MISCELLANEOUS Effect of Unilateral Iatrogenic Testicular Torsion on the Contralateral Testis in Rats: Prepubertal and Postpubertal Hassan Ahmadnia1, Mahmoud Dolati1, Alireza Ghanadi1, Mehdi YounesiRostami2, AlirezaAkhavan Rezayat3* Purpose: The present study was conducted to investigate the influence of hemicastration and age at hemicastration on the contralateral testis weight and function/testosterone production. Materials and Methods: 64 Wistar-derived male rats were divided randomly into 4 groups. Group 1 was named immature intervention, group 2 immaturecontrol, group 3 mature intervention, and group 4mature control. In group 1, rats were hemicastrated at 30 days of age (prepubertal). In group 2, sham surgery (midscrotal incision) was per- formed atthe same age. In group 3, rats were hemicastrated at 70 days of age (postpubertal) and in group 4,sham surgery was done at the same age. Twenty days after the first surgery, contralateral orchiectomy wasperformed in intervention groups and controls underwent random orchiectomy (left or right). Blood sampling for evaluation of serum testosterone was performed just before second surgery. Results:The mean testis weight (1692 ± 26.7 in group 1 versus 1375 ± 39.7 in group 2; P < .001 and 1760 ± 26.6 in group 3 versus 1425 ± 44.9 in Group 4; P < .001) and the mean testicular weight (mg) per 100 g of body weight (735.8 ± 82.3 in group 1 versus 634.8 ± 84.8 in group 2; P = .005 and 652.4 ± 61.4 in group 3 versus 572.6 ± 97.7 in group 4; P = .03) were significantly greater in hemicastrated rats as to their controls. Also, these parameters was greater in prepubertal group than postpubertalhemicastrated rats. There was no appreciable difference in serum testosterone levels across the 4 groups (P = .77). Conclusion: Our research demonstrated that hemicastration results in compensatory hypertrophy of the remaining testis and it decreases as the animals age. Hemicastration does not lead to reduction in serum testosterone levels and the remaining testis can retrieve a normal serum testosterone level. Keywords: compensatory hypertrophy; iatrogenic testicular torsion; rat; testis; unilateral orchiectomy INTRODUCTION In paired organs, the removal of or injury to one organ may result in compensatory hypertrophy of the re- maining organ. This phenomenon has previously been proved in the kidneys, thyroid glands, and ovaries(1-3). The time of unilateral testis removal (prior to and fol- lowing puberty) due to undescended testis (UDT), trauma, and testicular abscess affects the amount of compensatory hypertrophy. That is, compensatory hy- pertrophy of the testis occurs more frequently before puberty. Torsion of the spermatic cord is associated with restriction and interruption of testicular blood flow. Anatomical abnormalities (the bell-clapper de- formity caused by lack of normal attachment of the epididymis to the tunica vaginalis which leads to in- complete fixation of the testis and the epididymis to the scrotum or abnormally high attachment of the testis to the epididymis), cold weather, sudden movements or trauma which activate the cremasteric reflex, and rapid growth of the testis throughout puberty make individu- als prone to this medical condition. The symptoms of testicular torsion include a sudden and intense scrotal 1Mashhad University of Medical Sciences, Mashhad,Iran. 2Department of Urology, , Mazandaran University of Medical Sciences, Sari,Iran. 3Kidney Transplantation Complications Research Center, Mashhad University of Medical Sciences, Mashhad,Iran. *Correspondence: Kidney Transplantation Complications Research Center, Mashhad University of Medical Sciences, Mashhad,Iran. Tel: +98 9153148223. Fax : +98 5138436199. E-mail: alirezaakhavan30@yahoo.com. Received November 2017 & Accepted June 2018 pain which has started within the last six hours, vomit- ing, nausea, scrotal edema and erythema, fever as well as dysuria. On physical examination, tenderness of the scrotum, absence of cremasteric reflex, a higher testic- ular position, abnormal position of the epididymis on the anterior, thickening of the spermatic cord, testicular induration, loss of the grooves between the testis and the epididymis along with scrotal edema and erythema might also be observed(4,5). In order to diagnose testis torsion, radionuclide scanning, color doppler ultra- sound (CDUS), and high resolution ultrasound (HRUS) can be used(6-8). The presence of sexual findings is an in- dication for surgical exploration with detorsion and fix- ation as the treatment of choice(9-11). The prognosis and long-term outcomes of torsion are unknown. Neverthe- less, considering the recent studies it is indicated that ischemic injury is likely to occur quickly, even if the testis appears