Effects of hormonal treatment on nerve fibers in endometrium and myometrium in women with endometriosis

Transcription

1 Effects of hormonal treatment on nerve fibers in endometrium and myometrium in women with endometriosis Natsuko Tokushige, Ph.D., a Robert Markham, Ph.D., a Peter Russell, M.D., b and Ian S. Fraser, M.D. a a Department of Obstetrics and Gynaecology, Queen Elizabeth II Research Institute for Mothers and Infants, University of Sydney, Sydney; and b Department of Pathology, University of Sydney, Sydney, Australia Objective: To investigate how hormonal treatment can change nerve fiber density and to identify types of nerve fibers in endometrium and myometrium in women with endometriosis. Design: Laboratory study using human tissue. Setting: University-based laboratory. Patient(s): Hormonally treated and untreated women with endometriosis undergoing hysterectomy or curettage. Intervention(s): Endometrial and myometrial tissues were prepared from women with hormonally treated endometriosis and women with untreated endometriosis. Main Outcome Measure(s): Types and density of nerve fibers in endometrium and myometrium in women with hormonally treated and untreated endometriosis were determined immunohistochemically. Result(s): The nerve fiber density (mean density SD per square millimeter) in the functional and the basal layers of endometrium ( /mm 2 and /mm 2, respectively) and myometrium ( /mm 2 ) from women with hormonally treated endometriosis was much lower than that of endometrium (functional layer: 11 5/mm 2, basal layer: 18 8/mm 2, respectively) and myometrium (3 1/mm 2 ) from women with untreated endometriosis. Nerve growth factor and nerve growth factor receptor p75 expression was also significantly reduced in women with hormonally treated endometriosis compared with women with untreated endometriosis. Conclusion(s): Hormonal treatment significantly reduced nerve fiber density in endometrium and myometrium in women with endometriosis. (Fertil Steril Ò 2008;90: Ó2008 by American Society for Reproductive Medicine.) Key Words: Endometriosis, nerve fibers, hormonal therapy, endometrium, myometrium Endometriosis is a recurrent debilitating disease that affects predominantly reproductive-age women. It is characterized by the presence of endometrium-like tissue outside the uterus, and the symptoms of endometriosis include chronic dysmenorrhea, deep dyspareunia, and dyschezia. Endometriosis is an estrogen-dependent disease, and current medical treatments suppress estrogen synthesis. Drugs such as danazol, GnRH analogues, combined oral contraceptives (OCs), and progestogens have been used to treat endometriosis-associated pain (1). Danazol suppresses gonadotropin secretion, particularly LH, and inhibits ovarian estrogen production and endometrial growth (2). Gonadotropin-releasing hormone analogues decrease secretion of gonadotropins, resulting in dramatically decreased serum estrogen levels (3). Combined OCs inhibit ovulation (4) and cause decidualization, then marked endometrial thinning, and increase apoptosis and decrease Bcl-2 expression in eutopic endometrium in women with endometriosis (5). Progestogens also decrease Received June 21, 2007; revised August 24, 2007; accepted August 31, Supported by research funding from the Department of Obstetrics and Gynaecology, University of Sydney, Sydney, Australia. Reprint requests: Natsuko Tokushige, Ph.D., University of Sydney, Department of Obstetrics and Gynaecology, Queen Elizabeth II Research Institute for Mothers and Infants, NSW 2006, Australia (FAX: ; ntokushige@med.usyd.edu.au). serum estrogen levels by suppressing gonadotropin release (6), cause glandular atrophy and stromal decidualization in eutopic endometrium in women with endometriosis (4, 7), inhibit angiogenesis (8), and decrease intraperitoneal inflammation (9). Several studies have demonstrated the efficacy of drugs for endometriosis-associated pain; treatment with an OC, norethindrone, and dydrogesterone significantly reduced pain symptoms including deep dyspareunia, dysmenorrhea, and pelvic pain in women with endometriosis (10 13). It has been demonstrated that women with endometriosis have a greater density of nerve fibers in endometrium and myometrium than women without endometriosis (14) and that eutopic endometrium in women with endometriosis is innervated by sensory Ad, sensory C, and adrenergic fibers and myometrium by sensory Ad, sensory