Pain, mast cells, and nerves in peritoneal, ovarian, and deep infiltrating endometriosis

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1 ENDOMETRIOSIS Pain, mast cells, and nerves in peritoneal, ovarian, and deep infiltrating endometriosis Vincent Anaf, Ph.D., a Charles Chapron, M.D., d Issam El Nakadi, M.D., b Veronique De Moor, M.D., b Thierry Simonart, Ph.D., a and Jean-Christophe Noël, Ph.D. c a Department of Gynaecology, b Department of Digestive Surgery, and c Department of Pathology, Academic Hospital Erasme, Free University of Brussels, Brussels, Belgium; and d Department of Gynaecology, Université René Descartes (Paris V), Faculté de Médecine, Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Universitaire (GHU) Ouest, Service de Gynécologie Obstétrique II (Pr Chapron), Unité de chirurgie gynécologique, Clinique Universitaire Baudelocque, CHU Cochin, Paris, France Objective: To detect and quantify mast cells in peritoneal, ovarian, and deep infiltrating endometriosis and to study the relationship between mast cells and nerves in endometriosis. Design: Prospective histological and immunohistochemical study. Setting: University of Brussels, Belgium. Patient(s): Sixty-nine women undergoing laparoscopic excision of endometriosis for pain. Thirty-seven biopsies of normal tissue were obtained from women without endometriosis. Intervention(s): Excision of endometriosis from different anatomical locations. Main Outcome Measure(s): Immunohistochemistry with chymase and tryptase to confirm the presence of mast cells and activated mast cells, respectively, in endometriotic lesions. Quantification of mast cells, activated mast cells, and degranulating mast cells in the different locations of endometriosis. Study of the relationship between mast cells and nerves by quantifying mast cells located less than 25 m from nerves immunohistochemically stained with S-100 protein. Preoperative pain score evaluation by visual analogue scales. Result(s): Patients with deeply infiltrating lesions had significantly higher preoperative pain scores than patients with peritoneal or ovarian endometriosis. Mast cells and degranulating mast cells are significantly more abundant in endometriotic lesions than in nonaffected tissues. Deep infiltrating lesions show a significantly higher number of mast cells, activated mast cells, and mast cells located 25 m from nerves than peritoneal and ovarian lesions. We found significantly more degranulating mast cells in deep infiltrating lesions than in peritoneal lesions. Conclusion(s): The presence of increased activated and degranulating mast cells in deeply infiltrating endometriosis, which are the most painful lesions, and the close histological relationship between mast cells and nerves strongly suggest that mast cells could contribute to the development of pain and hyperalgesia in endometriosis, possibly by a direct effect on nerve structures. (Fertil Steril 2006;86: by American Society for Reproductive Medicine.) Key Words: Pain, endometriosis, mast cells, nerves, deep infiltrating endometriosis The most frequent complaints among women with endometriosis are dysmenorrhea, deep dyspareunia, and pelvic pain. Although peritoneal or ovarian endometriosis can be coincidentally diagnosed at laparoscopy in a woman without pain symptoms, deep infiltrating endometriosis is strongly associated with pain (1, 2). Deep infiltrating pelvic endometriosis is less frequent than peritoneal and ovarian endometriosis, but it can cause severe pain and discomfort and it can severely alter a patient s quality of life and sex life (3 5). The mechanisms by which deep infiltrating lesions cause pain and very often hyperalgesia are poorly understood. Most October 25, 2005; revised and accepted March 14, Reprint requests: Vincent Anaf, Department of Gynaecology, Hospital Erasme, 808 Route de Lennik, 1070 Brussels, Belgium (FAX: ; vincent.anaf@ulb.ac.be). probably the cause of pain in endometriosis is multifactorial (6). Deep lesions generally contain clusters of hemorrhages and hemosiderin-laden macrophages. Cyclical bleedings within the lesions can be responsible for hyperpressure and pain. This could explain why medical therapy inducing an amenorrhea (GnRh agonists, continued regimen of progestins, etc.) at least partially relieves a patient s symptoms. In severe cases, the rectum or rectosigmoid junction can be attached of fixed to the posterior aspect of the cervix or to the posterior vaginal wall. This fixed position of posterior pelvic structures (cervix, upper vagina, rectum) could also be a cause of pain occurring at defecation or during sexual intercourse. It has also been demonstrated that endometriosis itself can produce and release prostaglandins, inflammatory mediators such as kinins, histamine, interleukins, which can by themselves stimulate sensory nerve endings. Production 1336 Fertility and Sterility Vol. 86, No. 5, Month /06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 FIGURE 1 S-100 immunohistochemistry in a deep infiltrating endometriotic lesion. Note the close histological