoff value. MIF cutoff value (ng/mL) Number of endometriosis patients Number of control patients

⬍0.57 19

⬎0.57 36

25

13

The mechanisms underlying MIF involvement in endometriosis-associated pelvic pain remain to be further elucidated. Possible mechanisms may involve the release of pain mediators such as prostaglandins, which are known to be induced by MIF in other cell types (45). Research has been conducted in our laboratory to investigate such a possible mechanism.

Morin. Peripheral blood levels of MIF in endometriosis. Fertil Steril 2005.

proliferation of endometrial cells (19). Accordingly, it could be suggested that MIF may favor the ectopic growth of endometrial cells either directly through its proangiogenic properties or indirectly by its modulatory effects on immune cells. In agreement with this assertion is the ability of MIF to inhibit the activity of NK cells (35). This is particularly relevant, as a reduced NK activity toward autologous endometrial cells has consistently been reported in women with endometriosis (3–5). This may represent another plausible mechanism by which MIF may contribute to ectopic endometrial cell growth. An interesting finding of the present study is that MIF concentrations were more significantly increased in infertile than in fertile women with endometriosis as compared to normal women. This is consistent with our previous findings showing a more marked increase in MIF levels in the PF of infertile women with endometriosis (21) and provides further evidence for a possible role for MIF in endometriosisrelated infertility. Our data also showed that MIF concentrations were more significantly increased in women with endometriosis having pelvic pain, therefore suggesting a role for MIF in endometriosis-associated pain. There is substantial evidence that immunological alterations may play a role in endometriosis-related infertility (1, 2, 13). Some studies showed an impairment of implantation in patients with endometriosis (36, 37). Other investigators demonstrated that PF from women with endometriosis inhibits oocyte fertilization in vitro (38), and that macrophages phagocyte spermatozoa in vitro (39) and secrete numerous embryotoxic cytokines that may adversely affect the fecundity of women with endometriosis (40, 41). Infertility might also be due to endometriosis-related systemic changes as higher levels of autoantibodies specific to endometrial antigens were found in the peripheral blood of women having endometriosis (16, 42), and sera from patients with endometriosis adversely affect mouse embryo development in vitro (43). However, the identity of the circulating soluble factors involved remained unknown. It is still to be demonstrated whether MIF is able to exert direct embryotoxic effects. Nevertheless, one could speculate that MIF may adversely affect infertility in women with endometriosis because of its role in antigen-driven T-cell prolif870

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It is difficult to ascertain what would contribute to the increased concentration of MIF in the peripheral blood of women with endometriosis. Numerous cell types are known to synthesize and secrete MIF (44). In endometriosis, activated monocytes, which are known to secrete large amounts of MIF, may account for the increased concentration of this factor in the peripheral circulation. Our previous studies showed MIF to be mainly located in endometriotic glands and identified endothelial cells, macrophages, and T lymphocytes as cells markedly expressing MIF (20). Furthermore, MIF was found to be highly produced in the endometriotic lesions that were presenting noticeable vascularization and leukocyte infiltration (20), thereby making it plausible that these cells possibly contribute to MIF secretion in the serum of patients. The most significant elevation of circulating MIF in patients with endometriosis as compared to normal subjects was observed in the luteal phase of the menstrual cycle. However, statistical analysis of the results failed to demonstrate any significant difference in MIF levels between follicular and luteal phases within each control or endometriosis group, nor was there any correlation with the level of E2 or P found in the serum of patients. These results seem to rule out any hormonal modulation of MIF levels in the peripheral blood as it would have been expected on the basis of previous studies reporting high levels of human serum proinflammatory cytokines according to the menstrual cycle (46) and reduced serum concentrations of inflammatory and growth factors after hormone treatment in patients with endometriosis (47, 48). It is still, however, to be determined whether circulating MIF levels varied with hormone therapy in patients with endometriosis. Other growth and proinflammatory factors with growth or immunomodulatory properties such as vascular endothelial cell growth factor, IL-8, tumor necrosis factor (TNF)-␣, and monocyte chemotactic protein-1 have been shown to increase in the peripheral blood fluid of women with endometriosis (49, 50). The relationship between these factors and MIF and the mechanisms underlying such a systemic inflammatory process remains unknown. However, it is noteworthy that MIF was shown to override the glucocorticoid inhibition in monocyte secretions of TNF-␣, IL-1␤, IL-6, and IL-8 (29 –31), which further emphasizes the role of this factor in endometriosis-related inflammatory responses.

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In conclusion, our results demonstrate for the first time that MIF concentration is significantly increased in the peripheral blood of patients suffering from endometriosis and varied according to the severity of the disease and the infertility status of the patients. This is in keeping with our previous findings with regard to PF and makes plausible MIF as a key effector cell mediator involved in the pathophysiology of endometriosis through its proinflammatory and angiogenic properties. Acknowledgments: The authors hereby wish to thank François Belhumeur, M.D., Jacques Bergeron, M.D., Jean Blanchet, M.D., Marc Bureau, M.D., Simon Carrier, M.D., Elphège Cyr, M.D., Marléne Daris, M.D., Jean-Louis Dubé, M.D., Jean-Yves Fontaine, M.D., Céline Huot, M.D., Pierre Huot, M.D., Johanne Hurtubise, M.D., Jacques Mailoux, M.D., and Marc Villeneuve, M.D., for patient evaluation and providing peritoneal fluid samples; Madeleine Desaulniers, Monique Longpré, and Johanne Pelletier for technical assistance; and Lucile Turcot-Lemay, M.D., Ph.D., for her assistance with the statistical analyses.

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