- Email: [email protected]
Sperm caspase-9 in oligoasthenoteratozoospermic men with and without varicocele
Sperm caspase-9 in oligoasthenoteratozoospermic men with and without varicocele Adel Zalata, M.D.,a Mohamed El-Mogy, M.D.,b Ahmed Abdel-Khabir, M.D.,b Youssef El-Bayoumy, M.D.,b Mohamed El-Baz, M.D.,b and Taymour Mostafa, M.D.c a c
Medical Biochemistry Department and b Dermatology and Andrology Department, Mansoura University, Mansoura; and Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt
Objective: To assess sperm caspase-9 activity in infertile oligoasthenoteratozoospermic (OAT) men with and without varicocele. Design: Prospective. Setting: Academic setting. Patient(s): Eighty men: healthy fertile control subjects (n ¼ 20), OAT (n ¼ 25), and OAT associated with left-side varicocele (n ¼ 35). Intervention(s): History taking, clinical examination, semen analysis,assessment of seminal caspase-9. Main Outcome Measure(s): Semen analysis, sperm caspase-9. Result(s): Sperm caspase-9 was significantly increased in infertile OAT men associated with varicocele compared with OAT men without varicocele and healthy fertile control subjects. Sperm casapse-9 activity demonstrated significant negative correlation with sperm count, sperm motility, sperm velocity, sperm linear velocity, sperm linearity index, and sperm normal morphology. Conclusion(s): Sperm caspase-9 is exaggerated in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables. (Fertil Steril 2011;96:1097–9. 2011 by American Society for Reproductive Medicine.) Key Words: Male infertility, semen, sperm, apoptosis, caspase-9, varicocele
The role of apoptosis in the spermatogenesis process has attracted substantial research interest, because it is a dynamic process encompassing the mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, morphologic differentiation, maturation of spermatids, and finally sperm formation (1, 2). Caspases (cysteinyl aspartate– specific proteinases) are expressed as inactive proenzymes and participate in a cascade triggered in response to proapoptotic signals. The proper regulation of the caspase cascade plays an important role in sperm differentiation and testicular maturity. Caspases have been demonstrated to be implicated in the pathogenesis of impaired spermatogenesis, decreased sperm motility, and increased levels of sperm DNA fragmentation, varicocele, and immunologic infertility (3–5). In germ cells, caspase activity is consistently found at postmeiotic stages of development, particularly at the elongated spermatid stage being restricted to a postnuclear cytoplasmic compartment that is shed away from maturing spermatids as a residual body during spermiogenesis (6). Paasch et al. (7) demonstrated the presence of postacrosomal caspases to a lesser extent in ejaculated human spermatozoa with intact membranes compared with samples from infertile men, which were characterized by high numbers of cells with active caspases, especially in cytoplasmic residues. The presence of precursors and activated forms of initiator caspases 8 and 9 in conjunction with their Received June 7, 2011; revised and accepted August 2, 2011; published online August 24, 2011. A.Z. has nothing to disclose. M.E.-M. has nothing to disclose. A.A.-K. has nothing to disclose. Y.E.-B. has nothing to disclose. M.E.-B. has nothing to disclose. T.M. has nothing to disclose. Reprint requests to: Taymour Mostafa, M.D., Professor, Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.08.004
shared effector caspase-3 in human spermatozoa has also been confirmed (8). Varicocele has long been considered to be the most treatable cause of male infertility, with multiple pathophysiologic theories addressing low sperm count, decreased sperm motility, and increased sperm abnormal morphology (9–11). It has been suggested that apoptosis plays an important role in the development of oligozoospermia among varicocele patients with increased germ cell apoptosis demonstrated in either rat models with varicocele or human studies (12). Baccetti et al. (13) showed that up to 10% of spermatozoa in the ejaculate of infertile men associated with varicocele were apoptotic compared with 0.1% among healthy fertile control subjects. Therefore, a link between varicocele and apoptosis is proposed, because many pathways leading to apoptosis are associated with varicocele’s negative impact on spermatogenesis, such as heat stress, androgen deprivation, accumulated toxic stimuli, and increased eliminated reactive oxygen species (ROS) (14, 15). The present study aimed to assess sperm caspase-9 activity in infertile OAT men with and without varicocele.
