- Email: [email protected]
The Correlation of Biochemical and Morphologic Parameters in the Assessment of Sperm Maturity
Infertility The Correlation of Biochemical and Morphologic Parameters in the Assessment of Sperm Maturity Otas Durutovic, Natasa Lalic, Dragica Milenkovic-Petronic, Nebojsa Bojanic, Dejan Djordjevic, Bogomir Milojevic, Nebojsa Ladjevic, Ana Mimic, Lidija Tulic, Zoran Dzamic, and Sava Micic OBJECTIVE METHODS
RESULTS
CONCLUSION
To examine the relationship between biochemical markers and morphologic sperm characteristics, including head, neck, and tail changes. The study evaluated 154 patients who went to the Andrology Laboratory of the Clinic of Urology, Clinical Center of Serbia. Patients were divided into 4 groups: normozoospermic, oligozoospermic, severe oligozoospermic, and asthenozoospermic, according to the sperm concentration and motility. The differences in creatine kinase (CK) and CK-M levels between normozoospermic and the 2 groups of oligozoospermic patients were significantly different (P <.01). The CK and CK-M levels correlated negatively with sperm concentration and sperm motility, but correlated positively with the pathologic sperm form. Patients with CK values >0.093 have a total number of pathologic forms higher than 0.40 (87.5% sensitivity, 77.3% specificity, the area under the curve was 0.832, P <.001). Patients with CK values <0.09 U/L have normal spermatogenesis and pathologic disorder of the head <15%, neck <12%, and tail <10%. The relation between sperm morphology and biochemical markers included in the maturation process is established during the sperm genesis process. If the results of these markers are used together with the morphology of the spermatozoa in the interpretation of infertility, it would lead us to better insight of the fertility potential of the each patient. UROLOGY 82: 1296e1299, 2013. 2013 Elsevier Inc.
T
he parameters of conventional semen analysis (sperm concentration, motility, and morphology) have failed to show a consistent correlation with pregnancy rates.1,2 Therefore, biochemical markers are included as the objective indicator of biologic sperm powers. A number of independent studies have indicated that defective sperm function is associated with elevated levels of certain key enzymes, such as creatine kinase (CK),3-5 lactate dehydrogenase,6 and glucose-6-phosphate dehydrogenase.7 CK levels in human sperm are an objective biochemical marker of sperm maturity and fertilizing potential.3,8,9 Immunocytochemical studies of CK levels in individual spermatozoa have demonstrated that increased CK concentrations reflect residual cytoplasm in sperm that
Financial Disclosure: The authors declare that they have no relevant financial interests. From the Clinic of Urology, Clinical Center of Serbia, Belgrade, Serbia; the School of Medicine University of Belgrade, Belgrade, Serbia; the Center for Medical Biochemistry, Belgrade, Serbia; the Centre of Anesthesiology and Reanimatology, Clinical Centre of Serbia, Belgrade, Serbia; and the Clinic for Gynecology and Obstetrics, Clinical Centre of Serbia, Belgrade, Serbia Reprint requests: Bogomir Milojevic, Ph.D., Clinic of Urology, Clinical Center of Serbia, Resavska 51, 11000 Belgrade, Serbia. E-mail: [email protected] Submitted: April 1, 2013, accepted (with revisions): August 12, 2013
1296
ª 2013 Elsevier Inc. All Rights Reserved
was not extruded during late spermiogenesis.10,11 Interest in CK has been stimulated by studies suggesting that defective sperm function is associated with defects in spermatogenesis that leads to the release of immature spermatozoa from the germinal epithelium.3,7,8,12 Two isoforms of CK can be detected in the sperm: CK-B, which is representing the whole CK activity of the seminal plasma, and CK-M, the isoform of spermatozoa, which is mitochondrial origin.13 The biochemical parameters of CK activity and CK-M isoform ratio were evaluated in several studies.7,8 In couples with oligozoospermic husbands treated with intrauterine insemination, sperm CK activities differed between fertile and infertile men, although the sperm concentration and motility values between the 2 groups were similar or identical. A logistic regression analysis in fertile and infertile oligozospermic men further indicated that, whereas the occurrence of pregnancies correlated with CK activities, the sperm concentration values did not contribute to this correlation.14 The purpose of this study was to examine the relationship between biochemical CK and CK-M markers and morphologic sperm characteristics, including head, neck, and tail changes. 0090-4295/13/$36.00 http://dx.doi.org/10.1016/j.urology.2013.08.031
MATERIALS AND METHODS
Table 1. Correlation analysis of sperm characteristics with creatine kinase (CK) and CK-M activity
Patient Selection Semen samples of 154 men referred for semen analysis to the Andrology Laboratory of the Clinic of Urology, Clinical Center of Serbia were studied. Semen specimen was collected after a requested abstinence of 3-7 days. Median time of abstinence for normozoospermic group was 3.5 0.9 days, for oligozoospermic group was 3.8 0.8 days, for severe oligozoospermic group was 3.6 0.9 days, and for asthenozoospermic group was 3.6 0.5 days. The specimens were allowed to liquefy at 37 C for 30 minutes before evaluation of the sperm characteristics (concentration, motility, and morphology). Semen analysis was performed manually according to the World Health Organization guidelines, and morphology was examined using strict criteria (World Health Organization, 1999).15 According to the sperm concentration and motility, patients were divided into 4 groups: normozoospermic, oligozoospermic, severe oligozoospermic, and asthenozoospermic.