relationship between the nerve and the endometriotic stromal cells (the nerve is stained in brown). The arrows show the presence of stromal cells in the nerve structure. MATERIALS AND METHODS Patients Sixty-nine patients presenting with pain (mean age, 28 years 6 months) underwent laparoscopy or laparotomy for excision of endometriosis. Twenty-six patients presented with peritoneal and/or ovarian endometriosis. Fifteen patients presented with deep uterosacral nodules, 12 with a vaginal Douglas pouch endometriotic nodule, and 16 with large bowel deep endometriosis. Five different anatomic locations of endometriosis were studied (peritoneum, n 15; ovary, n 11; uterosacral ligaments, n 15; vaginal Douglas pouch, n 12; rectosigmoid, n 16). Vaginal Douglas pouch lesions correspond to nodular or indurated endometriotic lesions involving the Douglas pouch and the upper third of the posterior vagina but without involvement of the rectosigmoid junction or anterior rectal wall (17). and release of prostaglandins by endometriosis and inflammatory cells can explain the analgesic effect of nonsteroidal antiinflammatory drugs (7). It has been demonstrated that there is a close histological relationship between deep endometriotic lesions and nerves (Fig. 1) by means of perineural and intraneural invasion (mechanical nerve injury by nerve invasion) and that patients with the highest pain scores display significantly more neural invasion by endometriosis than patients with lower pain scores (8). Nerve growth factor (NGF), which plays a key role in the occurrence of pain, hyperalgesia, and neuropathic pain, is strongly expressed in deep infiltrating endometriosis, and its specific receptor (Trk-A) is expressed in nerves lying within deep lesions or in the vicinity of deep endometriotic lesions (9). There is cumulative evidence that mast cells play an important role in the pathogenesis of chronic pain and neuropathic pain in many pathological conditions (10 15). Mast cells can not only release mediators that increase excitability of neurons, but in turn, neurotransmitters such as substance P or NGF can trigger mast cell degranulation (16). To our knowledge this is the first study on mast cells and nerves in endometriosis and deep infiltrating endometriosis. In this work, we prospectively studied the presence, abundance, and number of degranulating mast cells in peritoneal, ovarian, and deep infiltrating endometriotic lesions. We also studied the histological relationship between mast cells and nerves in these lesions. Subjects were nonmenopausal women who were not currently under hormone treatment and who had stopped hormone treatment for 2 months. All patients were operated during the secretory phase between day 20 and day 23. Patients who were currently being treated for psychiatric or neurological disease and patients taking psychotropic drugs or antiepileptics were excluded. All patients who had another potential cause of chronic pain (chronic lumbar pain, arthritis, etc.) were also excluded from the study. Patients were asked not to take analgesics (paracetamol, nonsteroidal antiinflammatory drugs, etc.) antihistamines, or sodium cromoglycate from the first day of the cycle of the surgery. Controls were biopsies of normal tissues from patients without endometriosis. Biopsies of normal peritoneum (n 7) and ovary (n 8) were obtained from nonmenopausal women without endometriosis who underwent laparoscopic tubal ligation. Vaginal (n 6) and uterosacral ligament (n 8) biopsies were small biopsies 1cm 2 sampled in nonmenopausal women who underwent laparoscopic hysterectomy for uterine leiomyomas. Full thickness large bowel biopsies (n 8) originated from nonmenopausal women who underwent left colectomy for rectosigmoid adenocarcinoma. Biopsies were taken at a distance of 30 cm from the malignant lesion itself. Only consenting patients were included in the study, which was approved by the local institutional ethical committee. Histology and Immunohistochemistry After resection, all the tissues were fixed in 4% formaldehyde for 12 hours and then embedded in paraffin. All tissues were stained with hematoxylin and eosin to confirm the diagnosis of endometriosis. To identify granulated and degranulated mast cells, tissue sections were first stained with toluidine blue as described elsewhere (18). Immunohistochemistry was also applied on tissue sections with monoclonal antibodies against chymase and tryptase. Chymase is a significant marker for human mast cells and acts as a medi- Fertility and Sterility 1337

