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

Effects of hormonal treatment on nerve fibersin endometrium and myometrium in womenwith endometriosisNatsuko 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 nervefibers 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 treatedendometriosis and women with untreated endometriosis.Main Outcome Measure(s): Types and density of nerve fibers in endometrium and myometrium in women withhormonally 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 layersof endometrium (0.2� 0.7/mm2 and 0.9� 1.3/mm2, respectively) and myometrium (1.5� 0.8/mm2) from womenwith hormonally treated endometriosis was much lower than that of endometrium (functional layer: 11 � 5/mm2,basal layer: 18 � 8/mm2, respectively) and myometrium (3 � 1/mm2) from women with untreated endometriosis.Nerve growth factor and nerve growth factor receptor p75 expression was also significantly reduced in women withhormonally treated endometriosis compared with women with untreated endometriosis.Conclusion(s): Hormonal treatment significantly reduced nerve fiber density in endometrium and myometrium inwomen with endometriosis. (Fertil Steril� 2008;90:1589–98. �2008 by American Society for Reproductive Med-icine.)

Key Words: Endometriosis, nerve fibers, hormonal therapy, endometrium, myometrium

Endometriosis is a recurrent debilitating disease that affectspredominantly reproductive-age women. It is characterizedby the presence of endometrium-like tissue outside theuterus, and the symptoms of endometriosis include chronicdysmenorrhea, deep dyspareunia, and dyschezia. Endometri-osis is an estrogen-dependent disease, and current medicaltreatments suppress estrogen synthesis. Drugs such asdanazol, GnRH analogues, combined oral contraceptives(OCs), and progestogens have been used to treat endometri-osis-associated pain (1). Danazol suppresses gonadotropinsecretion, particularly LH, and inhibits ovarian estrogen pro-duction and endometrial growth (2). Gonadotropin-releasinghormone analogues decrease secretion of gonadotropins,resulting in dramatically decreased serum estrogen levels(3). Combined OCs inhibit ovulation (4) and cause decidual-ization, then marked endometrial thinning, and increase apo-ptosis and decrease Bcl-2 expression in eutopic endometriumin women with endometriosis (5). Progestogens also decrease

Received June 21, 2007; revised August 24, 2007; accepted August 31,

2007.

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, De-

partment of Obstetrics and Gynaecology, Queen Elizabeth II Research

Institute for Mothers and Infants, NSW 2006, Australia (FAX:

61-2-9351-4560; E-mail: [email protected]).

0015-0282/08/$34.00doi:10.1016/j.fertnstert.2007.08.074 Copyright ª2008 American

serum estrogen levels by suppressing gonadotropin release(6), cause glandular atrophy and stromal decidualization ineutopic endometrium in women with endometriosis (4, 7),inhibit angiogenesis (8), and decrease intraperitoneal inflam-mation (9).

Several studies have demonstrated the efficacy of drugs forendometriosis-associated pain; treatment with an OC, noreth-indrone, and dydrogesterone significantly reduced painsymptoms including deep dyspareunia, dysmenorrhea, andpelvic pain in women with endometriosis (10–13).

It has been demonstrated that women with endometriosishave a greater density of nerve fibers in endometrium andmyometrium than women without endometriosis (14) andthat eutopic endometrium in women with endometriosis isinnervated by sensory Ad, sensory C, and adrenergic fibersand myometrium by sensory Ad, sensory C, adrenergic,and cholinergic fibers (15). Several studies have shown the ef-ficacy of currently available hormonal treatments in alleviatingpain symptoms in women with endometriosis (16, 17). How-ever, no study has investigated yet whether hormonal treatmentchanges nerve fiber density in endometrium and myometriumin women with endometriosis who have pain symptoms.

We have studied innervation with different types of specificimmunohistochemical neuronal markers in endometrium

Fertility and Sterility� Vol. 90, No. 5, November 2008 1589Society for Reproductive Medicine, Published by Elsevier Inc.

mailto:[email protected]

and myometrium specimens from women with visually andbiopsy-proved endometriosis who were receiving hormonaltreatment until they underwent hysterectomy or endometrialsampling.