MATERIALS AND METHODS Eighty men attending the Andrology Department, University Hospital, were included in this study after Institutional Review Board approval and informed consents. They were grouped into: healthy fertile control subjects (n ¼ 20), oligoasthenoteratozoospermic (OAT; n ¼ 25), and OAT associated with leftside varicocele (n ¼ 35). Healthy fertile control subjects were cases that had fathered a child within the past year and were normozoospermic and devoid of varicocele. OAT cases had sperm count <20 106 sperm/mL, sperm motility <50%, and sperm normal forms >14%. Exclusion criteria were: smoking and leucocytospermia. They were subjected to history taking and clinical examination. Diagnosis of varicocele was clinically confirmed by scrotal ultrasound (Fukuda Denshi Tellus UF-850XTD) equipped with color flow
Fertility and Sterility Vol. 96, No. 5, November 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
1097
imaging when at least one scrotal vein had a maximum diameter of R3 mm and retrograde flow observed at rest or after Valsalva maneuver (16). All selected OAT cases with varicocele had grade II or III. Semen analysis was carried out using computer-assisted semen analysis (Autosperm; Ferti Pro) according to the World Health Organization guidelines (17). Sperm morphology was evaluated by phase-contrast microscope. Spermatozoa were separated by Sil-select gradient (Ferti Pro), and the purified spermatozoa were used for assessment of caspase-9 activity.
Estimation of Sperm Caspase-9 Activity Estimation of sperm caspase-9 activity was carried out by colorimetric assay (R&D Systems). Spermatozoa were lysed to collect their intracellular contents, and the cell lysate was tested for protease activity by adding a caspasespecific peptide conjugated to the color reporter molecule p-nitroaniline (pNA). Cleavage of the peptide by caspase-9 releases the chromophore pNA which was quantified spectrophotometrically at wavelength 405 nm. The optical density for caspase-9 activity was compared with a known standard of recombinant active caspase-9 (Chemicon).
Statistical Analyses Statistical analyses were carried out using SPSS version 17. Nonparametric data were expressed as median and range. Mann-Whitney test was used as a test of significance for comparison of two groups. Spearman rank correlation coefficient was used to study the relation between variables. P<.05 was set as significant.
RESULTS The mean level of seminal caspase-9 activity was significantly increased in infertile OAT cases and OAT cases associated with varicocele compared with healthy fertile control subjects. The mean level of seminal caspase-9 activity in OAT cases associated with varicocele was significantly increased compared with OAT cases without varicocele (Table 1). Seminal caspase-9 activity demonstrated significant negative correlation with sperm count (r ¼ 0.855; P¼.0001), sperm motility (r ¼ 0.777; P¼.0001), velocity (r ¼ 0.740, P¼.0001), linear velocity (r ¼ 0.659; P¼.0001), linearity index (r ¼ 0.466; P¼.0001), normal sperm morphology (r ¼ 0.760; P¼.0001).
