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Impact of intracystic ethanol instillation on ovarian cyst diameter and adjacent ovarian tissue
European Journal of Obstetrics & Gynecology and Reproductive Biology 174 (2014) 133–136
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Impact of intracystic ethanol instillation on ovarian cyst diameter and adjacent ovarian tissue Remzi Atilgan a, Zehra Sema Ozkan a,*, Tuncay Kuloglu b, Nevin Kocaman b, Melike Baspinar a, Behzat Can a, Mehmet S¸ims¸ek a, Ekrem Sapmaz a a b
Firat University School of Medicine, Department of Obstetrics and Gynecology, 23119 Elazig, Turkey Firat University School of Medicine, Department of Histology and Embryology, 23119 Elazig, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2013 Received in revised form 4 November 2013 Accepted 12 December 2013
Objective: To investigate the regression level of simple ovarian cyst size after local ethanol application and the damage level of adjacent ovarian reserve in rats. Study design: This study was conducted at Firat University Animal Laboratory with 18 mature (12–14 weeks old) female Wistar albino rats weighing 200–220 g, with regular cycles. Ovarian cyst induction was performed with unilateral salpingectomy. Fourteen rats with ovarian cysts after a second laparotomy were divided into two groups as follows: Group 1 (n = 7): cyst aspiration group, and Group 2 (n = 7): intracystic 95% ethanol application group. One month after the cyst aspiration procedure a third laparotomy was performed. The cyst number and size were recorded for each rat. Right ovariectomy was performed and formalin-fixed/paraffin-embedded tissues were sectioned at 5 mm thickness. Under light microscopy, ovarian total follicle reserve and fibrosis were evaluated with Masson trichrome staining and apoptosis was evaluated with TUNEL staining. The groups were compared with the Mann–Whitney U test and Wilcoxon Rank test. p < 0.05 was considered significant. Results: Ovarian cyst formation was observed in 85% (15/18) of rats. The mean diameter of ovarian cysts in Groups 1 and 2 were, respectively, 10.3 mm and 10.1 mm. After aspiration, there was no significant reduction in the cyst diameter (10.3 mm vs 8.1 mm), but after ethanol application the diameter significantly reduced (10.1 mm vs 3.4 mm, p < 0.05). Mean ovarian follicle count in Group 2 was significantly lower than in Group 1 (25 vs 42, p < 0.05), and mean fibrosis and apoptosis scores in Group 2 were significantly higher than in Group 1 (2.5 vs 0.9, p < 0.05). Conclusion: Local ethanol application reduces cyst diameter but concomitantly decreases ovarian reserve due to increased fibrosis in rats. In humans, intracystic ethanol application should be performed cautiously. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethanol sclerotherapy Ovarian reserve Salpingectomy Apoptosis
1. Introduction The incidence of adnexal masses among healthy women has been reported as 6%, of which 90% were cystic tumors, and the common form of these tumors was simple cysts [1]. Either laparoscopy or laparotomy can be performed for a cystectomy procedure, but on the other hand, ovarian cyst aspiration under ultrasound guidance could be the conservative treatment choice [2,3]. The aspiration procedure may bring the following complications: high recurrence, infection and lack of available histology [3–7]. As an option for treating benign cysts arising from various
organs, e.g. the thyroid [8], parathyroid [9], liver [10], kidney [11], spleen [12] and heart [13], ethanol sclerotherapy has been shown to be efficacious and cost-effective. Where the cyst volume has exceeded 100 ml, surgical treatment by laparoscopy or laparotomy has been advised [14]. Ethanol application on the epithelial surface of the cyst capsule induces sclerosis by reactional fibrosis due to coagulation necrosis [15,16]. In this study we aimed to investigate the effects of intracystic ethanol application on ovarian cyst diameter, ovarian follicular reserve and apoptosis in an experimental model. 2. Materials and methods
* Corresponding author at: Firat University Hospital, IVF Unit, 23119 Elazig, Turkey. Tel.: +90 5053983219. E-mail address: [email protected] (Z.S. Ozkan). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.12.019
This study was approved by Firat University Animal Use Committee and conducted at Firat University Animal Laboratory (FUTDAM). Twenty mature (12–14-week-old) female Wistar
134
R. Atilgan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 174 (2014) 133–136
Albino rats, weighing 200–220 g, with regular cycles were used for all experiments. (The study population number was determined by the Animal Use Committee.) The rats were housed individually in groups of five in plastic cages with chip bedding, and ad libitum access to rat chow (pellet) and water. They were maintained on a 12:12 light:dark cycle (lights on at 07:00 AM) at room temperature. Before surgical intervention, oral feeding except water intake was stopped. The rats on estrus cycle detected by vaginal smear were operated under intramuscular 60 mg/kg ketamine (Ketalar, Eczacibasi, Warner–Lambert, Istanbul, Turkey) and 7 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey) anesthesia and povidine–iodine surgical site antisepsis. After midline laparotomy, right total salpingectomy was performed for induction of ovarian cysts and then abdominal closure was done with 3/0 silk sutures. One month after first surgery, a second laparotomy was performed and a macroscopic ovarian cyst was observed in 15 rats (75%). The remaining five non-cystic rats were excluded from the study and one cystic rat was also excluded to equate the population number of groups. The 14 rats with ovarian cysts were randomly divided into two groups as follows; Group 1 (n = 7): the ovarian cyst was aspirated with an insulin injector and then the abdomen was closed. Group 2 (n = 7): the ovarian cyst was aspirated with an insulin injector and then the cystic cavity was irrigated with 95% ethanol equal to half of the aspirated cyst volume. After 10 min retention, the ethanol was aspirated and the abdomen was closed. One month after the second laparotomy, the 14 rats were decapitated after ketamine (75 mg/kg) + xylazine (10 mg/kg) intraperitoneal administration and exploratory laparotomy was performed. The presence and diameter of the ovarian cyst was recorded for each rat and then right oophorectomy was performed. 