3 ator of inflammation and tissue remodeling. It participates in matrix remodeling by activating procollagenase and controls blood flow by generating angiotensin 2. Chymase has also been shown to increase microvascular permeability and promote the accumulation of inflammatory cells. Human mast cell tryptase is an excellent marker for mast cell activation as well as being a mediator of inflammation and increases the accumulation of type 1 collagen in endometriosis. We have also studied the relationship between mast cells and nerves by using immunohistochemistry against S-100 protein, a specific marker of Schwann cells as we have described elsewhere (8, 19). In each specimen, eight serial sections of 4 m were obtained. The first section was stained with hematoxylin and eosin, and the second was stained with toluidine blue. The other sections were used for immunohistochemistry and controls. After deparaffinization in xylene and rehydration through graded concentrations of alcohol, the endogenous peroxydase activity was blocked in 0.3% H 2 O 2 in methanol for 30 minutes. For mast cell chymase, the antigen retrieval method was performed as described elsewhere (8). The normal serum was applied to minimize nonspecific reactivities. The sections were incubated at room temperature for 60 minutes with the specific monoclonal primary antibody. The characteristics and dilutions of the primary antibody were as follows: monoclonal antibody mast cell chymase (clone CC1, dilution 1/100; Novocastra Laboratories, Newcastle, United Kingdom), monoclonal antibody mast cell tryptase (clone 10 D 11, dilution 1/200; Novocastra Laboratories), and monoclonal antibody S-100 protein (clone 15 E2E2, dilution 1/100; Biogenex, San Ramon, CA). After rinsing with Tris-buffered saline (TBS), biotinylated antimouse immunoglobulin was applied for 30 minutes at room temperature. After rinsing with TBS, preformed avidin and biotinylated horseradish peroxydase macromolecular complex (Vectastain, Elite ABC kit, Vector Laboratories, Burlingame, CA) was applied for 30 minutes at room temperature. The antigen antibody reaction was visualized using diaminobenzidine. The sections were then slightly counterstained with hematoxylin, dehydrated, and coverslipped. To control for nonspecific binding of the primary antibodies, nonimmune mouse sera at the same concentration as the primary antibodies preparations were substituted as the first layers of the serial sections for mast cell chymase, mast cell tryptase, and S-100 protein immunohistochemistry. Positive controls consisted of schwannomas for S-100 protein and tonsils for mast cell chymase and tryptase. Because mast cells settle in all connective tissues it is not possible to find a human tissue that potentially contains no mast cells. Therefore, negative tissue controls were performed on hela (cervical adenocarcinoma) cultured cells, which are known to be free of mast cells. Immunohistochemistry performed on hela cultured cells was negative. Pain Evaluation All 69 patients (100%) answered mailed questionnaires in the preoperative phase. The corresponding author of the questionnaires was unknown to the patients and had not participated in the preoperative consultations or in the operations. The questionnaires were designed to obtain information about the intensity of preoperative pain using visual analogue pain scales for dysmenorrhea, deep dyspareunia, and rectalgia. The pain scales were ungraduated lines 10 cm long. No pain was indicated at the left side of the scale and the maximal pain you could imagine at the right side of the scale. Mast Cells Evaluation Mast cells were counted by two different investigators (JCN and VA) who were blinded to the site in 20 nonoverlapping randomly selected high power fields ( 400). When the difference in mean mast cell count per case between the two observers exceeded 10% (which occurred in 7.2% of cases), the cases were reevaluated using a multiheaded microscope and a final agreement was reached in all cases. To assess the state of activation of mast cells, we categorized the mast as granulated mast cells and degranulating mast cells as described elsewhere (18). Granulated mast cells are those containing densely packed cytoplasmic granules, and degranulating mast cells are those showing a typical release of granules. In addition, we investigated the spatial relationship between mast cells and nerves by counting the number of mast cells located 25 m from nerve structures as described elsewhere (20). The distance of 25 m was calculated by using a microscope grid (at 400) linked via a color CCD video camera (MW-F15-e, Panasonic, Matsushita Electrical Industrial, Osaka, Japan) to a large-screen monitor (Panasonic Quintrix; Matsushita Electrical Industrial, Pentwyn, Cardiff, United Kingdom). A grid (0.5 mm 2 ) was used to divide the endometriotic lesions in different fields. Microscopic fields were then digitized. The fields that were examined were randomly selected using a Chalkley grid (0.196 mm 2 )(21) containing 25 points that was overlaid on the digitized fields. The microscopic fields that were studied were those that were hit by one of the points of the Chalkley grid. This technique is considered to be a simple and acceptable method to randomly select fields or tissue elements for microscopic study (22). For chymase and tryptase immunohistochemistry, the results are expressed as mean number of mast cells/mm 2 SD. In addition, the results for degranulating mast cells are expressed as the mean number of degranulating mast cells/ mm 2 SD. The results for the spatial relationship between mast cells and nerves are expressed as mean number of mast cells located 25 m from nerve structures SD Anaf et al. Pain, mast cells, and endometriosis Vol. 86, No. 5, Month 2006

