Human Reproduction Vol.17, No.7 pp. 1895–1900, 2002
Hyperalgesia, nerve infiltration and nerve growth factor expression in deep adenomyotic nodules, peritoneal and ovarian endometriosis V.Anaf1,4, P.Simon1, I.El Nakadi3, I.Fayt2, T.Simonart2, F.Buxant1 and J-C.Noel2 Departments of 1Gynaecology, 2Pathology and 3Digestive Surgery, Hospital Erasme, Universite´ Libre de Bruxelles (ULB), Brussels, Belgium 4To
whom correspondence should be addressed at: Department of Gynaecology, Hospital Erasme, 808, Route de Lennik, 1070 Brussels, Belgium. E-mail:
[email protected]
BACKGROUND: The aim of this study was to investigate a possible role for nerve growth factor (NGF) in the mechanism of pain and hyperalgesia induced by deep adenomyotic nodules and other forms of endometriosis and to clarify the relationship between endometriotic lesions and the surrounding nerves. METHODS: Endometriotic lesions (deep adenomyotic nodules, peritoneal endometriosis, ovarian endometriosis) and eutopic endometrium were obtained from 51 patients presenting with pain. Patients were allocated to two groups (group 1: patients with a deep adenomyotic nodule (n ⍧ 23); group 2: patients with peritoneal and/or ovarian endometriosis but without deep adenomyotic nodule (n ⍧ 28). Immunohistochemistry with antibodies against NGF, NGF specific tyrosinekinase receptor (Trk-A) and S-100 protein was performed. Results were expressed as mean H-scores ⍨ SD, and correlated with the presence of hyperalgesia. RESULTS: The percentage of patients presenting hyperalgesia at physical examination was significantly higher in group 1 (96%) than in group 2 (11%) (P < 0.001). NGF expression was significantly stronger in deep adenomyotic nodules (DAN) than in ovarian (OE) and peritoneal endometriosis (PE), both in the proliferative phase in the glands [DAN: 226 ⍨ 18; OE: 140 ⍨ 9 (P < 0.001); PE: 110 ⍨ 7 (P < 0.001)] and in the stroma [(DAN: 204 ⍨ 21; OE: 125 ⍨ 15 (P < 0.001); PE: 100 ⍨ 9 (P < 0.01)]. NGF expression in DAN is also significantly stronger than in OE and PE in the secretory phase in the glands [DAN:181 ⍨ 32; OE: 85 ⍨ 3.3 (P < 0.001); PE: 65 ⍨ 9 (P < 0.001)] and in the stroma [DAN: 173 ⍨ 28; OE: 85 ⍨ 3.7 (P < 0.001); PE: 35 ⍨ 13 (P < 0.001)]. Perineurial and intraneurial invasion by endometriotic lesions were found only in deep adenomyotic nodules and not in the other forms of endometriosis. The specific receptor for NGF (TrkA) is expressed in all the nerves that were included in the biopsies. CONCLUSIONS: These results suggest a role of NGF in endometriotic pain and hyperalgesia in deep adenomyotic nodules. The strong expression of the NGFTrkA pathway in deep adenomyotic nodules could explain why this type of lesion infiltrates in richly innervated anatomical sites. Key words: adenomyosis/deep infiltrating endometriosis/endometriosis/hyperalgesia/nerve growth factor
Introduction The most common and most specific symptom of endometriosis is pain. Although all ectopic endometrial-like lesions can produce pain, the specific entity called ‘deep adenomyotic nodules’ can be responsible for severe symptoms. Indeed patients with untreated deep adenomyotic nodules very often relate high pre-operative pain scores (Clayton et al., 1999; Porpora et al., 1999; Anaf et al., 2000a). A deep adenomyotic nodule must be clearly differentiated from other forms of endometriosis. It represents a circumscribed nodule composed of abundant smooth muscles, endometrial stroma and endometrial glands and is similar to an adenomyoma (Donnez et al., 1995; Nisolle and Donnez, 1996, 1997; Anaf et al., 2000b). © European Society of Human Reproduction and Embryology
