Therapeutic Efficiency of Tibolone in a Rat Endometriosis Model

AN D METHODS The pre sent study was con duc ted at the Ex pe ri - men tal Ani mals La bo ra tory of the sur gery de part - ment of Gul ha ne Mi li tary Me di cal Aca demy , bet we en the July 04, 2 009 and Sep tem ber 18, 2009. The ap pro val of the ins ti tu ti o nal et hics com mit te e was ob ta i ned be fo re the study and all the pro ce du - res on the ani mals we re con duc ted in ac cor dan ce with the di rec ti ons of ‘Gu i de for the Ca re and Use of La bo ra tory Ani mals ’. The study was per for med on 30 fe ma le Swiss-Al bi no rats that we ig hed 200-250 grams, we re in the pro es trus pha se and non-preg - nant . The rats we re kept in ste el ca ges in a cli ma - te-con trol led en vi ron ment un der ad li bi tum fe e ding con di ti ons and pro vi ded with ac cess to day light bet - we en 06:00 am and 6 :00 pm . The study had three pha ses, the de ta ils of which are pre sen ted be low: PHA SE I The en do met ri o sis mo del in the rats was for med by the met hod pre vi o usly des cri bed by Ver non and Wil son :13 All the rats we re ad mi nis te red 100 mg/kg of ke ta min hydroch lo ri de (Ke ta lar ® fla kon) for anest - he si a . The tric ho tomy of the ab do mi nal re gi on was ma de by elec tric ra zor. A mid li ne in ci si on of 3 cm was ma de af ter di sin fec ti on of the ab do men with io di ne whi le the rats we re in su pi ne po si ti on. The cu ta ne o us, sub cu ta ne o us and musc le tis su es we re se pa ra ted and thus, the pe ri to ne al ca vity was ac ces - sed. A 15 mm seg ment of the right ute ri ne corn was re mo ved. This cylin dri cal seg ment was ope ned with lon gi tu di nal in ci si on and the en do met ri al la yer was ex po sed. The en do met ri al fa ce of the re mo ved seg - ment was su tu red with a sing le su tu re to me sen tery using non-ab sor bab le su tu re ma te ri al (No:4/0 silk su tu re ). The la yers of the ab do men we re con se cu ti - vely clo sed, and the pro ce du re was en ded. PHA SE II Twenty-eight days af ter the first ope ra ti on, the rats un der went a se cond la pa ro tomy un der ste ri le con - di ti ons as des cri bed abo ve. The in tra -ab do mi nal en - do met ri o tic fo ci and ad he si ons we re ob ser ved. The length and width of the imp lants we re me a su red and the sur fa ce are a of the imp lants was cal cu la ted . Turkiye Klinikleri J Med Sci 2011;31(6) 1352 Şimşek ve ark. Kadın Hastalıkları ve Doğum The ad he si ons we re clas si fi ed ac cor ding to the sco ring system of Bla u er, which was used pre vi o - usly in rat en do met ri o sis mo dels 14,15 as fol lows: AD HE SION SCO RE 0: No ad he si on 1: Thin ad he si ons 2: Thick ad he si on in one are a 3: Wi des pre ad thick ad he si ons 4: Ad he si ons of the in ter nal or gans to the ab - do mi nal wall The ab do men was clo sed and the pro ce du re was en ded . Thre e rats with no sus pec ted en do met - ri o sis fo ci mac ros co pi cally and three rats that di ed in the first pha se we re exc lu ded from the study. The re ma i ning 24 rats we re di vi ded in to three gro ups. Thre e days af ter the se cond pha se, the rats in the first gro up (the con trol gro up , n= 8) we re in - jec ted with 1 cc of 0.9% NaCl. The rats in the se cond gro up (n= 8) we re ad mi - nis te red 1 mg in tra mus cu lar le up ro lid ace ta te (Luc - rin De pot- 3M ®, Ab bott), as was used in pre vi o us stu di es as the tre at ment do se .5,9 The rats in the third gro up (n= 8) we re gi ven 1 mg/kg/day of ti bo lo ne (Li vi al ® tab let, Or ga non ) for four we eks with ga va ge . The do se of ti bo lo ne was cal cu la ted ba sed on the re com men da ti ons by Ge naz za ni et al. with the the ra pe u tic do se ran ge (0.2-2 mg/g/day) ar ran ged for fe ma le rats .16 PHA SE II I All rats we re sac ri fi ced by cer vi cal dis lo ca ti on and we re sub jec ted to la pa ro tomy pro ce du re as de fi ned ear li er. The are a of the en do met ri o tic fo ci and in - tra -ab do mi nal ad he si ons we re re cor ded . The chan ges in the are a of en do met ri o sis fo ci of all gro ups we re me a su red and com pa red . The in - tra -ab do mi nal ad he si on sco res we re al so com pa - red. STA TIS TI CAL ANALY SIS The da ta we re eva lu a ted using SPSS 15.0 prog ram. A ll the da ta we re ex pres sed as me an va lu es. The in - ter gro up com pa ri sons we re ma de wit pa i red sam - p le t test . A p va lu e <0.05 was con si de red as sta tis ti cally sig ni fi cant.

