- Email: [email protected]
A pilot study to evaluate the clinical relevance of endometriosis-associated nerve fibers in peritoneal endometriotic lesions
A pilot study to evaluate the clinical relevance of endometriosis-associated nerve fibers in peritoneal endometriotic lesions Sylvia Mechsner, M.D.,a Andrea Kaiser,a Andreas Kopf, M.D.,b Christine Gericke,c Andreas Ebert, M.D., Ph.D.,d and Julia Bartley, M.D.a a
Endometriosis Research Center Charite, Department of Gynaecology, b Department for Anaesthesiology and Intensive Care, Charite, Campus Benjamin Franklin; c Institute of Biometry und Clinical Epidemiology, Charite, Campus Mitte; and d Department of Obstetrics and Gynaecology, Endometriosis Center, Vivantes Humboldt-Klinikum, Berlin, Germany
Objective: To investigate the clinical relevance of endometriosis-associated nerve fibers in the development of endometriosis-associated symptoms. Design: Prospective nonrandomized study. Setting: University hospital endometriosis center. Patient(s): Fifty-one premenopausal patients underwent surgical laparoscopy because of chronic pelvic pain, dysmenorrhea, or for ovarian cysts. Endometriosis was diagnosed in 44 patients. Intervention(s): The preoperative and postoperative pain scores were determined using a standardized questionnaire with a visual analogue scale from 1–10. Patients with peritoneal endometriosis were divided into two groups depending on their preoperative pain score: group A with a pain score of at least 3 or more and group B with a pain score of 2 or less. Patients without peritoneal endometriosis were classified as group C and patients without endometriosis were classified as group D. Immunohistochemical analysis of neurofilament and protein gene product 9.5 were used for nerve fiber detection. Occurrence of endometriosis-associated nerve fibers was correlated with the severity of pelvic pain and/or dysmenorrhea. Result(s): Peritoneal endometriosis-associated nerve fibers were found significantly more frequently in group A than in group B (82.6% vs. 33.3%). Conclusion(s): The present study suggests that the presence of endometriosis-associated nerve fibers in the peritoneum is important for the development of endometriosis-associated pelvic pain and dysmenorrhea. (Fertil Steril 2009;92:1856–61. 2009 by American Society for Reproductive Medicine.) Key Words: Endometriosis, sensory nerve fibers, pelvic pain, dysmenorrhea, pain conduction, pathophysiology of endometriosis-related pain
Endometriosis is a chronic benign estrogen (E)-dependent gynecological disease affecting millions of women of reproductive-age worldwide (1). It is associated with infertility and pelvic pain’although, the severity of endometriosis-associated pelvic pain does not always correlate closely with the disease stage or lesion site (2–7). The pathophysiological basis for peritoneal endometriosisassociated pelvic pain is not known. Recently, pain-conducting substance P-positive nerve fibers were found to be directly colocalized with human peritoneal endometriotic lesions (8, 9). These nerve fibers could be characterized as sensory Ad, sensory C, cholinergic, and adrenergic nerve
Received March 5, 2008; revised August 25, 2008; accepted September 2, 2008; published online November 3, 2008. S.M. has nothing to disclose. A.K. has nothing to disclose. A.K. is a speaker or advisor for Grunenthal, Pfizer, BMS, and Cephalon. C.G. has nothing to disclose. A.E. has nothing to disclose. J.B. has nothing to disclose. Reprint requests: Sylvia Mechsner, M.D., Endometriosis Research Center , Department of Gynecology, Charite , Campus Benjamin Charite Franklin, Hindenburgdamm 30, 12200 Berlin, Germany (FAX: 49-30-8445-4477; E-mail: [email protected]).
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fibers (8). The endometriosis-associated nerve fibers are accompanied by immature blood vessels within the stroma. Nerve growth factors, such as nerve growth factor (NGF) and neutrophin-3 (NT-3), are expressed by endometriotic cells. A marker of neural outgrowth and regeneration, GAP-43, is expressed in endometriosis-associated nerve fibers but not in peritoneal nerves distant to the endometriotic lesion (9). These data suggest that peritoneal endometriotic cells exhibit neurotrophic properties and imply that the fibers play an important role in the etiology of endometriosis-associated pelvic pain (8, 9). In a rat model of surgically induced endometriosis, it has been shown that ectopic endometrial growth induces its own autonomic and sensory innervation (10). In rodents, the sensory and sympathetic nerve supply seem to develop within ectopic endometrial implants (11, 12). To investigate the clinical relevance of this observation we conducted a pilot study. The occurrence of peritoneal endometriosis-associated nerve fibers was correlated with the severity of symptoms, such as pelvic pain and/or dysmenorrhea, in patients undergoing surgery for endometriosis.
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.09.006
MATERIALS AND METHODS Patients Between March and October 2006, 51 patients underwent diagnostic laparoscopy because of chronic pelvic pain, dysmenorrhea, or for ovarian cysts with the presumption of endometriosis. All women were premenopausal with a mean age of 35 years (range 21–49 years). Thirty-six women had regular menstrual cycles and had received no hormonal treatment for at least 2 months before surgery. Fifteen patients were on hormonal treatment at the time of surgery (combined oral contraceptives [OC] or P-only pill). The respective phases of the cycle were defined by the date of the patients’ last menstrual period. In 44 patients (86.3%) endometriosis was diagnosed and classified by surgery according to the revised classification of the American Society of Reproductive Medicine (13) (I ¼ 11; II ¼ 6; III ¼ 9; IV ¼ 18). Of these 44 patients, 16 (36.4%) had peritoneal endometriosis only (in 2 cases without histologic confirmation), 7 (15.9%) peritoneal and ovarian endometriotic cysts, 13 (29.5%) peritoneal and deep infiltrating endometriosis (DIE) (7 with peritoneal endometriotic lesions and 6 with obliteration of the pouch of Douglas and adhesions), 1 (2.3%) peritoneal, ovarian, and DIE, 3 (6.8%) ovarian endometriotic cyst without peritoneal implants, and 4 (9.1%) ovarian and DIE without other peritoneal implants.
FIGURE 1 Study population (n ¼ 51). Patients with histologically confirmed peritoneal endometriosis (pEM) were divided into different groups depending on their preoperative pain score for dysmenorrhea and/or pelvic pain: groups A, A1, and B. Group A (n ¼ 23) consisted patients with peritoneal endometriotic lesion who also displayed other manifestations of endometriosis. Group A1 is a subpopulation of group A, only with peritoneal endometriotic lesions. Group A and A1 had a preoperative pain score of at least 3 or more. Group B (n ¼ 6) had a preoperative pain score of 2 or less. Patients with no peritoneal endometriosis but with other forms of endometriosis (ovarian or deep infiltrating endometriosis) were defined as group C (n ¼ 13). All patients with no endometriosis were classified as group D (n ¼ 7).