DISCUSSION In the present study there was a significant increase in seminal caspase-9 activity in OAT and OAT cases with varicocele compared with fertile control subjects. Also, seminal caspase-9 activity dem-
onstrated negative correlation with the investigated sperm parameters. Wang et al. (18) demonstrated that infertile men in general have decreased sperm variables induced by higher ROS levels in semen, which has a positive relation with increased sperm damage and higher levels of caspases 9 and 3, indicating positive apoptosis. The high percentage of apoptotic cells in infertile patients implies that more sperm earmarked to undergo apoptosis may escape and that these are the cells that should be eliminated if the apoptotic machinery functions properly during spermatogenesis (19, 20). Different studies have demonstrated high levels of seminal oxidative stress in men with varicocele, suggesting that sperm dysfunction may be related to oxidative stress (21–24). Varicocele has been demonstrated to increase testicular oxidative stress, which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis. Such stress conditions can cause changes in the dynamics of testicular microvascular blood blow, endocrine signaling, and germ cell apoptosis (25, 26). Tanaka et al. (27) and Sadek et al. (28) added that reduced DNA synthesis is one of the causes of spermatogeinc dysfunction in varicocele-associated testes compared with infertile cases devoid of such pathology. Fazlioglu et al. (29) showed that after creation of unilateral varicocele, the apoptotic process was increased in both testes and that it decreased markedly after varicocelectomy. Moustafa et al. (30) reported that apoptosis was significantly correlated with ROS within infertile patients in the whole ejaculate and in the mature and immature spermatozoa. In normal rats, Cam et al. (31) demonstrated that an increased rate of apoptosis with experimental varicocele suggests a molecular alteration involving ROS overproduction as a triggering mechanism. Pan et al. (32) demonstrated increased caspase-3 protein expression in germ cells and that Bcl2 overexpression in the internal spermatic vein of patients with varicocele may be one of the molecular mechanisms related to excessive testicular germ cell apoptosis in varicocele-associated testes. Chang et al. (33) suggested that certain protein-development apoptotic mechanisms that are increased in varicocele-associated cases might originate in the cytoplasmic droplet or mitochondria of spermatocytes and then might function within the nucleus of the cell. El-Domyati et al. (34) added that significant differences in DNA damage repair–associated proteins and apoptosis markers were observed in infertile men with varicocele compared with fertile young men. Old fertile men showed similar expression of the same markers compared with infertile varicocele patients.
TABLE 1 Data of the different studied groups, median (range).
Sperm count (million/mL) Sperm motility (%) Sperm velocity (mm/s) Sperm linear velocity (mm/s) Sperm linearity index (mm/s) Sperm normal morphology (%) Sperm caspase-9 (U/106 sperms)
Control subjects (n [ 20)
OAT (n [ 25)
OAT with varicocele (n [ 35)
68.8 (38.8–94.0) 55 (50–60) 80.2 (48.5–97.3) 57.8 (31–69.4) 69.1 (60–79.1) 58.0 (42–64) 1.09 (0.64–1.61)
13.7 (8.2–17.0)a 30 (5–45)a 54.5 (19.6–85.2)a 26.1 (8.3–53.0)a 50.6 (30.6–71.9)a 9 (2–13)a 1.18 (0.93–7.14)
11.5 (0.8–15.0)a 30 (5–45)a 48.9 (5–90.2)a 25.5 (5–56.9)a 55.1 (5–70.1) 7 (0–14)a 2.23 (0.63–14.81)a,b
Note: OAT ¼ oligoasthenoteratozoospermia. a Significant difference compared with the fertile control subjects. b Significant difference compared with the OAT men without varicocele. Zalata. Caspase-9 and varicocele. Fertil Steril 2011.
1098
Zalata et al.
Caspase-9 and varicocele
Vol. 96, No. 5, November 2011
Finally, although all cases in the present study were recruited devoid of smoking, leucocytospermia, and eminent medical disorders, occupational and/or environmental influences could not be totally assured to be eliminated. Nonetheless, it is concluded that sperm
caspase-9 is increased in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables.