C, adrenergic, and cholinergic fibers (15). Several studies have shown the efficacy of currently available hormonal treatments in alleviating pain symptoms in women with endometriosis (16, 17). However, no study has investigated yet whether hormonal treatment changes nerve fiber density in endometrium and myometrium in women with endometriosis who have pain symptoms. We have studied innervation with different types of specific immunohistochemical neuronal markers in endometrium /08/$34.00 Fertility and Sterility â Vol. 90, No. 5, November doi: /j.fertnstert Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 and myometrium specimens from women with visually and biopsy-proved endometriosis who were receiving hormonal treatment until they underwent hysterectomy or endometrial sampling. MATERIALS AND METHODS Tissue Collection This study was approved by the Human Ethics Committees of the Sydney South West Area Health Service and the University of Sydney, Sydney, Australia, and all women gave their informed consent for participation. Eighteen full-thickness uterine blocks, which included endometrium and contiguous myometrium (mean age of patient 40.2 years; range, years), and eight tissue samples from curettage (mean age of patient 26.8 years; range, years) were selected from women with laparoscopic evidence of endometriosis who underwent laparoscopy combined with hysteroscopy and were receiving hormonal treatment until they underwent hysterectomy or endometrial sampling. Of this same group of 26 women, 20 were taking oral progestogens and 7 were taking combined OCs (one woman was taking more than one form of medication). The types of progestogens were either medroxyprogesterone acetate or norethindrone, and the composition of the combined OCs was ethinylestradiol 30 mg and levonorgestrel 150 mg. The combined OCs were being taken continuously in this group of patients. The duration of medications varied from 1 month to 5 months before they underwent hysterectomy or endometrial sampling. Sixty-two percent of the women with hormonally treated endometriosis had peritoneal endometriosis alone, 23% had ovarian endometriosis alone, and 15% had both peritoneal and ovarian endometriosis. The locations of endometriosis were based on findings at the time of laparoscopy before treatment. The subsequent laparoscopy was performed because of persistent pain. The control group of 10 women (mean age 44.0 years; range, years) with proved endometriosis, who were not receiving any medication before hysterectomy, was part of an earlier study (14). Seventy percent of the women with untreated endometriosis had peritoneal endometriosis alone, 20% had ovarian endometriosis alone, and 10% had both peritoneal and ovarian endometriosis. The interval of time from the diagnosis of endometriosis till the hysterectomy was variable, and the diagnosis of endometriosis was originally made at a preceding laparoscopy, but the presence of endometriosis was confirmed at each hysterectomy. The patients with endometriosis all complained of dysmenorrhea and a range of other related pain symptoms. The severity of pain was not assessed systematically and prospectively in this study, but detailed clinical information was recorded in a standard format. Endometriosis in all patients was staged according to the revised American Fertility Society score, which ranged from I to IV (18). Immunohistochemistry After surgical removal, the specimens were immediately fixed in 10% neutral buffered formalin for approximately 18 to 24 hours, processed, and embedded in paraffin wax according to a standard protocol. Each section was cut at 4 mm and routinely stained with hematoxylin and eosin. Markers included polyclonal rabbit anti-protein gene product 9.5 (PGP9.5), a highly specific panneuronal marker, which recognizes all types of nerve fibers; monoclonal mouse anti-human neurofilament (NF); polyclonal rabbit anti-nerve growth factor (NGF); monoclonal mouse anti-human nerve growth factor receptor p75 (NGFRp75); polyclonal rabbit anti-substance P (SP); rabbit anti-calcitonin gene related peptide (CGRP); polyclonal rabbit anti-vesicular acetylcholine transporter (VAChT); monoclonal anti-tyrosine hydroxylase (TH); polyclonal rabbit anti-vasoactive intestinal polypeptide (VIP); and polyclonal rabbit anti-neuropeptide Y (NPY). Serial sections were immunostained with use of antibodies for PGP9.5 (dilution 1:1,400), NF (dilution 1:400), SP (dilution 1:7,000), CGRP (dilution 1:150), VAChT (dilution 1: 8,000), TH (dilution 1:1500), VIP (dilution 1:3,000), NPY (dilution 1:2,500), NGF (dilution 1:500), and NGFRp75 (dilution 1:1,700); incubated with REAL Detection System (DAKO, Carpinteria, CA), alkaline phosphatase/red, Link, biotinylated secondary antibodies, and REAL Detection System (DAKO), alkaline phosphatase/ RED, streptavidin alkaline phosphatase; and stained for REAL Detection System, chromogen (red) as described previously (15). All immunostaining was carried out on a DAKO Auto Stainer (model S3400; DAKO). Images of the sections were captured with use of an Olympus microscope BX51 and digital camera DP70 (Olympus, Tokyo, Japan). We used normal skin as a positive control because it reliably contains myelinated and unmyelinated nerve fibers expressing PGP9.5, NP, NGF, NGFRp75, SP, CGRP, TH, VAChT, VIP, and NPY. The functional layer of eutopic endometrium from women without endometriosis was used as a negative control because it does not contain any nerve fibers (19) expressing PGP9.5, NP, NGF, NGFRp75, SP, CGRP, TH, VAChT, VIP, or NPY. Statistical Analysis The images were captured by using an Olympus microscope BX51 and digital camera DP70, and an assessment of nerve fiber density was performed by Image Pro Plus Discovery (Media Cybernetics, Bethesda, MD). Once the images features were controlled at the original magnification (4), an orthogonal grid mask was sketched above the original images. The sections of the grid were 250 mm per side. Once the grid was in position, nerve fibers in the endometrial-myometrial sections and curettage samples and the total number of squares covering the endometrial-myometrial sections and curettage samples were counted. The total number of nerve fibers was divided by the total number of squares to obtain an average of nerve fibers per square (each square of 1590 Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008

3 mm). The results were expressed as the mean (SD) number of nerve fibers per square millimeter in each specimen from all endometrial-myometrial sections and curettage samples, stained for the polyclonal antibody against PGP9.5. The counting procedure was carried out twice by two independent observers (each blinded to the other) without any knowledge of the clinical parameters or other prognostic factors. The observers counted nerve fibers in the samples obtained from patients receiving hormonal therapies blindly. As previously recorded (14), at the time that the counting was done for the present study, the observers were also evaluating samples from patients not receiving hormonal therapy. The concordance rate was >95% between the observers. Nerve fiber density of endometrial-myometrial sections and curettage samples between women with hormonally treated endometriosis and women with untreated endometriosis (14) was compared with use of the Mann- Whitney test. Differences were considered to be significant at P<.05. RESULTS No nerve fibers (stained for PGP9.5) were detected in the functional layer of 8 curettage samples in women with treated endometriosis (Fig. 1A), and only 3 out of 18 hysterectomy samples in women with treated endometriosis showed nerve fibers in the functional layer of endometrium (mean density SD: /mm 2, range: 0 2.4/mm 2, Fig. 1B; Table 1). In those 3 treated women in whom endometrial nerve fibers were identified in the functional layer of endometrium, nerve fiber densities ranged between 2.1 and 2.4/mm 2.Of the three women in whom small numbers of nerve fibers were present, one patient was using combined OCs and two patients were using oral progestogens. Small numbers of nerve fibers were present in the basal layer of endometrium and in myometrium in patients with hormonally treated endometriosis (density: /mm 2, range: 0 4.3/mm 2, density: /mm 2, range: / mm 2, respectively, Fig. 1C; Table 1). Only 11 out of 18 had nerve fibers detectable in the basal layer, and the range for 11 treated women with identified nerve fibers in the basal layer was 0.1 to 4.3/mm 2. The nerve fiber density in the functional layer and the basal layer of endometrium and myometrium in women with hormonally treated endometriosis was much lower than that of women with untreated endometriosis who did not receive hormonal treatment for endometriosis before endometrial and hysterectomy sampling in our previous study (14) (functional layer: 11 5/mm 2, range: 5 23/ mm 2, basal layer: 18 8/mm 2, range: 