In all patients the visible peritoneal implants, endometriotic cysts, and deep infiltrating nodules were excised in toto. Patients with rectovaginal DIE underwent a resection of the lesion as described previously (14). In 8 (18.2%) cases it was necessary to remove part of the rectum because of bowel infiltration and in 1 case, a disc dissection of the anterior rectum wall was performed. In 29 of 44 patients (65.9%), peritoneal endometriotic lesions were confirmed by histology. In seven patients, with no apparent endometriosis, a peritoneal biopsy was taken. These patients suffered from adhesions (n ¼ 3), hydatid (n ¼ 1), and uterine fibroids (n ¼ 3).
Pain Evaluation Using a standardized questionnaire with a visual analogue scale (VAS), the severity of endometriosis-associated symptoms, such as pelvic pain and dysmenorrhea, was documented before and 3 months after surgery. The pain scale was subdivided into 10 grades. ‘‘No pain’’ was indicated at the left side of the scale and ‘‘the maximum pain you could imagine’’ at the right side of the scale. Furthermore, the presence of pain when defecating, micturating, and during intercourse (dyschezia, deep dyspareunia, and dysuria) was documented by a simple ‘‘Yes’’ and ‘‘No’’ on the questionnaire. Patients with histologically confirmed peritoneal endometriosis (n ¼ 29) were divided into two groups (A and B) depending on their preoperative pain score for dysmenorrhea Fertility and Sterility
Mechsner. Endometriosis-associated nerve fibers. Fertil Steril 2009.
and/or pelvic pain (Fig. 1). Group A (n ¼ 23) included all patients with peritoneal endometriotic lesions who coincidentally displayed additional manifestations of endometriosis (e.g., ovarian cysts or DIE). Group A1, a subgroup within group A, contained patients with only peritoneal endometriotic lesions and no other manifestation of endometriosis (Fig. 1). The definition of groups A and B is based on the cut of pain level of 3 on a 10-point scale. Groups A and A1 had a preoperative pain score of at least 3 or more (median preoperative pain score of 7). Group B (n ¼ 6) had a preoperative pain score of 2 or less (median preoperative pain score of 0.5). The cutoff point was set at the pain score of 3 (VAS) as this marks the border between patients who require therapy (15). Patients without peritoneal endometriosis but with other forms of endometriosis (ovarian or DIE) were defined as group C (n ¼ 13) with a median preoperative pain score of 6. All patients without endometriosis were classified as group D 1857
(n ¼ 7). They had a median preoperative pain score of 7 (Fig. 1). Tissue Samples We studied 45 typical peritoneal endometriotic lesions of 29 patients (5 active red lesions, 20 inactive black or white lesions, and 20 of unknown activity). The lesions were excised from peritoneum of the bladder (n ¼ 8), the pouch of Douglas (n ¼ 14), the ovarian fossa (n ¼ 5), the lateral pelvic wall (n ¼ 8), and the uterosacral ligament (n ¼ 10). All peritoneal biopsies had subperitoneal fat at the lower border. In addition, control biopsies of group D patients were taken from the peritoneum of the bladder (right side close to the round ligament) or of the pouch of the Douglas. All patients gave their consent for these biopsies. The study was approved by the Institutional Review Board (IRB; EA 4/023/05). Immunohistochemistry and Evaluation of Staining All tissues were fixed immediately in 4% buffered formalin for 12 hours and then embedded in paraffin. Serial sections 1–2 mm thick were cut for hematoxylin-eosin (H & E) staining and immunohistochemistry. Because the samples used were previously examined by the pathologist it was not possible to perform a three-dimensional analysis of the endometriotic lesion. After deparaffinization in xylene (twice for 5 minutes at room temperature [RT]) and rehydration (10 minutes at RT in acetone and acetone/tris-buffered saline, 1:1), a heat-induced epitope retrieval procedure was performed by heating the probes in citrate buffer (0.1 M citric acid and 0.1 M sodium citrate, pH 6.0 at 700 W, microwave, 17 minutes). After rinsing with tris-buffered saline, the sections were blocked with 10% fetal calf serum (FCS) for 30 minutes at RT. Neurofilament analysis, marker for myelinated nerve fibers (n [ 45) The sections were incubated for 1 hour at RT with monoclonal mouse antineurofilament antibody (clone 2F11, dilution 1:50; Dako, Hamburg, Germany). Rinsing with tris-buffered saline was followed by application of the labeled streptavidin-biotin kit (Dako). Fuchsin substrate (Dako) was used to visualize the specific immunoreactive staining. Protein gene-related product 9.5, marker for unmyelinated nerve fibers, n [ 45 The sections were incubated for 1 hour at RT with polyclonal rabbit antiprotein gene-related product 9.5 antibody (dilution 1:200; Dako). Rinsing with tris-buffered saline was followed by application of the labeled streptavidin-biotin kit (Dako). Fuchsin substrate (Dako) was used to visualize the specific immunoreactive staining. Peripheral nerve tissue was taken as a positive control. Samples of normal peritoneum were serially sectioned and examined to exclude the presence of endometriosis. Negative control sections were processed both by using nonspecific IgG (dilution 1:50; Dako) and by omitting the 1858
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primary antibody. Staining was detected using a radiophoto microscope (Carl Zeiss, Goettingen, Germany). Pictures were taken at different magnifications (40, 100, or 1,000) and further processed using Adobe Photoshop (Adobe Systems, Unterschleissheim, Germany). In cases where nerve fibers were not detectable, up to two more sections were taken from the paraffin block for reexamination. Using a grid of a surface of 1 mm2 on the microscope (at 40), the distance between endometrial glands/ stromal cells and the closest nerve fibers was graded as follows: 1 for close proximity with a space of less than 1.5 mm (defined as endometriosis-associated nerve fibers) and 2 for a space of more than 1.5 mm (9). The mean nerve score was analyzed for each sample. The sample size of the peritoneum was defined as 1 ¼ small, 2 ¼ moderate, and 3 ¼ large and the nerve scores was defined as 0 ¼ no nerves seen, 1 ¼ rare, 2 ¼ few, 3 ¼ many by two independent examiners (i.e., it was a semiquantitative analysis). Sections were evaluated and scored by two separate observers. The occurrence or absence of endometriosis-associated nerve fibers was correlated with the severity of symptoms of pelvic pain and dysmenorrhea, with the localization of peritoneal endometriotic lesions and with the activity of the peritoneal endometriotic lesions. Statistical Analysis Statistical analysis was performed using the SPSS program statistical software (V15.0.1.1; SPSS Inc., Chicago, IL). All tests were two-sided and the level of significance was .05. Frequencies of occurrence were tested with the Fisher’s exact test. RESULTS Nerve Fibers in Peritoneal Endometriotic Lesions Endometrial glands and stromal cells were detectable in 45 peritoneal endometriosis specimens taken from 29 patients. There was no histologic evidence of endometriosis in serial sections of specimens of group D. Nerve fibers were present in all specimens. The mean nerve score in peritoneum of patients with endometriosis was 1.7 (in the periphery of the endometriotic lesion, only 1.3), whereas the nerve score of healthy peritoneum was graded as 1.2. There was no significant difference in mean nerve score between peritoneal specimens with and without endometriosis. Nerve fibers were identified by immunohistochemical staining for neurofilament, a marker molecule for myelinated nerve fibers (16) and protein gene-related product 9.5, a panneuronal marker (17). The immunostaining pattern of both markers showed a comparable moderate staining. In group A, endometriosis-associated nerve fibers were found in 19 patients (82.6%). However, in group B, endometriosis-associated nerve fibers were present in only two cases (33.3%). The occurrence of endometriosis-associated nerve
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FIGURE 2 The occurrence of nerve fibers in peritoneal endometriotic lesions of patients with dysmenorrhea/pelvic pain (groups A and A1) and in those with either low or without dysmenorrhea/pelvic pain (group B). The difference remained significant (Fisher’s t-test).
Correlation of different symptoms (dysmenorrhea and/or pelvic pain; dyspareunia; dyschezia and dysuria) with the occurrence of peritoneal nerves The occurrence of nerve fibers in peritoneal endometriotic lesions was compared with the occurrence of different pain manifestations. Patients with dysmenorrhea and/or pelvic pain had a significantly higher frequency of nerve fibers in peritoneal endometriotic lesions than patients without those symptoms (P<.05). This correlation was not observed compared to the other pain manifestations (dyspareunia, dychezia, and dysuria). Hormonal treatment Fifteen patients developed symptoms under hormonal therapy. The distribution of patients on hormonal treatment was a followed: Group A: 5 of 23 (21.7%) (3 in group A1); group B: 0 of 6; group C: 6 of 13 (46.2%); and group D: 4 of 7 (57.1%). All 5 group A patients with hormonal treatment presented nerve fibers in close contact to the peritoneal endometriotic lesion. There was no significant difference regarding endometriosis-associated symptoms whether or not women were receiving hormonal treatment.
Mechsner. Endometriosis-associated nerve fibers. Fertil Steril 2009.
fibers in patients with severe pelvic pain and/or dysmenorrhea (groups A and A1) was significantly higher than in patients with low symptoms (group B) (P¼.034; P<.05) (Fig. 2). Occurrence of Nerve Fibers Compared to the Lesion Site, Activity, and Stage No significant correlation was detectable between the presence of peritoneal endometriosis-associated nerve fibers and the location of endometriotic lesions or active and inactive peritoneal lesions. In addition, the stage of endometriosis (defined according to the revised American Society of Reproductive Medicine) was not related to the presence of endometriotic-associated nerve fibers. There was no statistically significant intergroup difference. Analysis of the Questionnaire The analysis of the questionnaire showed that 43 of the 51 (84.3%) women had dysmenorrhea or pelvic pain. Thirtynine patients (76.5%) suffered from dysmenorrhea and 27 patients (52.9%) suffered from pelvic pain. Thirty-two women (628%; i.e., the majority) described both dysmenorrhea and pelvic pain. A total number of 36 (70.6%) patients suffered from dyspareunia, 28 (549%) women experienced dyschezia regularly, and 9 patients (17.65%) reported regular dysuria. Most of our patients (n ¼ 37, 72.6%) suffered from dysmenorrhea/pelvic pain and at least from one other symptom (dyspareunia, dyschezia, dysuria). A combination of dysmenorrhea/pelvic pain and all of the other complaints was experienced by only 7 (13.7%) women. Fertility and Sterility
DISCUSSION Recently, large series of laparoscopic resection of endometriotic lesions have shown promising results in terms of the reduction of chronic pelvic pain and reduction of severe dysmenorrhea (18). However, the fundamental pathological mechanism underlying endometriosis-related pain seems to be unclear, particularly the role of peritoneal lesions in generating pelvic pain (19, 20). Important data on innervation of endometriosis, endometrium, and myometrium have been delivered by different groups of investigators.(8–11, 19, 21–23). In this respect, the occurrence of nerve fibers in close topographical relationship to the peritoneal endometriotic lesion has been demonstrated (8, 9). Furthermore, evidence seems to suggest that these endometriosis-associated nerve fibers are of a new origin (9). The peritoneal endometriotic lesions are able to produce several pain-mediating substances, such as prostaglandins (PG) like PG E2 and F2a, histamine, kinins, NGF, and interleukins, which can activate peritoneal nociceptors (2, 24–26). However, there is no direct or indirect evidence that peritoneal endometriosis-associated nerve fibers are involved in conduction of endometriosis-related pain. In this pilot study we discussed a clinical correlation between the occurrence of endometriosis-associated nerve fibers and the severity of symptoms. All patients undergoing surgery either for symptoms of pelvic pain and/or dysmenorrhea or the ultrasound diagnoses of ovarian cysts have been included. To correlate the occurrence of endometriosis-associated nerve fibers in peritoneal endometriotic lesions, only specimens of peritoneal endometriotic lesions have been analyzed. Nerve fibers were detected using established markers against neurofilament (myelinated nerve fibers, such as Aa, Ab, Ag, Ad, and B fibers) and protein gene-related product 9.5 (a pan-neuronal marker against myelinated and 1859
unmyelinated nerve fibers such as Aa, Ab, Ag, Ad, B, and C fibers) (16, 17). The Ad fibers are small myelinated fibers and transmit sharp, prickling localized pain to the central nervous system. The C fibers are small unmyelinated fibers and transmit dull, aching, burning pain that is hard to locate (16, 17). In agreement with other investigators, myelinated and unmyelinated nerve fibers could be detected (8). We defined endometriosis-associated nerve fibers as ‘‘close’’ when the distance between the nerve fibers and the epithelial cells of the endometriotic lesion was less than 1.5 mm as done in previous studies by our group (9). This definition is indeed a random one; however, when using a magnification of 400 this distance can be evaluated with occurrence. Furthermore, it is unknown over which distance paracrine hormones can exert an effect on neighboring nerve fibers. As long as this question remains unanswered, the definition of endometriosis-associated nerve fibers can only be a random one. Our data suggest that in peritoneal endometriotic lesions of patients with severe symptoms (groups A and A1), there were significantly more endometriosis-associated nerve fibers than in those of patients with only few symptoms (group B) (P¼.031). Although statistical significance has been reached, such small number random fluctuation of data cannot be excluded. Because group C and D patients had no peritoneal endometriotic lesions, our study concentrated on the patients in groups A and B only. Importantly, Tokushige and colleagues (8) detected a higher number of nerve fibers in peritoneum of patients with endometriosis than on those without it. In agreement with Tulandi et al. (27), we could not detect any significant difference in the total nerve scores when comparing peritoneum from patients with and without endometriosis. The control collective in our study underwent diagnostic laparoscopy for symptoms like pelvic pain, but no apparent endometriotic lesions were detected. However, these women suffered from fibroids and adhesions. In contrast, the patients in the control group of Tokushige and colleagues (8) had no pain. They underwent laparoscopy for tubal sterilization or investigation of infertility. In this group no recognizable pathology was observed. This might explain the different findings of peritoneal nerve density. To study the clinical manifestation of symptoms in correlation to the occurrence of endometriosis-associated nerve fibers, we documented the severity of symptoms before surgical intervention using a questionnaire to reveal levels of endometriosis-related pain (VAS) and focusing particularly on pelvic pain and dysmenorrhea. Furthermore, the presence of dyschezia, dyspareunia, and dysuria was documented.