What is well known but rarely described by clinicians dealing with endometriosis is the important exacerbation of pain when pressure is exerted on deep nodular lesions at physical examination. This phenomenon of exquisite pain when a normally non-painful stimulus is applied is called ‘hyperalgesia’. Pain must be differentiated from hyperalgesia which is defined as an exacerbation of pain when a nonpainful stimulus is applied. It is characterized by a deplacement to the left of the curve stimulus/response which induces an increase in the pain sensation associated with a lower threshold of pain perception and an increased sensitivity of nociceptors (Bouaziz and Lombard, 1997). The most classical example of hyperalgesia is the pain exacerbation that occurs during or after deglutition in case of pharyngitis. 1895
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However the mechanisms by which endometriotic lesions produce pain and hyperalgesia are poorly understood. Numerous theories have been proposed to explain pain mediation by endometriotic tissue, including production and release of prostaglandins; inflammatory mediators such as kinins, histamine, interleukins, etc.; fibrosis; and cyclical haemorrhage. In the particular case of deep nodular adenomyosis involving the rectovaginal septum and/or the Douglas pouch, the immobility of the posterior pelvic structures (cervix, upper vagina and anterior rectal wall) could also explain the pain occurring at intercourse or at defecation. We recently showed that in adenomyotic nodules involving the Douglas pouch and/or the rectovaginal septum, there is a close histological relationship between the nerves and the lesion (Anaf et al., 2000a). In order to clarify this relationship and the observation that deep adenomyosis can infiltrate nerves and cause hyperalgesia, we studied the expression of the nerve growth factor (NGF) and its specific tyrosine-kinase receptor (TrkA), by immunohistochemistry, in different forms of endometriosis and the surrounding nerves. The results were correlated with the presence of hyperalgesia. Nerve growth factor (NGF) belongs to the neurotrophin family. The term neurotrophic means ‘neuron nourishing’ and classically refers to those proteins that promote the survival of specific populations of neurons. In recent years it has become apparent that NGF plays an important role in mediating neuropathological and nonneuropathological pain (Apfel, 2000). Trk-A is the high affinity receptor for NGF and is an essential component in the mediation of NGF response (Klein et al., 1991). The binding of NGF to Trk-A induces receptor autophosphorylation and induction of intracellular signalling pathways, resulting in diverse biological effects (Kaplan et al., 1991).
Materials and methods Patients A total of 51 consecutive Caucasian patients (mean age: 27.2 years; range: 24.3–37.8) with clinical or radiological suspicion of endometriosis underwent laparoscopy. Forty five percent of patients (n ⫽ 23) had a deep adenomyotic nodule: rectovaginal Douglas pouch (n ⫽ 8); rectovaginal Douglas pouch and rectovaginal septum (n ⫽ 12); uterosacral ligaments nodules (n ⫽ 3). All patients with rectovaginal Douglas pouch nodules with or without involvement of the rectovaginal septum had a partial involvement of the uterosacral ligaments. A total of 25% of patients (n ⫽ 13) had peritoneal endometriosis, 8% (n ⫽ 4) of patients had ovarian endometriosis and 22% (n ⫽ 11) had both ovarian and peritoneal endometriosis. Finally, 18% (n ⫽ 9) had a unilateral endometrioma and 12% of patients (n ⫽ 6) had bilateral endometriotic cysts. During the surgical procedure, an endometrial biopsy was performed in each patient for histological dating of the endometrium according to the criteria of Noyes (Noyes, 1973). The patients were allocated to two groups: group 1: patients with a deep endometriotic nodule (n ⫽ 23) and group 2: patients with peritoneal and/or ovarian endometriosis but without deep infiltrating endometriotic nodule (n ⫽ 28). The mean largest diameter of the ovarian endometriomas
