- Email: [email protected]
The Effect of Micronized Purified Flavonoid Fraction on the Prevention of Testicular Pathologies in Adolescent Rats with Experimentally Induced Varicocele
The Effect of Micronized Purified Flavonoid Fraction on the Prevention of Testicular Pathologies in Adolescent Rats with Experimentally Induced Varicocele Abdullah Armagan,* Faruk Dogan, Taylan Oksay, Tolga Akman, Hakan Darici, Firdevs Aylak and Osman Ergun From the Department of Urology, Faculty of Medicine, Bezmialem Vakif University (AA, TA), Departments of Urology (FD, TO, OE) and Histology and Embryology (HD), Faculty of Medicine, Suleyman Demirel University and Department of Biochemistry, Dr. Lütfi Kırdar Training and Research Hospital (FA), Istanbul, Turkey
Purpose: We investigated the effect of micronized purified flavonoid fraction on the prevention of testicular pathologies following varicocele induction. Materials and Methods: A total of 66 adolescent (6-week-old) male Wistar rats were included in study. Rats were divided into 7 groups, including group 1— control, group 2—sham operation, group 3—left varicocele induced, group 4 —varicocele induced, varicocelectomy done 4 weeks later and micronized purified flavonoid fraction administered for 4 weeks, group 5—varicocele induced and micronized purified flavonoid fraction administered for 8 weeks, group 6 —varicocele induced and beginning 4 weeks later micronized purified flavonoid fraction administered for 4 weeks, and group 7—varicocele induced and varicocelectomy done 4 weeks later. Before sacrifice bilateral real-time testicular microvascular perfusion of all rats was measured using the PeriFlux System 5000 PF 5010 LDPM Unit (Perimed, Järfälla, Sweden). All testes were graded according to the Johnsen scoring system. To assess apoptosis caspase-3 levels were measured. Results: Testicular weight in group 3 was markedly decreased and the extent of seminiferous tubular damage was significantly increased compared with the other groups. Bilateral testicular blood flow and the number of apoptotic germ cells were greater in group 3. Significantly higher Johnsen scores and a meaningful decrease in the apoptotic index were detected in groups 4 to 7 compared with group 3. Conclusions: We observed favorable effects of micronized purified flavonoid fraction on the regression of testicular damage secondary to varicocele.
Abbreviations and Acronyms MPFF ⫽ micronized purified flavonoid fraction Submitted for publication February 20, 2012. Study received animal care committee approval. * Correspondence: Department of Urology, Faculty of Medicine, Bezmialem Vakif University, 34093 Istanbul, Turkey (telephone: ⫹90 212 453 1700-1706; FAX: ⫹90 212 621 7580; e-mail: [email protected]).
Key Words: testis, varicocele, flavonoids, regional blood flow, apoptosis VARICOCELE is characterized by dilatation of the pampiniform plexus veins in the spermatic cord, and stasis and increased pressure in the associated veins.1 Varicocele is a common and correctible cause of male infertility. This condition causes progressive testicular damage that can result in infertility due to retarded testicular growth and impaired seminal parameters. How-
ever, the mechanisms of deterioration of the testicular structure and function as well as the impact on spermatogenesis have not yet been clearly characterized. Many mechanisms, including alterations in testicular blood flow, interaction between the testis and interstitial fluid, hyperthermia, venous hypertension, renal-adrenal reflux, hormonal dysfunction, autoim-
0022-5347/12/1885-2007/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2012.07.009 Vol. 188, 2007-2013, November 2012 RESEARCH, INC.GOL Printed in U.S.A.
AND
www.jurology.com
2007
2008
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
munity, the acrosome reaction, oxidative stress and apoptosis, have been proposed to explain the pathophysiology of varicocele.2 MPFF is a flavonoid vasoprotective venotonic agent whose active component is micronized flavonoid, which consists of 90% micronized diosmin and 10% flavonoids in the form of hesperidin.3 MPFF is an oral phlebotropic drug that improves venous tone and elasticity, increases lymphatic drainage and decreases capillary hyperpermeability. Furthermore, it increases O2 partial pressure and decreases CO2 partial pressure in patients with severe chronic venous insufficiency, attenuates the number of apoptotic cells, prolongs norepinephrine activity and increases Ca sensitivity of the contractile apparatus.4,5 Its anti-inflammatory effects decrease venous distensibility and venous emptying times in patients with functional or organic chronic venous insufficiency.6 Because of these characteristics, MPFF has been used successfully to treat many types of venous insufficiency, such as hemorrhoids and lymphedema.7,8 On the other hand, to our knowledge only a few clinical studies of its effect on varicocele are available and relevant experimental studies are lacking.9,10 We evaluated the impact of MPFF on testicular blood flow, spermatogenesis and testicular apoptosis in adolescent rats with experimentally induced varicocele.
MATERIALS AND METHODS The study was approved by the animal care committee at our institution. The study included 66 adolescent (6-weekold) male Wistar rats weighing between 172 and 252 gm. Care and use of the rats in this study were performed according to the recommendations of the Board of Registry Publication Guidelines.
Study Group Design The rats were divided into 7 groups. A standard midline incision 2 cm long was made for all surgical procedures. Surgery was performed with the rats under a single dose of intraperitoneal 5% ketamine (50 to 100 mg/kg) anesthesia. In group 1— control, 7 rats underwent no intervention but were sacrificed 8 weeks later. In group 2—sham operation, the left renal vein was exposed, rotated 360 degrees around its axis and left unligated. Nine rats were sacrificed 8 weeks later. In group 3—induced varicocele, the left renal vein was cleared from the overlying adipose and connective tissue. Adequate renal vein exposure was ensured. A 0.85 mm metal probe was placed along the left renal vein. The renal vein was constricted around the metal probe with 4-zero silk sutures placed proximal to the insertion point of the left internal spermatic vein. Subsequently, the metal probe was removed, leaving a 50% stenotic renal vein in 9 rats.11 In group 4 —varicocele plus varicocelectomy plus MPFF for 4 weeks, varicocele was induced using the technique described. Varicocelec-
tomy was performed 4 weeks after varicocele creation. MPFF was immediately administered for 4 weeks after the varicocelectomy procedure in 9 rats. To perform varicocelectomy a new incision was made on the previous midline incision line and the abdominal cavity was entered. Dilatation of the renal vein was observed in all rats. Varicocelectomy was performed using a high ligation technique. In group 5—varicocele plus MPFF for 8 weeks, beginning immediately after varicocele induction, as described, MPFF was administered for 8 weeks in 12 rats. In group 6 —varicocele plus MPFF for 4 weeks, varicocele was induced, as described. Beginning 4 weeks later, MPFF was administered for 4 weeks in 10 rats. In group 7—varicocele plus varicocelectomy, varicocele was induced, as described. Varicocelectomy was performed 4 weeks after varicocele creation in 10 rats.