9 80/mm 2, myometrium: 3 1/mm 2, range: 2 4/mm 2, respectively, P<.001 for all comparisons; Table 1). When stained for six neurotransmitter markers, the small number of nerve fibers in the functional layer (detected by PGP9.5) were stained for VIP and NPY (Fig. 2A and B; Table 1) but not with SP, CGRP, TH, or VAChT. Nerve fibers in the basal layer of endometrium were stained for SP, CGRP, VIP, and NPY (Fig. 2C through F; Table 1) but not with TH or VAChT. In myometrium, nerve fibers were stained for all six of the markers for SP, CGRP, TH, VAChT, VIP, and NPY (Fig. 2G and H; Table 1). However, after hormonal treatment for endometriosis, nerve fibers that stained for SP or CGRP were not observed in the functional layer of endometrium nor TH in the basal layer of endometrium. Endometrium from women with untreated endometriosis was strongly stained for both NGF and NGFRp75 (Fig. 3A and 3B), whereas endometrium from women with hormonally treated endometriosis was stained very weakly with both NGF and NGFRp75 (Fig. 3C and D). DISCUSSION This study has demonstrated highly significantly reduced nerve fiber density in the functional (mean, 0.4/mm 2 ) and basal (mean, 0.9/mm 2 ) layers of eutopic endometrium and myometrium (mean, 1.5/mm 2 ) from 26 women with endometriosis who were taking either oral progestogens or combined OCs compared with women who had laparoscopically confirmed endometriosis but were not receiving any treatment in our previous study (mean, 11/mm 2, 18/mm 2, and 3/mm 2, respectively)(14). Twenty-three out of 26 women with treated endometriosis did not have nerve fibers detectable in the functional layer of endometrium and only 11 out of 18 had nerve fibers detectable in the basal layer, whereas women with untreated endometriosis all had detectable nerve fibers in both the functional and basal layer (14). In myometrium in women with hormonally treated endometriosis, nerve fiber density was also greatly reduced by hormonal therapies. In our previous study, nerve fibers in the functional and basal layers of endometrium from women with untreated endometriosis stained for SP, CGRP, VIP, and NPY and SP, CGRP, TH, VIP, and NPY, respectively (15). In the three women in whom small numbers of nerve fibers were detected in the functional layer, these only stained for VIP and NPY but not with SP or CGRP observed in patients with untreated endometriosis in the functional layer (14). Of the three women in whom small numbers of nerve fibers were present, one patient was using a combined OC and two patients were using oral progestogens. In hormonally treated women, some SP- and CGRP-positive nerve fibers were seen in the basal layer of endometrium, but TH-positive nerve fibers observed in patients with untreated endometriosis were never seen (14). In myometrium, SP-, CGRP-, TH-, VAChT-, VIP-, and NPY-positive nerve fibers were present in patients with both hormonally treated and untreated endometriosis (14). In endometrium and myometrium from hormonally treated women with endometriosis, both NGF and NGFRp75 expression was greatly reduced compared with that in women with untreated endometriosis. These results indicate that both a combined OC and progestogens may specifically decrease sensory and adrenergic nerve fibers in endometrium in women with endometriosis. Many studies have shown the efficacy of currently available drugs to treat women with endometriosis-associated Fertility and Sterility â 1591

4 FIGURE 1 Nerve fibers in the functional layer and basal layer of endometrium from women with endometriosis who were receiving hormonal treatment. (A) The functional layer of endometrium from a patient with hormonally treated endometriosis, stained with PGP9.5. No nerve fibers were stained. (B) The functional layer of endometrium from a hormonally treated patient with endometriosis, stained with PGP9.5. Arrows denote several small nerve fibers in the functional layer of endometrium. (C) The basal layer of endometrium from a hormonally treated patient with endometriosis, stained with PGP9.5. Arrows denote nerve fibers in the basal layer. Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008

