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1 ORIGINAL ARTICLE Korean J Obstet Gynecol 2011;54(6): doi: /KJOG pissn eissn HISTOLOGIC COMPARISON OF POLYCYSTIC OVARY SYNDROME INDUCED BY ESTRADIOL VALERATE AND LETROZOLE Sung Chul Lim, MD 1*, Moon Jin Jeong, PhD 2*, Se Eun Kim, DVM 3, Seung Hyun Kim, DVM 3, Sam Cheol Kim, MD 4, Seung-Yong Seo, MD 1, Tak Kim, MD 5, Seong Soo Kang, DVM 3, Chun Sik Bae, DVM 3 1 Department of Pathology, Chosun University College of Medicine; 2 Department of Oral Histology, Chosun University College of Dentistry; 3 Department of Veterinary Surgery, Chonnam National University College of Veterinary Medicine; 4 Department of Medicine, Graduate School Chosun University, Gwangju; 5 Department of Obstetrics and Gynecology, Korea University College of Medicine, Seoul, Korea Objective To compare ovarian morphology of the estradiol valerate (EV)- and the letrozole (LE)-induced polycystic ovary syndrome (PCOS) in rat models. Methods EV were injected (IM) to 10 Sprague-Dawley rats and LE were administered (PO) to the other 10 Sprague-Dawley rats for 21 days. Sixty days after single injection of EV and 21 days after LE administration, the animals were sacrificed and the ovaries were sectioned, measured and analyzed histopathologically. Results LE-treated group showed larger ovary bearing more abundant cystic follicles with luteinized theca cells. These findings were more similar to human PCOS ovarian morphology. Conclusion LE-induced PCOS model is recommended to conduct PCOS-related animal experiments. Keywords: Polycystic ovary syndrome; Estradiol valerate; Letrozole; Ovary 다낭성난소증후군 (polycystic ovary syndrome, PCOS) 은호르몬의불균형에의하여여러가지기능적이상이초래되고비만, 다모증, 당뇨등과함께배란장애를일으켜불임을유발한다. 그러나 PCOS의발생기전은아직명확하게밝혀지지않았고명확한치료법또한아직개발되지못한실정이다 [1-4]. 이에인간의 PCOS에가장가까운모델을찾고자노력하고있으며여러실험동물모델을통하여발생기전및치료법개발에관한연구가활발하게진행되고있다 [5,6]. 임상에서 estradiol valerate (EV) 는피임, 갱년기증상치료나난소절제후에스트로겐결핍증상의개선에사용되며 [7,8], letrozole (LE) 는폐경후여성의호르몬대체요법이나에스트로겐또는프로게스테론수용체양성인전이성유방암의치료에사용되는약제로이들을과다투여하게되면호르몬의불균형이초래되어불임, 다모증, 부종등의 PCOS 와유사한증상이나타나게된다 [9,10]. 이에착안하여 PCOS를연구하기위해 EV와 LE를장기투여하여 PCOS를유발시킨동물실험모델을실험에이용하고있으나그발생기 전에관하여아직명확하게밝혀진바는없다. 그동안본연구자를비롯하여 Morales-Ledesma 등 [11] 은 EV를 Received: Revised: Accepted: Corresponding author: Chun Sik Bae, DVM Department of Veterinary Surgery, College of Veterinary Medicine, Chonnam National University, 300 Yongbong-dong, buk-gu, Gwangju , Korea Tel: , Fax: csbae210@chonnam.ac.kr * These two authors equally contributed to this work. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright Korean Society of Obstetrics and Gynecology 294