different manifestations (endometriosis genitalis externa, adenomyosis uteri, and DIE), it is very difficult to investigate one isolated parameter for pain conduction. Subgroup A1 had only peritoneal endometriotic lesions. As a result pain conduction dependent from peritoneal endometriotic lesions only could be analyzed. Endometriosis-associated nerve fibers could be detected in lesions of group A and A1 patients, but not in peritoneal endometriotic lesions of group B patients. This finding might be a possible co-factor in pain generation. In addition to endometriosis-associated nerve fibers, the activity of the peritoneal endometriotic lesions with the release of pain mediators and the lesion site are also factors for pain generation (2, 26). Furthermore, in the peritoneum of patients with endometriosis, endometriosis-associated smooth muscle cells could be demonstrated, which express oxytocin, E, and P receptors (21). It is possible that the cyclic activation of smooth muscle cells and endometriosis-associated nerve fibers may be involved in the development of cyclic pelvic pain and the sensation of dysmenorrhea-similar pain (21). The occurrence of nerve fibers in peritoneal endometriotic lesions was compared with the occurrence of different pain manifestations. Patients with dysmenorrhea with pelvic pain seem to have significant more nerve fibers in peritoneal endometriotic lesions than patients without these symptoms (P<.05), suggesting that nerve fibers are involved in pain conduction of dysmenorrhea and/or pelvic pain (8). Also in DIE cases only, it could be demonstrated that perineural invasion by endometriosis was associated with severe pain (22). One possible weak point of the study is the small number of patients in the subgroups. Interestingly, it has been demonstrated that there are significantly more sensory nerve fibers in the endometrium of patients with endometriosis than in those without endometriosis (23, 29). These important data suggested that nerve fibers in the endometrium may play an important role in generation of dysmenorrhea. Another important study demonstrated a persistence of dysmenorrhea and nonmenstrual pain after optimal endometriosis surgery in cases of adenomyosis (30). However, present data suggest, that both uterine and peritoneal factors are responsible for the development of dysmenorrhea and pelvic pain. In summary, this is the first evidence for a clinical correlation of nerve fibers in the development of endometriosis-related symptoms like pelvic pain and/or dysmenorrhea. REFERENCES
The analysis of the questionnaire demonstrated the typical distribution of endometriosis-related pain in our study group (28). However, due to the complexity of the endometriosis-related symptoms, the combined occurrence of the complaints (such as dysmenorrhea, pelvic pain, deep dyspareunia, dyschezia, and dysuria) and the coincidence of 1860
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1. Giudice LC, Kao LC. Endometriosis. Lancet 2004;364:1789–99. 2. Vernon MW, Beard JS, Graves K, Wilson EA. Classification of endometriotic implants by morphologic appearance and capacity to synthesize prostaglandin F. Fertil Steril 1986;46:801–6. 3. Fedele L, Parazzini F, Bianchi S, Arcaini L, Candiani GB. Stage and localization of pelvic endometriosis and pain. Fertil Steril 1990;53: 155–8. 4. Vercellini P, Bocciolone L, Vendola N, Colombo A, Rognoni M, Fedele L. Peritoneal endometriosis. Morphologic appearance in women with chronic pelvic pain. J Reprod Med 1991;36:533–6.
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5. Vercellini P, Trespidi L, De Giorgi O, Cortesi I, Parazzini F, Crosignani PG. Endometriosis and pelvic pain: relation to disease stage and localization. Fertil Steril 1996;65:299–304. 6. Porpora MG, Koninckx PR, Piazze J, Natili M, Colagrande S, Cosmi EV. Correlation between endometriosis and pelvic pain. J Am Assoc Gynecol Laparosc 1999;6:429–34. 7. Gruppo Italiano per lo Studio dell’ Endometriosi. Relationship between stage, site and morphological characteristics of pelvic endometriosis and pain. Hum Reprod 2001;16:2668–71. 8. Tokushige N, Markham R, Russell P, Fraser IS. Nerve fibres in peritoneal endometriosis. Hum Reprod 2006;21:3001–7. 9. Mechsner S, Schwarz J, Thode J, Loddenkemper C, Salomon DS, Ebert AD. Growth-associated Gap 43 positive sensory nerve fibers accompanied by immature vessels are located in or near peritoneal endometriotic lesions. Fertil Steril 2007;88:583–7. 10. Berkley KJ, Dmitrieva N, Curtis KS, Papka RE. Innervation of ectopic endometrium in a rat model of endometriosis. Proc Natl Acad Sci USA 2004;101:11094–8. 11. Berkley KJ, Rapkin AJ, Papka RE. The pains of endometriosis. Science 2005;308:1587–9. 12. Zhang G, Dmitrieva N, Liu Y, McGinty KA, Berkley KJ. Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factors content of ectopic endometrial cysts in the rat. Am J Physiol Regul Integr Comp Physiolol 2008;294:162–71. 13. The American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis. 1996. Fertil Steril 1997;67:817–21. 14. Zanetti-D€allenbach R, Bartley J, M€uller C, Schneider A, K€ohler C. Combined vaginal–laparoscopic–abdominal approach for the surgical treatment of rectovaginal endometriosis with bowel resection: a comparison of this new technique with various established approaches by laparoscopy and laparotomy. Surg Endosc 2008;22:995–1001. 15. Dworkin RH, Turk DC, Farrar JT, Haythornthwaite JA, Jensen MP, Katz NP. Core outcome measures for chronic pain clinical trial: IMMPACT recommendations. Pain 2005;113:9–19. 16. Lundberg LM, Alm P, Wharton J, Polak JM. Protein gene product 9.5 (PGP9.5): a new neuronal marker visualizing the whole innervation and pregnancy induced and developmental changes in the guinea pig. Histochemistry 1988;90:9–17.
