SEXUAL DYSFUNCTION AND INFERTILITY The Effect of Testicular Cryoablation on Testosterone Level in Rats: An Experimental Model of Histopathological Orchiectomy Serkan Ozcan,1 Emre Huri,1 Omer Gokhan Doluoglu,2* Tolga Karakan,1 Elif Ozer,2 Vildan Fidanci,3 Muzaffer Eroglu,1 Sema Hucumenoglu1 Purpose: To determine whether testicular cryoablation caused histopathological orchiectomy, and to show its effects on serum total testosterone (t-testosterone) levels in rats. Materials and Methods: A total of 12 Wistar albino male rats were used in this study. The animals were divided into two groups, as cryoablation (9 rats) and control (3 rats) groups. Bilateral cryoablation was performed in the cryoablation group. T-testosterone levels were measured in both groups before scrotal exploration. Bilateral or- chiectomy was performed in both groups 10 days after the cryoablation procedure. T-testosterone was measured immediately before orchiectomy. Mann-Whitney U test was used for intergroup comparisons. Results: Baseline t-testosterone levels were 1.31 (0.78-2.45) ng/mL and 0.98 (0.91-2.05) ng/mL in the cryoab- lation and the control groups, respectively (P = .92). T-testosterone levels were 0.23 (0.07-1.12) ng/mL and 2.87 (0.63-3.06) ng/mL in the cryoablation and the control groups, respectively, in the blood samples obtained at the time of orchiectomy (P = .03). Histopathological examination of rat testes revealed varying degrees of paratestic- ular inflammation and necrosis in 13 of 18 testes in the cryoablation group. None of 6 testes showed necrosis in the control group. Conclusion: Our study showed that histopathological orchiectomy could be obtained by cryoablation in rat testes. Keywords: disease models; animal; male; orchiectomy; adverse effects; rats, Wistar; testosterone. INTRODUCTION Prostate cells physiologically depend on androgens for stimulation of their growth, function, and pro- liferation. Testosterone is essential for the growth and perpetuation of tumor cells.(1) Testis is the source of most androgens, and adrenal biosynthesis provides only 5-10% of them. This is why androgen deprivation therapy (ADT) has been the main treatment option in advanced prostate cancer (PCa) after Huggins and col- leagues.(2,3) ADT can be provided through medical and surgical (bilateral orchiectomy) approaches. Surgical castration has still been accepted as the gold standard when compared to the other treatment options. It pro- vides a significant decrease in the testosterone level, and causes hypogonadism at the castration level. However, both treatment options have disadvantages. The most important disadvantage of the surgical castra- tion is its negative psychological effect. Some men may perceive this as an insult to their masculinity. The most important drawback of medical ADT is its cost and its burden to national health assurance since the treatment is administered for a long time. This is why it is logical to investigate new treatment modalities that do not ne- cessitate orchiectomy, and reach the castration level af- ter just one intervention or procedure. In this study, we investigated whether testicular cryoablation resulted in histopathological orchiectomy, and showed its effects on total testosterone levels in rats. MATERIALS AND METHODS Study Animals After the approval of local Ethics Committee, the study was performed in Ankara Education and Research Hospital’s Animal Experiment Laboratory, Ankara, Turkey, between August 1-20, 2013, in accordance with the directives on “Rules and Principles of Animal Experiment Laboratories” (NIH publication No. 85-23, revised 1985). A total of 12 Wistar albino male rats, weighing 250-350 g were used in this study. The rats were housed in a room at 20-24°C with a moisture of 60-70%. Twelve-hour light-darkness cycle (dark: 07:00 pm - 07:00 am) was provided. Standard pellets were used to feed the rats, and tap water was used as drinking water. The rats were divided into two groups, as cryo- ablation (9 rats) and control (3 rats) groups. First, scro- tal exploration was performed in both groups. Blood was drawn to measure total testosterone (t-testosterone) levels in both groups before scrotal exploration. Procedures The genital regions of the rats were shaved and washed