Characteristics Sperm concentration (106/mL) Motility (%) Morphology
Correlation Correlation P with CK-M P with CK (r*) Value (r*) Value 0.653
<.01
0.528
<.01
0.448 0.628
<.01 <.01
0.405 0.636
<.01 <.01
CK, creatine kinase. * Pearson’s correlation coefficient.
CK Estimation The CK values were defined by kinetic Olympus assay (CK test OSR 6279). The sperm cells were prepared using 250 mL ejaculated seminal plasmas extracted 3 times by the repeated flushing of sediment sperm cells with an imidazole (0.15 M NaCl and 0.03 M imidazole at pH 7.0). The residue is rinsed after the last buffer flushing, drained dry, and resuspended in 0.1% Triton X-100 solution with intense vortex stirring for approximately 20 seconds. The sample was centrifuged again, and the supernatant was analyzed for CK activity. The CK activity was expressed as international units/108 spermatozoa. The separation of CK-M isoenzymes from CK-B isoenzymes supernatant was obtained from the previously described procedure, which was performed by passing through a DEAE Sephadex A-50 column. The separation of CK isoenzyme with the help of an ion exchange column: the column is prepared by swelling 1-g Sephadex A-50 in 250 mL buffers at room temperature. The swelling Sephadex is than rinsed 3 times and the result is stored in the fridge at 4 C. A 100 mL of prepared sperm supernatant is added to the Eppendorff centrifuge tube of 50 mL Sephardexes. Seventy-five microliters of supernatants is used to determine the CK activity that originates from the CKM forms. CK-B activity is obtained by subtracting the CK-M activity from the total CK sperm activity. The obtained activity was expressed as international units/108 spermatozoa.
Statistical Analysis Statistical analysis was performed using the SPSS Windows statistical packet. The difference between patient groups was calculated using the Mann-Whitney U test. Correlation between CK and also CK-M with sperm concentration, motility, and morphology was done. Statistical significance was obtained using analysis of variance. Significant differences were accepted with values of P <.05. Receiver operating characteristic (ROC) test was used to determine predictive values of CK activities. As the CK distribution and sperm morphology are not uniform, a log transformation of these parameters was done to reduce the skew level.
RESULTS The differences in CK and CK-M levels between normozoospermic and the 2 groups of oligozoospermic UROLOGY 82 (6), 2013
Figure 1. The receiver operating characteristic test to determine the sensitivity and the specificity of CK-M activities in detecting pathologic sperm maturation, which is defined by disorder in sperm number. (Color version available online.)