REFERENCES 1. Kierszenbaum AL. Apoptosis during spermatogenesis: the thrill of being alive. Mol Reprod Dev 2001;58:30–8. 2. Tripathi R, Mishra DP, Shaha C. Male germ cell development: turning on the apoptotic pathways. J Reprod Immunol 2009;83:31–5. 3. Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol Hum Reprod 1998;4:757–62. 4. Lysiak JJ, Turner SD, Turner TT. Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis. Biol Reprod 2000;63:1465–72. 5. Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update 2004;10:39–51. 6. Blanco-Rodriguez J, Martinez-Garcia C. Apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium of the adult rat. J Reprod Fertil 1997;110:61–70. 7. Paasch U, Grunewald S, Agarwal A, Glander HJ. The activation pattern of caspases in human spermatozoa. Fertil Steril 2004;81:802–9. 8. Paasch U, Grunewald S, Fitz G, Glander HJ. Deterioration of plasma membrane is associated with activation of caspases in human spermatozoa. J Androl 2003;24:246–52. 9. Pasqualotto FF, Lucon AM, de Goes PM, Sobreiro BP, Hallak J, Pasqualotto EB, et al. Semen profile, testicular volume, and hormonal levels in infertile patients with varicoceles compared with fertile men with and without varicoceles. Fertil Steril 2005;83:74–7. 10. Roaiah MM, Mostafa T, Salem D, El-Nashar AR, Kamel II, El-Kashlan MS. Alpha-1,4-glucosidase activity in infertile oligoasthenozoospermic men with and without varicocele. Andrologia 2007;39:28–32. 11. Abdel Aziz MT, Mostafa T, Atta H, Kamal O, Kamel M, Hosni H, et al. Heme oxygenase enzyme activity in seminal plasma of oligoasthenoteratozoospermic males with varicocele. Andrologia 2010;42:236–41. 12. Caruso A, Walsh R, Ross L. Experimental varicocele induces testicular germ cell apoptosis in the rat. J Urol 1999;161:280–5.
Fertility and Sterility
13. Baccetti B, Collodel G, Piomboni P. Apoptosis in human ejaculated sperm. J Submicrosc Cytol Pathol 1996;28:587–96. 14. Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA. Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele. Int J Androl 2001;24:261–5. 15. Mostafa T, Anis T, Imam H, El-Nashar AR, Osman IA. Seminal reactive oxygen species–antioxidant relationship in fertile males with and without varicocele. Andrologia 2009;41:125–9. 16. Schurich M, Aigner F, Frauscher F, Pallwein L. The role of ultrasound in assessment of male fertility. Eur J Obstet Gynecol Reprod Biol 2009;144:S192–8. 17. World Health Organization. Laboratory manual for the examination of human semen and sperm–cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press; 1999. 18. Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 2003;80:531–5. 19. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7. 20. Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21:33–44. 21. Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr, Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol 1999;16:1831–4. 22. Zini A, Defreitas G, Freeman M, Hechter S, Jarvi K. Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa. Fertil Steril 2000;74:461–4. 23. Meucci E, Milardi D, Mordente A, Martorana GE, Giacchi E, De Marinis L, et al. Total antioxidant capacity in patients with varicoceles. Fertil Steril 2003;79:1577–83. 24. Mostafa T, Anis TH, Ghazi S, El-Nashar AR, Imam H, Osman IA. Reactive oxygen species and an-
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
tioxidants relationship in the internal spermatic vein blood of infertile men with varicocele. Asian J Androl 2006;8:451–4. Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl 2008;29:488– 98. Hassan A, el-Nashar EM, Mostafa T. Programmed cell death in varicocele-bearing testes. Andrologia 2009;41:39–45. Tanaka H, Fujisawa M, Okada H, Arakawa S, Kamidono S. Assessment of germ-cell kinetics in the testes of patients with varicocele using image analysis of immunostained proliferating cell nuclear antigen. Br J Urol 1996;78:769–71. Sadek A, Almohamdy AS, Zaki A, Aref M, Ibrahim SM, Mostafa T. Sperm chromatin condensation in infertile men with varicocele before and after surgical repair. Fertil Steril 2011;95:1705–8. Fazlioglu A, Yilmaz I, Mete O, Kurtulus F, Parlakilic O, Guctas O, et al. The effect of varicocele repair on experimental varicocele induced testicular germ cell apoptosis. J Androl 2008;29:29–34. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod 2009;19:129–38. Cam K, Simsek F, Yuksel M, Turkeri L, Haklar G, Yalcin S, 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;27:228–33. Pan LJ, Gao DJ, Zhang HP, Wang XY. Apoptosis and expression of caspase-3 protein in germ cells of varicocele rats. Zhonghua Nan Ke Xue 2006;12:53–6. Chang FW, Sun GH, Cheng YY, Chen IC, Chien HH, Wu GJ. Effects of varicocele upon the expression of apoptosis-related proteins. Andrologia 2010;42: 225–30. El-Domyati MM, Al-Din AB, Barakat MT, ElFakahany HM, Honig S, Xu J, et al. The expression and distribution of deoxyribonucleic acid repair and apoptosis markers in testicular germ cells of infertile varicocele patients resembles that of old fertile men. Fertil Steril 2010;93:795–801.