MATERIALS AND METHODS

Tissue Collection

This study was approved by the Human Ethics Committees ofthe Sydney South West Area Health Service and the Univer-sity of Sydney, Sydney, Australia, and all women gave theirinformed consent for participation. Eighteen full-thicknessuterine blocks, which included endometrium and contiguousmyometrium (mean age of patient 40.2 years; range, 31–50years), and eight tissue samples from curettage (mean ageof patient 26.8 years; range, 20–38 years) were selectedfrom women with laparoscopic evidence of endometriosiswho underwent laparoscopy combined with hysteroscopyand were receiving hormonal treatment until they underwenthysterectomy or endometrial sampling.

Of this same group of 26 women, 20 were taking oralprogestogens and 7 were taking combined OCs (one womanwas taking more than one form of medication). The types ofprogestogens were either medroxyprogesterone acetate ornorethindrone, and the composition of the combined OCswas ethinylestradiol 30 mg and levonorgestrel 150 mg. Thecombined OCs were being taken continuously in this groupof patients. The duration of medications varied from 1 monthto 5 months before they underwent hysterectomy or endome-trial sampling.

Sixty-two percent of the women with hormonally treatedendometriosis had peritoneal endometriosis alone, 23% hadovarian endometriosis alone, and 15% had both peritonealand ovarian endometriosis. The locations of endometriosiswere based on findings at the time of laparoscopy beforetreatment. The subsequent laparoscopy was performedbecause of persistent pain.

The control group of 10 women (mean age 44.0 years;range, 42–46 years) with proved endometriosis, who werenot receiving any medication before hysterectomy, was partof an earlier study (14). Seventy percent of the women withuntreated endometriosis had peritoneal endometriosis alone,20% had ovarian endometriosis alone, and 10% had bothperitoneal and ovarian endometriosis.

The interval of time from the diagnosis of endometriosistill the hysterectomy was variable, and the diagnosis ofendometriosis was originally made at a preceding laparos-copy, but the presence of endometriosis was confirmed ateach hysterectomy. The patients with endometriosis all com-plained of dysmenorrhea and a range of other related painsymptoms. The severity of pain was not assessed systemati-cally and prospectively in this study, but detailed clinical in-formation was recorded in a standard format. Endometriosisin all patients was staged according to the revised AmericanFertility Society score, which ranged from I to IV (18).

1590 Tokushige et al. Hormonal effects on endometrial ne

Immunohistochemistry

After surgical removal, the specimens were immediatelyfixed in 10% neutral buffered formalin for approximately18 to 24 hours, processed, and embedded in paraffin waxaccording to a standard protocol. Each section was cut at4 mm and routinely stained with hematoxylin and eosin.Markers included polyclonal rabbit anti-protein gene product9.5 (PGP9.5), a highly specific panneuronal marker, whichrecognizes all types of nerve fibers; monoclonal mouseanti-human neurofilament (NF); polyclonal rabbit anti-nervegrowth factor (NGF); monoclonal mouse anti-human nervegrowth factor receptor p75 (NGFRp75); polyclonal rabbitanti-substance P (SP); rabbit anti-calcitonin gene–relatedpeptide (CGRP); polyclonal rabbit anti-vesicular acetylcho-line transporter (VAChT); monoclonal anti-tyrosine hydrox-ylase (TH); polyclonal rabbit anti-vasoactive intestinalpolypeptide (VIP); and polyclonal rabbit anti-neuropeptideY (NPY). Serial sections were immunostained with use ofantibodies for PGP9.5 (dilution 1:1,400), NF (dilution1:400), SP (dilution 1:7,000), CGRP (dilution 1:150),VAChT (dilution 1: 8,000), TH (dilution 1:1500), VIP (dilu-tion 1:3,000), NPY (dilution 1:2,500), NGF (dilution 1:500),and NGFRp75 (dilution 1:1,700); incubated with REALDetection System (DAKO, Carpinteria, CA), alkaline phos-phatase/RED, Link, biotinylated secondary antibodies, andREAL Detection System (DAKO), alkaline phosphatase/RED, streptavidin alkaline phosphatase; and stained forREAL Detection System, chromogen (red) as described pre-viously (15).

