953Vol. 10 | No. 3 | Summer 2013 |U R O LO G Y J O U R N A L 1Department of Pediatric Surgery and Division of Pediatric Urology, Karad- eniz Technical University, Faculty of Medicine, Trab- zon, Turkey 2Department of Histology and Embryology, Karadeniz Technical University, Fac- ulty of Medicine, Trabzon, Turkey Mustafa Imamoğlu,1 Levent Sapan,1 Yavuz Tekelioğlu,2 Haluk Sarihan1 Long-Term Effects of Elevated Intra-Abdominal Pressure on Testes An Experimental Model of Laparoscopy Corresponding Author: Mustafa Imamoğlu, MD Department of Pediatric Surgery, Karadeniz Techni- cal University, Faculty of Medicine, Trabzon, Turkey Tel: + 90 462 325 5355 Fax: +90 462 325 0518 E-mail: mimamoglu61@ yahoo.com Received September 2011 Accepted April 2012 Purpose: To determine the long-term outcomes of pneumoperitoneum on the testes in an experi- mental laparoscopy model. Materials and Methods: Twenty-four rats were divided into three groups: Group A, the control group; Group B, exposed to a 10 mmHg intra-abdominal pressure (IAP); and Group C, exposed to a 20 mmHg IAP with CO2 pneumoperitoneum for 60 minutes. After 6 weeks, the testes were removed, and testicular injury score and Johnson score were examined histologically. Germ cell apoptosis was also detected using flow cytometry. Results: A significant difference was determined between all groups in terms of testicular injury scores, Johnson scores, and germ cell apoptosis percentages. For the testicular injury score, there were significant differences between the groups for the right testis (group A versus B, P = .009; group A versus C, P < .0001; and group B versus C, P = .001) and for the left testis (group A versus B, P = .001; group A versus C, P < .0001; and group B versus C, P = .002). Significant differences were determined in the Johnson scores for the right testis between all groups (group A versus B, P = .001; group A versus C, P < .0001; and group B versus C, P = .008, respectively). Percentage of apoptotic testis cells were significantly differed between all groups (P = .001 for each). Conclusion: This study shows that increased IAP during pneumoperitoneum causes histopathol- ogy and apoptotically-evident damage to the testes in the long-term, depending on the magnitude of IAP increase, which may cause sub/infertility. Considering the experimental nature of this study, further clinical studies are needed for a more decisive conclusion. Keywords: laparoscopy; pneumoperitoneum; fertility; testis; treatment outcome LAPAROSCOPIC UROLOGY 954 | INTRODUCTION Despite many potential advantages for the patients, laparoscopic surgery is not completely free of complications. Increased intra-abdominal pres- sure (IAP) is one of the main potential causes of laparos- copy-associated complications, which are subject to cur- rent investigations.(1-4) Studies have focused on changes in blood flow to intra- and extra-abdominal organs, including the intestine, liver, kidney, spleen, bladder, and even the tes- tes and ovaries, during pneumoperitoneum caused by lapa- roscopic procedure and its effects in both the early postop- erative period and the long-term.(1-10) During the laparoscopic procedure due to increased venous resistance, blood flow to the organs is reduced, which re- stores following desufflation of the abdominal cavity. This ischemia/reperfusion condition promotes the generation of various reactive oxygen species (ROS), causing oxidative tissue damage. In tissues subject to the ischemic-reperfusion, the increase in ROS production is suggested to involve two phases. Immediately after reperfusion, the first phase occurs, which extends for a few hours. This is a typical oxidative stress situation, “reversible” in terms of cellular injury. The first phase is followed by the second phase, which extends from hours to days. “Irreversible” tissue damage occurs dur- ing the second phase. This is the most important outcome, because the long-term consequences of these changes may affect organ functions. Since there is evidence of laparoscopic procedure-associated ischemia, which may underline pathogenesis of some early or long-term adverse clinical outcomes after laparoscopic procedures.