2.1. Histologic evaluation After oophorectomy, the adherent tissues were removed in culture medium, ovarian tissues were fixed with 10% formaldehyde and then paraffin-embedded tissue samples were cut into 4 mm sections for estimation of mean ovarian follicle count. The sections were stained with Masson trichrome to determine ovarian follicle reserve, fibrosis and angiogenesis in the corpus luteum
Table 1 Histopathological findings of right ovary in all rats. Parameters
Group 1 (n = 7)
Group 2 (n = 7)
p value
Primordial follicle count Primary follicle count Secondary follicle count Tertiary follicle count Ovarian follicle reserve Corpus luteum count Corpus albicans count Angiogenesis score in corpus luteum Fibrosis score Apoptosis score
17.1 2.9 13.2 1.8 9.0 1.6 3.1 0.7 42.5 6.5 1 0.6 00 1.8 0.4 0.9 0.2 0.4 0.5
7.0 3.3 9.7 4.3 6.0 2.7 2.5 0.8 25.2 10.9 0.8 0.4 00 0.6 0.2 2.5 0.5 2.3 0.7
* * Ns Ns * Ns Ns * * *
Note: Values are presented as mean SD. *p < 0.05, Mann–Whitney U test; Ns = non-significant.
under light microscopy. The 4 mm step sections were mounted at 50 mm intervals onto microscope slides to prevent counting the same structure twice, according to the method described previously [17]. Follicles were classified as primordial, primary, secondary, and tertiary follicles. The definitions of follicle types were as follows: primordial follicle = oocyte partially or completely surrounded by flattened pregranulosa cells; primary follicle = enlarged oocyte with one layer of cuboidal cells, or at least one cuboidal cell among flattened granulosa cells; secondary follicle = two layers of granulosa cells or at least one layer and one cell of the second layer; tertiary follicle = oocyte enclosed by more than two layers of granulosa cells with antrum formation. An atretic follicle was defined as a follicle that presented more than ten pyknotic nuclei per follicle; in the smallest follicles, the criterion for atresia was a degenerate oocyte, precocious antrum formation, or both [18–20]. The scoring for fibrosis was applied as follows: 0 = none, +1 = mild, +2 = moderate, +3 = intense [21,22]. Angiogenesis in the corpus luteum was defined according to P previous studies and H-SCORE ¼ Pi ði þ 1Þ, where i = intensity of staining with a value of (, minimal), (+, mild), (++, moderate) and (+++, strong) was applied [23,24]. To detect cells undergoing apoptosis, we applied the TUNEL method. We used the technique of Terminal-Transferase dUTP Nick End labeling (TUNEL Apoptag plus peroxidase in situ Apoptosis detection kit, S7101, Chemicon, USA). Sections were
Fig. 1. Masson trichrome staining under light microscope. Normal ovarian tissue of cyst aspiration group (1a, 1b, 1c). Decrement in ovarian follicle reserve, increment in fibrosis and decreased angiogenesis in corpus luteum of ethanol instillation group (1d, 1e, 1f). Red star, different types of follicles; black star, fibrosis areas; red arrow, angiogenesis; black arrow, primordial follicle; blue arrow, primary follicle; CL, corpus luteum; CH, corpus hemorrhagicum; FC, follicle cyst. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
R. Atilgan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 174 (2014) 133–136
pretreated with proteinase k (20 mg/ml) for 10 min. Endogenous hydrogen peroxidase activity was quenched in 3% hydrogen peroxide for 5 min. After a series of rinsing, nucleotides labeled with digoxigenin were enzymatically added to the DNA by terminal deoxy nucleotidyl transferase enzyme (TdT). The incubation was carried out for 60 min, and the labeled DNA was detected using anti-digoxigenin peroxidase for 30 min. The chromogen diaminobenzidine tetra hydrochloride (DAB) resulted in a brown reaction product. Mammary tissue was used for positive control. The staining intensity for TUNEL was scored semiquantitatively as follows: 0 = none, +1 = mild, +2 = moderate, and +3 = intense [25,26]. 2.2. Statistical analysis Comparison of groups was done with the Mann–Whitney U test, and repetitive comparison was done with the Wilcoxon Rank test. p values under 0.05 were considered as statistically significant. 3. Results Fifteen of the 20 rats (75%) developed an ovarian cyst. The mean cyst diameters before and after aspiration in Group 1 were 10.2 1.9 mm and 8.1 2.2 mm, respectively (p > 0.05). The mean cyst diameters before and after ethanol instillation in Group 2 were 10.1 2.4 mm and 3.4 2.7 mm, respectively (p < 0.05). The mean aspirated cyst volume was 0.62 0.46 ml and mean ethanol instillation volume was 0.31 0.23 ml. Primordial follicle count, primary follicle count and ovarian follicle reserve in Group 1 were significantly higher than in Group 2 (Table 1 and Fig. 1). The angiogenesis score in the corpus luteum in Group 1 was significantly higher than on Group 2, but the scores of apoptosis and fibrosis in Group 1 were significantly lower than in Group 2 (Table 1 and Fig. 1). 