1099
Medical Biochemistry Department and b Dermatology and Andrology Department, Mansoura University, Mansoura; and Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt
Objective: To assess sperm caspase-9 activity in infertile oligoasthenoteratozoospermic (OAT) men with and without varicocele. Design: Prospective. Setting: Academic setting. Patient(s): Eighty men: healthy fertile control subjects (n ¼ 20), OAT (n ¼ 25), and OAT associated with left-side varicocele (n ¼ 35). Intervention(s): History taking, clinical examination, semen analysis,assessment of seminal caspase-9. Main Outcome Measure(s): Semen analysis, sperm caspase-9. Result(s): Sperm caspase-9 was significantly increased in infertile OAT men associated with varicocele compared with OAT men without varicocele and healthy fertile control subjects. Sperm casapse-9 activity demonstrated significant negative correlation with sperm count, sperm motility, sperm velocity, sperm linear velocity, sperm linearity index, and sperm normal morphology. Conclusion(s): Sperm caspase-9 is exaggerated in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables. (Fertil Steril 2011;96:1097–9. 2011 by American Society for Reproductive Medicine.) Key Words: Male infertility, semen, sperm, apoptosis, caspase-9, varicocele
The role of apoptosis in the spermatogenesis process has attracted substantial research interest, because it is a dynamic process encompassing the mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, morphologic differentiation, maturation of spermatids, and finally sperm formation (1, 2). Caspases (cysteinyl aspartate– specific proteinases) are expressed as inactive proenzymes and participate in a cascade triggered in response to proapoptotic signals. The proper regulation of the caspase cascade plays an important role in sperm differentiation and testicular maturity. Caspases have been demonstrated to be implicated in the pathogenesis of impaired spermatogenesis, decreased sperm motility, and increased levels of sperm DNA fragmentation, varicocele, and immunologic infertility (3–5). In germ cells, caspase activity is consistently found at postmeiotic stages of development, particularly at the elongated spermatid stage being restricted to a postnuclear cytoplasmic compartment that is shed away from maturing spermatids as a residual body during spermiogenesis (6). Paasch et al. (7) demonstrated the presence of postacrosomal caspases to a lesser extent in ejaculated human spermatozoa with intact membranes compared with samples from infertile men, which were characterized by high numbers of cells with active caspases, especially in cytoplasmic residues. The presence of precursors and activated forms of initiator caspases 8 and 9 in conjunction with their Received June 7, 2011; revised and accepted August 2, 2011; published online August 24, 2011. A.Z. has nothing to disclose. M.E.-M. has nothing to disclose. A.A.-K. has nothing to disclose. Y.E.-B. has nothing to disclose. M.E.-B. has nothing to disclose. T.M. has nothing to disclose. Reprint requests to: Taymour Mostafa, M.D., Professor, Andrology and Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.08.004
shared effector caspase-3 in human spermatozoa has also been confirmed (8). Varicocele has long been considered to be the most treatable cause of male infertility, with multiple pathophysiologic theories addressing low sperm count, decreased sperm motility, and increased sperm abnormal morphology (9–11). It has been suggested that apoptosis plays an important role in the development of oligozoospermia among varicocele patients with increased germ cell apoptosis demonstrated in either rat models with varicocele or human studies (12). Baccetti et al. (13) showed that up to 10% of spermatozoa in the ejaculate of infertile men associated with varicocele were apoptotic compared with 0.1% among healthy fertile control subjects. Therefore, a link between varicocele and apoptosis is proposed, because many pathways leading to apoptosis are associated with varicocele’s negative impact on spermatogenesis, such as heat stress, androgen deprivation, accumulated toxic stimuli, and increased eliminated reactive oxygen species (ROS) (14, 15). The present study aimed to assess sperm caspase-9 activity in infertile OAT men with and without varicocele.