with diluted benzalkonium chloride solution before surgery. The surgical field was washed with povidone iodine. A single dose of intraperitoneal ketamine was used for anesthesia, at a dose of 30 mg/kg. Later, a ver- tical skin and subcutaneous tissue incision was done at the midline of scrotum. Testes were removed after freeing gubernaculum. Only scrotal exploration was Departments of Urology,1 Pathology,2 and Biochemistry,3 Ankara Training and Research Hospital, Ankara, Turkey. *Correspondence: Department of Urology, Ankara Training and Research Hospital, Sukriye Mahallesi Ulucanlar Caddesi. Tel: +90 533 215 7809. Fax: +90 312 362 4933. E-mail: drdoluoglu@yahoo.com.tr. Received November 2014 & Accepted April 2015 Vol 12 No 04 July-August 2015 2256 performed in the control group. Scrotal exploration was done immediately before cryoablation in the cryoabla- tion group, then bilateral cryoablation was performed using Brymill Cry-Ac Portable Cryo device (Figure 1). Tissue freezing procedure was done with liquid nitrogen. A 21G, green, 0.8 × 38 mm injector needle was placed on the device (Figure 1). The needle was stabbed to the testis, and the device was run for 5 min- utes until an ice ball was generated. After 15-minutes of thawing, a second nitrogen application was performed. Formation of the ice ball was seen and palpated. The testes were put back, and the incision was sutured. Bi- lateral orchiectomy was performed in both groups 10 days after the cryoablation procedure. T-testosterone level was measured immediately before orchiectomy. A third blood sample was obtained from the survived rats for t-testosterone measurement 72 hours after or- chiectomy (Figure 2, Flowchart). After macroscopic examination, removed testes were fixed in 10% neutral formalin solution. Tissues were stained with hematoxylin & eosin, and examined by an experienced uropathologist (EO) in Pathology Clinic of Ankara Training and Research Hospital. According to our experiences and observations, the tissues were analyzed for necrosis, inflammation, hyperemia, and edema on histopathological examination, and they were graded as 0, 1+, 2+, or 3+ for active inflammation, hyperemia, edema and paratesticular inflammation, and necrosis (0: none, 1+: ≤ 10% of the included tis- sue, 2+: 10-49% of the included tissue, 3+: ≥ 50% of the included tissue). The blood samples were stored at -20°C until t-testosterone measurement. T-testosterone measurement was done using Siemens ADVIA Centaur XP (Siemens; Erlangen, Germany) device, with direct chemiluminescence immunoassay method. The normal range of total testosterone was 0.66-5.4 ng/mL for the male rats in this device. Statistical Analysis Data analysis was performed using the Statistical Pack- age for the Social Science (SPSS Inc, Chicago, Illinois, USA) version 11.5. Mann-Whitney U test was used for the intergroup analysis of continuous variables. Cate- gorical variables were analyzed with Chi square test. Pearson correlation coefficient were used to compare testosterone and degree of necrosis. P values < .05 were considered as statistically significant. RESULTS Baseline median t-testosterone levels were 1.31 (0.78- 2.45) ng/mL and 0.98 (0.91-2.05) ng/mL in the cryo- ablation and the control groups, respectively (P = .92). The median t-testosterone levels measured at the time of orchiectomy (10 days after cryoablation) were 0.23 (0.07-1.12) ng/mL and 2.87 (0.63-3.06) ng/mL in the cryoablation and the control groups, respectively (P = .03) (Tables 1 and 2). Six rats survived after orchiec- tomy, and their median t-testosterone levels were 0.05 (0.04-0.43) ng/mL in cryoablation group (4 rats) and 0.01 (0.01-0.01) ng/mL in the control group (2 rats) (P = .38). Histopathological examination of the rat testes revealed various degrees of paratesticular inflammation and ne- crosis in 13 of 18 testes in cryoablation group. Necrosis was seen in 100% of 10 testes. Necrosis was not found in any of the 6 testes in the control group. There was a negative correlation between testosterone levels and the degree of inflammation and necrosis (P = .02, r = 0.74). In the control group, there were no significant findings except mild hyperemia in 3, and mild to mod- erate edema in 3 testes. However, high degrees