4. Comments In this study we found for the first time that intracystic ethanol instillation decreased ovarian cyst diameter, but showed a negative impact on ovarian follicle reserve in rats. Cyst aspiration alone has no meaningful efficacy on decreasing cyst diameter because of high recurrence rates, as we also observed in our study [3,4,6]. We performed total salpingectomy for ovarian cyst formation as in previous studies [21,27]. Sapmaz et al. reported that melatonin administration before salpingectomy decreased ovarian cyst development and conserved the ovarian reserve [21]. Because of our opinion that antioxidants have no effect after ischemia, we did not organize an antioxidant group for comparison. Ethanol instillation duration and retention volume for ovarian sclerotherapy is not yet clear. Tei et al. reported acute alcoholism after retention of 70 ml 99% ethanol in an endometriotic cyst under general anesthesia [28]. Intestinal perforation, intestinal stricture, shedding of neoplastic cells in the peritoneal cavity, extensive pelvic adhesions and pelvic bacterial infections were the reported complications of ethanol ovarian sclerotherapy [29,30]. In our study we did not observe any kind of these complications: this may be because of laparotomy and controlled ethanol instillation. Kukura et al. performed cyst puncture and 95% ethanol instillation with an amount of 50–75% of the evacuated volume for 5–20 min under transvaginal ultrasound guidance on 366 patients with simple, unilocular ovarian cysts. Their reported incidence of cyst rupture – alcohol spilt in pelvic cavity, pelvic pain and relapse were 0.8%, 8.1% and 8.2%, respectively. The relapse rate increased in the patients with cyst volume higher than 100 ml. The follow-up examinations revealed out no detrimental effect on menstrual cycle and ovarian reserve on
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the 3rd, 6th and 12th months after the procedure [15]. Koike et al. compared the reproductive performance of 45 subfertile women who underwent transvaginal ultrasound-guided ethanol sclerotherapy for ovarian endometrioma with those of 65 subfertile women without ovarian endometriotic cysts. Their instillation duration was 5 min and they observed no complications. There were no differences in the numbers of pregnancies, term deliveries, abortions, retrieved oocytes and quality of embryos between their endometriotic and non-endometriotic groups. They observed no increment in the levels of FSH and LH after sclerotherapy and their recurrence rate was 13.3% 5 months after sclerotherapy [31]. In an another study, the researchers compared the anti-mullerian hormone (AMH) levels between those of women who underwent ovarian sclerotherapy for recurrent endometrioma and those of women who underwent laparoscopic endometrioma extirpation. AMH levels were higher in the sclerotherapy group compared to the cystectomy group [32]. In our rat study, however, primordial follicle count and ovarian follicle reserve decreased after sclerotherapy. Researchers have reported lack of vitality of cyst epithelium 3 min after ethanol instillation and cyst capsule penetration 4–12 h after ethanol retention in kidney [33]. Hsieh et al. compared antral follicle number between those of 78 women who underwent 95% ethanol instillation for 0–10 min in ovarian endometriomas and those of 30 women who underwent ethanol retention in endometriomas under transvaginal ultrasound guidance. They performed ultrasonography at 3, 6, 9, and 12 months to determine persistence, size of cysts and the number of antral follicles. The 1year recurrence rates for the ethanol retention and ethanol aspiration groups were 13.3% and 32.1%, respectively, but they observed no difference in antral follicle count between the aspiration and retention groups [34]. In our study, however, ovarian primordial follicle counts decreased and apoptosis increased after ethanol instillation. The difference between our results and other studies may result from the following conditions: first, ethanol retention for 10 min may be a longer duration for rat ovary with simple cyst. Second, the thickness of the endometrioma cyst capsule in humans may protect against penetration of ethanol to ovarian tissue [35–37]. Livraghi et al. reported that ethanol diffusion into tissue induced cellular dehydration and protein denaturation that precipitated coagulation necrosis and reactive fibrosis [16]. The lifespan of corpus luteum is related to angiogenic activity mediated by vascular endothelial growth factor and hypoxia is the major stimulator of VEGF production [38–41]. In our study, the angiogenesis score in corpus luteum was higher in the cyst aspiration group than the ethanol sclerotherapy group. This might have arisen from ovarian hypoxia induced by total salpingectomy, and ethanol penetration might lead to decrement of angiogenesis in the corpus luteum. Persistence of ovarian cysts in the aspiration group might be the result of increased angiogenesis. In conclusion, ethanol instillation into simple ovarian cysts induced apoptosis and reactive fibrosis and caused decrement of ovarian reserve in rats. Conflict of interest statement All authors declare that they have no conflict of interest. References [1] Campbell S, Bhan V, Royston P, Whitehead MI, Collins WP. Transabdominal ultrasound screening for early ovarian cancer. BMJ 1989;299:1363–7. [2] Zanetta G, Trio D, Lissoni A, et al. Early and short-term complications after ultrasound-guided puncture of gynecologic lesions: evaluation after 1000 consecutive cases. Radiology 1993;183:161–4. [3] Marana R, Caruana P, Muzii L, Catalano GF, Mancuso S. Operative laparoscopy for ovarian cysts. Excision versus aspiration. J Reprod Med 1996;41:435–8.