5 TABLE 1 A quantitative assessment of nerve fiber density stained with PGP9.5 in endometrium and myometrium in women with hormonally treated and untreated endometriosis. Area Women with hormonally treated endometriosis Women with untreated endometriosis Functional layer of endometrium No. of specimens 26 (8 curettage þ hysterectomy) No. of positive staining with 3 10 PGP9.5 Total nerve fiber density a (mean SD/mm 2 ) stained with PGP9.5 Positive staining with six neurotransmitter markers VIP, NPY SP, CGRP, VIP, NPY Basal layer of endometrium No. of specimens No. of positive staining with PGP9.5 Total nerve fiber density (mean SD/mm 2 ) stained with PGP a Positive staining with 6 neurotransmitter markers SP, CGRP, VIP, NPY Myometrium No. of specimens No. of positive staining with PGP9.5 Total nerve fiber density a (mean SD/mm 2 ) stained with PGP9.5 Positive staining with 6 neurotransmitter markers SP, CGRP, TH, VAChT, VIP, NPY a P<.001 compared with women with hormonally treated endometriosis. Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril SP, CGRP, TH, VIP, NPY SP, CGRP, TH, VAChT, VIP, NPY pain. Two months of treatment with a GnRH analogue significantly reduced pain symptoms in women with endometriosis (17), and 6 months of treatment with a GnRH analogue reduced dysmenorrhea, dyspareunia, pelvic pain, pelvic tenderness, and pelvic induration in women with endometriosisrelated pain (20). Six months of treatment with danazol significantly alleviated endometriosis-associated pelvic pain, lower back pain, and defecation pain (18) and deep dyspareunia (10). Six to 12 months of treatment with a low-dose OC significantly reduced deep dyspareunia and dysmenorrhea in women with endometriosis (10, 11). Women with endometriosis who had dysmenorrhea, dyspareunia, and noncyclic pelvic pain had their pain significantly relieved by norethindrone acetate (12), and treatment with dydrogesterone for 6 months also reduced pelvic pain in women with endometriosis (13). However, the mechanisms by which these therapies reduce pain symptoms are unknown. There is significant evidence that estrogen and P can regulate neurotrophins. Ovariectomy induced a significant decrease in NGF protein, whereas estrogen treatment increased NGF protein significantly in the mouse uterus (21). Long-term estrogen treatment significantly increased NGF protein in the rat uterus (22, 23), and estrogen up-regulated both NGF and brain-derived neurotrophic factor protein in the rat endometrium (24). Decreased NGF concentrations were also shown in the rat uterus during pregnancy (25). Furthermore, estrogen promoted nerve fiber growth (26), but pregnancy reduced nerve fiber density in the rat uterus (27). It is of interest that P significantly increased uterine messenger RNA for NGF and NGF protein in mice (21), and the expression of uterine NGF was significantly stimulated by P in golden hamsters (28). Currently available progestogen and estrogen-progestogen therapies for endometriosis may cause these differing suppressive effects on NGF secretion because Fertility and Sterility â 1593

6 FIGURE 2 Nerve fibers in the functional layer and basal layer of endometrium and myometrium from women with endometriosis who were receiving hormonal treatment. (A) The functional layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stained with VIP. Arrows denote nerve fibers stained with VIP in the functional layer. (B) The functional layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stained with NPY. Arrow denotes nerve fibers stained with NPY in the functional layer. (C) The basal layer of endometrium from a woman with hormonally treated (GnRH analogue) endometriosis stained with SP. Arrow denotes nerve fibers stained with SP in the basal layer. (D) The basal layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stained with CGRP. Arrows denote nerve fibers stained with CGRP in the basal layer. Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008

7 FIGURE 2 CONTINUED (E) The basal layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stained with VIP. Arrow denotes nerve fibers stained with VIP in the basal layer. (F) The basal layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stained with NPY. Arrow denotes nerve fibers in the basal layer. (G) Myometrium from a woman with hormonally treated (progestogens) endometriosis stained with TH. Arrows denote nerve fibers in myometrium. (H) Myometrium from a woman with hormonally treated (progestogens) endometriosis stained with VAChT. Arrow denotes nerve fibers in myometrium. Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril Fertility and Sterility 1595