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17. Schlaepfer WW. Neurofilaments: structure, metabolism and implications in disease. J Neuropathol Exp Neurol 1987;46:117–29. 18. Garry R. The effectiveness of laparoscopic excision of endometriosis. Curr Opin Obstet Gynecol 2004;16:299–303. 19. Tamburro S, Canis M, Albuisson E, Dechelotte P, Darcha C, Mage G. Expression of transforming growth factor beta1 in nerve fibers is related to dysmenorrhea and laparoscopic appearance of endometriotic implants. Fertil Steril 2003;80:1131–6. 20. Whiteside JL, Falcone T. Endometriosis-related pelvic pain: what is the evidence? Clin Obstet Gynecol 2003;46:824–30. 21. Mechsner S, Bartley B, Loddenkemper C, Salomon DS, StarzinskiPowitz A, Ebert AD. Oxytocin receptor expression in smooth muscle cells of peritoneal endometriotic lesions and ovarian endometriotic cysts. Fertil Steril 2005;83:1220–31. 22. Anaf V, Simon P, El Nakadi I, Fayt I, Buxant F, Simonart T, et al. Relationship between endometriotic foci and nerves in rectovaginal endometriotic nodules. Hum Reprod 2000;15:1744–50. 23. Tokushige N, Markham R, Russell P, Fraser IS. High density od small nerve fibers in the functional layer of the endometrium in women with endometriosis. Hum Reprod 2006;21:782–7. 24. Jones HW Jr JG. Endometriosis. In: Gynecology. 3rd ed. Baltimore: Williams & Wilkins, 1982;352. 25. Drake TS, O’Brien WF, Ramwell PW, Metz SA. Peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F1 alpha in endometriosis. Am J Obstet Gynecol 1981;140:401–4. 26. Moon Y, Leung P, Ho Y. Prostaglandin F in human endometriotic tissue. Obset Gynecol 1981;141:344. 27. Tulandi T, Felemban A, Chen MF. Nerve fibers and histopathology of endometriosis-harboring peritoneum. J Am Assoc Gynecol Laparosc 2001;8:95–8. 28. Vercellini P, Fedele L, Aimi G, Pietropaolo G, Consonni D, Crosignani PG. Association between endometriosis stage, lesion type, patient characteristics and severita of pelvic pain symptoms: a multivariate analysis of over 1000 patients. Hum Reprod 2007;22:266–71. 29. Tokushige N, Markham R, Russell P, Fraser IS. Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis. Fertil Steril 2007;88:795–803. 30. Parker JD, Leondires M, Sinaii N, Premkumar A, Nieman LK, Stratton P. Persistence of dysmenorrhoea and nonmenstrual pain after optimal endometriosis surgery may indicate adenomyosis. Fertil Steril 2006;86:711–5.
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Endometriosis Research Center Charite, Department of Gynaecology, b Department for Anaesthesiology and Intensive Care, Charite, Campus Benjamin Franklin; c Institute of Biometry und Clinical Epidemiology, Charite, Campus Mitte; and d Department of Obstetrics and Gynaecology, Endometriosis Center, Vivantes Humboldt-Klinikum, Berlin, Germany
Objective: To investigate the clinical relevance of endometriosis-associated nerve fibers in the development of endometriosis-associated symptoms. Design: Prospective nonrandomized study. Setting: University hospital endometriosis center. Patient(s): Fifty-one premenopausal patients underwent surgical laparoscopy because of chronic pelvic pain, dysmenorrhea, or for ovarian cysts. Endometriosis was diagnosed in 44 patients. Intervention(s): The preoperative and postoperative pain scores were determined using a standardized questionnaire with a visual analogue scale from 1–10. Patients with peritoneal endometriosis were divided into two groups depending on their preoperative pain score: group A with a pain score of at least 3 or more and group B with a pain score of 2 or less. Patients without peritoneal endometriosis were classified as group C and patients without endometriosis were classified as group D. Immunohistochemical analysis of neurofilament and protein gene product 9.5 were used for nerve fiber detection. Occurrence of endometriosis-associated nerve fibers was correlated with the severity of pelvic pain and/or dysmenorrhea. Result(s): Peritoneal endometriosis-associated nerve fibers were found significantly more frequently in group A than in group B (82.6% vs. 33.3%). Conclusion(s): The present study suggests that the presence of endometriosis-associated nerve fibers in the peritoneum is important for the development of endometriosis-associated pelvic pain and dysmenorrhea. (Fertil Steril 2009;92:1856–61. 2009 by American Society for Reproductive Medicine.) Key Words: Endometriosis, sensory nerve fibers, pelvic pain, dysmenorrhea, pain conduction, pathophysiology of endometriosis-related pain
Endometriosis is a chronic benign estrogen (E)-dependent gynecological disease affecting millions of women of reproductive-age worldwide (1). It is associated with infertility and pelvic pain’although, the severity of endometriosis-associated pelvic pain does not always correlate closely with the disease stage or lesion site (2–7). The pathophysiological basis for peritoneal endometriosisassociated pelvic pain is not known. Recently, pain-conducting substance P-positive nerve fibers were found to be directly colocalized with human peritoneal endometriotic lesions (8, 9). These nerve fibers could be characterized as sensory Ad, sensory C, cholinergic, and adrenergic nerve
Received March 5, 2008; revised August 25, 2008; accepted September 2, 2008; published online November 3, 2008. S.M. has nothing to disclose. A.K. has nothing to disclose. A.K. is a speaker or advisor for Grunenthal, Pfizer, BMS, and Cephalon. C.G. has nothing to disclose. A.E. has nothing to disclose. J.B. has nothing to disclose. Reprint requests: Sylvia Mechsner, M.D., Endometriosis Research Center , Department of Gynecology, Charite , Campus Benjamin Charite Franklin, Hindenburgdamm 30, 12200 Berlin, Germany (FAX: 49-30-8445-4477; E-mail: [email protected]).