viable during detorsion(11,12). As testicular development in humans is akin to rats, it is assumed that the use of an experimental model in rats provides ben- eficial evidence for further research on the morphology and histology of the testis(13). In the present study, com- Urology Journal/Vol 16 No. 5/ September-October 2019/ pp. 501-505. [DOI: 10.22037/uj.v0i0.4289] pensatory hypertrophy caused by iatrogenic torsion of the testis on the contralateral side is investigated in an experimental rat model prior to and following puberty and then compared with the control group. MATERIALS AND METHODS Study Population In this investigation, the effect of iatrogenic torsion (in order to do a unilateral functional orchiectomy, but not surgical orchiectomy) was explored in the contralateral testis of either mature or immature rats. A total of 64 male Wistar rats with 20 days of age and an average weight of 60 ± 8 gr were purchased from an animal li- brary in Mashhad University of Medical Sciences. The experimental room was automatically air-conditioned once each 3 minutes and maintained in the standard temperature of 20-22 °C (SD: ± 2 °C),a humidity of 55%, and an 12 hour day-night cycle. The rats spent one week for quarantine and acclimation in the room before the start of the experiment. They were subsequently as- signed into four groups (n = 16) at random. Study Design and Procedures Group 1: After weighing, the immature rats in the first group underwent a unilateral iatrogenic torsion in 30 days of age. Either a right or left iatrogenic torsion was conducted randomly. After 20 days (at 50 days of age), their testis and body weight was measured again and general anesthesia was induced before collecting a supraorbital blood sample from the cavernous sinus to determine the plasma testosterone. Thereafter, the remaining testis was also removed and weighed(14)us- ing a high-precision balance (readable to the nearest 0.0001 g). Group 2: This group served as control immature rats. They were weighed on the 30th day and underwent sham surgery with scrotal incision on their skin after general anesthesia. Blood samples were obtained from the same vein and unilateral orchiectomy were carried out randomly before the second weight measurement of the testes. Group 3: Akin to Group 1, all mature rats in this group underwent a unilateral iatrogenic torsion on the 10th week. On the 90th day, they were weighed and under- went general anesthesia, blood sampling, as well as contralateral orchiectomy. In the long run, the weight of the testis was evaluated. Group 4: At the same time as group 3, mature rats in this group were treated with sham operation. A right (n = 8) or left (n = 6) orchiectomy was randomly per- formed after 20 days of sham surgery and weight of the testes was measured thereafter. An ELISA assay was used to determine the plasma lev- el of testosterone by a commercially available Testos- terone Rat/Mouse ELISA kit. This study was approved by Mashhad University of medical Sciences ethics committee and investigators had been certified to study on laboratory animals. Surgical technique All rats underwent general anesthesia before surgery. After making a unilateral incision on the scrotum, iat- rogenic torsion was performed and the testis was sub- sequently fixed by 4-0 nylon thread sutures in order to prevent detorsion. Incisions were sutured finally. We performed unilateral iatrogenic torsion to induce uni- lateral functional orchiectomy without any surgical or- chiectomy intervention in the first step. Surgical orchi- ectomies were only carried out in the last step in order to weigh the testes. Both of the control groups underwent sham surgery with scrotal incision on their skin after general anesthe- sia and then incisions were closed by sutures. Statistical Analysis The data was collected and entered into SPSS (Version 11.5, IBM, Chicago, IL, USA). The concentration of serum testosterone was compared among groups by analysis of variance (ANOVA) and independent sam- ple t-test. A P-value of less than .05 was considered significant. RESULTS In this study, rats were randomly divided into four groups, mature and immature ones underwent unilater- al iatrogenic torsion in addition to their corresponding controls. During the study period, two rats from Group Combined of thulium laser and bipolar in prostatectomy-Huang et al. Table 1. Comparison of body weights across groups before the intervention. Group Mean Weight (SD), g Minimum Weight, g Maximum Weight, g P-value G1 (n = 15) 151 (7.3) 140 165 .94a G2 (n = 16) 149 (13.1) 117 165 G3 (n = 16) 231 (12.3) 208 259 .34b G4 (n = 15) 224 (13.1) 195 241 Abbreviations: G: group; M: mean; SD: standard deviation, g: gram. a: Presents the comparison of body