MATERIALS AND METHODS Eighty men attending the Andrology Department, University Hospital, were included in this study after Institutional Review Board approval and informed consents. They were grouped into: healthy fertile control subjects (n ¼ 20), oligoasthenoteratozoospermic (OAT; n ¼ 25), and OAT associated with leftside varicocele (n ¼ 35). Healthy fertile control subjects were cases that had fathered a child within the past year and were normozoospermic and devoid of varicocele. OAT cases had sperm count <20 106 sperm/mL, sperm motility <50%, and sperm normal forms >14%. Exclusion criteria were: smoking and leucocytospermia. They were subjected to history taking and clinical examination. Diagnosis of varicocele was clinically confirmed by scrotal ultrasound (Fukuda Denshi Tellus UF-850XTD) equipped with color flow
Fertility and Sterility Vol. 96, No. 5, November 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
1097
imaging when at least one scrotal vein had a maximum diameter of R3 mm and retrograde flow observed at rest or after Valsalva maneuver (16). All selected OAT cases with varicocele had grade II or III. Semen analysis was carried out using computer-assisted semen analysis (Autosperm; Ferti Pro) according to the World Health Organization guidelines (17). Sperm morphology was evaluated by phase-contrast microscope. Spermatozoa were separated by Sil-select gradient (Ferti Pro), and the purified spermatozoa were used for assessment of caspase-9 activity.
Estimation of Sperm Caspase-9 Activity Estimation of sperm caspase-9 activity was carried out by colorimetric assay (R&D Systems). Spermatozoa were lysed to collect their intracellular contents, and the cell lysate was tested for protease activity by adding a caspasespecific peptide conjugated to the color reporter molecule p-nitroaniline (pNA). Cleavage of the peptide by caspase-9 releases the chromophore pNA which was quantified spectrophotometrically at wavelength 405 nm. The optical density for caspase-9 activity was compared with a known standard of recombinant active caspase-9 (Chemicon).
Statistical Analyses Statistical analyses were carried out using SPSS version 17. Nonparametric data were expressed as median and range. Mann-Whitney test was used as a test of significance for comparison of two groups. Spearman rank correlation coefficient was used to study the relation between variables. P<.05 was set as significant.
RESULTS The mean level of seminal caspase-9 activity was significantly increased in infertile OAT cases and OAT cases associated with varicocele compared with healthy fertile control subjects. The mean level of seminal caspase-9 activity in OAT cases associated with varicocele was significantly increased compared with OAT cases without varicocele (Table 1). Seminal caspase-9 activity demonstrated significant negative correlation with sperm count (r ¼ 0.855; P¼.0001), sperm motility (r ¼ 0.777; P¼.0001), velocity (r ¼ 0.740, P¼.0001), linear velocity (r ¼ 0.659; P¼.0001), linearity index (r ¼ 0.466; P¼.0001), normal sperm morphology (r ¼ 0.760; P¼.0001).