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fibers (8). The endometriosis-associated nerve fibers are accompanied by immature blood vessels within the stroma. Nerve growth factors, such as nerve growth factor (NGF) and neutrophin-3 (NT-3), are expressed by endometriotic cells. A marker of neural outgrowth and regeneration, GAP-43, is expressed in endometriosis-associated nerve fibers but not in peritoneal nerves distant to the endometriotic lesion (9). These data suggest that peritoneal endometriotic cells exhibit neurotrophic properties and imply that the fibers play an important role in the etiology of endometriosis-associated pelvic pain (8, 9). In a rat model of surgically induced endometriosis, it has been shown that ectopic endometrial growth induces its own autonomic and sensory innervation (10). In rodents, the sensory and sympathetic nerve supply seem to develop within ectopic endometrial implants (11, 12). To investigate the clinical relevance of this observation we conducted a pilot study. The occurrence of peritoneal endometriosis-associated nerve fibers was correlated with the severity of symptoms, such as pelvic pain and/or dysmenorrhea, in patients undergoing surgery for endometriosis.
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.09.006
MATERIALS AND METHODS Patients Between March and October 2006, 51 patients underwent diagnostic laparoscopy because of chronic pelvic pain, dysmenorrhea, or for ovarian cysts with the presumption of endometriosis. All women were premenopausal with a mean age of 35 years (range 21–49 years). Thirty-six women had regular menstrual cycles and had received no hormonal treatment for at least 2 months before surgery. Fifteen patients were on hormonal treatment at the time of surgery (combined oral contraceptives [OC] or P-only pill). The respective phases of the cycle were defined by the date of the patients’ last menstrual period. In 44 patients (86.3%) endometriosis was diagnosed and classified by surgery according to the revised classification of the American Society of Reproductive Medicine (13) (I ¼ 11; II ¼ 6; III ¼ 9; IV ¼ 18). Of these 44 patients, 16 (36.4%) had peritoneal endometriosis only (in 2 cases without histologic confirmation), 7 (15.9%) peritoneal and ovarian endometriotic cysts, 13 (29.5%) peritoneal and deep infiltrating endometriosis (DIE) (7 with peritoneal endometriotic lesions and 6 with obliteration of the pouch of Douglas and adhesions), 1 (2.3%) peritoneal, ovarian, and DIE, 3 (6.8%) ovarian endometriotic cyst without peritoneal implants, and 4 (9.1%) ovarian and DIE without other peritoneal implants.
FIGURE 1 Study population (n ¼ 51). Patients with histologically confirmed peritoneal endometriosis (pEM) were divided into different groups depending on their preoperative pain score for dysmenorrhea and/or pelvic pain: groups A, A1, and B. Group A (n ¼ 23) consisted patients with peritoneal endometriotic lesion who also displayed other manifestations of endometriosis. Group A1 is a subpopulation of group A, only with peritoneal endometriotic lesions. Group A and A1 had a preoperative pain score of at least 3 or more. Group B (n ¼ 6) had a preoperative pain score of 2 or less. Patients with no peritoneal endometriosis but with other forms of endometriosis (ovarian or deep infiltrating endometriosis) were defined as group C (n ¼ 13). All patients with no endometriosis were classified as group D (n ¼ 7).
In all patients the visible peritoneal implants, endometriotic cysts, and deep infiltrating nodules were excised in toto. Patients with rectovaginal DIE underwent a resection of the lesion as described previously (14). In 8 (18.2%) cases it was necessary to remove part of the rectum because of bowel infiltration and in 1 case, a disc dissection of the anterior rectum wall was performed. In 29 of 44 patients (65.9%), peritoneal endometriotic lesions were confirmed by histology. In seven patients, with no apparent endometriosis, a peritoneal biopsy was taken. These patients suffered from adhesions (n ¼ 3), hydatid (n ¼ 1), and uterine fibroids (n ¼ 3).
Pain Evaluation Using a standardized questionnaire with a visual analogue scale (VAS), the severity of endometriosis-associated symptoms, such as pelvic pain and dysmenorrhea, was documented before and 3 months after surgery. The pain scale was subdivided into 10 grades. ‘‘No pain’’ was indicated at the left side of the scale and ‘‘the maximum pain you could imagine’’ at the right side of the scale. Furthermore, the presence of pain when defecating, micturating, and during intercourse (dyschezia, deep dyspareunia, and dysuria) was documented by a simple ‘‘Yes’’ and ‘‘No’’ on the questionnaire. Patients with histologically confirmed peritoneal endometriosis (n ¼ 29) were divided into two groups (A and B) depending on their preoperative pain score for dysmenorrhea Fertility and Sterility
Mechsner. Endometriosis-associated nerve fibers. Fertil Steril 2009.
and/or pelvic pain (Fig. 1). Group A (n ¼ 23) included all patients with peritoneal endometriotic lesions who coincidentally displayed additional manifestations of endometriosis (e.g., ovarian cysts or DIE). Group A1, a subgroup within group A, contained patients with only peritoneal endometriotic lesions and no other manifestation of endometriosis (Fig. 1). The definition of groups A and B is based on the cut of pain level of 3 on a 10-point scale. Groups A and A1 had a preoperative pain score of at least 3 or more (median preoperative pain score of 7). Group B (n ¼ 6) had a preoperative pain score of 2 or less (median preoperative pain score of 0.5). The cutoff point was set at the pain score of 3 (VAS) as this marks the border between patients who require therapy (15). Patients without peritoneal endometriosis but with other forms of endometriosis (ovarian or DIE) were defined as group C (n ¼ 13) with a median preoperative pain score of 6. All patients without endometriosis were classified as group D 1857