Testicular Blood Flow Testicular tissue blood flow was measured using a PeriFlux 5000 laser Doppler flowmeter. Before the rats were sacrificed, the testes were removed from the scrotal sac and placed on the abdomen. Stable signals from 2 separate, minimally vascularized areas of the testicular surface were measured. The average value of the measurements was calculated.
Apoptotic Index and Johnsen Score Calculation Testicular tissues were fixed with 10% neutral buffered formaldehyde for at least 48 hours. Tissue specimens were washed, dehydrated and cleared in xylenes. Each tissue sample was embedded in paraffin and 4 to 5 m sections were prepared. Four consecutive sections of each 10 sections were taken. Two sections were stained with hematoxylin and eosin. The other 2 sections were stained immunohistochemically. Immunohistochemical analysis was performed using the streptavidin-peroxidase method. Slides reserved for immunohistochemistry were rehydrated and quenched in 3% H2O2 (Thermo Scientific®) for 5 minutes. Subsequently, heat activated antigen retrieval was performed in tris-ethylenediaminetetraacetic acid buffer (Merck, Darmstadt, Germany) at pH 9. Nonspecific binding was eliminated with Ultra V Block (Thermo Scientific). One section per set was stained with rabbit reactive caspase-3 antibody (ab44976, Abcam®). Another section was stained only with secondary antibody as a control. Tissues were incubated overnight at 4C in a humidified chamber. Goat anti-polyvalent solution (Thermo Scientific) was used as secondary antibody. Diaminobenzidine staining was performed with the UltraVision Plus Large Volume Detection System (Thermo Scientific). Slides were counterstained with hematoxylin and eosin (Merck). To evaluate the apoptotic index, caspase-3 positive germ cells were counted and examined under 400⫻ magnification with a light microscope. The total number was divided by the total number of seminiferous tubules. Cross-sections of 20 biopsy specimens of seminiferous tubules retrieved from the right and left testes of each animal were examined to evaluate seminiferous epithelial quality. We used the Johnsen scoring system, which rates the quality of spermatogenesis on a 10-point scale based on the presence or absence of certain spermatogenic cell types.11,12
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
2009
Figure 1. Left and right testicular weight in all groups
Statistical Analysis All statistical analyses were done using SPSS®, version 16.0. A normality test (Kolmogorov-Smirnov test with significance considered at p ⬎0.05) was performed to evaluate the distribution of numerical variables. Numerical variables with a skewed distribution were compared using the Kruskal-Wallis test. Post hoc analyses were done using the Mann-Whitney U test, as needed.
RESULTS Figure 1 shows mean testicular weight in all groups. Mean left and right testicular weight was lower in groups 3 and 6 compared with the other groups. However, mean left testicular weight was higher in group 4 than in group 3 (p ⫽ 0.003), while right testicular weight was similar in groups 4 and 3. Compared with the control group, mean bilateral testicular weight was similar in the sham operated group and group 7, and in groups 4 and 5. The table lists mean microvascular blood flow, Johnsen scores and apoptotic indexes. No statistically significant difference was found in mean microvascular blood flow measurements of left testicular tissue among groups 1, 2, 4, 5 and 7. Mean microvascular blood flow of left testicular tissue in group 3 was significantly higher than in the other groups. Mean left testicular tissue measurements in
group 6 were significantly higher than in group 1 but lower than in group 3 (p ⫽ 0.002 and ⬍0.0001, respectively), while left testicular values were significantly greater in group 6 than in group 5 (p ⫽ 0.003). In all groups mean microvascular blood flow measurements of right testicular tissue were highly similar to left testicular tissue values. Mean Johnsen scores for the left testis in groups 3 to 7 were significantly lower than in group 1. Mean scores for the left and right testes in group 3 were significantly lower than in the other groups (p ⬍0.05, fig. 2), while scores for the left and right testes in group 5 were significantly higher than in group 6 (p ⬍0.05). Mean scores for the right testes in groups 2, 4 and 7 were similar to those in group 1 (p ⬎0.05). However, mean scores for the right testes in groups 3, 5 and 6 were significantly lower than the corresponding values in group 1, while mean right testicular scores in group 1 were significantly higher than the corresponding values in groups 3, 5 and 6 (p ⬍0.05). The mean apoptotic index of left testes was statistically significantly higher in groups 5 to 7 than in group 1 (p ⬍0.05). The mean index of left testes in group 3 was significantly higher than in the other groups, excluding group 6 (p ⬍0.05, fig. 3). No significant difference was noted in the mean left testic-
Testicular blood flow, Johnsen score and apoptotic index in all groups (Kruskal-Wallis test p ⬍0.0001) Mean ⫾ SD Testicular Blood Flow (ml/min/100 gm) Group No. (intervention) 1 2 3 4 5 6 7
(control) (sham operation) (varicocele) (varicocele, varicocelectomy ⫹ MPFF) (varicocele ⫹ 8-wk MPFF) (varicocele ⫹ 4-wk MPFF) (varicocele ⫹ varicocelectomy)
Mean ⫾ SD Johnsen Score
Mean ⫾ SD Apoptotic Index
Lt
Rt
Lt
Rt
Lt
Rt
94 ⫾ 5.03 92.44 ⫾ 2.69 119.66 ⫾ 6.44 94.66 ⫾ 3.84 95.50 ⫾ 6.12 103.50 ⫾ 5.46 94.60 ⫾ 5.08
92.85 ⫾ 6.2 93.44 ⫾ 3.28 120.77 ⫾ 6.05 94.77 ⫾ 4.57 96.25 ⫾ 5.52 104 ⫾ 4.29 95.20 ⫾ 4.15
9.68 ⫾ 0.09 9.72 ⫾ 0.08 7.48 ⫾ 0.28 9.56 ⫾ 0.10 9.32 ⫾ 0.11 8.80 ⫾ 0.24 9.51 ⫾ 0.13
9.75 ⫾ 0.05 9.66 ⫾ 0.13 8.76 ⫾ 0.21 9.60 ⫾ 0.18 9.5 ⫾ 0.12 9.28 ⫾ 0.21 9.63 ⫾ 0.14
0.154 ⫾ 0.012 0.167 ⫾ 0.014 0.398 ⫾ 0.015 0.164 ⫾ 0.008 0.174 ⫾ 0.024 0.316 ⫾ 0.030 0.193 ⫾ 0.011
0.172 ⫾ 0.011 0.143 ⫾ 0.021 0.491 ⫾ 0.019 0.184 ⫾ 0.011 0.212 ⫾ 0.015 0.478 ⫾ 0.018 0.195 ⫾ 0.019
2010
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
Figure 2. Seminiferous epithelium staining to evaluate spermatogenesis in groups 1— control (A), 2—sham operation (B), 3—varicocele (C and D), 4 —varicocele plus varicocelectomy plus MPFF (E), 5—varicocele plus 8-week MPFF (F), 6 —varicocele plus 4-week MPFF (G) and 7—varicocele plus varicocelectomy (H). Seminiferous tubules were normal in groups 1 (A) and 2 (B). Seminiferous tubular structure was damaged. Disorganization in most tubules (thin arrow), spermatids in some tubules (thick arrow) and spermatozoa (star) were not observed in group 3 (C) but germ cell sloughing was seen (D). Seminiferous tubules in group 4 were similar to those in group 1 (E). In groups 5 and 7 there was minimal damage to seminiferous tubular structure, which did not differ from that in group 1 (F). Moderate damage to seminiferous tubules was observed in group 6 (G). H&E, reduced from ⫻200.