DISCUSSION In the present study there was a significant increase in seminal caspase-9 activity in OAT and OAT cases with varicocele compared with fertile control subjects. Also, seminal caspase-9 activity dem-
onstrated negative correlation with the investigated sperm parameters. Wang et al. (18) demonstrated that infertile men in general have decreased sperm variables induced by higher ROS levels in semen, which has a positive relation with increased sperm damage and higher levels of caspases 9 and 3, indicating positive apoptosis. The high percentage of apoptotic cells in infertile patients implies that more sperm earmarked to undergo apoptosis may escape and that these are the cells that should be eliminated if the apoptotic machinery functions properly during spermatogenesis (19, 20). Different studies have demonstrated high levels of seminal oxidative stress in men with varicocele, suggesting that sperm dysfunction may be related to oxidative stress (21–24). Varicocele has been demonstrated to increase testicular oxidative stress, which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis. Such stress conditions can cause changes in the dynamics of testicular microvascular blood blow, endocrine signaling, and germ cell apoptosis (25, 26). Tanaka et al. (27) and Sadek et al. (28) added that reduced DNA synthesis is one of the causes of spermatogeinc dysfunction in varicocele-associated testes compared with infertile cases devoid of such pathology. Fazlioglu et al. (29) showed that after creation of unilateral varicocele, the apoptotic process was increased in both testes and that it decreased markedly after varicocelectomy. Moustafa et al. (30) reported that apoptosis was significantly correlated with ROS within infertile patients in the whole ejaculate and in the mature and immature spermatozoa. In normal rats, Cam et al. (31) demonstrated that an increased rate of apoptosis with experimental varicocele suggests a molecular alteration involving ROS overproduction as a triggering mechanism. Pan et al. (32) demonstrated increased caspase-3 protein expression in germ cells and that Bcl2 overexpression in the internal spermatic vein of patients with varicocele may be one of the molecular mechanisms related to excessive testicular germ cell apoptosis in varicocele-associated testes. Chang et al. (33) suggested that certain protein-development apoptotic mechanisms that are increased in varicocele-associated cases might originate in the cytoplasmic droplet or mitochondria of spermatocytes and then might function within the nucleus of the cell. El-Domyati et al. (34) added that significant differences in DNA damage repair–associated proteins and apoptosis markers were observed in infertile men with varicocele compared with fertile young men. Old fertile men showed similar expression of the same markers compared with infertile varicocele patients.
TABLE 1 Data of the different studied groups, median (range).
Sperm count (million/mL) Sperm motility (%) Sperm velocity (mm/s) Sperm linear velocity (mm/s) Sperm linearity index (mm/s) Sperm normal morphology (%) Sperm caspase-9 (U/106 sperms)
Control subjects (n [ 20)
OAT (n [ 25)
OAT with varicocele (n [ 35)
68.8 (38.8–94.0) 55 (50–60) 80.2 (48.5–97.3) 57.8 (31–69.4) 69.1 (60–79.1) 58.0 (42–64) 1.09 (0.64–1.61)
13.7 (8.2–17.0)a 30 (5–45)a 54.5 (19.6–85.2)a 26.1 (8.3–53.0)a 50.6 (30.6–71.9)a 9 (2–13)a 1.18 (0.93–7.14)
11.5 (0.8–15.0)a 30 (5–45)a 48.9 (5–90.2)a 25.5 (5–56.9)a 55.1 (5–70.1) 7 (0–14)a 2.23 (0.63–14.81)a,b
Note: OAT ¼ oligoasthenoteratozoospermia. a Significant difference compared with the fertile control subjects. b Significant difference compared with the OAT men without varicocele. Zalata. Caspase-9 and varicocele. Fertil Steril 2011.
1098
Zalata et al.
Caspase-9 and varicocele
Vol. 96, No. 5, November 2011
Finally, although all cases in the present study were recruited devoid of smoking, leucocytospermia, and eminent medical disorders, occupational and/or environmental influences could not be totally assured to be eliminated. Nonetheless, it is concluded that sperm
caspase-9 is increased in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables.
REFERENCES 1. Kierszenbaum AL. Apoptosis during spermatogenesis: the thrill of being alive. Mol Reprod Dev 2001;58:30–8. 2. Tripathi R, Mishra DP, Shaha C. Male germ cell development: turning on the apoptotic pathways. J Reprod Immunol 2009;83:31–5. 3. Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol Hum Reprod 1998;4:757–62. 4. Lysiak JJ, Turner SD, Turner TT. Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis. Biol Reprod 2000;63:1465–72. 5. Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update 2004;10:39–51. 6. Blanco-Rodriguez J, Martinez-Garcia C. Apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium of the adult rat. J Reprod Fertil 1997;110:61–70. 7. Paasch U, Grunewald S, Agarwal A, Glander HJ. The activation pattern of caspases in human spermatozoa. Fertil Steril 2004;81:802–9. 8. Paasch U, Grunewald S, Fitz G, Glander HJ. Deterioration of plasma membrane is associated with activation of caspases in human spermatozoa. J Androl 2003;24:246–52. 9. Pasqualotto FF, Lucon AM, de Goes PM, Sobreiro BP, Hallak J, Pasqualotto EB, et al. Semen profile, testicular volume, and hormonal levels in infertile patients with varicoceles compared with fertile men with and without varicoceles. Fertil Steril 2005;83:74–7. 10. Roaiah MM, Mostafa T, Salem D, El-Nashar AR, Kamel II, El-Kashlan MS. Alpha-1,4-glucosidase activity in infertile oligoasthenozoospermic men with and without varicocele. Andrologia 2007;39:28–32. 11. Abdel Aziz MT, Mostafa T, Atta H, Kamal O, Kamel M, Hosni H, et al. Heme oxygenase enzyme activity in seminal plasma of oligoasthenoteratozoospermic males with varicocele. Andrologia 2010;42:236–41. 12. Caruso A, Walsh R, Ross L. Experimental varicocele induces testicular germ cell apoptosis in the rat. J Urol 1999;161:280–5.