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[4] Aboulghar MA, Mansour RT, Serour GI, Rizk B. Ultrasonic transvaginal aspiration of endometriotic cysts: an optional line treatment in selected cases of endometriosis. Hum Reprod 1991;6:1408–10. [5] Larsen JF, Pedersen OD, Gregersen E. Ovarian cyst fenestration via the laparoscope. Acta Obstet Gynecol Scand 1986;65:529. [6] Zanetta G, Lissoni A, Torri V, et al. Role of puncture and aspiration in expectant management of simple ovarian cysts: a randomized study. BMJ 1996;313: 1110–3. [7] Trimbos JB, Hacker NF. The case against aspirating ovarian cysts. Cancer 1993;72:828–31. [8] Antonelli A, Campatelli A, Di Vito A, et al. Comparison between ethanol sclerotherapy and emptying with injection of saline in treatment of thyroid cysts. Clin Investig 1994;72:971–4. [9] Zingrillo M, Ghiggi MR, Liuzzi A. A large, nonfunctioning parathyroid cyst recurring after aspiration and subsequently cured by percutaneous ethanol injection. J Clin Ultrasound 1996;24:378–82. [10] Ikeda Y, Kawasaki K, Ikeda T, Uchida H, Morinaga K. Treatment of large liver cyst evaluated with CA 19-9 in the cystic fluid. HPB Surg 1996;9: 179–82. [11] el-Diasty TA, Shokeir AA, Tawfeek HA, Mahmoud NA, Nabeeh A, Ghoneim MA. Ethanol sclerotherapy for symptomatic simple renal cysts. J Endourol 1995;9: 273–6. [12] Akhan O, Baykan Z, Og˘uzkurt L, Sayek I, Ozmen MN. Percutaneous treatment of a congenital splenic cyst with alcohol: a new therapeutic approach. Eur Radiol 1997;7:1067–70. [13] Kinoshita Y, Shimada T, Murakami Y, et al. Ethanol sclerosis can be a safe and useful treatment for pericardial cyst. Clin Cardiol 1996;19:833–5. [14] Kukura V, Canic´ T, Duic´ Z, Podgajski M, Drinkovic´ I. Transvaginal sclerosing therapy of cystic ovarian lesions with alcohol. Ultraschall Med 2004;25: 363–6. [15] Kukura V, Krivak-Bolanca I, Sentija K, Katalanic-Simon S. Alcohol sclerosing ovarian cystic lesions, 20 years experience. Coll Antropol 2010;34:37–40. [16] Livraghi T, Paracchi A, Ferrari C, et al. Treatment of autonomous thyroid nodules with percutaneous ethanol injection: preliminary results. Work in progress. Radiology 1990;175:827–9. [17] Woodruff TK, D’Agostino J, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. Science 1988;239:1296–9. [18] Mazaud S, Guigon CJ, Lozach A, et al. Establishment of the reproductive function and transient fertility of female rats lacking primordial follicle stock after fetal gamma-irradiation. Endocrinology 2002;143:4775–87. [19] Souza AZ, Fonseca AM, Izzo VM, Clauzet RM, Salvatore CA. Ovarian histology and function after total abdominal hysterectomy. Obstet Gynecol 1986;68: 847–9. [20] Anzalone CR, Hong LS, Lu JK, LaPolt PS. Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat. Biol Reprod 2001;64:1056–62. [21] Sapmaz E, Kale A, Akpolat N. Melatonin use in unilateral total salpingectomy in rats. Clin Exp Obstet Gynecol 2012;39:186–90. [22] Samrao D, Wang D, Ough F, et al. Histologic parameters predictive of disease outcome in women with advanced stage ovariancarcinoma treated with neoadjuvant chemotherapy. Transl Oncol 2012;5:469–74.