ular apoptotic index of groups 4 and 1, while in group 5 it was significantly lower than in group 6 (p ⬍0.05), and in groups 4 and 7 it did not significantly differ (p ⬎0.05). The mean right testicular apoptotic index in groups 3 and 5 to 7 was significantly higher than the estimated index in group 1, while in group 3 it was significantly higher than that calculated in the other groups (each p ⬍0.05). The mean right testicular apoptotic index in group 5 was significantly lower than in group 6 and in group 7 it was significantly lower than in group 4 (each p ⬍0.05).
DISCUSSION The incidence of varicocele, which can cause testicular growth retardation, atrophy and impaired seminal parameters, was estimated at 40% for men with primary infertility and 71% for men with secondary infertility.13 The incidence of testicular atrophy secondary to varicocele ranges from 10% to 75% in various studies.14 Testicular atrophy is defined as the loss of seminiferous tubular components. This pathological condition is the main etiological factor responsible for
abnormal semen parameters and subfertility. Enlargement of the hypovolemic testis at a rate of 53% to 100% was reported after surgical intervention.15 On the other hand, to our knowledge no medical therapy preventing testicular atrophy has been developed. In the current study the left testicular weight of rats treated with MPFF for 8 weeks after varicocele induction was significantly higher than that in the varicocele induced group and it was comparable to that in the control group. Furthermore, no statistically significant difference was detected between this group and group 7. The left testicular weight of rats in group 6 was significantly lower than in the other treated groups as well as the control and sham treated groups but significantly greater than in the varicocele group. The difference between the testicular weight in group 6, which received MPFF beginning 4 weeks after varicocele induction, and the other treatment groups can be explained by certain hypotheses. 1) Short-term MPFF use has a weaker effect on the prevention of testicular volume loss. 2) MPFF leads to testicular hypovolemia, decreasing interstitial fluid
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
2011
Figure 3. Caspase-3 positive labeled germ cells (arrows) were not seen in seminiferous epithelium in control group (A) but they were seen in varicocele (B), varicocele plus varicocelectomy plus MPFF (C) and varicocele plus 8-week MPFF (D) groups. Reduced from ⫻200.
formation or limiting the increase in testicular volume during the early posttreatment period. In experimental animal studies conflicting results have been reported regarding varicocele and alterations in blood flow. Some studies have shown decreased testicular blood flow in varicocele models16 but many have indicated an increase in blood flow after varicocele formation.17,18 Increased blood flow inhibits spermatogenesis because of enhanced testicular temperature. In a human study Wright et al found that testicular temperature increased bilaterally in individuals with varicocele and decreased after varicocelectomy.19 Additionally, a color Doppler study performed by Tarhan et al revealed an increase in testicular blood flow in men with varicocele.20 In our study we detected significantly increased microvascular blood flow in the left testes of rats in the varicocele induced group compared with other groups, while no significant difference was found in microvascular blood flow between the control group, and groups 4, 5 and 7. The attenuating effect of MPFF on microvascular blood flow in the 2 testes can be explained by its protective and regulatory effects on testicular microcirculation. Apoptosis, a regulated form of cell death, is a physiological process essential for normal testicular physiology.21 Germ cell apoptosis is a physiological testicular process that is necessary to maintain spermatogenesis. However, an increase in germ cell apoptosis can induce a spermatogenetic defect, resulting in infertility.
Various studies indicate the importance of apoptosis in the pathogenesis of varicocele.12,22 Cam et al found that the number of apoptotic cells in the testicular tissue of the varicocele group was significantly higher than in the control group.22 Fazlioglu et al reported that in an experimental varicocele model the apoptotic activity in germ cells had increased twofold in comparison with the control group 14 days after varicocele development and the apoptotic rate attained its maximal level at 28 days.23 In that study cellular apoptosis decreased after varicocelectomy and mean apoptotic indexes approached those of the control group. Clinical studies have been performed of the correlation between apoptosis and varicocele. Lin et al detected an increase in germ cell apoptosis in testicular biopsy materials from infertile men with idiopathic testicular failure.24 The group suggested that this increase might lead to testicular dysfunction and infertility. Baccetti et al evaluated the number of apoptotic spermatocytes during seminal analyses and detected an almost hundredfold increase in the apoptotic index relative to that in men without varicocele.25 This correlation between varicocele and testicular apoptosis could be explained by the local temperature increase in the testis and hypoxia due to venous stasis. The mentioned studies demonstrate that testicular superoxide levels increased, in addition to the increase in apoptotic activity, and indicate that substances such as vitamin E and mel-
2012
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
atonin, which decrease testicular superoxide levels, could decrease testicular germ cell apoptosis.21,26 In our series varicocele was induced experimentally and the germ cell apoptotic index was assessed using caspase-3 analysis. The apoptotic index was almost threefold higher in the varicocele group (group 3) than in the control group. MPFF administration for 8 weeks immediately after the induction of varicocele decreased the apoptotic index more than 2.3 times. On the other hand, the apoptotic index of the right testis in the varicocele group was statistically higher compared with the other groups. This impact of MPFF can be explained by its influence on apoptosis. Alterations related to spermatogenesis in patients with varicocele are associated with various factors, such as testicular hypoxia secondary to stasis, abnormal regulation of testicular temperature, reflux of toxic adrenal products and renal metabolic processes, and spermatic venous hypertension.27 Eliminating these factors by varicocelectomy resulted in improved spermatogenesis in many series.27 In an experimental study Tek et al investigated the effect of vascular endothelial growth factor injection in the testes on spermatogenesis and apoptosis in a varicocele induced adolescent rat model.12 They concluded that vascular endothelial growth factor may have a positive
role in improving the testicular damage that occurs after varicocele in ipsilateral testes. In our study the Johnsen score was assessed by hematoxylin and eosin staining after the induction of a varicocele model. Lower Johnsen scores were obtained in the varicocele induced group relative to those in all other groups with a statistically significant difference between the groups.