of active inflammation, hyperemia, and edema were seen in cry- oablation group (Table 3) (Figures 3 and 4). DISCUSSION Androgens are needed for growth and proliferation of the prostate cells. Although testosterone is not tumor- igenic, tumor cells need it to proliferate and survive. Androgens are mainly produced by testes. Ninety to ninety-five percent of androgens are produced in testes, and 5-10% is released by the adrenal glands. Apoptosis occurs in prostate cells if androgenic stimulation of the prostate cells is blocked (programmed cell death). Any treatment modality that suppresses androgenic stimula- tion is called as ADT. ADT can be performed both by suppression of androgen release and by decreasing the effects of circulating androgens (androgen blockage). The treatment is called as complete androgen blockage if both methods are used simultaneously. The standard castration level is determined as < 50 ng/mL for tes- tosterone. Current chemiluminescence tests have found the mean testosterone level as 15 ng/mL after surgical castration. Therefore, castration level was accepted as 20 ng/mL. ADT is the primary treatment choice in advanced PCa. Table 1. Comparison of total testosterone (t-testosterone) levels in cryoablation and the control groups. Variables Cryoablation Group (9 rats) Control Group (3 rats) P Value Total testosterone levels before cryoablation (ng/mL) 1.31 (0.78-2.45) 0.98 (0.91-2.05) .92 Total testosterone levels at the time of orchiectomy (ng/mL) 0.23 (0.07-1.12) 2.87 (0.63-3.06) .03 Total testosterone levels 72 hours after orchiectomy (ng/mL) 0.05 (0.04-0.43) 0.01 (0.01-0.01) .38 Variables Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Rat 7 Rat 8 Rat 9 Total testosterone levels in 0.065 0.23 0.076 0.67 0.13 0.29 0.12 0.38 1.12 cryoablation group (ng/mL) Total testosterone levels in 0.63 2.87 3.06 _____ _____ _____ _____ _____ _____ the control group (ng/mL) Table 2. Total testosterone (t-testosterone) levels at the time of orchiectomy in the cryoablation and the control groups. Sexual Dysfunction and Infertility 2257 Testicular Cryoablation and Testosterone Level-Ozcan et al. (2,4) ADT can be performed by medical or surgical meth- ods. Bilateral orchiectomy has currently been accepted as the gold standard among other ADT methods. The advantages of orchiectomy are its low cost, and easy application under local anesthesia. However, some men may perceive this operation as an insult to their mascu- linity.(5) Long acting luteinizing-hormone releasing hormone (LH-RH) agonists are the primary agents used for medical ADT. Continuous stimulation of the receptors by these agents caused downregulation of LH-RH re- ceptors, and hence, testosterone levels are suppressed. (6) However, the castration levels cannot be reached in approximately 10% of the patients. If castration level is accepted as 20 ng/mL, this value reaches to 15%. (7) Flare phenomenon can be seen at the initiation of this treatment, leading to increased bone pain, bladder output obstruction, kidney obstruction and spinal cord compression. Long term use of these agents brings a large economic burden to national health insurance. In Europe, the money spent for the PCa patients diagnosed in 2006 was 106.7-179.0 million Euros. A formal meta-analysis estimated the cost-effectiveness of different ADT methods in advanced PCa. This study showed that the most cost-effective treatment for ADT was bilateral orchiectomy. The cost-effectiveness of medical hormone therapies was found worse.(8) There- fore, it may be logical to investigate cost-effective, new treatment modalities that do not necessitate orchiecto- my, and reach the castration level after just one inter- vention or procedure. Testicular cryoablation has not been performed before in order to suppress androgens. Cryotherapy freezes and kills the cells. Cell death oc- curs by protein denaturation due to dehydration, direct laceration of the cell membrane by ice crystals, and is- chemia caused by vascular stasis and microthrombi. (9- 12) Cryotherapy was first used by Gonder and colleagues in the field of urology in 1964 for benign prostatic hy- perplasia and PCa.(13) This modality has been the pre- ferred method for treatment of localized PCa in elderly patients with comorbidities.