D u ring the study three rats di ed and anot her three rats with no sus pec ted en do met ri o sis fo ci mac ros - co pi cally we re exc lu ded from the study. The study was comp le ted with 24 rats. In the se cond pha se, the me an are a of en do - m et ri o tic fo ci and in tra -ab do mi nal ad he si on sco res among the gro ups we re si mi lar (Tab le 1). En do - m et ri o tic fo ci we re mac ros co pi cally ob ser ved as cystic or vas cu lar le si ons . At the end of the tre at - m ent, the are a of the en do met ri o tic fo ci and ad he - si on sco res we re fo und to dec re a se in all thre e gro ups (Fi gu re 1). In Gro ups 2 and 3, the re duc ti on was mo re mar ked com pa red to that in the con trol gro up and the difference was sta tis ti cally sig ni fi - cant (p< 0. 05) (Tab le 2).

N o vel tre at ment met hods of en do met ri o sis, a chro - nic di se a se with unc le ar eti o logy, are fre qu ently used in ex pe ri men tal rat, mi ce, mon key, and rab bit m o dels. One of the se met hods was used by Ver non and Wil son for the first ti me in a rat en do met ri o - sis mo del be ca u se rats are inex pen si ve and ha ve a short 4-day mens tru al cycle, short lu te al pha se of the cycle, and pre do mi nant es trus pha se .13,17 GnRHa is wi dely used in the me di cal tre at - m ent of en do met ri o sis with a pro ven ef fi ci ency .18- Turkiye Klinikleri J Med Sci 2011;31(6) 1353 Gynecology and Obstetrics Şimşek et al Area of endometriotic foci (mm 2) Adhesion score Pre Post P value Pre Post P value Control (n= 8)36.6 ± 11.5 28.8 ± 7.8 0.070 3.1 ± 0.8 1.6 ± 0.5 0.005 Leuprolid (n= 8)35.2 ± 9.9 9.3 ± 8.9 0.003 2.8 ± 0.8 1 ± 1.06 0.004 Tibolone (n= 8)33.1 ± 12.1 15.3 ± 8.2 0.000 2.5 ± 0.9 1.1 ± 1.1 0.001 TABLE 1: Pre- and posttreatment area of endometriotic foci and adhesion scores in the three groups. 20 In the use of GnRHa , FSH and LH sec re ti on from the hypoph ysis are tem po ra rily in cre a sed . Ho we - ver, with its sup raph ysi o lo gi cal do se, ago nis tic ac - ti vity is sus ta i ned, re cep tors are down-re gu la ted, and hypoph ysis-go nad axis is in hi bi ted. In con se - qu en ce of inac ti va ti on of the oral form , it is used in the form of sub cu ta ne o us or in tra mus cu lar in - jec ti ons. GnRHa has be en shown to in du ce at rophy in the en do met ri o tic fo ci, in cre a se the ac ti vity of pe ri to ne al na tu ral kil ler cells, and re du ce the le vels of pe ri to ne al IL-6, IL-1 β, TNF α, and Ca- 125 in rat en do met ri o sis mo dels .21,22 In our study, post tre at - m ent mac ros co pic chan ges and ad he si on sco res we - re eva lu a ted in a rat en do met ri o sis mo del. Pa ral lel to the fin dings in the li te ra tu re, the gro up that re - ce i ved GnRHa tre at ment had sig ni fi cant re duc ti - ons in the are as of the en do met ri o tic fo ci and ad he si on sco res com pa red to the rats in the con trol gro up .5-7,21 Ho we ver, in ad di ti on to the high costs, it car ri es the risk of os te o po ro sis when used for long pe ri ods of ti me. A d di ti o nally, GnRHa tre at - m ent pro du ces se ve ral tro ub le so me si de ef fects be - ca u se of its strong an ti es tro ge nic ef fects, and re lap se might oc cur af ter the ces sa ti on of tre at - ment. Du e to the se re a sons, al ter na ti ve