(11-14) Therefore, various strategies, such as minimization of the IAP, ischemic preconditioning, and pre- treatment with erythropoietin, mesna and/or antioxidants, are needed and indeed currently under consideration for the pre- vention of these laparoscopy-associated adverse outcomes. (15-18) The blood supply of the testis is through the “testicular ar- tery”, which has a high vascular resistance. Therefore cap- illary hydrostatic pressure in the testis low and is prone to affected from increase in venous pressure. Oxygen tension in the testis is low. The seminiferous tubules are apparently adapted to this condition of low oxygen, low vascular per- fusion pressure, and high metabolic activity, and normally this vasculature is sufficient to supply the testis with adequate amounts of perfusion. This suggests that moderate distur- bances in the blood supply to the organ may affect testicular function, resulting in sub/infertility.(19,20) It has been well documented that elevated intraabdominal pressure during laparoscopic surgery may cause ischemia/ reperfusion injury in primarily affected and also in distant or- gans and tissues; the effect is time- and pressure-dependent. (1,8,10,11,14,15,18) Thus, it is not surprising that testicular dam- age may develop and lead to, as a long-term consequence of laparoscopy, sub/infertility. Indeed, the experimental models have demonstrated that the increased IAP during laparoscopy reduces testis blood flow, which in turn causes a significant rise in oxidative stress in the testis tissue and, secondary to this, various degrees of damage occur in the parenchyma in the early stage.(19,20) However, the long-term effects of in- creased IAP on the testes have not been studied to date. This study was planned with the aim of investigating whether damage arising in the early period had a negative impact on the testis function in the long term, which is the most impor- tant potential cause of sub/infertility. If it is determined that damage arises in the testis tissue in conventional pressures used in laparoscopy, such as 10 mmHg, then it may be nec- essary to review the range of pressures used during laparos- copy, especially in patients with conditions for subfertility. MATERIALS AND METHODS Experimental Protocol and Operative Technique The study protocol was approved by the University Animal Care and Ethics Committee. Anesthesia was inducted by in- tramuscular injection of ketamine hydrochloride 90 mg/kg (Ketalar, Parke-Davis, Berlin, Germany) plus xylazine 10 mg/kg (Rompun, Bayer, Germany). After tracheostomy, a 16 G cannula was inserted in the trachea. Thereafter, main- tenance was performed by intramuscular injection of 10 mg/ kg xylazine at hourly intervals. Continuous intravenous in- fusion was performed through cannula in the right femoral vein. The animals were continuously infused with physiolog- ical saline at 4 mL/kg/h. Muscle relaxation was attained and maintained by hourly intramuscular injections of 2.0 mg/kg pancuronium bromide (Pavulon; Organon Teknika, Boxtel, Laparoscopic Urology 955Vol. 10 | No. 3 | Summer 2013 |U R O LO G Y J O U R N A L the Netherlends). Then, the rats were mechanically ventilated with a pressure-controlled mode (peak inspiratory pressure of 12 cm H2O, a positive end-expiratory pressure of 8 cm H2O, a fraction of inspired oxygen of 1.0, a frequency of 50 beats per minute, a tidal volume of 10 mL/kg, and an inspira- tion/expiration ratio of 1:1. By placing an 18-gauge angiocatheter caudally to the ster- num a peritoneal cavity puncture was achieved for perform- ing the pneumoperitoneum. Insufflation of carbon dioxide was also performed through this catheter. The targeted IAP was achieved and controlled for 60 min using an electronic laparoflator (KARL STORZ GmbH, Tuttlingen, Germany). Groups Twenty-four adult male Sprague-Dawley rats (weighing be- tween 400 and 450 g) were randomly allocated to one of three groups: group A (n = 8), the gasless group (control group, an angiocatheter alone being inserted into the abdomen); group B (n = 8), 10 mmHg IAP with pneumoperitoneum for 60 min; and group C (n = 8), 20 mmHg IAP with pneumoperi- toneum for 60 min. After six weeks, the testes were removed and longitudinally bisected. Half of the testes were fixed in Bouin’s solution for histological examination. The other half were fixed in 10% buffered formalin solution and then em- bedded in paraffin blocks for apoptosis evaluation. Histology Light microscopic examination of the tissue sections was performed after Hematoxylin and Eosin (H & E) staining. Histological evaluations were performed by a histologist blind to the protocol of the study. Two microscopic parameters were used for this evaluation; testicular injury score and maturation of the germinative epi- thelium. Quantification of the histological injury was performed by a 4-level grading scale.(21) Testicular histology with oorderly arrangement of germinal cells was rated as Grade 1. Inju- ries presenting less orderly, with noncohesive germinal cells and seminiferous tubules in the form of tightly-packed were rated as Grade 2. Tissues in the form of disordered sloughed germinal cells with shrunken pyknotic nuclei and unclear seminiferous tubule borders were considered as Grade 3. In- juries characterised by seminiferous tubules closely packed with coagulative necrosis of the germinal cells were rated as Grade 4. The mean injury score for each testis was then cal- culated. Figure 1. A) Histopathological image of grade 2 testis damage in group B (showing histological testis structure comprising a small number of closely packed, orderly, damaged seminiferous tubules and non-cohesive germinal cells) (Hematoxylin and Eo- sin stain, original magnification ×4); B) Histopathological image of grade 3 testis damage in group C (showing histological testis structure with mildly damaged seminiferous tubules and slightly damaged germinal cells and pyknotic cells in the tubules) (Hema- toxylin and Eosin stain, original magnification ×10) Figure 2. A) Histopathological image of testicular biopsy score 8 in group B (a large number of spermatid and spermatogenetic cells appear undifferentiated in the seminiferous tubule) (Hematoxylin and Eosin stain, original magnification ×25); B) Histopathological testicular biopsy score 6 image in group C (with a small number of spermatogonia cells and spermatids) (Hematoxylin and Eosin stain, original magnification ×40) Long-Term Effects of Laparoscopy on Testes | Imamoğlu et al 956 | Second, testicular biopsy score (TBS) modified by Johnson was used for grading the maturity of the germinative epithe- lium of the seminiferous tubules.(22) For this purpose evalua- tion of 25-50 tubules were performed for each section under 25×objective. For each testis the mean TBS was calculated by scoring system ranging from 1-10. Apoptosis Tissue preparation was performed according to Hedley’s method.(23) Material was treated with pepsin solution at room temperature, and thereafter, lysis and permeability reactive DNA-prep (PN 4238055-B) was added to lyse erythrocytes and increase the permeability of cell membranes. DNA prep stain (PN 4238055-BR) including propidium iodide was fi- nally added to stain cell DNAs. Flow cytometric analysis was carried out with EPICS ELITE ESP (Coulter). For each his- togram, 5000 to 10 000 cells were analyzed. Chicken eryth- rocyte nuclei (Coulter DNA prep PN 6604453) were used as DNA-diploid standard. Results of the histograms were ana- lyzed using a multicycle DNA analysis program.