(14) In our study, normal testicular tissue was tried to be destructed by cryoablation, and the histological and bi- ochemical results of cryoablation were analyzed. The t-testosterone level measured at the time of orchiecto- my was 0.23 (0.07-1.12) ng/mL in the cryoablation, and 2.87 (0.63-3.06) ng/mL in the control groups. The values obtained in the cryoablation group have suggest- ed that t-testosterone level can be significantly reduced after cryoablation. Histopathological examination re- vealed varying degrees of paratesticular inflammation and necrosis in 13 of 18 rat testes in the cryoablation group. One hundred percent of 10 testes showed ne- crosis. Necrosis is followed by fibrosis. Since fibrotic testes will remain in their position, the patients will not have a perception of “testicular absence”. Our results suggest that cryoablation can result in histopathological orchiectomy. When cryotherapy is used for cancer, it is important to preserve neighboring tissues while de- structing the cancerous tissue. Since there are no neigh- boring structures of testes, we suppose that cryotherapy may be a suitable option for ADT. Systemic inflammatory response may develop after cry- oablation,(15,16) and may cause hypotension, respiratory distress, multi-organ dysfunction and intravascular co- agulation due to cytokine production.(17) This response usually develops after cryoablation of masses with large Table 3. Histopathological findings in study groups. Variables Active Inflammation Hyperemia Edema Paratesticular Inflammation and Necrosis Group 0 +1 +2 +3 0 +1 +2 +3 0 +1 +2 +3 0 +1 +2 +3 Cryoablation (n = 18) 0 4 2 12 0 5 8 5 0 4 13 1 5 1 1 11 Control (n = 6) 5 1 0 0 3 3 0 0 3 2 1 0 6 0 0 0 P value .0001 .001 .003 .004 Figure 1. Brymill Cry-Ac Portable Cryo device. Figure 2. Study flowchart. Vol 12 No 04 July-August 2015 2258 Testicular Cryoablation and Testosterone Level-Ozcan et al. volumes. None of the rats died after cryotherapy, until the time of orchiectomy in our study. CONCLUSIONS Our study showed that histopathological orchiectomy could be obtained in testes by cryoablation. In our opin- ion, cryoablation can be an alternative to medical and surgical (orchiectomy) castration in patients with ad- vanced PCa. Since orchiectomy is not performed, the perception of orchiectomy as an insult to masculinity may not appear. In addition, the economic burden of the disease to national health assurance may decrease since castration levels can be reached by a single procedure, and the patients do not have to use LH-RH analogs for a long time. Cryoablation can be performed in the office, under local anesthesia. As we have done, if cheap and easy-to-use systems can be made available, we suppose that cryoablation can be a logical option to achieve cas- tration. Our study is the first study one on this topic, and prospective randomized studies on a larger subject Figure 3. Extensive necrosis and normal histological findings in testis (Hematoxylin and eosin stain × 10). Figure 4. Findings of mild (A), moderate (B), and severe necrosis (C). (D) Extensive necrosis and severe inflammation (Hematoxylin and eosin stain × 40). group are needed to further clarify this issue. CONFLICT OF INTEREST None declared. REFERENCES 1. Berman DM, Rodriguez R, Veltri RW. Development, molecular biology and physiology of the prostate. 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Cryoablation in Sexual Dysfunction and Infertility 2259 Testicular Cryoablation and Testosterone Level-Ozcan et al. prostate cancer. Turkiye Klinikleri J Urol. 2008;1:79-89. 15. Chapman WC, Debelak JP, Blackwell TS, et al. Hepatic cryoablation-induced acute lung injury: pulmonary hemodynamic and permeability effects in a sheep model. Arch Surg. 2000;135:667-72. 16. Washington K, Debelak JP, Gobbell C, et al. Hepatic cryoablation-induced acute lung injury: Histopathologic findings. J Surg Res. 2001;95:1-7. 17. Seifert JK, Stewart GJ, Hewitt PM, Bolton EJ, Junginger T, Morris DL. Interleukin-6 and tumor necrosis factor- alpha levels following hepatic cryotherapy: association with volume and duration of freezing. World J Surg. 1999;23:1019-26. Vol 12 No 04 July-August 2015 2260 Testicular Cryoablation and Testosterone Level-Ozcan et al.