[23] Altungu¨l AC, Kale A, Sapmaz E, Akpolat N. Examination of the effect of melatonin use in Pomeroy method of tubal ligation on ovarian histology in rats. Clin Exp Obstet Gynecol 2012;39:103–6. [24] Inan S, Vatansever S, Celik-Ozenci C, Sanci M, Dicle N, Demir R. Immunolocalizations of VEGF, its receptors flt-1, KDR and TGF-beta’s in epithelial ovarian tumors. Histol Histopathol 2006;21:1055–64. [25] Suzuki S, Toledo-Pereyra LH, Rodriguez FJ. Role of neutrophils during the first 24 hours after liver ischemia and reperfusion injury. Transpl Proc 1994;26: 3695–700. [26] Li AM, Quan Y, Guo YP, Li WZ, Cui XG. Effects of therapeutic hypercapnia on inflammation and apoptosis after hepatic ischemia–reperfusion injury in rats. Chin Med J (Engl) 2010;123:2254–8. [27] Atilgan R, Celik A, Sapmaz E, et al. The effect of unilateral salpingectomy on ovarian histopathological examination: an experimental study. Turkiye Klinikleri J Gynecol Obst 2005;15:235–42. [28] Tei A, Ueki M, Yokono S, Oguri K. Acute alcoholism after ethanol fixation for ovarian chocolate cyst. Masui Jpn J Anesthesiol 1996;45:496–9. [29] Zanetta G, Trio D, Lissoni A, Pittelli M, Valle CD, Rangoni G. Ultrasound-guided aspiration of endometriomas: possible applications and limitations. Fertil Steril 1995;64:709–13. [30] Okagaki R, Osuga Y, Momoeda M, Tsutsumi O, Taketani Y. Laparoscopic findings after ultrasound-guided transvaginal ethanol sclerotherapy for ovarian endometrial cyst. Hum Reprod 1999;14:270. [31] Koike T, Minakami H, Motoyama M, Ogawa S, Fujiwara H, Sato I. Reproductive performance after ultrasound-guided transvaginal ethanol sclerotherapy for ovarian endometriotic cysts. Eur J Obstet Gynecol Reprod Biol 2002;105:39. [32] Yazbeck C, Madelenat P, Ayel JP, et al. Ethanol sclerotherapy: a treatment option for ovarian endometriomas before ovarian stimulation. Reprod Biomed Online 2009;19:121–5. [33] Bean WJ. Renal cysts: treatment with alcohol. Radiology 1981;138:329–31. [34] Hsieh CL, Shiau CS, Lo LM, Hsieh TT, Chang MY. Effectiveness of ultrasoundguided aspiration and sclerotherapy with 95% ethanol for treatment of recurrent ovarian endometriomas. Fertil Steril 2009;91:2709–13. [35] Sapmaz E, Akpolat N. Examination of the effect of trichloroacetic acid attachment and instillation methods on ovarian reserve and dysplasic changes in ovarian surface epithelium. Fırat Tıp Dergisi 2012;17:71–5. [36] Solloway MJ, Dudley AT, Bikoff EK, Lyons KM, Hogan BL, Robertson EJ. Mice lacking Bmp6 function. Dev Genet 1998;22:321–39. [37] Yan C, Wang P, DeMayo J, et al. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol Endocrinol 2001;15:854–66. [38] Trollmann R, Amann K, Schoof E, et al. Hypoxia activates the human placental vascular endothelial growth factor system in vitro and in vivo: up-regulation of vascular endothelial growth factor in clinically relevant hypoxic ischemia in birth asphyxia. Am J Obstet Gynecol 2003;188:517–23. [39] Neeman M, Abramovitch R, Schiffenbauer YS, Tempel C. Regulation of angiogenesis by hypoxic stress: from solid tumours to the ovarian follicle. Int J Exp Pathol 1997;78:57–70. [40] Abulafia O, Sherer DM. Angiogenesis of the ovary. Am J Obstet Gynecol 2000; 182:240–6. [41] Geva E, Jaffe RB. Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril 2000;74:429–38.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Impact of intracystic ethanol instillation on ovarian cyst diameter and adjacent ovarian tissue Remzi Atilgan a, Zehra Sema Ozkan a,*, Tuncay Kuloglu b, Nevin Kocaman b, Melike Baspinar a, Behzat Can a, Mehmet S¸ims¸ek a, Ekrem Sapmaz a a b
Firat University School of Medicine, Department of Obstetrics and Gynecology, 23119 Elazig, Turkey Firat University School of Medicine, Department of Histology and Embryology, 23119 Elazig, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2013 Received in revised form 4 November 2013 Accepted 12 December 2013
Objective: To investigate the regression level of simple ovarian cyst size after local ethanol application and the damage level of adjacent ovarian reserve in rats. Study design: This study was conducted at Firat University Animal Laboratory with 18 mature (12–14 weeks old) female Wistar albino rats weighing 200–220 g, with regular cycles. Ovarian cyst induction was performed with unilateral salpingectomy. Fourteen rats with ovarian cysts after a second laparotomy were divided into two groups as follows: Group 1 (n = 7): cyst aspiration group, and Group 2 (n = 7): intracystic 95% ethanol application group. One month after the cyst aspiration procedure a third laparotomy was performed. The cyst number and size were recorded for each rat. Right ovariectomy was performed and formalin-fixed/paraffin-embedded tissues were sectioned at 5 mm thickness. Under light microscopy, ovarian total follicle reserve and fibrosis were evaluated with Masson trichrome staining and apoptosis was evaluated with TUNEL staining. The groups were compared with the Mann–Whitney U test and Wilcoxon Rank test. p < 0.05 was considered significant. Results: Ovarian cyst formation was observed in 85% (15/18) of rats. The mean diameter of ovarian cysts in Groups 1 and 2 were, respectively, 10.3 mm and 10.1 mm. After aspiration, there was no significant reduction in the cyst diameter (10.3 mm vs 8.1 mm), but after ethanol application the diameter significantly reduced (10.1 mm vs 3.4 mm, p < 0.05). Mean ovarian follicle count in Group 2 was significantly lower than in Group 1 (25 vs 42, p < 0.05), and mean fibrosis and apoptosis scores in Group 2 were significantly higher than in Group 1 (2.5 vs 0.9, p < 0.05). Conclusion: Local ethanol application reduces cyst diameter but concomitantly decreases ovarian reserve due to increased fibrosis in rats. In humans, intracystic ethanol application should be performed cautiously. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethanol sclerotherapy Ovarian reserve Salpingectomy Apoptosis