CONCLUSIONS Our study shows that in rats with experimentally induced varicoceles, testicular weight was significantly lower on the side with the varicocele and microvascular blood flow increased in each testis. Based on the results of our histopathological analyses, the Johnsen score decreased on each side, while the apoptotic index of each testis increased after varicocele was induced. Microvascular blood flow decreased considerably, while testicular weight increased after varicocelectomy and long-term MPFF treatment. Additionally, a significant increase in the Johnsen score and a marked decrease in germ cell apoptosis were observed. The favorable results suggest that long-term MPFF might be a good treatment option to prevent testicular damage secondary to varicocele.
REFERENCES 1. Greenfield SP, Seville P and Wan J: Experience with varicoceles in children and young adults. J Urol 2002; 168: 1684. 2. Fretz PC and Sandlow JI: Varicocele: current concepts in pathophysiology, diagnosis, and treatment. Urol Clin North Am 2002; 29: 921. 3. Struckmann JR: Clinical efficacy of micronized purified flavonoid fraction: an overview. J Vasc Res, suppl., 1999; 36: 37. 4. Porter JM and Moneta GL: Reporting standards in venous disease: an update. International Consensus Committee on Chronic Venous Disease. J Vasc Surg 1995; 21: 635. 5. Cesarone MR, Belcaro G, Rohdewald P et al: Comparison of Pycnogenol and Daflon in treating chronic venous insufficiency: a prospective, controlled study. Clin Appl Thromb Hemost 2006; 12: 205. 6. Lyseng-Williamson KA and Perry CM: Micronised purified flavonoid fraction. Drugs 2003; 63: 71. 7. Jiang ZM and Cao JD: The impact of micronized purified flavonoid fraction on the treatment of acute haemorrhoidal episodes. Curr Med Res Opin 2006; 22: 1141. 8. Pecking AP: Evaluation by lymphoscintigraphy of the effect of a micronized flavonoid fraction (Da-
flon 500 mg) in the treatment of upper limb lymphedema. Int Angiol 1995; 14: 39. 9. Söylemez H, Kiliç S, Atar M et al: Effects of micronised purified flavonoid fraction on pain, semen analysis and scrotal color Doppler parameters in patients with painful varicocele; results of a randomized placebo-controlled study. Int Urol Nephrol 2012; 44: 401. 10. Zampieri N, Pellegrino M, Ottolenghi A et al: Effects of bioflavonoids in the management of subclinical varicocele. Pediatr Surg Int 2010; 26: 505. 11. Aggarwal A, Misro MM, Maheshwari A et al: Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo. Mol Cell Endocrinol 2012; 348: 155. 12. Tek M, Cayan S, Yilmaz N et al: The effect of vascular endothelial growth factor on spermatogenesis and apoptosis in experimentally varicocele-induced adolescent rats. Fertil Steril 2009; 91: 2247. 13. La Vignera S, Condorelli R, Vicari E et al: Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis. J Androl 2012; 33: 389.
14. Çayan S, Akbay E, Bozlu M et al: The effect of varicocele repair on testicular volume in children and adolescents with varicocele. J Urol 2002; 168: 731. 15. Hadziselimoviç F, Herzog B, Liebundgut B et al: Testicular and vascular changes in children and adults with varicocele. J Urol 1989; 142: 583. 16. Li H, Dubocq F, Jiang Y et al: Effect of surgically induced varicocele on testicular blood flow and Sertoli cell function. Urology 1999; 53: 1258. 17. Turner TT: The study of varicocele through the use of animal models. Hum Rep Update 2001; 7: 78. 18. Saypol DC, Howards SS, Turner TT et al: Influence of surgically induced varicocele on testicular blood flow, temperature, and histology in adult rats and dogs. J Clin Invest 1981; 68: 39. 19. Wright EJ, Young GP and Goldstein M: Reduction in testicular temperature after varicocelectomy in infertile men. Urology 1997; 50: 257. 20. Tarhan S, Ucer O, Sahin MO et al: Long-term effect of microsurgical inguinal varicocelectomy on testicular blood flow. J Androl 2011; 32: 33. 21. Aktas C, Kanter M and Kocak Z: Antiapoptotic and proliferative activity of curcumin on ovarian follicles in mice exposed to whole body ionizing
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
radiation. Toxicol Ind Health, Epub ahead of print November 10, 2011.
duced testicular germ cell apoptosis. J Androl 2008; 29: 29.
22. Cam K, Simsek F, Yuksel M et al: The role of reactive oxygen species and apoptosis in the pathogenesis of varicocele in a rat model and efficiency of vitamin E treatment. Int J Androl 2004; 27: 228.
24. Lin WW, Lamb DJ, Lipshultz LI et al: The role of autosomal cell apoptosis regulator genes in human spermatogenesis. Int Urol Nephrol 1999; 31: 237.
23. Fazlioglu A, Yılmaz I, Mete O et al: The effect of varicocele repair on experimental varicocele in-
25. Baccetti B, Collodel G and Piomboni P: Apoptosis in human ejaculated sperm cells. J Submicrosc Cytol Pathol 1996; 28: 587.