(n ¼ 7). They had a median preoperative pain score of 7 (Fig. 1). Tissue Samples We studied 45 typical peritoneal endometriotic lesions of 29 patients (5 active red lesions, 20 inactive black or white lesions, and 20 of unknown activity). The lesions were excised from peritoneum of the bladder (n ¼ 8), the pouch of Douglas (n ¼ 14), the ovarian fossa (n ¼ 5), the lateral pelvic wall (n ¼ 8), and the uterosacral ligament (n ¼ 10). All peritoneal biopsies had subperitoneal fat at the lower border. In addition, control biopsies of group D patients were taken from the peritoneum of the bladder (right side close to the round ligament) or of the pouch of the Douglas. All patients gave their consent for these biopsies. The study was approved by the Institutional Review Board (IRB; EA 4/023/05). Immunohistochemistry and Evaluation of Staining All tissues were fixed immediately in 4% buffered formalin for 12 hours and then embedded in paraffin. Serial sections 1–2 mm thick were cut for hematoxylin-eosin (H & E) staining and immunohistochemistry. Because the samples used were previously examined by the pathologist it was not possible to perform a three-dimensional analysis of the endometriotic lesion. After deparaffinization in xylene (twice for 5 minutes at room temperature [RT]) and rehydration (10 minutes at RT in acetone and acetone/tris-buffered saline, 1:1), a heat-induced epitope retrieval procedure was performed by heating the probes in citrate buffer (0.1 M citric acid and 0.1 M sodium citrate, pH 6.0 at 700 W, microwave, 17 minutes). After rinsing with tris-buffered saline, the sections were blocked with 10% fetal calf serum (FCS) for 30 minutes at RT. Neurofilament analysis, marker for myelinated nerve fibers (n [ 45) The sections were incubated for 1 hour at RT with monoclonal mouse antineurofilament antibody (clone 2F11, dilution 1:50; Dako, Hamburg, Germany). Rinsing with tris-buffered saline was followed by application of the labeled streptavidin-biotin kit (Dako). Fuchsin substrate (Dako) was used to visualize the specific immunoreactive staining. Protein gene-related product 9.5, marker for unmyelinated nerve fibers, n [ 45 The sections were incubated for 1 hour at RT with polyclonal rabbit antiprotein gene-related product 9.5 antibody (dilution 1:200; Dako). Rinsing with tris-buffered saline was followed by application of the labeled streptavidin-biotin kit (Dako). Fuchsin substrate (Dako) was used to visualize the specific immunoreactive staining. Peripheral nerve tissue was taken as a positive control. Samples of normal peritoneum were serially sectioned and examined to exclude the presence of endometriosis. Negative control sections were processed both by using nonspecific IgG (dilution 1:50; Dako) and by omitting the 1858
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primary antibody. Staining was detected using a radiophoto microscope (Carl Zeiss, Goettingen, Germany). Pictures were taken at different magnifications (40, 100, or 1,000) and further processed using Adobe Photoshop (Adobe Systems, Unterschleissheim, Germany). In cases where nerve fibers were not detectable, up to two more sections were taken from the paraffin block for reexamination. Using a grid of a surface of 1 mm2 on the microscope (at 40), the distance between endometrial glands/ stromal cells and the closest nerve fibers was graded as follows: 1 for close proximity with a space of less than 1.5 mm (defined as endometriosis-associated nerve fibers) and 2 for a space of more than 1.5 mm (9). The mean nerve score was analyzed for each sample. The sample size of the peritoneum was defined as 1 ¼ small, 2 ¼ moderate, and 3 ¼ large and the nerve scores was defined as 0 ¼ no nerves seen, 1 ¼ rare, 2 ¼ few, 3 ¼ many by two independent examiners (i.e., it was a semiquantitative analysis). Sections were evaluated and scored by two separate observers. The occurrence or absence of endometriosis-associated nerve fibers was correlated with the severity of symptoms of pelvic pain and dysmenorrhea, with the localization of peritoneal endometriotic lesions and with the activity of the peritoneal endometriotic lesions. Statistical Analysis Statistical analysis was performed using the SPSS program statistical software (V15.0.1.1; SPSS Inc., Chicago, IL). All tests were two-sided and the level of significance was .05. Frequencies of occurrence were tested with the Fisher’s exact test. RESULTS Nerve Fibers in Peritoneal Endometriotic Lesions Endometrial glands and stromal cells were detectable in 45 peritoneal endometriosis specimens taken from 29 patients. There was no histologic evidence of endometriosis in serial sections of specimens of group D. Nerve fibers were present in all specimens. The mean nerve score in peritoneum of patients with endometriosis was 1.7 (in the periphery of the endometriotic lesion, only 1.3), whereas the nerve score of healthy peritoneum was graded as 1.2. There was no significant difference in mean nerve score between peritoneal specimens with and without endometriosis. Nerve fibers were identified by immunohistochemical staining for neurofilament, a marker molecule for myelinated nerve fibers (16) and protein gene-related product 9.5, a panneuronal marker (17). The immunostaining pattern of both markers showed a comparable moderate staining. In group A, endometriosis-associated nerve fibers were found in 19 patients (82.6%). However, in group B, endometriosis-associated nerve fibers were present in only two cases (33.3%). The occurrence of endometriosis-associated nerve
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FIGURE 2 The occurrence of nerve fibers in peritoneal endometriotic lesions of patients with dysmenorrhea/pelvic pain (groups A and A1) and in those with either low or without dysmenorrhea/pelvic pain (group B). The difference remained significant (Fisher’s t-test).
Correlation of different symptoms (dysmenorrhea and/or pelvic pain; dyspareunia; dyschezia and dysuria) with the occurrence of peritoneal nerves The occurrence of nerve fibers in peritoneal endometriotic lesions was compared with the occurrence of different pain manifestations. Patients with dysmenorrhea and/or pelvic pain had a significantly higher frequency of nerve fibers in peritoneal endometriotic lesions than patients without those symptoms (P<.05). This correlation was not observed compared to the other pain manifestations (dyspareunia, dychezia, and dysuria). Hormonal treatment Fifteen patients developed symptoms under hormonal therapy. The distribution of patients on hormonal treatment was a followed: Group A: 5 of 23 (21.7%) (3 in group A1); group B: 0 of 6; group C: 6 of 13 (46.2%); and group D: 4 of 7 (57.1%). All 5 group A patients with hormonal treatment presented nerve fibers in close contact to the peritoneal endometriotic lesion. There was no significant difference regarding endometriosis-associated symptoms whether or not women were receiving hormonal treatment.
Mechsner. Endometriosis-associated nerve fibers. Fertil Steril 2009.