All immunostaining was carried out on a DAKO AutoStainer (model S3400; DAKO). Images of the sectionswere captured with use of an Olympus microscope BX51and digital camera DP70 (Olympus, Tokyo, Japan). Weused normal skin as a positive control because it reliablycontains myelinated and unmyelinated nerve fibers express-ing PGP9.5, NP, NGF, NGFRp75, SP, CGRP, TH, VAChT,VIP, and NPY. The functional layer of eutopic endometriumfrom women without endometriosis was used as a negativecontrol 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 microscopeBX51 and digital camera DP70, and an assessment of nervefiber 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 originalimages. The sections of the grid were 250 mm per side.Once the grid was in position, nerve fibers in the endome-trial-myometrial sections and curettage samples and the totalnumber of squares covering the endometrial-myometrial sec-tions and curettage samples were counted. The total numberof nerve fibers was divided by the total number of squares toobtain an average of nerve fibers per square (each square of

rve fibers Vol. 90, No. 5, November 2008

250 � 250 mm). The results were expressed as the mean(�SD) number of nerve fibers per square millimeter ineach specimen from all endometrial-myometrial sectionsand curettage samples, stained for the polyclonal antibodyagainst PGP9.5. The counting procedure was carried outtwice by two independent observers (each blinded to theother) without any knowledge of the clinical parameters orother prognostic factors. The observers counted nerve fibersin the samples obtained from patients receiving hormonaltherapies blindly. As previously recorded (14), at the timethat the counting was done for the present study, the observerswere also evaluating samples from patients not receivinghormonal therapy. The concordance rate was >95% betweenthe observers. Nerve fiber density of endometrial-myometrialsections and curettage samples between women with hor-monally treated endometriosis and women with untreatedendometriosis (14) was compared with use of the Mann-Whitney test. Differences were considered to be significantat P<.05.

RESULTS

No nerve fibers (stained for PGP9.5) were detected in thefunctional layer of 8 curettage samples in women with treatedendometriosis (Fig. 1A), and only 3 out of 18 hysterectomysamples in women with treated endometriosis showed nervefibers in the functional layer of endometrium (mean density� SD: 0.4 � 0.9/mm2, range: 0–2.4/mm2, Fig. 1B; Table1). In those 3 treated women in whom endometrial nerve fi-bers were identified in the functional layer of endometrium,nerve fiber densities ranged between 2.1 and 2.4/mm2. Ofthe three women in whom small numbers of nerve fiberswere present, one patient was using combined OCs and twopatients were using oral progestogens.

Small numbers of nerve fibers were present in the basallayer of endometrium and in myometrium in patients withhormonally treated endometriosis (density: 0.9 � 1.3/mm2,range: 0–4.3/mm2, density: 1.5 � 0.8/mm2, range: 0.3–3.1/mm2, respectively, Fig. 1C; Table 1). Only 11 out of 18 hadnerve fibers detectable in the basal layer, and the range for11 treated women with identified nerve fibers in the basallayer was 0.1 to 4.3/mm2. The nerve fiber density in the func-tional layer and the basal layer of endometrium and myome-trium in women with hormonally treated endometriosis wasmuch lower than that of women with untreated endometriosiswho did not receive hormonal treatment for endometriosisbefore endometrial and hysterectomy sampling in our previ-ous study (14) (functional layer: 11 � 5/mm2, range: 5–23/mm2, basal layer: 18 � 8/mm2, range: 9–80/mm2, myome-trium: 3 � 1/mm2, range: 2–4/mm2, respectively, P<.001for all comparisons; Table 1).