(24) Statistical Analysis Comparisons among the groups were performed using Kruskal-Wallis ANOVA (Mann-Whitney U test with Bonfer- roni correction as a post hoc test) for testicular injury score, TBS, and apoptosis values. Measured data are presented as median for testicular injury scores and arithmetic mean ± standard deviation for TBS and apoptosis, and data obtained by counting are shown as percentage. P < .05 was considered statistically significant. RESULTS Testicular Injury Score Group analysis revealed significant differences between all groups with regard to the degree of damage for both right and left testis (Table 1). For the right testis, this difference arose from the differences between group A versus group B and group A versus group C, and group B versus group C (P = .009, P < .0001, and P = .001, respectively). For the left testis, this difference arose from the comparisons between group A versus group B and group A versus group C, and group B versus group C (P < .001, and P < .0001, and P = .002, respectively). Median values of damage was grade 1 in group A, grade 2 in group B (Figure 1A), and grade 3 in group C (Figure 1B). In the 10 mmHg IAP group specimens, seminiferous tubules were packed closely, and germinal cells were noncohesive and less orderly, particularly in the peripheral testicular paren- chyma. In the 20 mm Hg IAP group, the specimens showed sloughed germinal cells with shrunken pyknotic nuclei and less distinct seminiferous tubule borders, these changes oc- curring diffusely in the testicular parenchyma. Testicular Biopsy Score Mean TBS revealed almost full spermatogenesis in both right and left testes in group A, whereas disturbance of spermatid differentiation was determined in both right and left testes in groups B and C. For the right testis, this difference arose from Group A versus group B, group A versus group C, and group B versus group C (P = .001, P < .0001, and P = .008, respectively). For the left testis, this difference arose from group A versus group B and group A versus group C (P = .001 and P = .001, respectively; Table 1). Median TBS was 10 in group A and 8 in group B (Figure 2a), which was as low as 6 in group C (Figure 2b). Apoptosis There was a statistically significant difference in both right and left testes apoptosis percentages between all groups (Ta- ble 1). For both the left and right testes, this difference arose from group A versus group B and group A versus group C, and group B versus group C (P = .001 and P = .001, and P = .001, respectively). In group A, apoptosis level was over 5% in both testes in two rats, and below 5% in both testes in others. Total apoptosis level was 4.4%. In group B, apoptosis level was over 20% in both testes in one rat, over 18% in both testes in two, over 14% in both testes in three, and below 14% in the right testis and above 14% in the left testis in one rat. Total apoptosis level was 22.4%. In group C, apoptosis level was above 30% in both testes in one rat, above 28% in both testes in three, above 24% in both testes in three, and below 24% in both testes in one rat. Total apoptosis level was 31.0%. Laparoscopic Urology 957Vol. 10 | No. 3 | Summer 2013 |U R O LO G Y J O U R N A L Long-Term Effects of Laparoscopy on Testes | Imamoğlu et al Table. Testicular injury score, Testicular biopsy score and Apopitosis results Parametres and Groups Right Testis Left Testis Testicular injury scores Median (min-max) Median (min-max) A 1 (1-1) 1 (1-1) B 2 (1-2) 2 (1-2) C 3 (2-3) 3 (2-3) P .0005 .0005 Testicular biopsy scores Mean ± SD Median (min-max) Mean ± SD Median (min-max) A 9.9 ± 0.4 10 (9-10) 9.8 ± 0.5 10 (9-10) B 7.9 ± 0.8 8 (7-9) 8.0 ± 0.8 8 (7-9) C 6.3 ± 0.4 6 (5-8) 6.8 ± 1.0 6 (5-8) P .0005 .0005 Apopitosis Mean ± SD (min-max) Mean ± SD (min-max) A 4.5 ± 0.7 (3.9-5.7) 4.6 ± 0.6 (3.5-5.5) B 16.6 ± 3.3 (12.2-22.4) 16.7 ± 3.0 (13.4-21.5) C 27.3 ± 2.8 (23.4-30.8) 27.2 ± 2.7 (22.8-31) P .0005 .0005 DISCUSSION By aiming obtaining information on the long-term effects of laparoscopy-associated IAP increase on testicular function, this experimental study evaluated impacts of a low (10 mmHg reflecting conventional abdominal pressure during laparos- copy) and