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was evaluated by a pre-operative endovaginal ultrasonography and was 3.5 cm (range: 2.1–6.8 cm). Hyperalgesia Hyperalgesia is defined as an exacerbation of pain when a nonpainful stimulus is applied (Bouaziz and Lombard, 1997). Patients who presented an intense pain when a pressure was exerted in the posterior fornix were considered as presenting hyperalgesia. In those patients, the pain could be reproduced by touching the posterior fornix with a sponge on forceps. The comparison of the number of patients presenting with hyperalgesia in group 1 and in group 2 was made using odds ratio (OR), corresponding 95% confidence intervals (CI) and contingency table analysis based on χ2 summary statistics. Surgical procedures and sampling technique All patients underwent resection of all visible peritoneal implants, endometriotic cysts and deep adenomyotic nodules by laparoscopy (n ⫽ 51). Patients presenting with a nodular lesion in the Douglas pouch with or without involvement of the rectovaginal septum underwent a resection of the lesion as previously described by Donnez et al., (Donnez et al., 1995). In three patients (6%), it was necessary to remove the rectum (anterior resection of the rectum) because of the presence of an important rectal stenosis and rectorrhagia. The resection of the rectum was performed by a laparoscopically-assisted technique as previously described (Anaf et al., 2000c). Histology and immunohistochemistry After resection, the lesions were immediately fixed in 4% formaldehyde for 12 h and then embedded in paraffin. For each specimen, seven serial sections of 4 µm were performed. The first section was stained with haematoxylin and eosin, the other sections were used for immunohistochemistry and controls. For immunohistochemistry, the antigen retrieval method was performed for NGF and NGF receptor, but not for S-100 protein, as has been previously described (Anaf et al., 2000d). The characteristics and dilution of the primary antibodies were respectively: monoclonal antibody S100 protein (clone 15E2E2; dilution 1/100, Biogenex, San Ramon, USA); the polyclonal rabbit anti-TrkA immunoglobulin (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against an epitope corresponding to amino acids 763 to 777 mapping adjacent to the carboxyl terminus of human Trk p 140 (1/600 dilution); and the polyclonal antibody against NGF (NGF H-20, dilution 1/400; Santa Cruz Biotechnology) raised against ultrapure 2.5 S NGF. To control for non-specific binding of the primary antibodies, nonimmune mouse serum at the same concentration as the primary antibodies preparation were substituted as the first layer of the serial sections for S100, NGF and TrkA immunohistochemistry. Positive controls were Schawnnoma and neurofibroma previously proven to express NGF-receptor, NGF and S-100 protein. The immunohistological expression of NGF and TrkA were assessed by using the semiquantitative H-score as previously described for the same antibodies (Zhu et al., 1999; Friess et al., 1999). The H-score is based on a summation of the proportion of lesion cells showing different degrees of reactivity as follows: 0% of negative cells ⫹ 1% of weakly positive cells ⫹ 2% of moderately positive cells ⫹ 3% of strongly positive cells. The H-score ranges from 0–300. The maximum score (300) represents 100% of cells showing strong positivity. In each of the different anatomical locations of endometriosis and in eutopic endometrium, we compared NGF expression between the proliferative and the secretory phase, between the glands and the chorion and between the different anatomical locations of endometriosis (peritoneal lesions versus ovarian lesions; peritoneal lesions
Hyperalgesia and nerve growth factor in deep endometriosis
Figure 1. Typical example of peri- and intraneurial invasion by endometriotic stromal cells (S-100 immunostaining) (magnification ⫻40).
Figure 2. Strong glandular and moderate stromal NGF expression (NGF immunostaining; detail of a rectovaginal adenomyotic nodule) (magnification ⫻10).
versus deep adenomyotic nodules; ovarian lesions versus deep adenomyotic nodules). For NGF expression H-scores, data were compared using Student’s t-test (two-tailed).
Comparison of nerve density between the different topographical regions was performed using the Student’s t-test (two-tailed).