1. Introduction The incidence of adnexal masses among healthy women has been reported as 6%, of which 90% were cystic tumors, and the common form of these tumors was simple cysts [1]. Either laparoscopy or laparotomy can be performed for a cystectomy procedure, but on the other hand, ovarian cyst aspiration under ultrasound guidance could be the conservative treatment choice [2,3]. The aspiration procedure may bring the following complications: high recurrence, infection and lack of available histology [3–7]. As an option for treating benign cysts arising from various
organs, e.g. the thyroid [8], parathyroid [9], liver [10], kidney [11], spleen [12] and heart [13], ethanol sclerotherapy has been shown to be efficacious and cost-effective. Where the cyst volume has exceeded 100 ml, surgical treatment by laparoscopy or laparotomy has been advised [14]. Ethanol application on the epithelial surface of the cyst capsule induces sclerosis by reactional fibrosis due to coagulation necrosis [15,16]. In this study we aimed to investigate the effects of intracystic ethanol application on ovarian cyst diameter, ovarian follicular reserve and apoptosis in an experimental model. 2. Materials and methods
* Corresponding author at: Firat University Hospital, IVF Unit, 23119 Elazig, Turkey. Tel.: +90 5053983219. E-mail address: [email protected] (Z.S. Ozkan). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.12.019
This study was approved by Firat University Animal Use Committee and conducted at Firat University Animal Laboratory (FUTDAM). Twenty mature (12–14-week-old) female Wistar
134
R. Atilgan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 174 (2014) 133–136
Albino rats, weighing 200–220 g, with regular cycles were used for all experiments. (The study population number was determined by the Animal Use Committee.) The rats were housed individually in groups of five in plastic cages with chip bedding, and ad libitum access to rat chow (pellet) and water. They were maintained on a 12:12 light:dark cycle (lights on at 07:00 AM) at room temperature. Before surgical intervention, oral feeding except water intake was stopped. The rats on estrus cycle detected by vaginal smear were operated under intramuscular 60 mg/kg ketamine (Ketalar, Eczacibasi, Warner–Lambert, Istanbul, Turkey) and 7 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey) anesthesia and povidine–iodine surgical site antisepsis. After midline laparotomy, right total salpingectomy was performed for induction of ovarian cysts and then abdominal closure was done with 3/0 silk sutures. One month after first surgery, a second laparotomy was performed and a macroscopic ovarian cyst was observed in 15 rats (75%). The remaining five non-cystic rats were excluded from the study and one cystic rat was also excluded to equate the population number of groups. The 14 rats with ovarian cysts were randomly divided into two groups as follows; Group 1 (n = 7): the ovarian cyst was aspirated with an insulin injector and then the abdomen was closed. Group 2 (n = 7): the ovarian cyst was aspirated with an insulin injector and then the cystic cavity was irrigated with 95% ethanol equal to half of the aspirated cyst volume. After 10 min retention, the ethanol was aspirated and the abdomen was closed. One month after the second laparotomy, the 14 rats were decapitated after ketamine (75 mg/kg) + xylazine (10 mg/kg) intraperitoneal administration and exploratory laparotomy was performed. The presence and diameter of the ovarian cyst was recorded for each rat and then right oophorectomy was performed. 2.1. Histologic evaluation After oophorectomy, the adherent tissues were removed in culture medium, ovarian tissues were fixed with 10% formaldehyde and then paraffin-embedded tissue samples were cut into 4 mm sections for estimation of mean ovarian follicle count. The sections were stained with Masson trichrome to determine ovarian follicle reserve, fibrosis and angiogenesis in the corpus luteum
Table 1 Histopathological findings of right ovary in all rats. Parameters
Group 1 (n = 7)
Group 2 (n = 7)
p value
Primordial follicle count Primary follicle count Secondary follicle count Tertiary follicle count Ovarian follicle reserve Corpus luteum count Corpus albicans count Angiogenesis score in corpus luteum Fibrosis score Apoptosis score
17.1 2.9 13.2 1.8 9.0 1.6 3.1 0.7 42.5 6.5 1 0.6 00 1.8 0.4 0.9 0.2 0.4 0.5
7.0 3.3 9.7 4.3 6.0 2.7 2.5 0.8 25.2 10.9 0.8 0.4 00 0.6 0.2 2.5 0.5 2.3 0.7
* * Ns Ns * Ns Ns * * *
Note: Values are presented as mean SD. *p < 0.05, Mann–Whitney U test; Ns = non-significant.