When stained for six neurotransmitter markers, the smallnumber of nerve fibers in the functional layer (detected byPGP9.5) were stained for VIP and NPY (Fig. 2A and B; Table1) but not with SP, CGRP, TH, or VAChT. Nerve fibers in thebasal layer of endometrium were stained for SP, CGRP, VIP,

Fertility and Sterility�

and NPY (Fig. 2C through F; Table 1) but not with TH orVAChT. In myometrium, nerve fibers were stained for allsix of the markers for SP, CGRP, TH, VAChT, VIP, andNPY (Fig. 2G and H; Table 1).

However, after hormonal treatment for endometriosis, nervefibers that stained for SP or CGRP were not observed in thefunctional layer of endometrium nor TH in the basal layer ofendometrium. Endometrium from women with untreatedendometriosis was strongly stained for both NGF andNGFRp75 (Fig. 3A and 3B), whereas endometrium fromwomen with hormonally treated endometriosis was stainedvery weakly with both NGF and NGFRp75 (Fig. 3C and D).

DISCUSSION

This study has demonstrated highly significantly reducednerve fiber density in the functional (mean, 0.4/mm2) andbasal (mean, 0.9/mm2) layers of eutopic endometrium andmyometrium (mean, 1.5/mm2) from 26 women with endome-triosis who were taking either oral progestogens or combinedOCs compared with women who had laparoscopically con-firmed endometriosis but were not receiving any treatmentin our previous study (mean, 11/mm2, 18/mm2, and 3/mm2,respectively)(14). Twenty-three out of 26 women with treatedendometriosis did not have nerve fibers detectable in thefunctional layer of endometrium and only 11 out of 18 hadnerve fibers detectable in the basal layer, whereas womenwith untreated endometriosis all had detectable nerve fibersin both the functional and basal layer (14). In myometriumin women with hormonally treated endometriosis, nerve fiberdensity was also greatly reduced by hormonal therapies.

In our previous study, nerve fibers in the functional and basallayers of endometrium from women with untreated endometri-osis stained for SP, CGRP, VIP, and NPY and SP, CGRP, TH,VIP, and NPY, respectively (15). In the three women inwhom small numbers of nerve fibers were detected in the func-tional layer, these only stained for VIP and NPY but not with SPor CGRP observed in patients with untreated endometriosis inthe functional layer (14). Of the three women in whom smallnumbers of nerve fibers were present, one patient was usinga combined OC and two patients were using oral progestogens.In hormonally treated women, some SP- and CGRP-positivenerve fibers were seen in the basal layer of endometrium, butTH-positive nerve fibers observed in patients with untreatedendometriosis were never seen (14). In myometrium,SP-, CGRP-, TH-, VAChT-, VIP-, and NPY-positive nerve fi-bers were present in patients with both hormonally treatedand untreated endometriosis (14). In endometrium and myome-trium from hormonally treated women with endometriosis,both NGF and NGFRp75 expression was greatly reduced com-pared with that in women with untreated endometriosis. Theseresults indicate that both a combined OC and progestogens mayspecifically decrease sensory and adrenergic nerve fibers inendometrium in women with endometriosis.

Many studies have shown the efficacy of currently avail-able drugs to treat women with endometriosis-associated

1591

FIGURE 1

Nerve fibers in the functional layer and basal layer of endometrium from women with endometriosis who werereceiving hormonal treatment. (A) The functional layer of endometrium from a patient with hormonally treatedendometriosis, stained with PGP9.5. No nerve fibers were stained. (B) The functional layer of endometrium froma hormonally treated patient with endometriosis, stained with PGP9.5. Arrows denote several small nerve fibersin the functional layer of endometrium. (C) The basal layer of endometrium from a hormonally treated patient withendometriosis, stained with PGP9.5. Arrows denote nerve fibers in the basal layer.

Tokushige. Hormonal effects on endometrial nerve fibers. Fertil Steril 2008.

1592 Tokushige et al. Hormonal effects on endometrial nerve fibers Vol. 90, No. 5, November 2008

TABLE 1A quantitative assessment of nerve fiber density stained with PGP9.5 in endometrium and myometriumin women with hormonally treated and untreated endometriosis.