8 FIGURE 3 Expression of NGF and NGFRp75 in endometrium from a woman with endometriosis who was receiving hormonal treatment and a woman with endometriosis who was not receiving hormonal treatment. (A) Endometrium from a woman with untreated endometriosis stained with NGF. (B) Endometrium from a woman with untreated endometriosis stained with NGFRp75. (C) Endometrium from a woman with hormonally treated (progestogens) endometriosis stained with NGF. (D) Endometrium from a woman with hormonally treated (progestogens) endometriosis stained with NGFRp75. Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008

9 of the prolonged and continuous mode of therapeutic delivery required in women. The suppression of NGF and greatly reduced endometrial nerve fiber density may be important in the response to the treatment. On the other hand, it is surprising that NGF and NGFRp75 expression was greatly reduced by exogenous hormonal therapy in the women with endometriosis. Bcl-2 is a neuronal survival promoting protein (29, 30) and inhibits neuronal apoptosis and promotes nerve fiber growth (29). Vascular endothelial growth factor (VEGF) is an angiogenic factor and induces axonal outgrowth (31, 32). Combined OCs increased apoptosis and decreased Bcl-2 expression in eutopic endometrium in women with endometriosis (5), and progestogen combined with E 2 decreased the expression of Bcl-2 in the rat endometrium (33). Gonadotropin-releasing hormone analogues also induced apoptosis (34) and decreased Bcl-2 expression in eutopic endometrium in women with endometriosis (35). Combined OCs also significantly reduced glandular VEGF expression in human endometrium (36, 37). A GnRH analogue (leuprolide acetate) down-regulated VEGF release in eutopic endometrium in women with endometriosis (38). Because Bcl-2 and VEGF are expressed in nerve fibers (39, 40), hormonal therapies for endometriosis may also decrease the expression of molecules that regulate nerve fiber growth in eutopic endometrium and myometrium in women with endometriosis. In summary, hormonal therapies may substantially reduce nerve fiber density in eutopic endometrium and myometrium in women with endometriosis by decreasing the synthesis and expression of molecules that have neurotrophic and neuroprotective effects in women with endometriosis. Further studies are needed to explore the effects of hormonal therapy on nerve fibers in endometriotic plaques and nodules in women with endometriosis-associated pain. REFERENCES 1. Amsterdam LL, Gentry W, Jobanputra S, Wolf M, Rubin SD, Bulun SE. Anastrazole and oral contraceptives: a novel treatment for endometriosis. Fertil Steril 2005;84: Valle RF, Sciarra JJ. Endometriosis: treatment strategies. Ann N Y Acad Sci 2003;997: Child TJ, Tan SL. Endometriosis: aetiology, pathogenesis and treatment. Drugs 2001;61: Rice VM. Conventional medical therapies for endometriosis. Ann N Y Acad Sci 2002;955: Meresman GF, Auge L, Bara~nao RI, Lombardi E, Tesone M, Sueldo C. Oral contraceptives suppress cell proliferation and enhance apoptosis of eutopic endometrial tissue from patients with endometriosis. Fertil Steril 2002;77: Luciano AA, Turksoy RN, Carleo J. Evaluation of oral medroxyprogesterone acetate in the treatment of endometriosis. Obstet Gynecol 1988;72: Schweppe KW. Current place of progestins in the treatment of endometriosis-related complaints. Gynecol Endocrinol 2001;15: Blei F, Wilson EL, Mignatti P, Rafkin DB. Mechanism of action of angiostatic steroids: suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis. J Cell Physiol 1993;155: ESHRE Capri Workshop Group. Ovarian and endometrial function during hormonal contraception. Hum Reprod 2001;16: Vercellini P, Trespidi L, Colombo A, Vendola N, Marchini M, Crosignani PG. A gonadotropin-releasing hormone agonist versus a low-dose oral contraceptive for pelvic pain associated with endometriosis. Fertil Steril 1993;60: Parazzini F, Cintio ED, Chatenoud L, Moroni S, Ardovino I, Struzziero E, et al. Estroprogestin vs. gonadotrophin agonists plus estroprogestin in the treatment of endometriosis-related pelvic pain: a randomized trial. Gruppo Italiano per lo Studio dell Endometriosi 2000;88: Muneyyirci-Delale O, Karacan M. Effect of norethindrone acetate in the treatment of symptomatic endometriosis. Int J Fertil Womens Med 1988;43: Overton CE, Lindsay PC, Johal B, Collins SA, Siddle NC, Shaw RW, et al. A randomized, double-blind, placebo-controlled study of luteal phase dydrogesterone (Duphaston) in women with minimal to mild endometriosis. Fertil Steril 1994;62: Tokushige N, Markham R, Russell P, Fraser IS. High density of small nerve fibers in the functional layer of the endometrium in women with endometriosis. Hum Reprod 2006;21: Tokushige N, Markham R, Russell P, Fraser IS. Different types of nerve fibers in eutopic endometrium and myometrium in women with endometriosis. Fertil Steril. In press. 