4 TABLE 1 Tryptase mast cells count. Peritoneum Ovary Rectovaginal septum and Douglas pouch Uterosacral ligament Large bowel E C E C E C E C E C N Mast cells SD P NS NS.001 Note: C controls; E endometriosis; NS not significant. P values obtained after Bonferroni s correction. Statistical Analysis The comparisons among the numbers of chymase-positive, tryptase-positive, degranulating mast cells, and mast cells located 25 m from nerves in endometriotic and in nonaffected tissues were performed using the Student s t-test (two-tailed). P.05 was considered statistically significant. As there were multiple comparisons and with regard to the highly significant P values reported, the P value was reported after Bonferroni s correction (Tables 1 4). Comparison of preoperative pain scores between patients with deep infiltrating endometriosis (large bowel endometriosis, uterosacral ligament nodules, vaginal Douglas pouch nodules) and patients without deep infiltrating endometriosis (peritoneal endometriosis, ovarian endometriomas) was performed using the nonparametric Wilcoxon signed rank test. RESULTS Chymase-Positive Mast Cells Count Endometriotic lesions contain significantly more chymasepositive mast cells than the unaffected tissues in all the different studied locations. Deep infiltrating lesions contain significantly more chymase-positive mast cells than peritoneal and ovarian endometriosis (P.001) (Table 4). Tryptase-Positive Mast Cells Count Endometriotic lesions contain significantly more tryptase-positive mast cells (activated) than the unaffected tissues in all the different studied locations (P.001), except for peritoneal and uterosacral lesions, where it was not significant (Table 1). Deep infiltrating lesions contain significantly more tryptase-positive mast cells than peritoneal and ovarian endometriosis except between uterosacral lesions and ovarian lesions (Table 4). Mast Cells Located <25 m from Nerve Structures Endometriotic lesions contain significantly more tryptasepositive mast cells (activated) located 25 m from nerve structures than the unaffected tissues (Table 2; Fig. 2). Deep infiltrating lesions contain significantly more mast cells in close relationship with nerves than peritoneal and ovarian endometriosis (P.001) (Table 4). TABLE 2 Number of mast cells located <25 m from nerves. Peritoneum Ovary Rectovaginal septum and Douglas pouch Uterosacral ligament Large bowel E C E C E C E C E C N Mast cells SD P Note: C controls; E endometriosis. P values obtained after Bonferroni s correction. Fertility and Sterility 1339

5 TABLE 3 Number of degranulating mast cells/mm 2. Peritoneum Ovary Rectovaginal septum and Douglas pouch Uterosacral ligament Large bowel E C E C E C E C E C N Mast cells SD P Note: C controls; E endometriosis. P values obtained after Bonferroni s correction. Degranulating Mast Cells in Endometriosis and Controls Endometriotic lesions contain significantly more degranulating mast cells than the unaffected tissues in all the different studied locations (Table 3). Deep infiltrating lesions contain significantly more degranulating mast cells than peritoneal endometriosis (P.001) but not more than ovarian endometriotic lesions (Table 4). Pain Evaluation The mean preoperative pain scores for patients with peritoneal and ovarian endometriosis were 5 3, 4 1.5, and 3 1, respectively, for dysmenorrhea, deep dyspareunia, and rectalgia. The mean preoperative pain scores for patients with deep infiltrating endometriosis were 9 1, 9 1, and 8 1, respectively, for dysmenorrhea, deep dyspareunia, and rectalgia. Patients with deep infiltrating endometriosis had significantly higher pain scores than patients without deep infiltrating endometriosis (P.001). DISCUSSION Mast cells settle in connective tissues and usually do not circulate in the bloodstream. These cells play a central role in inflammatory reactions and contain special cytoplasmic granules that store mediators of inflammation such as histamine, proteases (chymase and tryptase), cytokines, chemotactic factors, and metabolites of arachidonic acid that act on the vasculature, smooth muscle, connective tissue, mucous glands, and inflammatory cells. Recently, a few studies have been published on mast cells and peritoneal and ovarian endometriosis. These studies showed an increased number of mast cells in peritoneal and ovarian endometriosis and suggested a possible role for mast cells in the occurrence of fibrosis and adhesions in these lesions (18, 23, 24). This is the first study on mast cells and deep infiltrating endometriosis and on the relationship between mast cells and nerves in endometriotic lesions. Endometriosis, but especially deep infiltrating endometriosis, is strongly associated with pain (1). However, the neurobiological basis of pain in these lesions remains poorly understood. Mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological find- TABLE 4 Comparisons between deep infiltrating and peritoneal and ovarian lesions (P values) in endometriosis. Chymase mast cells Tryptase mast cells Mast cells <25 m Degranulating mast cells Peritoneum/rectovaginal septum Peritoneum/uterosacral ligament.001 NS Peritoneum/large bowel Ovary/rectovaginal septum NS Ovary/uterosacral ligament.01 NS.001 NS Ovary/large bowel NS Note: NS not significant. P values obtained after Bonferroni s correction Anaf et al. Pain, mast cells, and endometriosis Vol. 86, No. 5, Month 2006