Area

Women withhormonally treated

endometriosis

Women withuntreated

endometriosis

Functional layer of endometriumNo. of specimens 26 (8 curettage þ 18

hysterectomy)10

No. of positive staining withPGP9.5

3 10

Total nerve fiber density(mean � SD/mm2) stainedwith PGP9.5

0.4 � 0.9 11 � 5a

Positive staining with sixneurotransmitter markers

VIP, NPY SP, CGRP,VIP, NPY

Basal layer of endometriumNo. of specimens 18 10No. of positive staining withPGP9.5

11 10


0.9 � 1.3 18 � 8a

Positive staining with 6neurotransmitter markers

SP, CGRP,VIP, NPY

SP, CGRP, TH,VIP, NPY

MyometriumNo. of specimens 18 10No. of positive staining withPGP9.5

18 10


1.5 � 0.8 3 � 1a

Positive staining with 6neurotransmitter markers

SP, CGRP, TH,VAChT, VIP, NPY

SP, CGRP, TH,VAChT, VIP, NPY

a P< .001 compared with women with hormonally treated endometriosis.


pain. Two months of treatment with a GnRH analogue signif-icantly reduced pain symptoms in women with endometriosis(17), and 6 months of treatment with a GnRH analoguereduced dysmenorrhea, dyspareunia, pelvic pain, pelvic ten-derness, and pelvic induration in women with endometriosis-related pain (20). Six months of treatment with danazolsignificantly alleviated endometriosis-associated pelvicpain, lower back pain, and defecation pain (18) and deep dys-pareunia (10). Six to 12 months of treatment with a low-doseOC significantly reduced deep dyspareunia and dysmenor-rhea in women with endometriosis (10, 11). Women withendometriosis who had dysmenorrhea, dyspareunia, andnoncyclic pelvic pain had their pain significantly relievedby norethindrone acetate (12), and treatment with dydroges-terone for 6 months also reduced pelvic pain in women withendometriosis (13). However, the mechanisms by whichthese therapies reduce pain symptoms are unknown.


There is significant evidence that estrogen and P canregulate neurotrophins. Ovariectomy induced a significantdecrease in NGF protein, whereas estrogen treatment in-creased NGF protein significantly in the mouse uterus (21).Long-term estrogen treatment significantly increased NGFprotein in the rat uterus (22, 23), and estrogen up-regulatedboth NGF and brain-derived neurotrophic factor protein inthe rat endometrium (24). Decreased NGF concentrationswere also shown in the rat uterus during pregnancy (25). Fur-thermore, estrogen promoted nerve fiber growth (26), butpregnancy reduced nerve fiber density in the rat uterus (27).It is of interest that P significantly increased uterine messen-ger RNA for NGF and NGF protein in mice (21), and the ex-pression of uterine NGF was significantly stimulated by P ingolden hamsters (28). Currently available progestogen andestrogen-progestogen therapies for endometriosis may causethese differing suppressive effects on NGF secretion because

1593

FIGURE 2

Nerve fibers in the functional layer and basal layer of endometrium and myometrium from women with endometriosiswho were receiving hormonal treatment. (A) The functional layer of endometrium from a woman with hormonallytreated (progestogens) endometriosis stained with VIP. Arrows denote nerve fibers stained with VIP in the functionallayer. (B) The functional layer of endometrium from a woman with hormonally treated (progestogens) endometriosisstained with NPY. Arrow denotes nerve fibers stained with NPY in the functional layer. (C) The basal layer ofendometrium from a woman with hormonally treated (GnRH analogue) endometriosis stained with SP. Arrowdenotes nerve fibers stained with SP in the basal layer. (D) The basal layer of endometrium from a woman withhormonally treated (progestogens) endometriosis stained with CGRP. Arrows denote nerve fibers stained withCGRP in the basal layer.