2 Sung Chul Lim, et al. Polycystic ovary syndrome induced by estradiol valerate and letrozole Sprague-Dawley 랫드에주사하여성공적으로 PCOS를유발한적이있는데, 이모델을만드는데 60일이라는비교적긴시간이필요한단점이있다. 그러나최근 LE를이용하여 21일후 PCOS를성공적으로유발할수있다는보고가있다 [12]. EV는 1회근육주사후 60일째 [5,6,11], LE 는 21일간경구투여후 [12] PCOS가유발되기때문에두가지질환유발동물모델간장단점이존재한다. 또한 EV와 LE에의해유발된 PCOS동물모델중어느것이인체에발생하는 PCOS와형태학적으로더유사한지는밝혀진바가없다. 본실험은 EV와 LE 장기투여로 PCOS를유발시켜난소의형태학적변화를조직학적으로비교관찰하여인체의 PCOS에가까운모델을찾아효과적인 PCOS 실험모델을확립하고자한다. 을절단하여파라핀포매에의해 4 μm 두께의절편으로제작하여 H&E 염색을실시하여관찰하였다. 제작된슬라이드를대상으로광학현미경하에서 MagnaFire digital camera system (Optronics, Goleta, CA, USA) 으로디지털이미지를얻어 Visus Image Analysis System (Image & Microscope Technology, Daejeon, Korea) 으로난소의전체단면적을구하고난포낭 (follicular cyst) 의크기및수를측정하였다. 난자변성이나과립막세포의농축이하나라도관찰되면폐쇄난포로분류하였으며, 그렇지않으면건강하다고분류하였다. 또한폐쇄난포의형태학적특성을과립막세포내에서농축핵이흩어져있거나기저막으로부터과립막세포층의박리, 기저판 (basal lamina) 의균열및난포강내세포잔여물의존재로특정하였다. 정상적인월경주기를보이며체중이 g인 7주령의암컷 Sprague-Dawley 랫드 20마리를 Samtako (Osan, Korea) 에서구입하였고실험실환경적응을위하여 1주간예비사육후실험을실시하였다. 본실험은전남대학교동물실험윤리위원회의승인하에표준작업지침서에따라수행되었으며사육실조건은 23 ± 2 o C, 50 ± 5% 의상대습도와 12시간의명암주기를유지하고물과사료 (Samyangfeed, Daejeon, Korea) 는자유롭게섭취하도록하였다. 실험동물은 EV 투여군과 LE 투여군으로나누고각군마다 10마리씩배치하였다. EV 투여군은 4 mg의 EV (Sigma-Aldrich, St. Louis, MO, USA) 를 0.2 ml의 sesame oil (Sigma, USA) 에용해시켜 1회근육주사를실시하고 60일후에실험을시작하였다. LE 투여군은 letrozole (Femara, Novartis, Seoul, Korea) 1 mg/kg를 1% carboxymethylcellulose 용액 1 ml에녹인후 21일간경구투여하였다. 인간에서 PCOS를진단하기위한난소의형태변화기준은다양하지만 Rotterdam consensus에따르면초음파소견상난포낭 (follicular cyst) 의수가 12개이상이고, 난포낭의크기는직경이 10 mm 이하이며, 난소의크기는 10 ml 이상이어야한다 [13-18]. 저자들의선행연구에따르면 EV 1회투여로 Sprague-Dawley 랫드에서성공적인 PCOS가발생하였는데 [5,6], 랫드에서인간의기준을그대로따르는것은문제가있어본연구에서는난포낭의수를기준으로그룹간비교하는것이객관적일것으로판단되었다. 실험종료시, 모든랫드에 chloral hydrate (500 mg/kg) 를복강주사하여전신마취를실시한후난소를적출하여 0.1 M sodium cacodylate buffer (ph 7.4) 에고정액 4% paraformaldehyde-4% sucrose를처치하여 12시간동안고정시켰으며난소의최대단면을관찰할수있게조직 수치는평균 ± 표준편차로나타내었다. 측정된항목의결과에대한통계분석은 Student s t-test와다중비교를위한 Bonferroni 검정법을사용하였다. 또한 1% 의유의수준 (P < 0.05) 에서유의성을검증하였다. 난소의크기및형태변화뿐만아니라호르몬변화및대사과정의이상을파악하기위하여정상대조군과함께 testosterone, estradiol, progesterone 등의변화에대한연구를병행한결과유의한변화가관찰되어 (data not shown), 본실험에서 EV와 LE 처치군모두 PCOS가발생하는것으로판단되었다. EV와 LE의 PCOS 유발률을비교한결과각각 50% 와 80% 로 EV에비하여 LE가통계학적으로유의하게 (P < 0.05) 높은 PCOS 유발률을나타내었다. 난소의크기는 EV 처리군의경우 ± cm 2, LE 처리군의 ± cm 2 으로 LE 처리군이유의하게증가하였으며, 난소의최대단면적을대상으로난포낭의수및크기를비교한결과 EV 처리군의경우는평균 3.4 ± 1.73개의난포낭이관찰되었으나, LE 처리군의경우는 17.5 ± 6.21개의난포낭이관찰되었고, 대부분의난포낭은 EV 처리군, LE 처리군관계없이직경이 1 mm 미만이었다 (Table 1). Table 1. Comparison of ovary between estradiol valerate (EV) and letrozole (LE) treatment EV group LE group P-value Ovary size (cm²) ± ± <0.001 PCOS induction (%) <0.05 Follicular cyst (No.) 3.4 ± ± 6.21 <0.001 Follicular size (mm) <1.0 <1.0 Follicular luteinization - + Stromal hyperthecosis + - Corpus luteum - + (a few) PCOS, polycystic ovary syndrome