Results Perineurial and endoneurial infiltration Perineurial invasion was defined as the presence of stromal cells that have penetrated the perineurium, which is composed of concentric flattened cells separated by layers of collagen at the periphery of the myelinated neural cells (Sternberg, 1992). Endoneurial or intrafascicular invasion was defined as the presence of stromal and/or glandular cells within the nerve fascicle (Sternberg, 1992) (Figure 1). Nerve density Biopsies of the following four different unaffected anatomical sites [peritoneum of the ovarian fossa (n ⫽ 5), ovary (n ⫽ 4), rectovaginal septum (n ⫽ 6), uterosacral ligament (n ⫽ 5)] were taken in nonmenopausal patients without endometriosis who underwent vaginal hysterectomy (n ⫽ 5) or laparoscopically assisted vaginal hysterectomy (n ⫽ 6) for an enlarged uterus with several symptomatic myomas. Four patients underwent unilateral adnexectomy for a persisting unilateral ovarian cyst after a 3 month course of combined oral contraception. All patients gave consent for these biopsies. Using a grid of a surface of 1 mm2 on the microscope (at ⫻40) linked via a colour CCD video camera (MW-FI5-e, Panasonic; Matsushita Electrical Industrial Co Ltd, Osaka, Japan) to a large screen monitor (Panasonic Quintrix; Matsushita Electrical Company of UK, Pentwyn, Cardiff, UK), we calculated the number of nerve sections in 10 nonoverlapping randomly selected areas of 1 mm2 surface in the four respective tissues, previously stained with the monoclonal antibody against protein S-100. The results are expressed as mean numbers of nerve sections per surface of 1 mm2 ⫾ SD.
Hyperalgesia In total, 96% (n ⫽ 22) of patients in group 1 and 11% (n ⫽ 3) of patients in group 2 presented with hyperalgesia at physical examination. The pain could be reproduced by touching the posterior fornix with a sponge on a forceps. The percentage of patients with hyperalgesia was significantly higher in group 1 (96%) than in group 2 (11%) (OR, 183.3; 95% CI, 33.7–995.2; P ⬍ 0.001). Immunohistochemistry Table I represents the NGF immunoreactivity (expressed as mean H-scores ⫾ SD) in the eutopic and ectopic endometrial glands and stroma according to the phase of the menstrual cycle. Comparison of the NGF expression between the glands and the stroma in the different anatomical locations When the expression of NGF (mean H-scores ⫾ SD) was compared between the glands and the stroma in the proliferative and the secretory phase there was no statistically significant difference in the eutopic endometrium (EE), ovarian endometriosis (OE) and deep adenomyotic nodules (DAN). However in peritoneal endometriosis (PE), this difference was found to be significant in the proliferative phase (glands: 110 ⫾ 7; stroma: 100 ⫾ 9; P ⬍ 0.05) and in the secretory phase (glands: 65 ⫾ 9; stroma: 35 ⫾ 13; P ⬍ 0.001). 1897
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Table I. NGF expression in endometriosis and endometrium
Eutopic endometrium (proliferative) Eutopic endometrium (secretory) Peritoneal endometriosis (proliferative) Peritoneal endometriosis (secretory) Ovarian endometriosis (proliferative) Ovarian endometriosis (secretory) Deep endometriotic nodule (proliferative) Deep endometriotic nodule (secretory)
n⫽
Glands
8 8 10 15 10 11 12 11
85 ⫾ 2010a 1107 659a 140 ⫾ 85 ⫾ 226 ⫾ 181 ⫾
15
9 3.3a 18 32
Stroma
P-value
7013 30 14a 1009 3513a 125 ⫾ 85 ⫾ 204 ⫾ 173 ⫾
NS NS ⬍ 0.05 ⬍ 0.001 NS NS NS NS
15 3.7a 21 28
aSignificantly
different when comparing secretory with proliferative. Results are expressed as means ⫾ SD. Nerves: ⫹, endothelial cells: ⫹⫹; muscle layers arteries: ⫹ (⫹: weak staining; ⫹⫹: moderate staining; ⫹⫹⫹: strong staining). NGF expression H-scores in the glands and the stroma were compared using Student’s t-test (two-tailed).
Figure 3. Section of the same lesion. NGF specific receptor (Trk-A) immunostaining. Presence of a strong staining of the nerve. The nerve is in narrow contact with the stromal cells of the adenomyotic lesion (magnification ⫻20).