FIGURE 2 CONTINUED

(E) The basal layer of endometrium from a woman with hormonally treated (progestogens) endometriosis stainedwith VIP. Arrow denotes nerve fibers stained with VIP in the basal layer. (F) The basal layer of endometrium froma woman with hormonally treated (progestogens) endometriosis stained with NPY. Arrow denotes nerve fibers inthe basal layer. (G) Myometrium from a woman with hormonally treated (progestogens) endometriosis stainedwith 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.


Fertility and Sterility� 1595

FIGURE 3

Expression of NGF and NGFRp75 in endometrium from a woman with endometriosis who was receivinghormonal 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 womanwith 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.



of the prolonged and continuous mode of therapeutic deliveryrequired in women. The suppression of NGF and greatly re-duced endometrial nerve fiber density may be important inthe response to the treatment. On the other hand, it is surpris-ing that NGF and NGFRp75 expression was greatly reducedby exogenous hormonal therapy in the women with endome-triosis.

Bcl-2 is a neuronal survival–promoting protein (29, 30)and inhibits neuronal apoptosis and promotes nerve fibergrowth (29). Vascular endothelial growth factor (VEGF) isan angiogenic factor and induces axonal outgrowth (31,32). Combined OCs increased apoptosis and decreasedBcl-2 expression in eutopic endometrium in women with en-dometriosis (5), and progestogen combined with E2 de-creased the expression of Bcl-2 in the rat endometrium(33). Gonadotropin-releasing hormone analogues also in-duced apoptosis (34) and decreased Bcl-2 expression in eu-topic endometrium in women with endometriosis (35).Combined OCs also significantly reduced glandular VEGFexpression in human endometrium (36, 37). A GnRH ana-logue (leuprolide acetate) down-regulated VEGF release ineutopic endometrium in women with endometriosis (38). Be-cause Bcl-2 and VEGF are expressed in nerve fibers (39, 40),hormonal therapies for endometriosis may also decrease theexpression of molecules that regulate nerve fiber growth ineutopic endometrium and myometrium in women with endo-metriosis.

In summary, hormonal therapies may substantially reducenerve fiber density in eutopic endometrium and myometriumin women with endometriosis by decreasing the synthesis andexpression of molecules that have neurotrophic and neuro-protective effects in women with endometriosis. Further stud-ies are needed to explore the effects of hormonal therapy onnerve fibers in endometriotic plaques and nodules in womenwith endometriosis-associated pain.

REFERENCES1. Amsterdam LL, Gentry W, Jobanputra S, Wolf M, Rubin SD, Bulun SE.

Anastrazole and oral contraceptives: a novel treatment for endometriosis.

Fertil Steril 2005;84:300–4.

2. Valle RF, Sciarra JJ. Endometriosis: treatment strategies. Ann N Y Acad

Sci 2003;997:229–39.

3. Child TJ, Tan SL. Endometriosis: aetiology, pathogenesis and treatment.

Drugs 2001;61:1735–50.

4. Rice VM. Conventional medical therapies for endometriosis. Ann N Y

Acad Sci 2002;955:343–52.

5. Meresman GF, Aug�e 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;77:1141–7.

6. Luciano AA, Turksoy RN, Carleo J. Evaluation of oral medroxyproges-

terone acetate in the treatment of endometriosis. Obstet Gynecol

1988;72:323–7.

7. Schweppe KW. Current place of progestins in the treatment of endome-

triosis-related complaints. Gynecol Endocrinol 2001;15:22–8.

8. Blei F, Wilson EL, Mignatti P, Rafkin DB. Mechanism of action of angio-

static steroids: suppression of plasminogen activator activity via stimula-

tion of plasminogen activator inhibitor synthesis. J Cell Physiol

1993;155:568–78.


9. ESHRE Capri Workshop Group. Ovarian and endometrial function dur-

ing hormonal contraception. Hum Reprod 2001;16:1527–35.

10. 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 endometri-

osis. Fertil Steril 1993;60:75–9.