KJOG Vol. 54, No. 6, 2011 1 mm Fig. 1. Scan image of the ovary, estradiol valerate-treated group.

Relatively large ovary bearing many cystic follicles (asterisks) in the thickened cortex (H&E, 40). 200 μm Fig. 2. Higher magnification of the ovary, estradiol valerate-treated group.

난소의조직학적변화를관찰한결과, EV 처리군은두꺼워진난소피질내에서 3-4개의난포낭이관찰되고난포막세포및과립막세포모두에서난포의황체화는거의발견되지않으나기질내황체화가현저하여기질난포증 (stromal hyperthecosis) 소견을보였다. 황체나백체는거의관찰되지않았다 (Figs. 1, 2).

. Fig. 4. Higher magnifi cation of the ovary, letrozole-treated group. Many cystic follicles (asterisks) consisted of luteinized theca cells are noted.

3 KJOG Vol. 54, No. 6, mm Fig. 1. Scan image of the ovary, estradiol valerate-treated group. Relatively small ovary bearing four cystic follicles (asterisks) in the cortex is demonstrated (H&E, 40). 1 mm Fig. 3. Scan image of the ovary, letrozole-treated group. Relatively large ovary bearing many cystic follicles (asterisks) in the thickened cortex (H&E, 40). 200 μm Fig. 2. Higher magnification of the ovary, estradiol valerate-treated group. Two cystic follicles (asterisks) devoid of luteinized theca cells and stromal hyperthecosis (arrows) are identifi ed. Neither corpus luteum nor corpus albicans are found (H&E, 100). 난소의조직학적변화를관찰한결과, EV 처리군은두꺼워진난소피질내에서 3-4개의난포낭이관찰되고난포막세포및과립막세포모두에서난포의황체화는거의발견되지않으나기질내황체화가현저하여기질난포증 (stromal hyperthecosis) 소견을보였다. 황체나백체는거의관찰되지않았다 (Figs. 1, 2). 한편 LE 처리군은난소의크기가전체적으로증가하여단면적이평균약 3배커졌으며, 두꺼워진난소피질내에서 EV 처리군에비하여현저하게많은수의난포낭이관찰되었다. 난포낭의경우난포막세포의황체화가잘관찰되지만기질내황체화세포는거의관찰되지않았다. 또한황체가소수관찰되지만백체는확인할수없었다 (Figs. 3, 4). Fig. 4. Higher magnifi cation of the ovary, letrozole-treated group. Many cystic follicles (asterisks) consisted of luteinized theca cells are noted. A corpus luteum (pyramid) is demonstrated (H&E, 100). 200 μm 다낭성난소증후군 (PCOS) 은불임원인의 40-50% 를차지하는난소배란장애의주요원인중하나로매우중요한질환이지만, 그원인이나발생기전에관하여아직명확하게밝혀지지않고있다 [1-4]. 따라서, 실험동물을이용한질환모델의개발이중요한데약제를이용한 PCOS 모델이알려져있다. 대표적인것은 EV와 LE을이용한모델이다 [5,6,11,12]. EV는에스트로겐제제로서저에스트로겐증에대한치료제로사용되는데갱년기, 폐경기증상치료및난소절제술후저에스트로겐증치료및예방에사용된다 [7,8]. LE는 aromatase 억제제로유방암의 296