patients were significantly different (P <.01). Patients with normozoospermia had CK values of 0.097 0.026 UI/108 sperm cells and CK-M 0.041 0.015 UI/108 sperm cells, whereas patients with oligozoospermia had CK values of 0.46 0.30 UI/108 sperm cells and CK-M 0.11 0.07 UI/108 sperm cells. Patients with severe oligozoospermia had CK values of 1.90 2.2 UI/108 sperm cells and CK-M 0.18 0.1 UI/108 sperm cells, respectively. The CK and CK-M levels correlated negatively with sperm concentration and sperm motility. The CK and CK-M levels correlated positively with the pathologic sperm forms (Table 1). Using the ROC curves, cut-off values were obtained for the CK and CK-M that significantly correlate with the sperm concentration. The calculated cut-off value for CK from 0.093 UI/108 sperm had a sensitivity of 93.75% and a specificity of 86.36%. The area under the curve (AUC) was 0.957 (P <.001). 1297
Table 2. Cut-off values, specificities, and sensitivities and the area under the curve for defining head, neck, and tail sperm pathology Pathologic Sperm Forms
Cut- Sensitivity Specificity P off (%) (%) AUC Value
Head Neck Tail
0.15 0.12 0.12
62.5 67 75
52.5 65 61
0.623 <.05 0.701 <.05 0.671 <.05
AUC, area under the curve.
Figure 2. The receiver operating characteristic test to determine the sensitivity and the specificity of morphology in detecting pathologic sperm maturation, which is defined by disorder in CK activities. (Color version available online.)
Comparing the CK-M values with the sperm concentration, a cut-off value for CK-M with 0.048 UI/108 sperm was calculated with 85.70% sensitivity and 72.73% specificity. AUC below ROC curve was 0.862 (P <.001; Fig. 1). Examining the number of pathologic forms of sperm cells whose presence is the marker of morphologic spermatogenesis disorder, which correlates with the established cut-off values for CK of 0.093, it has been established that patients with CK values >0.093 have a total number of pathologic forms higher than 0.40 (87.5% sensitivity, 77.3% specificity, AUC was 0.832, P <.001; Fig. 2). When the pathologic sperm forms are defined as head, neck, and tail disorders and compared with the cut-off CK values established for normal spermatogenesis with the help of the ROC curve, results are obtained that indicate that patients with CK values <0.09 U/L have normal spermatogenesis and pathologic disorder of the head in <15% (62.5% sensitivity, 52.5% specificity, AUC was 0.623, P <.05), neck <12% (67% sensitivity, 65% specificity, AUC was 0.701, P <.05), and tail <10% (75% sensitivity, 61% specificity, AUC was 0.671, P <.05; Table 2).
COMMENT Previous studies have shown an inverse correlation between CK activities in sperm cells and the number of sperm cells, pointing that there is a metabolic difference between sperm cells in normozoospermic and oligozoospermic group of patients.4,5,16 Such findings indicate a disorder related to sperm maturation, which is why CK 1298
is considered the predictor of sperm maturity.17 Mature spermatozoa show a higher concentration of the CK-M isoform that is expressed only during the last phase of spermiogenesis in elongated spermatids and in mature sperm.4,5,18 Studies suggest that the plasma membrane changes take place in spermiogenesis simultaneously with cytoplasmic extrusion and the expression of the new sperm-specific CK-M.4,18 On the basis of the results of previous studies, one can ask a theoretical question about which marker is better for predicting male fertility, CK, or determining sperm morphology. Measuring CK activities is an objective marker, whereas determining sperm morphology is a subjective marker, subject to major variations depending on the technician. Sperm cells that substantially completed the cytoplasmic extrusions could be subject, during spermatogenesis, to the interruption or halt of biochemical maturation in later development. Thus, biochemical sperm maturation and functional sperm ability can be reduced without any visible changes in sperm morphology, which in some cases explains the disharmony between CK values and the number of pathologic sample forms.17 The analysis of the relationship between the CK and sperm morphology indicates that there is a relationship between raised levels of CK and the increased retention of cytoplasm and morphologic disorders of the sperm head and tail.16,19 We compared the presence of pathologic changes in the head, neck, and tail with the CK and CK-M values in normozoospermic and oligozoospermic patients. The level of maturation is defined by CK and CK-M values, whose cut-off values were established previously. It was shown that CK and CK-M values were increased in patients who had >15% of morphologic changes to the head, >12% present cytoplasmic residue, and >12% of sperm tail disorder. Huszar et al3,9 have shown that there is a relationship between elevated cytoplasmic retention and head disorder, followed by increased number of big, round sperm heads or increased incidence of amorphous sperm heads. It must be taken into consideration that the cytoplasmic residue is sometimes not noticed in native and stained slides, so one gets the impression that the sperm heads are of changed dimensions. It has been shown that sperm tail, which arises during the genesis of immature sperm cells and which has not completed cytoplasmic extrusion, is shorter because of a standstill in sperm genetic development.18 Therefore, in these cases, a short tail and the failed remodeling of UROLOGY 82 (6), 2013
cytoplasm is a cause of reduced interaction with the zona pellucida. The process of sperm genesis was followed by cytoplasmic extrusion and the expression of CK-M isoforms by remodeling the sperm plasma membrane, which facilitates the formation of the space required for sperm and egg cell interaction,18 so that a possibility for a binding-zone site is considered a part of the remodeling process of the plasma membrane in mature sperm cells. If the morphologic changes to sperm cells are regarded in the light of related events in sperm genesis, the connection between sperm immaturity and the increasing number of changes in the sperm head, neck, and tail clearly suggest the connection of morphologic and biochemical parameters. Morphology alone, even following strict criteria does not have a major predictive significance, but morphology with biochemical markers can be used as predictive markers in the assessment of sperm maturity. The difference between cell maturation, which is a gene regulated process, and epididymal maturation, which includes modifications to improve motility, functional membrane integrity to increase resistance to premature acrosome reaction, and efficiency in the female reproductive tract, is important in the maturation process.