tre at ment op ti ons are be ing in ves ti ga ted. Ti bo lo ne is a ste ro id mo le cu le from the gro up of mo le cu les that act as es tro ge nic ac ti va tor re gu la - tor in se lec ti ve tis su es .11 It has an es tro ge nic ac ti - vity in the cen tral ner vo us system, bo ne, and ge ni tal tis su es, whi le it does not have such ef fects in the bre ast tis su e and en do met ri um , so it is ma inly used for the ma na ge ment of cli mac te ric symptoms in post me no pa u sal wo men .11,23 Ti bo lo ne in the en - do met ri um is ir re ver sibly con ver ted to its ∆-4 iso - m er that binds to both pro ges te ro ne and an dro gen re cep tors. 11 Ti bo lo ne and its ∆-4 iso mer in du ce es - tro gen inac ti va ting enz ymes 17 β- hydroxy ste ro id dehy dro ge na se and sul fot rans fe ra se, in hi bit sul fa - ta se and en han ce lo cally the de ac ti va ti on of bi o lo - gi cally ac ti ve es tro ge nic me ta bo li tes. 11,12 Nu me ro us stu di es ha ve shown that to ac hi e ve add-back tre at - m ent of en do met ri o sis, the use of ti bo lo ne in cre a se the bo ne mi ne ral den sity, re du ces an ti-es tro ge nic si de-ef fects, however do es not af fect the de ve lop - m ent of en do met ri o sis fo ci .24-26 In the me no pa u sal pe ri od, the use of ti bo lo ne has be en shown to in du ce en do met ri al at rophy .27 Et tin ger et al., in ves ti ga ted the en do met ri al ef fects of ti bo lo ne in 3519 post-me no pa u sal pa ti ents, and they emp ha si zed the neg li gib le ef fect of ti bo lo ne on en do met ri al pro li fe ra ti on for ma ti on in the first three ye ars of its use .28 On the ot her hand, li te ra - tu re re ve als en do met ri o sis de ve lop ment with ti bo - lo ne used for post me no pa u sal hor mo ne tre at ment in some ca ses .29 To the best of our know led ge, the pri mary ef fi ci ency of ti bo lo ne in en do met ri o sis tre - at ment has not be en stu di ed to da te in a rat en do - Turkiye Klinikleri J Med Sci 2011;31(6) 1354 Şimşek ve ark. Kadın Hastalıkları ve Doğum FIGURE 1: Comparison of delta values (postreatment-pretreatment) of areas of endometriotic foci among the three groups. Control (n= 8) Leuprolid acetate (n= 8) Tibolone (n= 8) p value Areas of endometriotic foci (mm 2) 8 (-3-30) 25.5 (3-54) 16.5 (9-30) 0.018* Adhesion scores 1 (0-3) 2 (0-4) 1.5 (0-2) 0.683 TABLE 2: Comparison of delta values (posttreatment -pretreatment) of areas of endometriotic foci and adhesion scores among the three groups. *p<0.05 when comparison between control vs leuprolid acetate groups and control vs tibolon groups. met ri o sis mo del. The re fo re, our study might be con si de red as the first to in ves ti ga te the re la ti ons - hip bet we en ti bo lo ne and en do met ri o sis in a rat mo del. In our study, ti bo lo ne ca u sed a reg res si on in the en do met ri o tic fo ci in the early pe ri od as with GnRHa use. The are as of en do met ri o tic fo ci we re sig ni fi cantly re du ced in the gro up of rats that re - ce i ved ti bo lo ne (Gro up-3) when com pa red to the rats in the con trol gro up (The me an are as of en do - met ri o tic fo ci we re 33. 