4 Sung Chul Lim, et al. Polycystic ovary syndrome induced by estradiol valerate and letrozole 치료는물론예방 (chemoprevention) 에도사용되지만 PCOS 치료제로도사용되고있다 [9,10,12,19]. EV나 LE와같은치료약제를과량투여하게되면부작용으로 PCOS와유사한난소변화및임상증상을초래하게되어이를이용하여실험동물을이용한질환모델을개발하게되었다. 즉, 랫드에 4 mg의 EV를 1회근육주사후 60일이경과하면 PCOS가발생하지만 [7,8] LE는 21일간매일경구투여를하여야 PCOS가발생한다 [12]. 이런이유로 EV는 1회근육주사의간편성은있지만 60일을기다려야하는단점이있고, LE는매일경구투여하는번거로움이있는반면 EV에비하여 1/3의짧은경과후실험을할수있다는장점이있다. 그러나 EV와 LE에의한 PCOS 유발률이나인간에서발생하는 PCOS와의유사성등을비교한보고는아직없다. 본실험에서 LE는 EV에비하여 PCOS 유발률이유의하게 (P < 0.05) 높을뿐만아니라현저한낭성변화를보여비침습적방법으로성공적인 PCOS 동물모델이성립되었음을더욱더용이하게평가할수있을것으로생각되었다. 다시말하면 PCOS 질환모델은효과적으로 PCOS를유발한후여러가지약제나시술등의치료효과를형태학적으로또는기능적으로평가하기위하여사용하게되는데성공적인실험을위해서는선제적으로 PCOS가초래되는것이중요하다. 그러나 PCOS가제대로초래되었는지를평가하기위하여개복하여난소를확인한후치료효과실험을하는것은여러가지제약이뒤따른다. 따라서비침습적인방법, 가령초음파등으로난소를평가하여 PCOS가초래되었음을확인한후실험을하게되면후속실험의결과에대한신뢰를더욱높일수있을것으로생각된다. 인체에발생한 PCOS를진단하기위한난소의형태변화기준이다양하여일부논란이있지만 Rotterdam consensus에따르면초음파소견상난포낭 (follicular cyst) 의수가 12개이상이고난포낭의크기는직경이 10 mm 이하이며, 난소의크기는 10 ml 이상이어야한다 [13-18]. 본실험의경우는랫드를이용한동물모델인관계로난포의직경이나난소의크기와같은내용은인체의초음파소견을그대로적용하는것은한계가있겠으나난포낭의수는도움이될것으로판단된다. 난소의최대단면적을대상으로난포낭의수및크기를비교하면 EV 처리군의경우는평균 3.4 ± 1.73개의난포낭이관찰되었으나, LE 처리군의경우는 17.5 ± 6.21개의난포낭이관찰되었고, 대부분의난포낭은 EV 처리군, LE 처리군관계없이직경이 1 mm 미만으로관찰되었다. 인체에발생한 PCOS 난소의조직학적소견은다수의작은난포낭이피막밑에서관찰되고피질의바깥쪽은두껍고배란으로생긴황체 (corpus luteum) 나백체 (corpus albicans) 가나타나지않는다. 난포는두껍고부분적으로황체화 (lutenized) 된난포막세포 (theca cell) 를보이나과립막세포 (granulosa cell) 의발달은미약하고황체화하지않는다. 또한기질내에서도황체화된세포를볼수있다 [20,21]. 본실험의경우 EV 처리군은두꺼워진난소피질내에서 3-4개의난포낭이관찰되고난포의황체화는난포막세포및과립막세포모두에서거의발견되지않으나기질내황체화가현저하여기질난포증 (stromal hyperthecosis) 소견을보였다. 황체나백체는거의관찰되지않았다. 한편, LE 처리군은난소의크기가전체적으로증가하여단면적이평균약 3배커졌으며, 두꺼워진난소피질내에서 EV 처리군에비하여현저하게많은수의난포낭이관찰되었다. 난포낭의경우난포막세포의황체화가잘관찰되지만기질내황체화세포는거의관찰되지않았다. 또한황체가소수관찰되지만백체는확인할수없었다. 이상과같은소견을토대로인간에발생하는 PCOS에가까운동물모델이 EV 처리군인지, LE 처리군인지를명확하게정의할수는없지만난소가커지고난포낭의수가증가하여야한다는 Rotterdam consensus 등의기준에따르면 LE 처리군이인간 PCOS에더가까운질환동물모델임을알수있다. 또한 LE 처리에의한 PCOS 모델은비교적짧은기간에질환을유발할수있고, 비침습적방법으로질환유발을더욱용이하게확인할수있어추후 PCOS 연구에적극활용할수있을것으로생각된다. Acknowledgments 이논문은 2006년정부 ( 교육인적자원부 ) 의재원으로한국학술진흥재단의지원을받아수행된연구임 (KRF E00126). References 1. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83: Diamanti-Kandarakis E, Kouli CR, Bergiele AT, Filandra FA, Tsianateli TC, Spina GG, et al. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profi le. J Clin Endocrinol Metab 1999;84: Asunción M, Calvo RM, San Millán JL, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin Endocrinol Metab 2000;85: Moran C, Tena G, Moran S, Ruiz P, Reyna R, Duque X. Prevalence of polycystic ovary syndrome and related disorders in mexican women. Gynecol Obstet Invest 2010;69: Bai YH, Lim SC, Song CH, Bae CS, Jin CS, Choi BC, et al. Electro-acupuncture reverses nerve growth factor abundance in experimental polycystic ovaries in the rat. Gynecol Obstet Invest 2004;57: Lee JC, Pak SC, Lee SH, Lim SC, Bai YH, Jin CS, et al. The effect of herbal medicine on nerve growth factor in estradiol valerate-induced polycystic ovaries in rats. Am J Chin Med 297