under light microscopy. The 4 mm step sections were mounted at 50 mm intervals onto microscope slides to prevent counting the same structure twice, according to the method described previously [17]. Follicles were classified as primordial, primary, secondary, and tertiary follicles. The definitions of follicle types were as follows: primordial follicle = oocyte partially or completely surrounded by flattened pregranulosa cells; primary follicle = enlarged oocyte with one layer of cuboidal cells, or at least one cuboidal cell among flattened granulosa cells; secondary follicle = two layers of granulosa cells or at least one layer and one cell of the second layer; tertiary follicle = oocyte enclosed by more than two layers of granulosa cells with antrum formation. An atretic follicle was defined as a follicle that presented more than ten pyknotic nuclei per follicle; in the smallest follicles, the criterion for atresia was a degenerate oocyte, precocious antrum formation, or both [18–20]. The scoring for fibrosis was applied as follows: 0 = none, +1 = mild, +2 = moderate, +3 = intense [21,22]. Angiogenesis in the corpus luteum was defined according to P previous studies and H-SCORE ¼ Pi ði þ 1Þ, where i = intensity of staining with a value of (, minimal), (+, mild), (++, moderate) and (+++, strong) was applied [23,24]. To detect cells undergoing apoptosis, we applied the TUNEL method. We used the technique of Terminal-Transferase dUTP Nick End labeling (TUNEL Apoptag plus peroxidase in situ Apoptosis detection kit, S7101, Chemicon, USA). Sections were
Fig. 1. Masson trichrome staining under light microscope. Normal ovarian tissue of cyst aspiration group (1a, 1b, 1c). Decrement in ovarian follicle reserve, increment in fibrosis and decreased angiogenesis in corpus luteum of ethanol instillation group (1d, 1e, 1f). Red star, different types of follicles; black star, fibrosis areas; red arrow, angiogenesis; black arrow, primordial follicle; blue arrow, primary follicle; CL, corpus luteum; CH, corpus hemorrhagicum; FC, follicle cyst. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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pretreated with proteinase k (20 mg/ml) for 10 min. Endogenous hydrogen peroxidase activity was quenched in 3% hydrogen peroxide for 5 min. After a series of rinsing, nucleotides labeled with digoxigenin were enzymatically added to the DNA by terminal deoxy nucleotidyl transferase enzyme (TdT). The incubation was carried out for 60 min, and the labeled DNA was detected using anti-digoxigenin peroxidase for 30 min. The chromogen diaminobenzidine tetra hydrochloride (DAB) resulted in a brown reaction product. Mammary tissue was used for positive control. The staining intensity for TUNEL was scored semiquantitatively as follows: 0 = none, +1 = mild, +2 = moderate, and +3 = intense [25,26]. 2.2. Statistical analysis Comparison of groups was done with the Mann–Whitney U test, and repetitive comparison was done with the Wilcoxon Rank test. p values under 0.05 were considered as statistically significant. 3. Results Fifteen of the 20 rats (75%) developed an ovarian cyst. The mean cyst diameters before and after aspiration in Group 1 were 10.2 1.9 mm and 8.1 2.2 mm, respectively (p > 0.05). The mean cyst diameters before and after ethanol instillation in Group 2 were 10.1 2.4 mm and 3.4 2.7 mm, respectively (p < 0.05). The mean aspirated cyst volume was 0.62 0.46 ml and mean ethanol instillation volume was 0.31 0.23 ml. Primordial follicle count, primary follicle count and ovarian follicle reserve in Group 1 were significantly higher than in Group 2 (Table 1 and Fig. 1). The angiogenesis score in the corpus luteum in Group 1 was significantly higher than on Group 2, but the scores of apoptosis and fibrosis in Group 1 were significantly lower than in Group 2 (Table 1 and Fig. 1). 4. Comments In this study we found for the first time that intracystic ethanol instillation decreased ovarian cyst diameter, but showed a negative impact on ovarian follicle reserve in rats. Cyst aspiration alone has no meaningful efficacy on decreasing cyst diameter because of high recurrence rates, as we also observed in our study [3,4,6]. We performed total salpingectomy for ovarian cyst formation as in previous studies [21,27]. Sapmaz et al. reported that melatonin administration before salpingectomy decreased ovarian cyst development and conserved the ovarian reserve [21]. Because of our opinion that antioxidants have no effect after ischemia, we did not organize an antioxidant group for comparison. Ethanol instillation duration and retention volume for ovarian sclerotherapy is not yet clear. Tei et al. reported acute alcoholism after retention of 70 ml 99% ethanol in an endometriotic cyst under general anesthesia [28]. Intestinal perforation, intestinal