1 mm 2 and 15.3 mm 2, res - pec ti vely) . The le vel of the reg res si on in the en do met ri o tic fo ci was si mi lar to the one that ob - ta i ned with GnRHa tre at ment. Ad di ti o nally, af ter a 4-we ek tre at ment pe ri od, the ad he si on sco res of the rats we re sig ni fi cantly smaller com pa red to the sco res of the rats in the con trol gro up. The pri mary aim of this study was the de ter mi na ti on and eva - lu a ti on of mac ros co pic ap pe a ran ce and ad he si ons sco res . Ho we ver, the reg res si on in the en do met ri - o tic fo ci was not his to pat ho lo gi cally gra ded, which is a li mi ta ti on of our study. In conc lu si on, the pre li mi nary in for ma ti on ob - ta i ned in this study sug gests that des pi te es tro ge nic ef fects, ti bo lo ne can be used as an al ter na ti ve agent in the tre at ment of en do met ri o sis du e to its an dro - ge nic and pro ges ta ge nic bi o ac ti ve pro ducts . Ho we - ver, our fin dings ne ed to be con fir med by furt her de ta i led stu di es with his to pat ho lo gic cor re la ti ons. Turkiye Klinikleri J Med Sci 2011;31(6) 1355 Gynecology and Obstetrics Şimşek et al 1. Vignali M, Infantino M, Matrone R, Chiodo I, Somigliana E, Busacca M, et al. Endometriosis: novel etiopathogenetic concepts and clinical perspectives. Fertil Steril 2002;78 (4): 665-78. 2. Elagöz Ş, Aker H, Eğilmez R, Çetin A.[Histopatho - logical diagnostic criteria of endometriosis and asso - ciated lesions: Review of 63 cases]. Turkiye Klinikleri J Gynecol Obst 2000;10(4):253-9. 3. Matarese G, De Placido G, Nikas Y, Alviggi C. Patho - genesis of endometriosis: natural immunity dysfunc - tion or autoimmune disease? Trends Mol Med 2003;9(5):223-8. 4. Nap AW, Groothuis PG, Demir AY, Evers JL, Dun - selman GA. Pathogenesis of endometriosis. Best Pract Res Clin Obstet Gynaecol 2004;18(2):233-44. 5. Altintas D, Kokcu A, Tosun M, Cetinkaya MB, Kan - demir B. Comparison of the effects of cetrorelix, a GnRH antagonist, and leuprolide, a GnRH agonist, on experimental endometriosis. J Obstet Gynaecol Res 2008; 34(6):1014-9. 6. Akkaya P, Onalan G, Haberal N, Bayraktar N, Mülayim B, Zeyneloglu HB. Doxycycline causes re - gression of endometriotic implants: a rat model. Hum Reprod 2009;24(8):1900-8. 7. Mohammadzadeh A, Heidari M, Soltanghoraee H, Jeddi-Tehrani M, Ghaffari Novin M, Akhondi MM, et al. Evaluation of the effect of pentoxifylline on white blood cell count in serum and peritoneal fluid in fe - male rats with endometriosis. J Obstet Gynaecol Res 2008; 34(3):307-13. 8. Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of en - dometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999;72(1):135-41. 9. Henig I, Rawlins RG, Weinrib HP, Dmowski WP. Ef- fects of danazol, gonadotropin-releasing hormone ag - onist, and estrogen/progestogen combination on experimental endometriosis in the ovariectomized rat. Fertil Steril 1988;49(2):349-55. 10. Plosker GL, Brogden RN. Leuprorelin. A review of its pharmacology and therapeutic use in prostatic can - cer, endometriosis and other sex hormone-related disorders. Drugs 1994; 48(6):930-67. 11. Modelska K, Cummings S. Tibolone for post- menopausal women: systematic review of random - ized trials. J Clin Endocrinol Metab 2002; 87(1):16-23. 12. Kloosterboer HJ. Tissue-selectivity: the mechanism of action of tibolone. Maturitas 2004; 48(Suppl 1):S30- 40. 13. Vernon MW, Wilson EA. Studies on the surgical in - duction of endometriosis in the rat. Fertil Steril 1985;44(5):684-94. 