6 FIGURE 2 Presence of activated and degranulating mast cells within or near the perineurium. The black arrows show a nerve structure. The white arrows show degranulating mast cells. ings (20, 25). In both conditions, an increase of mast cells has been described in the bladder and the colonic mucosa, respectively. Moreover, mast cells are also increased and in close relationship with nerves in other very painful conditions such as acute and chronic pancreatitis (26). In this study, we found a significantly greater number of mast cells in endometriotic lesions (Fig. 3) than in controls, which is consistent with previous findings in peritoneal and ovarian endometriosis (18, 23, 24). Moreover, the number of mast cells in deep infiltrating endometriosis whatever the anatomical location is significantly more important than in peritoneal and ovarian endometriosis. In these lesions we also found a greater proportion of mast cells in close proximity with the nerves (Fig. 2) of the different layers of the large bowel but also with the nerves of the uterosacral ligaments and the posterior vaginal wall. Very interestingly, this study also demonstrated the presence of degranulating mast cells within the nerve structures, especially in the perineurium (Fig. 2). Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. NGF is one such product (11, 27 31). NGF is released and functions as a chemoattractant for mast cells, but it can also trigger mast cell degranulation. The capacity of NGF to attract mast cells could explain the higher number of mast cells in endometriotic lesions since it has been shown that the content of mast cells in the eutopic endometrium of patients with endometriosis is very low and is not significantly different from that of the endometrium of unaffected patients (18). What is well known by physicians dealing with deep infiltrating endometriosis is the important exacerbation of pain when pressure is exerted on deep nodular or indurated lesions at physical examination. This phenomenon of exquisite pain occurring when a nonpainful stimulus is applied is called hyperalgesia. Hyperalgesia is a major characteristic of neuropathic pain, which corresponds to a pain sensation that is out of proportion with the intensity of nociceptors stimulation (30). Neuropathic pain is usually accompanied by a nerve injury (invasion of the brachial plexus in Pancoast s syndrome, postzoosterian pain, etc.). This phenomenon also occurs in deep infiltrating endometriosis where nerve invasion by endometriotic stromal cells is frequently observed (8). However, neuropathic pain symptoms can also be induced by inflammatory stimuli in the absence of nerve injury (31, 32). Histamine is a key mediator released by activated mast cells. It can sensitize nociceptors (14, 15), and neuronal histamine receptors are upregulated or modulated by nerve injury (33, 34). In addition, histamine plays a critical role in leukocyte recruitment after mast cell activation (35). However, activated mast cells contribute directly to neuropathic hyperalgesia by releasing mediators other than histamine, including tryptase (36, 37), tumor necrosis factor-alpha (38 40), prostaglandins (41), serotonin (11), and interleukin-1 (42). Activation of mast cells may also contribute indirectly to the development of neuropathic pain by the recruitment of leukocytes that release algesic mediators. Neutrophils and macrophages release mediators such as prostaglandin E 2, eicosanoids, and reactive oxygen intermediates, FIGURE 3 Uterosacral ligament endometriotic lesion and tryptase immunohistochemistry. Abundant tryptase (activated mast cells) in the endometriotic stromal cells. Fertility and Sterility 1341

7 which can sensitize nociceptors and induce hyperalgesia (12, 13, 41, 43, 44). Our findings are not only of interest from the neurophysiological point of view, but perhaps, in the future, also from the therapeutic point of view. Indeed, it has been demonstrated in animal models that the stabilization of mast cells by sodium cromoglycate suppressed the development of hyperalgesia. Treatment with histamine receptor antagonists also suppressed the development of hyperalgesia after nerve injury and alleviated hyperalgesia once it was established (45). Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in endometriosis and especially in deep infiltrating lesions contribute to the development of pain and hyperalgesia by direct effects on neurons. REFERENCES 1. Cornillie FJ, Oosterlynck J, Lauweryns M, Koninckx PR. 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