stricture, shedding of neoplastic cells in the peritoneal cavity, extensive pelvic adhesions and pelvic bacterial infections were the reported complications of ethanol ovarian sclerotherapy [29,30]. In our study we did not observe any kind of these complications: this may be because of laparotomy and controlled ethanol instillation. Kukura et al. performed cyst puncture and 95% ethanol instillation with an amount of 50–75% of the evacuated volume for 5–20 min under transvaginal ultrasound guidance on 366 patients with simple, unilocular ovarian cysts. Their reported incidence of cyst rupture – alcohol spilt in pelvic cavity, pelvic pain and relapse were 0.8%, 8.1% and 8.2%, respectively. The relapse rate increased in the patients with cyst volume higher than 100 ml. The follow-up examinations revealed out no detrimental effect on menstrual cycle and ovarian reserve on
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the 3rd, 6th and 12th months after the procedure [15]. Koike et al. compared the reproductive performance of 45 subfertile women who underwent transvaginal ultrasound-guided ethanol sclerotherapy for ovarian endometrioma with those of 65 subfertile women without ovarian endometriotic cysts. Their instillation duration was 5 min and they observed no complications. There were no differences in the numbers of pregnancies, term deliveries, abortions, retrieved oocytes and quality of embryos between their endometriotic and non-endometriotic groups. They observed no increment in the levels of FSH and LH after sclerotherapy and their recurrence rate was 13.3% 5 months after sclerotherapy [31]. In an another study, the researchers compared the anti-mullerian hormone (AMH) levels between those of women who underwent ovarian sclerotherapy for recurrent endometrioma and those of women who underwent laparoscopic endometrioma extirpation. AMH levels were higher in the sclerotherapy group compared to the cystectomy group [32]. In our rat study, however, primordial follicle count and ovarian follicle reserve decreased after sclerotherapy. Researchers have reported lack of vitality of cyst epithelium 3 min after ethanol instillation and cyst capsule penetration 4–12 h after ethanol retention in kidney [33]. Hsieh et al. compared antral follicle number between those of 78 women who underwent 95% ethanol instillation for 0–10 min in ovarian endometriomas and those of 30 women who underwent ethanol retention in endometriomas under transvaginal ultrasound guidance. They performed ultrasonography at 3, 6, 9, and 12 months to determine persistence, size of cysts and the number of antral follicles. The 1year recurrence rates for the ethanol retention and ethanol aspiration groups were 13.3% and 32.1%, respectively, but they observed no difference in antral follicle count between the aspiration and retention groups [34]. In our study, however, ovarian primordial follicle counts decreased and apoptosis increased after ethanol instillation. The difference between our results and other studies may result from the following conditions: first, ethanol retention for 10 min may be a longer duration for rat ovary with simple cyst. Second, the thickness of the endometrioma cyst capsule in humans may protect against penetration of ethanol to ovarian tissue [35–37]. Livraghi et al. reported that ethanol diffusion into tissue induced cellular dehydration and protein denaturation that precipitated coagulation necrosis and reactive fibrosis [16]. The lifespan of corpus luteum is related to angiogenic activity mediated by vascular endothelial growth factor and hypoxia is the major stimulator of VEGF production [38–41]. In our study, the angiogenesis score in corpus luteum was higher in the cyst aspiration group than the ethanol sclerotherapy group. This might have arisen from ovarian hypoxia induced by total salpingectomy, and ethanol penetration might lead to decrement of angiogenesis in the corpus luteum. Persistence of ovarian cysts in the aspiration group might be the result of increased angiogenesis. In conclusion, ethanol instillation into simple ovarian cysts induced apoptosis and reactive fibrosis and caused decrement of ovarian reserve in rats. Conflict of interest statement All authors declare that they have no conflict of interest. References [1] Campbell S, Bhan V, Royston P, Whitehead MI, Collins WP. Transabdominal ultrasound screening for early ovarian cancer. BMJ 1989;299:1363–7. [2] Zanetta G, Trio D, Lissoni A, et al. Early and short-term complications after ultrasound-guided puncture of gynecologic lesions: evaluation after 1000 consecutive cases. Radiology 1993;183:161–4. [3] Marana R, Caruana P, Muzii L, Catalano GF, Mancuso S. Operative laparoscopy for ovarian cysts. Excision versus aspiration. J Reprod Med 1996;41:435–8.
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