weights before the intervention between Group1 and Group 2 b: Presents the comparison of body weights before the intervention between Group3 and Group 4 Group Mean Weight (SD), g Minimum Weight, g Maximum Weight, g P-value G1 (n = 15) 231 (19.4) 194 263 .17a G2 (n = 16) 217 (15.2) 191 248 G3 (n = 16) 271 (17.5) 248 310 .02b G4 (n = 15) 250 (22.2) 203 285 Abbreviations: G: group; M: mean; SD: standard deviation, g: gram. a: Presents the comparison of body weights after the intervention between Group1 and Group 2 b: Presents the comparison of body weights after the intervention between Group3 and Group 4 Table 2. Comparison of body weights a across groups after the intervention. Miscellaneus 502 Vol 16 No 04 September-October 2019 503 1 and 4 died, thus 62 were included in the final analysis. Table 1 and Table 2 show the mean body weight of the study groups prior to and following intervention. There was no remarkable difference across the groups before intervention (P > .05)(Table 1). It was revealed that a unilateral iatrogenic torsion did not have any impact on body growth and rats’ weight at prepubertal age (P = .17). On the contrary, performing this surgery follow- ing puberty was found to significantly accelerate body growth and increase rats’ weight from 250 ± 22.2 g in Group 4 to 271 ± 17.5 g in Group 3 (P =.02). Moreover, the weight of the contralateral testis was remarkably higher in Group 3 compared with Group 1 (1760 (26.6)mg vs.1692( 26.7) mg; P < .001), and Group 4 was also higher than Group 2 (1425 (44.9) mg vs.1375 (39.7) mg; p < .001). Therefore, it could be con- cluded that unilateral iatrogenic torsion possibly causes hypertrophy in the contralateral testis and is associated with a higher degree of compensatory hypertrophy at postpubertal age (Table 2& Table 3). A significant difference was observed in the mean weight of the remaining testis in Group 1when com- pared to Group 2 (1692 ± 26.7 vs. 1375 ± 39.7; P < .001). A similar increase was evident in Groups 3 and 4 (from 1425 ± 44.9 mg to 1760 ± 26.6 mg; P < .001). Pairwise comparisons between Groups 1 and 3, as well as Groups 2 and 4 indicated no statistically significant difference (P = 0.52 and P = 0.75, respectively). As the weight of the testis is contingent on the body weight of the animal, the ratio of testis weight (mg) to total body weight was measured in each rat in order to minimize any possible errors in estimating compensa- tory hypertrophy of the remaining testis. This ratio was defined as the mean testis weight (mg) per 100 gram of body weight. Table 4 summarizes the results of this analysis. As shown, groups 1 and 3 presented with a no- tably higher ratio than their corresponding controls (P < .05). Furthermore, this ratio was significantly higher in Group 1 compared to Group 3; P =.02), however, the other two groups demonstrated comparable ratios (P = .15). Additionally, the plasma testosterone levels were statistically shown to be alike across the study groups (P =.77) (Table 5). Thus, unilateral iatrogenic torsion and the time of which it is induced does not seem to have an effect on the level of testosterone as evident by the remaining testis being able to compensate for desirable testosterone levels. DISCUSSION The removal of or injury to one pair of a paired organ may result in compensatory hypertrophy of the re- maining organ. This phenomenon has been previously proved in the kidneys, thyroid glands, and ovaries(2,3,15). Removal of unilateral testis due to undescended testis (UDT), trauma, and testicular abscess causes great dif- ficulties in future. It is hypothesized that the unilateral testis removal ends up in compensatory hypertrophy of the contralateral testis. However, this occurs under normal circumstances when the testis is palpable in the usual position(3). On the other hand, testicular re- moval prior to and following puberty is influential on the degree of compensatory hypertrophy. If the testis is removed at prepubertal age, more compensatory hy- pertrophy occurs than after puberty(16).Orchiectomy is recommended for prepubertal patients who are afflict- ed with UDT, torsion, and testicular trauma and whose probability of testicular loss is high. Nowadays, some surgeons, in spite of testicular loss, still insist on re- taining the appearance of the testes. The removal of an injured testis which has lost its viability and spermato- genesis is more likely to increase FSH and then cause compensatory hypertrophy of the contralateral tes- tis(17,18). Furthermore, contralateral testicular injury on affinity of anti-sperm antibody may induce damage to the healthy testis and cause hypofertility in future(12,17). As testicular development in humans is akin to rats, it is assumed that the use of an experimental rat model provides beneficial evidence for further research on the morphology and histology of the testis. Considering the results of the present study it is concluded that a uni- lateral functional orchiectomy at postpubertal age leads to an increase in body growth, which is corroborated by Putra et al.