CONCLUSION In conclusion, the relation between sperm morphology and biochemical markers included in the maturation process is established during the sperm genesis process. If the results of these markers are used together with the morphology of the spermatozoa in the interpretation of infertility, it would lead us to better insight of the fertility potential of the each patient. References 1. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol. 1991;164:542-551. 2. Bourgeron T. Mitochondrial function and male infertility. Results and Problems in Cell Differentiation. 2000;28:187-210. 3. Huszar G, Vigue L. Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology. Mol Reprod Dev. 1993;34:292-298. 4. Huszar G, Patrizio P, Vigue L, et al. Cytoplasmic extrusion and the switch from creatine kinase B to M isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa. J Androl. 1998;19:11-20.
UROLOGY 82 (6), 2013
5. Rolf C, Behre HM, Cooper TG, et al. Creatine kinase activity in human spermatozoa and seminal plasma lacks predictive value for male fertility in in vitro fertilization. Fertil Steril. 1998;69:727-734. 6. Casano R, Orlando C, Serio M, et al. LDH and LDH-X activity in sperm from normospermic and oligospermic men. Int J Androl. 1991; 14:257-263. 7. Aitken RJ, Krausz C, Buckingham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Mol Reprod Dev. 1994;39: 268-279. 8. Huszar G, Vigue L. Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa. J Androl. 1994;15:71-77. 9. Gergely A, Kovanci E, Senturk L, et al. Morphometric assessment of mature and diminished maturity human spermatozoa: sperm regions that reflect differences in maturity. Hum Reprod. 1999;14:20072014. 10. Huszar G, Stone K, Dix D, et al. Putative creatine kinase M-isoform in human sperm is identified as the 70-kilodalton heat shock protein HspA2. Biol Reprod. 2000;63:925-932. 11. Lalwani S, Sayme N, Vigue L, et al. Biochemical markers of early and late spermatogenesis: relationship between the lactate dehydrogenase-X and creatine kinase-M isoform concentrations in human spermatozoa. Mol Reprod Dev. 1996;43:495-502. 12. Cayli S, Sakkas D, Vigue L, et al. Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm. Mol Hum Reprod. 2004; 10(5):365-372. 13. Wallimann T, Hemmer W. Creatine kinase in non-muscle tissues and cells. Mol Cell Biochem 1994:133-134. 14. Huszar G, Vigue L, Corrales M. Sperm creatine kinase activity in fertile and infertile oligozospermic men. J Androl. 1990;11:40-46. 15. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed. Cambridge: Cambridge University Press; 1999. 16. Gomez E, Buckingham DW, Brindle J, et al. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl. 1996;17(3):276-287. 17. Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003;79(4):829-843. 18. Huszar G, Sbracia M, Vigue L, et al. Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol Reprod. 1997;56(4):1020-1024. 19. Twigg J, Fulton N, Gomez E, et al. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum Reprod. 1998;13(6):1429-1436.