14. Blauer KL, Collins RL. The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits. Fertil Steril 1988; 49(1):144-9. 15. Koçak I, Unlü C, Akçan Y, Yakin K. Reduction of ad - hesion formation with cross-linked hyaluronic acid after peritoneal surgery in rats. Fertil Steril 1999;72(5):873-8. 16. Genazzani AR, Bernardi F, Pluchino N, Giretti MS, Begliuomini S, Casarosa E, et al. Effect of tibolone administration on central and peripheral levels of al- lopregnanolone and beta-endorphin in female rats. Menopause 2006; 13 (1):57-64. 17. Story L, Kennedy S. Animal studies in endometriosis: a review. ILAR J 2004;45(2):132-8. 18. Dlugi AM, Miller JD, Knittle J. Lupron depot (leupro - lide acetate for depot suspension) in the treatment of endometriosis: a randomized, placebo-controlled, double-blind study. Lupron Study Group. Fertil Steril 1990;54(3):419-27. 19. Heinrichs WL, Henzl MR. Human issues and medical economics of endometriosis. Three- vs. six-month GnRH-agonist therapy. J Reprod Med 1998;43(3 Suppl):299-308. 20. Hornstein MD, Yuzpe AA, Burry K, Buttram VC Jr, Heinrichs LR, Soderstrom RM, et al. Retreatment with nafarelin for recurrent endometriosis symptoms: effi- cacy, safety, and bone mineral density. Fertil Steril 1997;67 (6): 1013-8. 21. Zanagnolo VL, Beck R, Schlaff WD, Damewood MD, Bobbie D, Rock JA. Time-related effects of go - nadotropin-releasing hormone analog treatment in ex - perimentally induced endometriosis in the rat. Fertil Steril 1991; 55(2):411-5. 22. Ocal G, Kokcu A, Cetinkaya MB, Tosun M, Kefeli M, Kandemir B. Efficacy of levamisole on experimental endometriosis. Int J Gynaecol Obstet 2007;99(1):38- 42. 23. Conner P, Christow A, Kersemaekers W, Söderqvist G, Skoog L, Carlström K, et al. A comparative study of breast cell proliferation during hormone replace - ment therapy: effects of tibolon and continuous com - bined estrogen-progestogen treatment. Climacteric 2004;7 (1):50-8. 24. Lindsay PC , Shaw RW, Bennink HJ, Kicovic P. The effect of add-back treatment with tibolone (Livial) on patients treated with the gonadotropin-releasing hor- mone agonist triptorelin (Decapeptyl). Fertil Steril 1996; 65(2):342-8. 25. Shin SY, Min JA, Yoon BK, Bae DS, Choi DS. The incidence and characteristics of uterine bleeding dur- ing postoperative GnRH agonist treatment combined with tibolone add-back therapy in endometriosis pa - tients of reproductive age. Eur J Obstet Gynecol Re - prod Biol 2007;133(1):90-4. 26. Agorastos T, Vaitsi V, Paschopoulos M, Vakiani A, Zournatzi-Koiou V, Saravelos H, et al. Prolonged use of gonadotropin-releasing hormone agonist and ti- bolone as add-back therapy for the treatment of en - dometrial hyperplasia. Maturitas 2004;48(2):125-32. 27. Szlendak-Sauer K, Wierzba W, Radowicki S. [The in - fluence of a tibolone therapy on endometrium in post- menopausal women]. Ginekol Pol 2008;79(11): 758-61. 28. Ettinger B, Kenemans P, Johnson SR, Mol-Arts M, Van Os S, Seifert W, et al. Endometrial effects of ti- bolone in elderly, osteoporotic women. Obstet Gy - necol 2008;112(3):653-9. 29. Sundar SS, Gornall RJ, Kerr-Wilson R, Swingler GR, Kinder RB, McCarthy K. A case report of recurrent endometriosis following Tibolone hormone replace - ment therapy. J Obstet Gynaecol 2007;27(4):433-4. REFERE NCES