11. Parazzini F, Cintio ED, Chatenoud L, Moroni S, Ardovino I,

Struzziero E, et al. Estroprogestin vs. gonadotrophin agonists plus estro-

progestin in the treatment of endometriosis-related pelvic pain: a random-

ized trial. Gruppo Italiano per lo Studio dell’Endometriosi 2000;88:

11–4.

12. Muneyyirci-Delale O, Karacan M. Effect of norethindrone acetate in the

treatment of symptomatic endometriosis. Int J Fertil Womens Med

1988;43:24–7.

13. 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;62:701–7.

14. 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;21:782–7.

15. Tokushige N, Markham R, Russell P, Fraser IS. Different types of nerve

fibers in eutopic endometrium and myometrium in women with endome-

triosis. Fertil Steril. In press.

16. Baxter N, Black J, Duffy S. The effect of a gonadotrophin-releasing hor-

mone analogue as first-line management in cyclical pelvic pain. J Obstet

Gynaecol 2004;24:64–6.17. 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;1:13–23.18. American Society for Reproductive Medicine. Revised American Soci-

ety for Reproductive Medicine classification of endometriosis: 1996.

Fertil Steril 1997;67:817–21.19. Quinn MJ, Kirk N. Differences in uterine innervation at hysterectomy.

Am J Obstet Gynecol 2002;187:1515–9.

20. Schlaff WD, Carson SL, Luciano A, Ross D, Bergqvist A. Subcutaneous

injection of depot medroxyprogesterone acetate compared with leupro-

lide acetate in the treatment of endometriosis-associated pain. Fertil

Steril 2006;85:314–25.

21. Bjorling DE, Beckman M, Clayton MK, Wang ZY. Modulation of nerve

growth factor in peripheral organs by estrogen and progesterone. Neuro-

science 2002;110:155–67.

22. Ch�avez-Genaro R, Crutcher K, Viettro L, Richeri A, Coirolo N,

Burnstock G, et al. Differential effects of estrogen on developing and ma-

ture uterine sympathetic nerves. Cell Tissue Res 2002;308:61–73.

23. Chalar C, Richeri A, Viettro L, Ch�avez-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;314:191–205.

24. 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:

2760–8.

25. Varol FG, Duchemin AM, Neff NH, Hadjiconstantinou M. Nerve growth

factor (NGF) and NGF mRNA change in rat uterus during pregnancy.

Neurosci Lett 2000;294:58–62.

26. Marshall JM. Effects of ovarian steroids and pregnancy on adrenergic

nerves of uterus and oviduct. Am J Physiol 1981;240:C165–74.

27. Haase EB, Buchman J, Tietz AE, Schramm LP. Pregnancy-induced

uterine neuronal degeneration in the rat. Cell Tissue Res 1997;288:

293–306.

28. 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;74:850–6.

29. Chen DF, Schneider GE, Martinou JC, Tonegawa S. Bcl-2 promotes regen-

eration of severed axons in mammalian CNS. Nature 1997;385:434–9.

30. 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;17:9172–82.

1597

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;19:5731–40.

32. Lin G, Chen KC, Hsien PS, Yeh CH, Lue TF, Lin CS. Neurotrophic ef-

fects of vascular endothelial growth factor and neurotrophins on cultured

major pelvic ganglia. BJU Int 2003;92:631–5.

33. Akcali KC, Khan SA, Moulton BC. Effect of decidualization on the ex-

pression of bax and bcl-2 in the rat uterine endometrium. Endocrinology

1996;137:3123–31.

34. 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 endo-

metriosis. Fertil Steril 2003;80(Suppl 2):702–7.

35. 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;22:644–53.

1598 Tokushige et al. Hormonal effects on endometrial ne

36. 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;14:3080–7.

37. 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):85–95.

38. 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;18:1767–71.

39. Merry DE, Korsmeyer SJ. Bcl-2 gene family in the nervous system. Annu

Rev Neurosci 1997;20:245–67.

40. 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;109:693–705.

rve fibers Vol. 90, No. 5, November 2008

Documents

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