fibers in patients with severe pelvic pain and/or dysmenorrhea (groups A and A1) was significantly higher than in patients with low symptoms (group B) (P¼.034; P<.05) (Fig. 2). Occurrence of Nerve Fibers Compared to the Lesion Site, Activity, and Stage No significant correlation was detectable between the presence of peritoneal endometriosis-associated nerve fibers and the location of endometriotic lesions or active and inactive peritoneal lesions. In addition, the stage of endometriosis (defined according to the revised American Society of Reproductive Medicine) was not related to the presence of endometriotic-associated nerve fibers. There was no statistically significant intergroup difference. Analysis of the Questionnaire The analysis of the questionnaire showed that 43 of the 51 (84.3%) women had dysmenorrhea or pelvic pain. Thirtynine patients (76.5%) suffered from dysmenorrhea and 27 patients (52.9%) suffered from pelvic pain. Thirty-two women (628%; i.e., the majority) described both dysmenorrhea and pelvic pain. A total number of 36 (70.6%) patients suffered from dyspareunia, 28 (549%) women experienced dyschezia regularly, and 9 patients (17.65%) reported regular dysuria. Most of our patients (n ¼ 37, 72.6%) suffered from dysmenorrhea/pelvic pain and at least from one other symptom (dyspareunia, dyschezia, dysuria). A combination of dysmenorrhea/pelvic pain and all of the other complaints was experienced by only 7 (13.7%) women. Fertility and Sterility
DISCUSSION Recently, large series of laparoscopic resection of endometriotic lesions have shown promising results in terms of the reduction of chronic pelvic pain and reduction of severe dysmenorrhea (18). However, the fundamental pathological mechanism underlying endometriosis-related pain seems to be unclear, particularly the role of peritoneal lesions in generating pelvic pain (19, 20). Important data on innervation of endometriosis, endometrium, and myometrium have been delivered by different groups of investigators.(8–11, 19, 21–23). In this respect, the occurrence of nerve fibers in close topographical relationship to the peritoneal endometriotic lesion has been demonstrated (8, 9). Furthermore, evidence seems to suggest that these endometriosis-associated nerve fibers are of a new origin (9). The peritoneal endometriotic lesions are able to produce several pain-mediating substances, such as prostaglandins (PG) like PG E2 and F2a, histamine, kinins, NGF, and interleukins, which can activate peritoneal nociceptors (2, 24–26). However, there is no direct or indirect evidence that peritoneal endometriosis-associated nerve fibers are involved in conduction of endometriosis-related pain. In this pilot study we discussed a clinical correlation between the occurrence of endometriosis-associated nerve fibers and the severity of symptoms. All patients undergoing surgery either for symptoms of pelvic pain and/or dysmenorrhea or the ultrasound diagnoses of ovarian cysts have been included. To correlate the occurrence of endometriosis-associated nerve fibers in peritoneal endometriotic lesions, only specimens of peritoneal endometriotic lesions have been analyzed. Nerve fibers were detected using established markers against neurofilament (myelinated nerve fibers, such as Aa, Ab, Ag, Ad, and B fibers) and protein gene-related product 9.5 (a pan-neuronal marker against myelinated and 1859
unmyelinated nerve fibers such as Aa, Ab, Ag, Ad, B, and C fibers) (16, 17). The Ad fibers are small myelinated fibers and transmit sharp, prickling localized pain to the central nervous system. The C fibers are small unmyelinated fibers and transmit dull, aching, burning pain that is hard to locate (16, 17). In agreement with other investigators, myelinated and unmyelinated nerve fibers could be detected (8). We defined endometriosis-associated nerve fibers as ‘‘close’’ when the distance between the nerve fibers and the epithelial cells of the endometriotic lesion was less than 1.5 mm as done in previous studies by our group (9). This definition is indeed a random one; however, when using a magnification of 400 this distance can be evaluated with occurrence. Furthermore, it is unknown over which distance paracrine hormones can exert an effect on neighboring nerve fibers. As long as this question remains unanswered, the definition of endometriosis-associated nerve fibers can only be a random one. Our data suggest that in peritoneal endometriotic lesions of patients with severe symptoms (groups A and A1), there were significantly more endometriosis-associated nerve fibers than in those of patients with only few symptoms (group B) (P¼.031). Although statistical significance has been reached, such small number random fluctuation of data cannot be excluded. Because group C and D patients had no peritoneal endometriotic lesions, our study concentrated on the patients in groups A and B only. Importantly, Tokushige and colleagues (8) detected a higher number of nerve fibers in peritoneum of patients with endometriosis than on those without it. In agreement with Tulandi et al. (27), we could not detect any significant difference in the total nerve scores when comparing peritoneum from patients with and without endometriosis. The control collective in our study underwent diagnostic laparoscopy for symptoms like pelvic pain, but no apparent endometriotic lesions were detected. However, these women suffered from fibroids and adhesions. In contrast, the patients in the control group of Tokushige and colleagues (8) had no pain. They underwent laparoscopy for tubal sterilization or investigation of infertility. In this group no recognizable pathology was observed. This might explain the different findings of peritoneal nerve density. To study the clinical manifestation of symptoms in correlation to the occurrence of endometriosis-associated nerve fibers, we documented the severity of symptoms before surgical intervention using a questionnaire to reveal levels of endometriosis-related pain (VAS) and focusing particularly on pelvic pain and dysmenorrhea. Furthermore, the presence of dyschezia, dyspareunia, and dysuria was documented.
different manifestations (endometriosis genitalis externa, adenomyosis uteri, and DIE), it is very difficult to investigate one isolated parameter for pain conduction. Subgroup A1 had only peritoneal endometriotic lesions. As a result pain conduction dependent from peritoneal endometriotic lesions only could be analyzed. Endometriosis-associated nerve fibers could be detected in lesions of group A and A1 patients, but not in peritoneal endometriotic lesions of group B patients. This finding might be a possible co-factor in pain generation. In addition to endometriosis-associated nerve fibers, the activity of the peritoneal endometriotic lesions with the release of pain mediators and the lesion site are also factors for pain generation (2, 26). Furthermore, in the peritoneum of patients with endometriosis, endometriosis-associated smooth muscle cells could be demonstrated, which express oxytocin, E, and P receptors (21). It is possible that the cyclic activation of smooth muscle cells and endometriosis-associated nerve fibers may be involved in the development of cyclic pelvic pain and the sensation of dysmenorrhea-similar pain (21). The occurrence of nerve fibers in peritoneal endometriotic lesions was compared with the occurrence of different pain manifestations. Patients with dysmenorrhea with pelvic pain seem to have significant more nerve fibers in peritoneal endometriotic lesions than patients without these symptoms (P<.05), suggesting that nerve fibers are involved in pain conduction of dysmenorrhea and/or pelvic pain (8). Also in DIE cases only, it could be demonstrated that perineural invasion by endometriosis was associated with severe pain (22). One possible weak point of the study is the small number of patients in the subgroups. Interestingly, it has been demonstrated that there are significantly more sensory nerve fibers in the endometrium of patients with endometriosis than in those without endometriosis (23, 29). These important data suggested that nerve fibers in the endometrium may play an important role in generation of dysmenorrhea. Another important study demonstrated a persistence of dysmenorrhea and nonmenstrual pain after optimal endometriosis surgery in cases of adenomyosis (30). However, present data suggest, that both uterine and peritoneal factors are responsible for the development of dysmenorrhea and pelvic pain. In summary, this is the first evidence for a clinical correlation of nerve fibers in the development of endometriosis-related symptoms like pelvic pain and/or dysmenorrhea. REFERENCES
The analysis of the questionnaire demonstrated the typical distribution of endometriosis-related pain in our study group (28). However, due to the complexity of the endometriosis-related symptoms, the combined occurrence of the complaints (such as dysmenorrhea, pelvic pain, deep dyspareunia, dyschezia, and dysuria) and the coincidence of 1860
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