a high increase (20 mmHg) in IAP, obtained by pneumoperitoneum, on testicular morphology after 6 weeks, and documented pressure-dependent histopathological and apoptotic evidences implicating ischemia/reperfusion related damage in IAP increased groups compared to the control. Although not investigated in detail, the obtained effects on testes are most likely due to reduction of its blood flow secondary to increased IAP occurring through two possible main mechanisms. The first is the direct effect of IAP on the testicular artery and vein, with their long course. Veins are subjected to greater pressure than arteries, and high venous resistance develops related to the increased hydrostatic pres- sure in the testes. The non-elasticity of the testis tunica al- buginea also makes an additional contribution to the venous congestion, and then to impaired arterial circulation. Further- more, this state of hypoperfusion may persist even after IAP has returned to normal. In a previous study, we determined, by color Doppler ultrasonography, that a level of 10 mmHg IAP at 30% to 35% and 20 mmHg IAP at 40% to 45% caused a decrease in testicular blood flow.(19) In that study, we also showed that 10 minutes after desufflation, testicular reperfu- sion blood flow levels were still lower than basal testicular blood flow levels. Second, it has been demonstrated that vena cava inferior pres- sure and vascular resistance significantly increased during prolonged periods of pneumoperitoneum, and remained so during the whole post pneumoperitoneum period.(25) Valve- less testicular veins having to pump blood to a higher pres- sure venous system after a long course will further increase the hydrostatic pressure reflected in the testes. This high ve- nous hydrostatic pressure contributes to the impairment of arterial circulation and deepening of the hypoxia. Sweeney and associates reported that in conditions of increased tes- ticular venous pressure, microvascular fluid changes declines dramatically, and spermatogenesis and gametogenesis are harmed in consequence.(26) In our previous study, oxidative stress response and histologi- cal outcomes of testes in 10 and 20 mmHg IAP groups were also compared with the control group. In that study, we dem- onstrated that 10 mmHg IAP, which is accepted as conven- tional abdominal pressure, in association with the testicular hypoperfusion, increased free radical production, and subse- quent testicular damage occurred in the early period that is 30 958 | Laparoscopic Urology minutes after pneumoperitoneum deflation.(19) Recently, Istanbulluoğlu and coworkers also showed in a por- cine model that laparoscopic nephrectomy caused ischemic changes in the testes in the acute stage.(20) Most importantly, they found that germ cell apoptosis was increased. No signif- icant difference was noted in Johnson’s scores between their two groups; however, congestion and necrosis, which were not documented in the control group, were observed in the increased IAP group. The key question is that whether these changes in the testicular tissue might be correlated with long- term testicular function. With respect to the consequences of sub-/infertility, long-term effects of different IAP levels in as- sociation with laparoscopy on spermatogenesis and testicular tissue histology was evaluated, and the results indicated the need for further studies. The evidence from these two studies leads us to design the present study to investigate the long- term effects of increased IAP on testes. In the present study, two microscopic parameters were used for histological examination. As can be seen in Table 1, compared with the control group (grade 1), the mean “testicular injury score” was significantly increased in both, the 10 (grade 2) and 20 mmHg IAP groups (grade 3). In the 10 mmHg IAP group, injury was observed, especially in the peripheral portion of the testicular paren- chyma; however, in the 20 mmHg IAP group, the changes occurred diffusely