1299
RESULTS
CONCLUSION
To examine the relationship between biochemical markers and morphologic sperm characteristics, including head, neck, and tail changes. The study evaluated 154 patients who went to the Andrology Laboratory of the Clinic of Urology, Clinical Center of Serbia. Patients were divided into 4 groups: normozoospermic, oligozoospermic, severe oligozoospermic, and asthenozoospermic, according to the sperm concentration and motility. The differences in creatine kinase (CK) and CK-M levels between normozoospermic and the 2 groups of oligozoospermic patients were significantly different (P <.01). The CK and CK-M levels correlated negatively with sperm concentration and sperm motility, but correlated positively with the pathologic sperm form. Patients with CK values >0.093 have a total number of pathologic forms higher than 0.40 (87.5% sensitivity, 77.3% specificity, the area under the curve was 0.832, P <.001). Patients with CK values <0.09 U/L have normal spermatogenesis and pathologic disorder of the head <15%, neck <12%, and tail <10%. The relation between sperm morphology and biochemical markers included in the maturation process is established during the sperm genesis process. If the results of these markers are used together with the morphology of the spermatozoa in the interpretation of infertility, it would lead us to better insight of the fertility potential of the each patient. UROLOGY 82: 1296e1299, 2013. 2013 Elsevier Inc.
T
he parameters of conventional semen analysis (sperm concentration, motility, and morphology) have failed to show a consistent correlation with pregnancy rates.1,2 Therefore, biochemical markers are included as the objective indicator of biologic sperm powers. A number of independent studies have indicated that defective sperm function is associated with elevated levels of certain key enzymes, such as creatine kinase (CK),3-5 lactate dehydrogenase,6 and glucose-6-phosphate dehydrogenase.7 CK levels in human sperm are an objective biochemical marker of sperm maturity and fertilizing potential.3,8,9 Immunocytochemical studies of CK levels in individual spermatozoa have demonstrated that increased CK concentrations reflect residual cytoplasm in sperm that
Financial Disclosure: The authors declare that they have no relevant financial interests. From the Clinic of Urology, Clinical Center of Serbia, Belgrade, Serbia; the School of Medicine University of Belgrade, Belgrade, Serbia; the Center for Medical Biochemistry, Belgrade, Serbia; the Centre of Anesthesiology and Reanimatology, Clinical Centre of Serbia, Belgrade, Serbia; and the Clinic for Gynecology and Obstetrics, Clinical Centre of Serbia, Belgrade, Serbia Reprint requests: Bogomir Milojevic, Ph.D., Clinic of Urology, Clinical Center of Serbia, Resavska 51, 11000 Belgrade, Serbia. E-mail: [email protected] Submitted: April 1, 2013, accepted (with revisions): August 12, 2013
1296
ª 2013 Elsevier Inc. All Rights Reserved
was not extruded during late spermiogenesis.10,11 Interest in CK has been stimulated by studies suggesting that defective sperm function is associated with defects in spermatogenesis that leads to the release of immature spermatozoa from the germinal epithelium.3,7,8,12 Two isoforms of CK can be detected in the sperm: CK-B, which is representing the whole CK activity of the seminal plasma, and CK-M, the isoform of spermatozoa, which is mitochondrial origin.13 The biochemical parameters of CK activity and CK-M isoform ratio were evaluated in several studies.7,8 In couples with oligozoospermic husbands treated with intrauterine insemination, sperm CK activities differed between fertile and infertile men, although the sperm concentration and motility values between the 2 groups were similar or identical. A logistic regression analysis in fertile and infertile oligozospermic men further indicated that, whereas the occurrence of pregnancies correlated with CK activities, the sperm concentration values did not contribute to this correlation.14 The purpose of this study was to examine the relationship between biochemical CK and CK-M markers and morphologic sperm characteristics, including head, neck, and tail changes. 0090-4295/13/$36.00 http://dx.doi.org/10.1016/j.urology.2013.08.031
MATERIALS AND METHODS
Table 1. Correlation analysis of sperm characteristics with creatine kinase (CK) and CK-M activity
Patient Selection Semen samples of 154 men referred for semen analysis to the Andrology Laboratory of the Clinic of Urology, Clinical Center of Serbia were studied. Semen specimen was collected after a requested abstinence of 3-7 days. Median time of abstinence for normozoospermic group was 3.5 0.9 days, for oligozoospermic group was 3.8 0.8 days, for severe oligozoospermic group was 3.6 0.9 days, and for asthenozoospermic group was 3.6 0.5 days. The specimens were allowed to liquefy at 37 C for 30 minutes before evaluation of the sperm characteristics (concentration, motility, and morphology). Semen analysis was performed manually according to the World Health Organization guidelines, and morphology was examined using strict criteria (World Health Organization, 1999).15 According to the sperm concentration and motility, patients were divided into 4 groups: normozoospermic, oligozoospermic, severe oligozoospermic, and asthenozoospermic.