5 KJOG Vol. 54, No. 6, ;31: Jensen JT. Evaluation of a new estradiol oral contraceptive: estradiol valerate and dienogest. Expert Opin Pharmacother 2010;11: Zacharieva S, Kirilov G, Kalinov K, Shigarminova R, Nachev E, Orbetzova M, et al. Effect of different hormone replacement therapy regimens on circadian blood pressure profile and active renin in postmenopausal women. Gynecol Endocrinol 2002;16: Torrisi R, Bagnardi V, Rotmensz N, Scarano E, Iorfi da M, Veronesi P, et al. Letrozole plus GnRH analogue as preoperative and adjuvant therapy in premenopausal women with ER positive locally advanced breast cancer. Breast Cancer Res Treat 2011;126: Iwata H. Neoadjuvant endocrine therapy for postmenopausal patients with hormone receptor-positive early breast cancer: a new concept. Breast Cancer 2011;18: Morales-Ledesma L, Linares R, Rosas G, Morán C, Chavira R, Cárdenas M, et al. Unilateral sectioning of the superior ovarian nerve of rats with polycystic ovarian syndrome restores ovulation in the innervated ovary. Reprod Biol Endocrinol 2010;8: Kafali H, Iriadam M, Ozardali I, Demir N. Letrozole-induced polycystic ovaries in the rat: a new model for cystic ovarian disease. Arch Med Res 2004;35: Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19: Jonard S, Robert Y, Cortet-Rudelli C, Pigny P, Decanter C, Dewailly D. Ultrasound examination of polycystic ovaries: is it worth counting the follicles? Hum Reprod 2003;18: Murphy MK, Hall JE, Adams JM, Lee H, Welt CK. Polycystic ovarian morphology in normal women does not predict the development of polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91: Hassa H, Tanir HM, Yildiz Z. Comparison of clinical and laboratory characteristics of cases with polycystic ovarian syndrome based on Rotterdam s criteria and women whose only clinical signs are oligo/anovulation or hirsutism. Arch Gynecol Obstet 2006;274: Mortensen M, Rosenfi eld RL, Littlejohn E. Functional signifi - cance of polycystic-size ovaries in healthy adolescents. J Clin Endocrinol Metab 2006;91: Welt CK, Gudmundsson JA, Arason G, Adams J, Palsdottir H, Gudlaugsdottir G, et al. Characterizing discrete subsets of polycystic ovary syndrome as defined by the Rotterdam criteria: the impact of weight on phenotype and metabolic features. J Clin Endocrinol Metab 2006;91: Lanham MS, Lebovic DI, Domino SE. Contemporary medical therapy for polycystic ovary syndrome. Int J Gynaecol Obstet 2006;95: Young RH, Scully RE. Ovarian pathology in infertility. In: Kraus FT, Damjanov I, Kaufman N, editors. Pathology of reproductive failure. Baltimore: Williams and Wilkins; p Homburg R. Polycystic ovary syndrome - from gynaecological curiosity to multisystem endocrinopathy. Hum Reprod 1996;11:

6 Sung Chul Lim, et al. Polycystic ovary syndrome induced by estradiol valerate and letrozole Estradiol valerate 와 letrozole 에의해유발되는다낭성난소증후군의난소변화비교 1 조선대학교의과대학병리학교실, 2 조선대학교치과대학구강조직학교실, 3 전남대학교수의과대학, 4 조선대학교대학원의학과, 5 고려대학교의과대학산부인과학교실임성철 1*, 정문진 2*, 김세은 3, 김승현 3, 김삼철 4, 서승용 1, 김탁 5, 강성수 3, 배춘식 3 목적다낭성난소증후군 (polycystic ovary syndrome, PCOS) 을 estradiol valerate (EV) 와 letrozole (LE) 로유발할수있는데인간의다낭성난소증후군과가장유사한질환동물모델이무엇인가를알아보고자하였다. 연구방법 EV와 LE를각각랫드에처리하여 PCOS를유발시킨후희생시켜난소를적출하였다. 난소를조직학적으로관찰하여다낭성난소변화유발유무, 난소의크기, 난포낭의크기및수를측정하고난포및간질세포의황체화등을비교관찰하였다. 결과 LE처리군은 EV처리군에비하여난소의크기및난포낭의수가유의하게증가하고난포막세포의황체화가뚜렷하여인간의 PCOS와더유사한소견을보였다. 결론랫드에서 LE처리에의한 PCOS는 PCOS질환유발동물모델로더적합한것으로평가된다. 중심단어 : 다낭성난소증후군, estradiol valerate, letrozole, 난소 299

3차_JKDA 16권3호 05_( ) 성연아_저자-간행 교정.indd

3차_JKDA 16권3호 05_( ) 성연아_저자-간행 교정.indd J Korean Diabetes 2015;16:189-193 Vol.16, No.3, 2015 ISSN 2233-7431, 이화여자대학교의과대학내과학교실 Epidemiology and Diagnostic Criteria of Polycystic Ovary Syndrome Hyejin Lee, Yeon-Ah Sung Department of Internal Medicine,