in the testicular parenchyma. The testicu- lar injury scores were also confirmed by TBS. Almost full spermatogenesis was observed in the control group, whereas disturbance of spermatid differentiation was determined in the 10 and 20 mmHg IAP groups. The mean TBS values of the study groups (the 20 mmHg group being even lower) were significantly lower than the control group (Table 1). Testicular injury and TBS scores are excellent parameters in the study of spermatogenesis, being widely used in the evaluation of testicular atrophy. Therefore, our experimental histopathological results indicate that increased IAP during laparoscopy may cause testicular damage, which could lead to sub-/infertility. Khoury and colleagues recently suggested that the pneumo- peritoneum induces apoptosis, increasing in parallel with ris- ing IAP and pneumoperitoneum exposure duration, and this may be a mechanism involved in renal “delayed graft dys- function’’ in recipients of laparoscopically harvested kidneys. (27) Bergh and associates observed that in conditions of tes- ticular hypoperfusion, apoptosis may develop in Sertoli and germ cells in the seminal tubules, as a result of which fertility will be negatively affected.(28) Our study also indicates simi- lar outcomes for apoptosis evaluation. In the 10 mmHg IAP group, total apoptosis level from the testis tissues was sig- nificantly higher than that of the control group (22.4% versus 4.4%). In the 20 mmHg IAP group, the level of apoptosis rose even higher (total apoptosis level of 31.0%; Table 1); indicating that the damage had a pressure-dependence. We therefore suggest that this importantly increased apoptosis levels exhibits an additional risk for impairment of fertility, considered in association with our histopathological results. One might wonder whether the scrotal pressure increased similar to intraperitoneal pressure and played a role in the obtained results. Although we did not perform any pressure measurements during the experiments, we did not notice any scrotal swelling and therefore we believe that the scro- tal pressure remained normal during the abdominal insuffla- tion. Furthermore, communication between the peritoneum and scrotum with patent processus vaginalis is necessary for increase in testicular pressure in parallel to intraperitoneal pressure. Therefore, the obtained results are more likely due to the increased pressure exerted on the vessels supplying the testes. CONCLUSION This experimental study shows that increased IAP during pneumoperitoneum may cause potential adverse effects on fertility by causing gradual testicular hypoperfusion and re- lated oxidative stress in the long-term, evidenced by histopa- thology and apoptosis index, depending on the magnitude of IAP increase. The results of this experimental study warrant further clinical studies to evaluate fertility of boys undergo- ing laparoscopy. CONFLICT OF INTEREST None declared. 959Vol. 10 | No. 3 | Summer 2013 |U R O LO G Y J O U R N A L Long-Term Effects of Laparoscopy on Testes | Imamoğlu et al REFERENCES 1. Schafer M, Sagesser H, Reichen J, Krahenbuhl L. Alterations in hemodynamics and hepatic and splanchnic circulation during lap- aroscopy in rats. Surg Endosc. 2001;15:1197–1201. 2. Unsal MA, Imamoglu M, Cay A, et al. The acute alterations in bio- chemistry, morphology and contractility of the rat isolated urinary bladder induced by increased intra-abdominal pressure. Gynecol Obstet Invest. 2006;61:179-87. 3. Unsal MA, Imamoglu M, Kadıoğlu M, et al. The acute alterations in biochemistry, morphology, and contractility of rat-isolated termi- nal ileum via increased intra-abdominal pressure. Pharmacol Res. 2006;53:135-41. 4. Schilling MK, Redaelli C, Krahenbühl, et al. Splanchnic microcircula- tory changes during CO2 laparoscopy. J Am Coll Surg 1997;184:378- 82. 5. Jakimowicz J, Stultiens G, Smulders F. Laparoscopic insuffla- tion of the abdomen reduces portal venous flow. Surg Endosc 1998;12:129-32. 