2013
26. Espino J, Ortiz Á, Bejarano I et al: Melatonin protects human spermatozoa from apoptosis via melatonin receptor- and extracellular signal-regulated kinase-mediated pathways. Fertil Steril 2011; 95: 2290. 27. Naughton CK, Nangia AK and Agarwal A: Varicocele and male infertility: part II. Pathophysiology of varicoceles in male infertility. Hum Reprod Update 2001; 7: 473.
Purpose: We investigated the effect of micronized purified flavonoid fraction on the prevention of testicular pathologies following varicocele induction. Materials and Methods: A total of 66 adolescent (6-week-old) male Wistar rats were included in study. Rats were divided into 7 groups, including group 1— control, group 2—sham operation, group 3—left varicocele induced, group 4 —varicocele induced, varicocelectomy done 4 weeks later and micronized purified flavonoid fraction administered for 4 weeks, group 5—varicocele induced and micronized purified flavonoid fraction administered for 8 weeks, group 6 —varicocele induced and beginning 4 weeks later micronized purified flavonoid fraction administered for 4 weeks, and group 7—varicocele induced and varicocelectomy done 4 weeks later. Before sacrifice bilateral real-time testicular microvascular perfusion of all rats was measured using the PeriFlux System 5000 PF 5010 LDPM Unit (Perimed, Järfälla, Sweden). All testes were graded according to the Johnsen scoring system. To assess apoptosis caspase-3 levels were measured. Results: Testicular weight in group 3 was markedly decreased and the extent of seminiferous tubular damage was significantly increased compared with the other groups. Bilateral testicular blood flow and the number of apoptotic germ cells were greater in group 3. Significantly higher Johnsen scores and a meaningful decrease in the apoptotic index were detected in groups 4 to 7 compared with group 3. Conclusions: We observed favorable effects of micronized purified flavonoid fraction on the regression of testicular damage secondary to varicocele.
Abbreviations and Acronyms MPFF ⫽ micronized purified flavonoid fraction Submitted for publication February 20, 2012. Study received animal care committee approval. * Correspondence: Department of Urology, Faculty of Medicine, Bezmialem Vakif University, 34093 Istanbul, Turkey (telephone: ⫹90 212 453 1700-1706; FAX: ⫹90 212 621 7580; e-mail: [email protected]).
Key Words: testis, varicocele, flavonoids, regional blood flow, apoptosis VARICOCELE is characterized by dilatation of the pampiniform plexus veins in the spermatic cord, and stasis and increased pressure in the associated veins.1 Varicocele is a common and correctible cause of male infertility. This condition causes progressive testicular damage that can result in infertility due to retarded testicular growth and impaired seminal parameters. How-
ever, the mechanisms of deterioration of the testicular structure and function as well as the impact on spermatogenesis have not yet been clearly characterized. Many mechanisms, including alterations in testicular blood flow, interaction between the testis and interstitial fluid, hyperthermia, venous hypertension, renal-adrenal reflux, hormonal dysfunction, autoim-
0022-5347/12/1885-2007/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2012.07.009 Vol. 188, 2007-2013, November 2012 RESEARCH, INC.GOL Printed in U.S.A.
AND
www.jurology.com
2007
2008
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
munity, the acrosome reaction, oxidative stress and apoptosis, have been proposed to explain the pathophysiology of varicocele.2 MPFF is a flavonoid vasoprotective venotonic agent whose active component is micronized flavonoid, which consists of 90% micronized diosmin and 10% flavonoids in the form of hesperidin.3 MPFF is an oral phlebotropic drug that improves venous tone and elasticity, increases lymphatic drainage and decreases capillary hyperpermeability. Furthermore, it increases O2 partial pressure and decreases CO2 partial pressure in patients with severe chronic venous insufficiency, attenuates the number of apoptotic cells, prolongs norepinephrine activity and increases Ca sensitivity of the contractile apparatus.4,5 Its anti-inflammatory effects decrease venous distensibility and venous emptying times in patients with functional or organic chronic venous insufficiency.6 Because of these characteristics, MPFF has been used successfully to treat many types of venous insufficiency, such as hemorrhoids and lymphedema.7,8 On the other hand, to our knowledge only a few clinical studies of its effect on varicocele are available and relevant experimental studies are lacking.9,10 We evaluated the impact of MPFF on testicular blood flow, spermatogenesis and testicular apoptosis in adolescent rats with experimentally induced varicocele.
MATERIALS AND METHODS The study was approved by the animal care committee at our institution. The study included 66 adolescent (6-weekold) male Wistar rats weighing between 172 and 252 gm. Care and use of the rats in this study were performed according to the recommendations of the Board of Registry Publication Guidelines.
Study Group Design The rats were divided into 7 groups. A standard midline incision 2 cm long was made for all surgical procedures. Surgery was performed with the rats under a single dose of intraperitoneal 5% ketamine (50 to 100 mg/kg) anesthesia. In group 1— control, 7 rats underwent no intervention but were sacrificed 8 weeks later. In group 2—sham operation, the left renal vein was exposed, rotated 360 degrees around its axis and left unligated. Nine rats were sacrificed 8 weeks later. In group 3—induced varicocele, the left renal vein was cleared from the overlying adipose and connective tissue. Adequate renal vein exposure was ensured. A 0.85 mm metal probe was placed along the left renal vein. The renal vein was constricted around the metal probe with 4-zero silk sutures placed proximal to the insertion point of the left internal spermatic vein. Subsequently, the metal probe was removed, leaving a 50% stenotic renal vein in 9 rats.11 In group 4 —varicocele plus varicocelectomy plus MPFF for 4 weeks, varicocele was induced using the technique described. Varicocelec-
tomy was performed 4 weeks after varicocele creation. MPFF was immediately administered for 4 weeks after the varicocelectomy procedure in 9 rats. To perform varicocelectomy a new incision was made on the previous midline incision line and the abdominal cavity was entered. Dilatation of the renal vein was observed in all rats. Varicocelectomy was performed using a high ligation technique. In group 5—varicocele plus MPFF for 8 weeks, beginning immediately after varicocele induction, as described, MPFF was administered for 8 weeks in 12 rats. In group 6 —varicocele plus MPFF for 4 weeks, varicocele was induced, as described. Beginning 4 weeks later, MPFF was administered for 4 weeks in 10 rats. In group 7—varicocele plus varicocelectomy, varicocele was induced, as described. Varicocelectomy was performed 4 weeks after varicocele creation in 10 rats.