(19) Also, a unilateral functional orchiec- tomy came up with compensatory hypertrophy of the Combined of thulium laser and bipolar in prostatectomy-Huang et al. Table 3. Comparison of the contralateral testis weight ofthe rats after the intervention. Group Mean Weight ( SD), mg Minimum Weight, mg Maximum Weight, mg P-value G1 (n = 15) 1692( 26.7) 1540 1860 < .001a G2 (n = 16) 1375 (39.7) 1060 1650 G3 (n = 16) 1760 (26.6) 1540 1900 < .001b G4 (n = 15) 1425 (44.9) 980 1680 Abbreviations: G: group; M: dean; SD: standard deviation, mg: milligram. a: Presents the comparison of the remaining testis weightafter the intervention between Group1 and Group 2 b: Presents the comparison of the remaining testis weightafter the intervention between Group3 and Group 4 Group Mean testis weight Minimum testis weight (mg) / Maximum testis weight (mg) / P-value (mg)/100g ofbody weight (SD) 100g of body weight 100g of body weight G1 (n = 15) 735.8(82.3) 618.5 853.7 .005a G2 (n = 16) 634.8(84.8) 495.3 774.5 G3 (n = 16) 652.4(61.4) 496.8 746.0 .03b G4 (n = 15) 572.6(97.7) 343.9 704.4 Abbreviations: G: group; M: mean; SD: standard deviation a: Presents the comparison of the ratio of the remaining testis weight (mg) per 100g of body weight between Group 1 and Group 2 b: Presents the comparison of the ratio of the remaining testis weight (mg) per 100g of body weight between Group 3 and Group 4 Table 4. Comparison of the contralateraltestis weight (mg)per 100g ofbody weight after the intervention. contralateral testis, which is in agreement with Putra, Lin, Simorangkir, and SaneFuji’s findings yet contra- dicts Romero’s findings(3,17,19,20,21.22,23).Having a unilater- al functional orchiectomy prior to puberty is associated with more compensatory hypertrophy in the contralat- eral testis. This is supported by Putra, Furuya, Cunning- ham, and Tusti where as some researchers have report- ed the opposite effect(19,24,25-28). Besides, in this study, a unilateral functional orchiectomy was not correlated to the plasma level of testosterone, with the remaining testis being able to compensate for diminished testos- terone concentrations. In spite of Ahmadi’s results, this is confirmed by Furuye et al.(25,29) It has been reported that the size of the testis is directly related to the amount of spermatogenesis. Given the occurrence of compensa- tory hypertrophy in the contralateral testis, it would be expected that normal spermatogenesis and fertility can be preserved(21). There were some limitations to our study including ab- sence of histopathologic study, and fertility status as- sessment. Also there was no evaluation of testis weight before the intervention. Compensatory hypertrophy could be the consequence of multiple factors including unilateral torsion and we did not assess the role of other possible factors. CONCLUSIONS Our research demonstrated that hemicastration results in compensatory hypertrophy of the remaining testis and the degree of compensation is inversely associated with the age of rats (prepubertal or postpubertal). Hem- icastration did not lead to reduction in serum testoster- one levels and the remaining testis retrieved a normal serum testosterone level. It is recommended that future studies investigate histopathologic changes, fertility status and also other possible factors which could lead to compensatory hypertrophy of the contralateral testis. This study paved the way for further research on larger animals such as dogs, cats, rabbits, and goats. ACKNOWLEDGMENT We are thankful of the research council of Mashhad University of Medical Science for their financial sup- port. CONFLICT OF INTEREST The authors report no conflict of interest. REFERENCES 1. Gibadulin R. Compensatory hypertrophy of the thyroid gland. BullExper BiolMed. 1963;54:790-3. 2. Khan Z, Gada RP, Tabbaa ZM, et al. Unilateral oophorectomy results in compensatory follicular recruitment in the remaining ovary at time of ovarian stimulation for in vitro fertilization. FertilSteril. 2014;101:722-7. 3. Grinspon RP, Habib C, Bedecarrás P, et al. Compensatory function of the remaining testis is dissociated in boys and adolescents with monorchidism. 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Effects of unilateral and bilateral orchidectomy on serum levels of carcinogenic embryonic antigen in male rats. Bull Env Pharmacol Life Sci. 2014;3:164-7. Combined of thulium laser and bipolar in prostatectomy-Huang et al.