Comparison of the NGF expression in the glands between the different anatomical locations NGF expression in the glands is significantly stronger in DAN than in OE and in PE in the proliferative phase [DAN: 226 ⫾ 18; OE: 140 ⫾ 9 (P ⬍ 0.001); PE: 110 ⫾ 7 (P ⬍ 0.001)] (Figure 2). NGF expression was significantly stronger in ovarian than in peritoneal endometriosis [OE: 140 ⫾ 9; PE: 110 ⫾ 7 (P ⬍ 0.001)]. In the secretory phase in the glands, NGF H-scores in deep adenomyotic nodules were also significantly stronger than in ovarian and peritoneal endometriosis [DAN: 181 ⫾ 32; OE: 85 ⫾ 3.3 (P ⬍ 0.001); PE: 65 ⫾ 9 (P ⬍ 0.001)]. Also in the 1898
secretory phase NGF expression in OE was stronger than in PE [OE: 85 ⫾ 3,3; PE: 65 ⫾ 9 (P ⬍ 0.001)]. Comparison of the NGF expression in the stroma between the different anatomical locations NGF expression in the stroma is significantly stronger in DAN than in OE and PE in the proliferative phase [DAN: 204 ⫾ 21; OE: 125 ⫾ 15 (P ⬍ 0.001); PE: 100 ⫾ 9 (P ⬍ 0.001)]. NGF expression in OE was also significantly stronger than in PE [OE: 125 ⫾ 15; PE: 100 ⫾ 9 (P ⬍ 0.01)]. In the secretory phase in the stroma, the expression of NGF was also significantly stronger in DAN than in OE and PE [DAN: 173 ⫾ 28; OE: 85 ⫾ 3.7 (P ⬍ 0.001); PE: 35 ⫾ 13 (P ⬍ 0.001)]. The same significant stronger NGF expression was found in OE when compared with PE [OE: 85 ⫾ 3.7; PE: 35 ⫾ 13 (P ⬍ 0.001)]. NGF expression in the non-endometriotic tissue NGF was weakly expressed in the nerves (⫹) that were surrounded and/or invaded by endometriotic stromal cells. The smooth muscle cells of the arteries (⫹) also weakly expressed NGF. Endothelial cells moderately expressed NGF (⫹⫹) as previously described (Friess et al., 1999; Zhu et al., 1999). NGF specific receptor (Trk-A) expression NGF specific receptor (Trk-A) was immunohistochemically strongly expressed (⫹⫹⫹) in all the nerves that were surrounded and/or invaded by endometriosis or that were near endometriotic lesions (Figure 3). NGF receptor was not expressed in the endometriotic glands and stroma of the different anatomical locations, and it was not expressed in the non-endometriotic tissues of the biopsies (arteries, endothelium, peritoneum). Perineurial and endoneurial invasion Perineurial and intraneurial invasion by endometriotic stroma and/or glands were found only in deep infiltrating adenomyotic nodules (Figures 1 and 3). Using S-100 and NGF receptor immunohistochemistry in ovarian and peritoneal endometriosis, the nerves were always located at a distance from the endometriotic lesions. Nerve density The non-affected rectovaginal septum (RVS) contained significantly higher nerve density (expressed as mean number of
Hyperalgesia and nerve growth factor in deep endometriosis
nerve sections per ml2 of tissue ⫾ SD) than the non-affected ovary (O) or peritoneum (P) [RVS: 6 ⫾ 2; O: 1 ⫾ 1 (P ⬍ 0.001); P: 2 ⫾ 1 (P ⬍ 0.001)]. The non-affected uterosacral ligament (UL) also contained a significantly higher nerve density than the non-affected ovary (O) or peritoneum (P) [UL: 5 ⫾ 2; O: 1 ⫾ 1 (P ⬍ 0.001); P: 2 ⫾ 1 (P ⬍ 0.01)]. There was no significant difference between the nerve density of the unaffected rectovaginal septum and that of the unaffected uterosacral ligament (RVS: 6 ⫾ 2; UL: 5 ⫾ 2; NS). Also between the unaffected peritoneum and ovary there was no significant difference in terms of nerve density (O: 1 ⫾ 1; P: 2 ⫾ 1; NS). Discussion Why do deep adenomyotic nodules almost always occur in the same anatomical sites? Why are deep sub-peritoneal nodules often more symptomatic than the other forms of endometriosis? Both these two questions remain unanswered in the abundant literature on endometriosis. In the present study we have demonstrated for the first time that NGF is expressed in all the different forms of endometriosis with a particular strong expression in deep adenomyotic nodules which are considered as the most symptomatic form of the disease (Anaf et al., 2000a). Interestingly, NGF is well known to play a key role in the occurrence of