Testicular Blood Flow Testicular tissue blood flow was measured using a PeriFlux 5000 laser Doppler flowmeter. Before the rats were sacrificed, the testes were removed from the scrotal sac and placed on the abdomen. Stable signals from 2 separate, minimally vascularized areas of the testicular surface were measured. The average value of the measurements was calculated.
Apoptotic Index and Johnsen Score Calculation Testicular tissues were fixed with 10% neutral buffered formaldehyde for at least 48 hours. Tissue specimens were washed, dehydrated and cleared in xylenes. Each tissue sample was embedded in paraffin and 4 to 5 m sections were prepared. Four consecutive sections of each 10 sections were taken. Two sections were stained with hematoxylin and eosin. The other 2 sections were stained immunohistochemically. Immunohistochemical analysis was performed using the streptavidin-peroxidase method. Slides reserved for immunohistochemistry were rehydrated and quenched in 3% H2O2 (Thermo Scientific®) for 5 minutes. Subsequently, heat activated antigen retrieval was performed in tris-ethylenediaminetetraacetic acid buffer (Merck, Darmstadt, Germany) at pH 9. Nonspecific binding was eliminated with Ultra V Block (Thermo Scientific). One section per set was stained with rabbit reactive caspase-3 antibody (ab44976, Abcam®). Another section was stained only with secondary antibody as a control. Tissues were incubated overnight at 4C in a humidified chamber. Goat anti-polyvalent solution (Thermo Scientific) was used as secondary antibody. Diaminobenzidine staining was performed with the UltraVision Plus Large Volume Detection System (Thermo Scientific). Slides were counterstained with hematoxylin and eosin (Merck). To evaluate the apoptotic index, caspase-3 positive germ cells were counted and examined under 400⫻ magnification with a light microscope. The total number was divided by the total number of seminiferous tubules. Cross-sections of 20 biopsy specimens of seminiferous tubules retrieved from the right and left testes of each animal were examined to evaluate seminiferous epithelial quality. We used the Johnsen scoring system, which rates the quality of spermatogenesis on a 10-point scale based on the presence or absence of certain spermatogenic cell types.11,12
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
2009
Figure 1. Left and right testicular weight in all groups
Statistical Analysis All statistical analyses were done using SPSS®, version 16.0. A normality test (Kolmogorov-Smirnov test with significance considered at p ⬎0.05) was performed to evaluate the distribution of numerical variables. Numerical variables with a skewed distribution were compared using the Kruskal-Wallis test. Post hoc analyses were done using the Mann-Whitney U test, as needed.
RESULTS Figure 1 shows mean testicular weight in all groups. Mean left and right testicular weight was lower in groups 3 and 6 compared with the other groups. However, mean left testicular weight was higher in group 4 than in group 3 (p ⫽ 0.003), while right testicular weight was similar in groups 4 and 3. Compared with the control group, mean bilateral testicular weight was similar in the sham operated group and group 7, and in groups 4 and 5. The table lists mean microvascular blood flow, Johnsen scores and apoptotic indexes. No statistically significant difference was found in mean microvascular blood flow measurements of left testicular tissue among groups 1, 2, 4, 5 and 7. Mean microvascular blood flow of left testicular tissue in group 3 was significantly higher than in the other groups. Mean left testicular tissue measurements in
group 6 were significantly higher than in group 1 but lower than in group 3 (p ⫽ 0.002 and ⬍0.0001, respectively), while left testicular values were significantly greater in group 6 than in group 5 (p ⫽ 0.003). In all groups mean microvascular blood flow measurements of right testicular tissue were highly similar to left testicular tissue values. Mean Johnsen scores for the left testis in groups 3 to 7 were significantly lower than in group 1. Mean scores for the left and right testes in group 3 were significantly lower than in the other groups (p ⬍0.05, fig. 2), while scores for the left and right testes in group 5 were significantly higher than in group 6 (p ⬍0.05). Mean scores for the right testes in groups 2, 4 and 7 were similar to those in group 1 (p ⬎0.05). However, mean scores for the right testes in groups 3, 5 and 6 were significantly lower than the corresponding values in group 1, while mean right testicular scores in group 1 were significantly higher than the corresponding values in groups 3, 5 and 6 (p ⬍0.05). The mean apoptotic index of left testes was statistically significantly higher in groups 5 to 7 than in group 1 (p ⬍0.05). The mean index of left testes in group 3 was significantly higher than in the other groups, excluding group 6 (p ⬍0.05, fig. 3). No significant difference was noted in the mean left testic-
Testicular blood flow, Johnsen score and apoptotic index in all groups (Kruskal-Wallis test p ⬍0.0001) Mean ⫾ SD Testicular Blood Flow (ml/min/100 gm) Group No. (intervention) 1 2 3 4 5 6 7
(control) (sham operation) (varicocele) (varicocele, varicocelectomy ⫹ MPFF) (varicocele ⫹ 8-wk MPFF) (varicocele ⫹ 4-wk MPFF) (varicocele ⫹ varicocelectomy)
Mean ⫾ SD Johnsen Score
Mean ⫾ SD Apoptotic Index
Lt
Rt
Lt
Rt
Lt
Rt
94 ⫾ 5.03 92.44 ⫾ 2.69 119.66 ⫾ 6.44 94.66 ⫾ 3.84 95.50 ⫾ 6.12 103.50 ⫾ 5.46 94.60 ⫾ 5.08
92.85 ⫾ 6.2 93.44 ⫾ 3.28 120.77 ⫾ 6.05 94.77 ⫾ 4.57 96.25 ⫾ 5.52 104 ⫾ 4.29 95.20 ⫾ 4.15
9.68 ⫾ 0.09 9.72 ⫾ 0.08 7.48 ⫾ 0.28 9.56 ⫾ 0.10 9.32 ⫾ 0.11 8.80 ⫾ 0.24 9.51 ⫾ 0.13
9.75 ⫾ 0.05 9.66 ⫾ 0.13 8.76 ⫾ 0.21 9.60 ⫾ 0.18 9.5 ⫾ 0.12 9.28 ⫾ 0.21 9.63 ⫾ 0.14
0.154 ⫾ 0.012 0.167 ⫾ 0.014 0.398 ⫾ 0.015 0.164 ⫾ 0.008 0.174 ⫾ 0.024 0.316 ⫾ 0.030 0.193 ⫾ 0.011
0.172 ⫾ 0.011 0.143 ⫾ 0.021 0.491 ⫾ 0.019 0.184 ⫾ 0.011 0.212 ⫾ 0.015 0.478 ⫾ 0.018 0.195 ⫾ 0.019
2010
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
Figure 2. Seminiferous epithelium staining to evaluate spermatogenesis in groups 1— control (A), 2—sham operation (B), 3—varicocele (C and D), 4 —varicocele plus varicocelectomy plus MPFF (E), 5—varicocele plus 8-week MPFF (F), 6 —varicocele plus 4-week MPFF (G) and 7—varicocele plus varicocelectomy (H). Seminiferous tubules were normal in groups 1 (A) and 2 (B). Seminiferous tubular structure was damaged. Disorganization in most tubules (thin arrow), spermatids in some tubules (thick arrow) and spermatozoa (star) were not observed in group 3 (C) but germ cell sloughing was seen (D). Seminiferous tubules in group 4 were similar to those in group 1 (E). In groups 5 and 7 there was minimal damage to seminiferous tubular structure, which did not differ from that in group 1 (F). Moderate damage to seminiferous tubules was observed in group 6 (G). H&E, reduced from ⫻200.