Fertility and Sterility
13. Baccetti B, Collodel G, Piomboni P. Apoptosis in human ejaculated sperm. J Submicrosc Cytol Pathol 1996;28:587–96. 14. Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA. Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele. Int J Androl 2001;24:261–5. 15. Mostafa T, Anis T, Imam H, El-Nashar AR, Osman IA. Seminal reactive oxygen species–antioxidant relationship in fertile males with and without varicocele. Andrologia 2009;41:125–9. 16. Schurich M, Aigner F, Frauscher F, Pallwein L. The role of ultrasound in assessment of male fertility. Eur J Obstet Gynecol Reprod Biol 2009;144:S192–8. 17. World Health Organization. Laboratory manual for the examination of human semen and sperm–cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press; 1999. 18. Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 2003;80:531–5. 19. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602–7. 20. Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21:33–44. 21. Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr, Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol 1999;16:1831–4. 22. Zini A, Defreitas G, Freeman M, Hechter S, Jarvi K. Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa. Fertil Steril 2000;74:461–4. 23. Meucci E, Milardi D, Mordente A, Martorana GE, Giacchi E, De Marinis L, et al. Total antioxidant capacity in patients with varicoceles. Fertil Steril 2003;79:1577–83. 24. Mostafa T, Anis TH, Ghazi S, El-Nashar AR, Imam H, Osman IA. Reactive oxygen species and an-
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
tioxidants relationship in the internal spermatic vein blood of infertile men with varicocele. Asian J Androl 2006;8:451–4. Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl 2008;29:488– 98. Hassan A, el-Nashar EM, Mostafa T. Programmed cell death in varicocele-bearing testes. Andrologia 2009;41:39–45. Tanaka H, Fujisawa M, Okada H, Arakawa S, Kamidono S. Assessment of germ-cell kinetics in the testes of patients with varicocele using image analysis of immunostained proliferating cell nuclear antigen. Br J Urol 1996;78:769–71. Sadek A, Almohamdy AS, Zaki A, Aref M, Ibrahim SM, Mostafa T. Sperm chromatin condensation in infertile men with varicocele before and after surgical repair. Fertil Steril 2011;95:1705–8. Fazlioglu A, Yilmaz I, Mete O, Kurtulus F, Parlakilic O, Guctas O, et al. The effect of varicocele repair on experimental varicocele induced testicular germ cell apoptosis. J Androl 2008;29:29–34. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod 2009;19:129–38. Cam K, Simsek F, Yuksel M, Turkeri L, Haklar G, Yalcin S, 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;27:228–33. Pan LJ, Gao DJ, Zhang HP, Wang XY. Apoptosis and expression of caspase-3 protein in germ cells of varicocele rats. Zhonghua Nan Ke Xue 2006;12:53–6. Chang FW, Sun GH, Cheng YY, Chen IC, Chien HH, Wu GJ. Effects of varicocele upon the expression of apoptosis-related proteins. Andrologia 2010;42: 225–30. El-Domyati MM, Al-Din AB, Barakat MT, ElFakahany HM, Honig S, Xu J, et al. The expression and distribution of deoxyribonucleic acid repair and apoptosis markers in testicular germ cells of infertile varicocele patients resembles that of old fertile men. Fertil Steril 2010;93:795–801.
1099