6. Alexakis N, Gakiopoulou H, Dimitriou C, et al. Liver histology al- terations during carbon dioxide pneumoperitoneum in a porcine model. Surg Endosc. 2008;22:415-20. 7. Guven S, Muci E, Unsal MA, Yulug E, Alver A, Kadioglu Duman M, Mentese A.The effects of carbon dioxide pneumoperitoneum on ovarian blood flow, oxidative stress markers, and morphology dur- ing laparoscopy: a rabbit model. Fertil Steril. 2010;93:1327-32. 8. Szold A, Weinbroum AA. Carbon dioxide pneumoperitoneum- related liver injury is pressure dependent: A study in an isolated- perfused organ model. Surg Endosc. 2008;22:365-71. 9. Demyttenaere S, Feldman LS, Fried GM. Effect of pneumoperito- neum on renal perfusion and function: A systematic review. Surg Endosc. 2007;21:152-60. 10. Unsal MA, Guven S, Imamoglu M, Aydin S, Alver A. The effect of CO2 insufflation-desufflation attacks on tissue oxidative stress markers during laparoscopy: a rat model. Fertil Steril. 2009;92:363-8. 11. Polat C, Yılmaz S, Serteteser M, et al. The effects of different intra- abdominalpressures on lipid peroxidation and protein oxida- tion status during laparoscopic cholecystectomy. Surg Endosc 2003;17:1719-22. 12. Eleftheriadis E, Kotzampassi K, Botsios D, et al. Splanchnic ischemia during laparoscopic cholecystectomy. Surg Endos 1996;10:324-6. 13. Richards WO, Scovill W, Shin B, Reed W. Acute renal failure associated with increased intra-abdominal pressure. Ann Surg 1983;197:183-7. 14. Glantzounis GK, Tselepis AD, Tambaki AP, et al. Laparoscopic sur- gery-induced changes in oxidative stress markers in human plas- ma. Surg Endosc 2001;15:1315-9. 15. Cay A, Imamoglu M, Unsal MA, et al. Does anti-oxidant prophylaxis with melatonin prevent adverse outcomes related to increased oxi- dative stress caused by laparoscopy in experimental rat model? J Surg Research. 2006;135:2-8. 16. Ypsilantis P, Tentes I, Anagnostopoulos K, et al. Mesna protects splanchnic organs from oxidative stress induced by pneumoperito- neum. Surg Endosc. 2009;23:583-9. 17. Yılmaz S, Köken T, Tokyol C, et al. Can preconditioning reduce lapa- roscopy-induced tissue injury? Surg Endosc. 2003;17:819-24 18. Cevrioglu AS, Yılmaz S, Koken T, Tokyol C, Yılmazer M, Fenkci IV. Comparison of the effects of low intra-abdominal pressure and ischaemic preconditioning on the generation of oxidative stress markers and inflammatory cytokines during laparoscopy in rats. Hum Reprod. 2004;19:2144-51. 19. Imamoglu M, Cay A, Unsal MA, et al. The effects of increased in- traabdominal pressure on testicular blood flow, oxidative stress markers, and morphology. J Pediatr. Surg. 2006;41:1118–24. 20. Istanbulluoğlu MO, Pişkin M, Zor M, et al. The acute effects of in- creased intra-abdominal pressure on testicular tissue: an experi- mental study in pigs. Urol. 2011;77:510.e12-510.e16. 21. Cosentino MJ, Nishida M, Robinowitz R, et al. Histopathology of pre- pubertal rat testes subjected to various durations of spermatic cord torsion. J Androl 1986;7:23-31. 22. Johnsen SG. Testicular biopsy score count – a method for registra- tion of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1:2-25. 23. Hedley DW, Shankey TV and Wheeless LL. DNA Cytometry Consen- sus Conference. Cytometry 1993; 14: 471–500. 24. Shapiro HM. Practical Flow Cytometry. Extrinsic Parameters. 3rd ed.: Wiley-Liss, New York, 1995; 251–262. 25. Lindberg F, Bergqvist D, Rasmussen I, et al. Hemodynamic changes in the inferior caval vein during pneumoperitoneum: an experi- mental study in pigs. Surg Endosc. 1997;11:431-37. 26. Sweeney TE, Rozum JS, Desjardins C, Gore RW. Microvascular pres- sure distribution in the hamster testis. Am J Physiol. 260:H1581-89. 27. Khoury W, Jakowlev K, Fein A, Orenstein H, Weinbroum AA. Renal apoptosis following carbon dioxide pneumoperitoneum in a rat model. J Urol. 2008;180:1554-8. 28. Bergh A, Collin O, Lissbrant E. Effects of Acute Graded Reductions in Testicular Blood Flow on Testicular Morphology in the Adult Rat. Biol Reprod. 2001;64:13-20.