ular apoptotic index of groups 4 and 1, while in group 5 it was significantly lower than in group 6 (p ⬍0.05), and in groups 4 and 7 it did not significantly differ (p ⬎0.05). The mean right testicular apoptotic index in groups 3 and 5 to 7 was significantly higher than the estimated index in group 1, while in group 3 it was significantly higher than that calculated in the other groups (each p ⬍0.05). The mean right testicular apoptotic index in group 5 was significantly lower than in group 6 and in group 7 it was significantly lower than in group 4 (each p ⬍0.05).
DISCUSSION The incidence of varicocele, which can cause testicular growth retardation, atrophy and impaired seminal parameters, was estimated at 40% for men with primary infertility and 71% for men with secondary infertility.13 The incidence of testicular atrophy secondary to varicocele ranges from 10% to 75% in various studies.14 Testicular atrophy is defined as the loss of seminiferous tubular components. This pathological condition is the main etiological factor responsible for
abnormal semen parameters and subfertility. Enlargement of the hypovolemic testis at a rate of 53% to 100% was reported after surgical intervention.15 On the other hand, to our knowledge no medical therapy preventing testicular atrophy has been developed. In the current study the left testicular weight of rats treated with MPFF for 8 weeks after varicocele induction was significantly higher than that in the varicocele induced group and it was comparable to that in the control group. Furthermore, no statistically significant difference was detected between this group and group 7. The left testicular weight of rats in group 6 was significantly lower than in the other treated groups as well as the control and sham treated groups but significantly greater than in the varicocele group. The difference between the testicular weight in group 6, which received MPFF beginning 4 weeks after varicocele induction, and the other treatment groups can be explained by certain hypotheses. 1) Short-term MPFF use has a weaker effect on the prevention of testicular volume loss. 2) MPFF leads to testicular hypovolemia, decreasing interstitial fluid
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
2011
Figure 3. Caspase-3 positive labeled germ cells (arrows) were not seen in seminiferous epithelium in control group (A) but they were seen in varicocele (B), varicocele plus varicocelectomy plus MPFF (C) and varicocele plus 8-week MPFF (D) groups. Reduced from ⫻200.
formation or limiting the increase in testicular volume during the early posttreatment period. In experimental animal studies conflicting results have been reported regarding varicocele and alterations in blood flow. Some studies have shown decreased testicular blood flow in varicocele models16 but many have indicated an increase in blood flow after varicocele formation.17,18 Increased blood flow inhibits spermatogenesis because of enhanced testicular temperature. In a human study Wright et al found that testicular temperature increased bilaterally in individuals with varicocele and decreased after varicocelectomy.19 Additionally, a color Doppler study performed by Tarhan et al revealed an increase in testicular blood flow in men with varicocele.20 In our study we detected significantly increased microvascular blood flow in the left testes of rats in the varicocele induced group compared with other groups, while no significant difference was found in microvascular blood flow between the control group, and groups 4, 5 and 7. The attenuating effect of MPFF on microvascular blood flow in the 2 testes can be explained by its protective and regulatory effects on testicular microcirculation. Apoptosis, a regulated form of cell death, is a physiological process essential for normal testicular physiology.21 Germ cell apoptosis is a physiological testicular process that is necessary to maintain spermatogenesis. However, an increase in germ cell apoptosis can induce a spermatogenetic defect, resulting in infertility.
Various studies indicate the importance of apoptosis in the pathogenesis of varicocele.12,22 Cam et al found that the number of apoptotic cells in the testicular tissue of the varicocele group was significantly higher than in the control group.22 Fazlioglu et al reported that in an experimental varicocele model the apoptotic activity in germ cells had increased twofold in comparison with the control group 14 days after varicocele development and the apoptotic rate attained its maximal level at 28 days.23 In that study cellular apoptosis decreased after varicocelectomy and mean apoptotic indexes approached those of the control group. Clinical studies have been performed of the correlation between apoptosis and varicocele. Lin et al detected an increase in germ cell apoptosis in testicular biopsy materials from infertile men with idiopathic testicular failure.24 The group suggested that this increase might lead to testicular dysfunction and infertility. Baccetti et al evaluated the number of apoptotic spermatocytes during seminal analyses and detected an almost hundredfold increase in the apoptotic index relative to that in men without varicocele.25 This correlation between varicocele and testicular apoptosis could be explained by the local temperature increase in the testis and hypoxia due to venous stasis. The mentioned studies demonstrate that testicular superoxide levels increased, in addition to the increase in apoptotic activity, and indicate that substances such as vitamin E and mel-
2012
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
atonin, which decrease testicular superoxide levels, could decrease testicular germ cell apoptosis.21,26 In our series varicocele was induced experimentally and the germ cell apoptotic index was assessed using caspase-3 analysis. The apoptotic index was almost threefold higher in the varicocele group (group 3) than in the control group. MPFF administration for 8 weeks immediately after the induction of varicocele decreased the apoptotic index more than 2.3 times. On the other hand, the apoptotic index of the right testis in the varicocele group was statistically higher compared with the other groups. This impact of MPFF can be explained by its influence on apoptosis. Alterations related to spermatogenesis in patients with varicocele are associated with various factors, such as testicular hypoxia secondary to stasis, abnormal regulation of testicular temperature, reflux of toxic adrenal products and renal metabolic processes, and spermatic venous hypertension.27 Eliminating these factors by varicocelectomy resulted in improved spermatogenesis in many series.27 In an experimental study Tek et al investigated the effect of vascular endothelial growth factor injection in the testes on spermatogenesis and apoptosis in a varicocele induced adolescent rat model.12 They concluded that vascular endothelial growth factor may have a positive
role in improving the testicular damage that occurs after varicocele in ipsilateral testes. In our study the Johnsen score was assessed by hematoxylin and eosin staining after the induction of a varicocele model. Lower Johnsen scores were obtained in the varicocele induced group relative to those in all other groups with a statistically significant difference between the groups.