16. Baxter N, Black J, Duffy S. The effect of a gonadotrophin-releasing hormone analogue as first-line management in cyclical pelvic pain. J Obstet Gynaecol 2004;24: Telimaa S, Puolakka J, Ronnberg L, Kauppila A. Placebo-controlled comparison of danazol and high-dose medroxyprogesterone acetate in the treatment of endometriosis. Gynecol Endocrinol 1987;1: American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: Fertil Steril 1997;67: Quinn MJ, Kirk N. Differences in uterine innervation at hysterectomy. Am J Obstet Gynecol 2002;187: Schlaff WD, Carson SL, Luciano A, Ross D, Bergqvist A. Subcutaneous injection of depot medroxyprogesterone acetate compared with leuprolide acetate in the treatment of endometriosis-associated pain. Fertil Steril 2006;85: Bjorling DE, Beckman M, Clayton MK, Wang ZY. Modulation of nerve growth factor in peripheral organs by estrogen and progesterone. Neuroscience 2002;110: Chavez-Genaro R, Crutcher K, Viettro L, Richeri A, Coirolo N, Burnstock G, et al. Differential effects of estrogen on developing and mature uterine sympathetic nerves. Cell Tissue Res 2002;308: Chalar C, Richeri A, Viettro L, Chavez-Genaro R, Bianchimano P, Marmol NM, et al. Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA. Cell Tissue Res 2003;314: Krizsan-Agbas D, Pedchenko T, Hasan W, Smith PG. Estrogen regulates sympathetic neurite outgrowth by modulating brain derived neurotrophic factor synthesis and release by the rodent uterus. Eur J Neurosci 2003;18: Varol FG, Duchemin AM, Neff NH, Hadjiconstantinou M. Nerve growth factor (NGF) and NGF mrna change in rat uterus during pregnancy. Neurosci Lett 2000;294: Marshall JM. Effects of ovarian steroids and pregnancy on adrenergic nerves of uterus and oviduct. Am J Physiol 1981;240:C Haase EB, Buchman J, Tietz AE, Schramm LP. Pregnancy-induced uterine neuronal degeneration in the rat. Cell Tissue Res 1997;288: Zhanquan S, Arai KY, Jin W, Weng Q, Watanabe G, Suzuki AK, et al. Expression of nerve growth factor and its receptors NTRK1 and TNFRSF1B is regulated by estrogen and progesterone in the uteri of golden hamsters. Biol Reprod 2006;74: Chen DF, Schneider GE, Martinou JC, Tonegawa S. Bcl-2 promotes regeneration of severed axons in mammalian CNS. Nature 1997;385: Clark RS, Chen J, Watkins SC, Kochanek PM, Chen M, Stetler RA, et al. Apoptosis-suppressor gene bcl-2 expression after traumatic brain injury in rats. J Neurosci 1997;17: Fertility and Sterility â 1597

10 31. Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system. J Neurosci 1999;19: Lin G, Chen KC, Hsien PS, Yeh CH, Lue TF, Lin CS. Neurotrophic effects of vascular endothelial growth factor and neurotrophins on cultured major pelvic ganglia. BJU Int 2003;92: Akcali KC, Khan SA, Moulton BC. Effect of decidualization on the expression of bax and bcl-2 in the rat uterine endometrium. Endocrinology 1996;137: Meresman GF, Bilotas M, Buquet RA, Bara~nao RI, Sueldo C, Tesone M. Gonadotropin-releasing hormone agonist induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril 2003;80(Suppl 2): Bilotas M, Baranao RI, Buquet R, Sueldo C, Tesone M, Meresman GF. Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum Reprod 2006;22: Macpherson AM, Archer DF, Leslie S, Charnock-Jones DS, Makkink WK, Smith SK. The effect of etonogestrel on VEGF, oestrogen and progesterone receptor immunoreactivity and endothelial cell number in human endometrium. Hum Reprod 1999;14: Charnock-Jones DS, Macpherson AM, Archer DF, Leslie S, Makkink WK, Sharkey AM, et al. The effect of progestins on vascular endothelial growth factor, oestrogen receptor and progesterone receptor immunoreactivity and endothelial cell density in human endometrium. Hum Reprod 2000;15(Suppl 3): Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, Baranao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 2003;18: Merry DE, Korsmeyer SJ. Bcl-2 gene family in the nervous system. Annu Rev Neurosci 1997;20: Mukouyama YS, Shin D, Britsch S, Taniguchi M, Anderson DJ. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell 2002;109: Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008