hyperalgesia (Apfel, 2000). Firstly, because NGF itself can serve as a pain mediator and it can induce the expression of substance P and calcitonin gene related peptide (CGRP), neuropeptides involved in modulating central pain transmission (Kessler and Black, 1980; McLean et al., 1989). Secondly, it can increase the number of sensory neurons and is selectively trophic for small fibre sensory neurons and sympathetic ganglion neurons which participate in mediating pain sensation (Apfel, 2000). Transgenic animals lacking the gene for NGF or for its specific receptor TrkA are born with no small diameter nociceptive sensory neurons and are hypoalgesic. In contrast, animals that over-express NGF are behaviourally hyperalgesic (Davis et al., 1993). Therefore the over-expression of NGF in all the forms of endometriosis, but particularly in deep adenomyotic nodules, could explain at least partially the occurrence of pain and hyperalgesia which appear as the major symptoms of the disease. However, until now there has been no clear explanation for the significantly stronger expression of NGF in deep adenomyotic nodules when compared to the other forms of endometriosis. It can be due to the possible different aetiopathogenies of these different forms of endometriosis. Indeed rectovaginal adenomyosis could result from the metaplasia of Mu¨ llerian remnants located deep in the rectovaginal septum. Peritoneal endometriosis could result from implantation of endometrial cells and stroma and ovarian endometriosis from the metaplasia of the invaginated ovarian mesothelium into the ovarian cortex (Nisolle and Donnez, 1996, 1997). Therefore the natural history of deep adenomyotic nodules could be different from that of the other forms of the disease. A second possible explanation consists in the inflammation process which is frequently present in deep adenomyotic nodules. It has been shown that levels
of NGF are significantly elevated at sites of inflammation (Weskamp and Otten, 1987) where its production may be increased by inflammatory mediators such as interleukin-1, tumour necrosis factor-α or other cytokines (Lindholm et al., 1987; Friedman et al., 1990; Aloe et al., 1992; Donnerer et al., 1992; Safieh-Garabedian et al., 1995). In turn, NGF can induce hyperplasia and degranulation of mast cells, releasing additional inflammatory mediators such as serotonin which sensitize peripheral nociceptors (Marshall et al., 1990; Lewin et al., 1994). The second interesting observation of this study is that the specific receptor for NGF is not expressed in endometriotic lesion but is mainly present in sub-peritoneal, peritoneal and ovarian nerves. Therefore it seems that NGF might interact with NGF receptors and that there may exist some attraction between endometriotic cells and the surrounding nerves. Because NGF can act as a positive chemotaxin for neurons and may facilitate their contact with target tissues (Gundersen and Barrett, 1980; Yamamoto and Iseki, 1992), this might explain why deep nodular lesions essentially occur in richly innervated anatomical sites (rectovaginal septum, uterosacral ligaments) (Figures 1 and 3) and not in anatomical sites with scanty underlying nerves. These data are similar to those recently described in pancreatic cancer and chronic pancreatitis, suggesting that the NGF/Trk-A interaction might influence the pain syndrome in these painful diseases as well as the particular form of invasion (perineurial invasion) of pancreatic cancer cells (Friess et al., 1999; Zhu et al., 1999). In conclusion, the higher expression of NGF in deep adenomyotic nodules correlated with the presence of hyperalgesia in patients with deep adenomyotic nodules, and the proclivity of adenomyotic lesions to invade along, around (perineurial invasion) and into nerves (endoneurial or intrafascicular invasion) in richly innervated anatomical sites, suggest an important role of NGF in the expression of the pain syndrome and possibly the capacity of some lesions to infiltrate.
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