CONCLUSIONS Our study shows that in rats with experimentally induced varicoceles, testicular weight was significantly lower on the side with the varicocele and microvascular blood flow increased in each testis. Based on the results of our histopathological analyses, the Johnsen score decreased on each side, while the apoptotic index of each testis increased after varicocele was induced. Microvascular blood flow decreased considerably, while testicular weight increased after varicocelectomy and long-term MPFF treatment. Additionally, a significant increase in the Johnsen score and a marked decrease in germ cell apoptosis were observed. The favorable results suggest that long-term MPFF might be a good treatment option to prevent testicular damage secondary to varicocele.
REFERENCES 1. Greenfield SP, Seville P and Wan J: Experience with varicoceles in children and young adults. J Urol 2002; 168: 1684. 2. Fretz PC and Sandlow JI: Varicocele: current concepts in pathophysiology, diagnosis, and treatment. Urol Clin North Am 2002; 29: 921. 3. Struckmann JR: Clinical efficacy of micronized purified flavonoid fraction: an overview. J Vasc Res, suppl., 1999; 36: 37. 4. Porter JM and Moneta GL: Reporting standards in venous disease: an update. International Consensus Committee on Chronic Venous Disease. J Vasc Surg 1995; 21: 635. 5. Cesarone MR, Belcaro G, Rohdewald P et al: Comparison of Pycnogenol and Daflon in treating chronic venous insufficiency: a prospective, controlled study. Clin Appl Thromb Hemost 2006; 12: 205. 6. Lyseng-Williamson KA and Perry CM: Micronised purified flavonoid fraction. Drugs 2003; 63: 71. 7. Jiang ZM and Cao JD: The impact of micronized purified flavonoid fraction on the treatment of acute haemorrhoidal episodes. Curr Med Res Opin 2006; 22: 1141. 8. Pecking AP: Evaluation by lymphoscintigraphy of the effect of a micronized flavonoid fraction (Da-
flon 500 mg) in the treatment of upper limb lymphedema. Int Angiol 1995; 14: 39. 9. Söylemez H, Kiliç S, Atar M et al: Effects of micronised purified flavonoid fraction on pain, semen analysis and scrotal color Doppler parameters in patients with painful varicocele; results of a randomized placebo-controlled study. Int Urol Nephrol 2012; 44: 401. 10. Zampieri N, Pellegrino M, Ottolenghi A et al: Effects of bioflavonoids in the management of subclinical varicocele. Pediatr Surg Int 2010; 26: 505. 11. Aggarwal A, Misro MM, Maheshwari A et al: Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo. Mol Cell Endocrinol 2012; 348: 155. 12. Tek M, Cayan S, Yilmaz N et al: The effect of vascular endothelial growth factor on spermatogenesis and apoptosis in experimentally varicocele-induced adolescent rats. Fertil Steril 2009; 91: 2247. 13. La Vignera S, Condorelli R, Vicari E et al: Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis. J Androl 2012; 33: 389.
14. Çayan S, Akbay E, Bozlu M et al: The effect of varicocele repair on testicular volume in children and adolescents with varicocele. J Urol 2002; 168: 731. 15. Hadziselimoviç F, Herzog B, Liebundgut B et al: Testicular and vascular changes in children and adults with varicocele. J Urol 1989; 142: 583. 16. Li H, Dubocq F, Jiang Y et al: Effect of surgically induced varicocele on testicular blood flow and Sertoli cell function. Urology 1999; 53: 1258. 17. Turner TT: The study of varicocele through the use of animal models. Hum Rep Update 2001; 7: 78. 18. Saypol DC, Howards SS, Turner TT et al: Influence of surgically induced varicocele on testicular blood flow, temperature, and histology in adult rats and dogs. J Clin Invest 1981; 68: 39. 19. Wright EJ, Young GP and Goldstein M: Reduction in testicular temperature after varicocelectomy in infertile men. Urology 1997; 50: 257. 20. Tarhan S, Ucer O, Sahin MO et al: Long-term effect of microsurgical inguinal varicocelectomy on testicular blood flow. J Androl 2011; 32: 33. 21. Aktas C, Kanter M and Kocak Z: Antiapoptotic and proliferative activity of curcumin on ovarian follicles in mice exposed to whole body ionizing
MICRONIZED PURIFIED FLAVONOID FRACTION AND PREVENTION OF TESTICULAR PATHOLOGIES
radiation. Toxicol Ind Health, Epub ahead of print November 10, 2011.
duced testicular germ cell apoptosis. J Androl 2008; 29: 29.
22. Cam K, Simsek F, Yuksel M et al: The role of reactive oxygen species and apoptosis in the pathogenesis of varicocele in a rat model and efficiency of vitamin E treatment. Int J Androl 2004; 27: 228.
24. Lin WW, Lamb DJ, Lipshultz LI et al: The role of autosomal cell apoptosis regulator genes in human spermatogenesis. Int Urol Nephrol 1999; 31: 237.
23. Fazlioglu A, Yılmaz I, Mete O et al: The effect of varicocele repair on experimental varicocele in-
25. Baccetti B, Collodel G and Piomboni P: Apoptosis in human ejaculated sperm cells. J Submicrosc Cytol Pathol 1996; 28: 587.
2013
26. Espino J, Ortiz Á, Bejarano I et al: Melatonin protects human spermatozoa from apoptosis via melatonin receptor- and extracellular signal-regulated kinase-mediated pathways. Fertil Steril 2011; 95: 2290. 27. Naughton CK, Nangia AK and Agarwal A: Varicocele and male infertility: part II. Pathophysiology of varicoceles in male infertility. Hum Reprod Update 2001; 7: 473.