Characteristics Sperm concentration (106/mL) Motility (%) Morphology
Correlation Correlation P with CK-M P with CK (r*) Value (r*) Value 0.653
<.01
0.528
<.01
0.448 0.628
<.01 <.01
0.405 0.636
<.01 <.01
CK, creatine kinase. * Pearson’s correlation coefficient.
CK Estimation The CK values were defined by kinetic Olympus assay (CK test OSR 6279). The sperm cells were prepared using 250 mL ejaculated seminal plasmas extracted 3 times by the repeated flushing of sediment sperm cells with an imidazole (0.15 M NaCl and 0.03 M imidazole at pH 7.0). The residue is rinsed after the last buffer flushing, drained dry, and resuspended in 0.1% Triton X-100 solution with intense vortex stirring for approximately 20 seconds. The sample was centrifuged again, and the supernatant was analyzed for CK activity. The CK activity was expressed as international units/108 spermatozoa. The separation of CK-M isoenzymes from CK-B isoenzymes supernatant was obtained from the previously described procedure, which was performed by passing through a DEAE Sephadex A-50 column. The separation of CK isoenzyme with the help of an ion exchange column: the column is prepared by swelling 1-g Sephadex A-50 in 250 mL buffers at room temperature. The swelling Sephadex is than rinsed 3 times and the result is stored in the fridge at 4 C. A 100 mL of prepared sperm supernatant is added to the Eppendorff centrifuge tube of 50 mL Sephardexes. Seventy-five microliters of supernatants is used to determine the CK activity that originates from the CKM forms. CK-B activity is obtained by subtracting the CK-M activity from the total CK sperm activity. The obtained activity was expressed as international units/108 spermatozoa.
Statistical Analysis Statistical analysis was performed using the SPSS Windows statistical packet. The difference between patient groups was calculated using the Mann-Whitney U test. Correlation between CK and also CK-M with sperm concentration, motility, and morphology was done. Statistical significance was obtained using analysis of variance. Significant differences were accepted with values of P <.05. Receiver operating characteristic (ROC) test was used to determine predictive values of CK activities. As the CK distribution and sperm morphology are not uniform, a log transformation of these parameters was done to reduce the skew level.
RESULTS The differences in CK and CK-M levels between normozoospermic and the 2 groups of oligozoospermic UROLOGY 82 (6), 2013
Figure 1. The receiver operating characteristic test to determine the sensitivity and the specificity of CK-M activities in detecting pathologic sperm maturation, which is defined by disorder in sperm number. (Color version available online.)
patients were significantly different (P <.01). Patients with normozoospermia had CK values of 0.097 0.026 UI/108 sperm cells and CK-M 0.041 0.015 UI/108 sperm cells, whereas patients with oligozoospermia had CK values of 0.46 0.30 UI/108 sperm cells and CK-M 0.11 0.07 UI/108 sperm cells. Patients with severe oligozoospermia had CK values of 1.90 2.2 UI/108 sperm cells and CK-M 0.18 0.1 UI/108 sperm cells, respectively. The CK and CK-M levels correlated negatively with sperm concentration and sperm motility. The CK and CK-M levels correlated positively with the pathologic sperm forms (Table 1). Using the ROC curves, cut-off values were obtained for the CK and CK-M that significantly correlate with the sperm concentration. The calculated cut-off value for CK from 0.093 UI/108 sperm had a sensitivity of 93.75% and a specificity of 86.36%. The area under the curve (AUC) was 0.957 (P <.001). 1297
Table 2. Cut-off values, specificities, and sensitivities and the area under the curve for defining head, neck, and tail sperm pathology Pathologic Sperm Forms
Cut- Sensitivity Specificity P off (%) (%) AUC Value
Head Neck Tail
0.15 0.12 0.12
62.5 67 75
52.5 65 61
0.623 <.05 0.701 <.05 0.671 <.05
AUC, area under the curve.
Figure 2. The receiver operating characteristic test to determine the sensitivity and the specificity of morphology in detecting pathologic sperm maturation, which is defined by disorder in CK activities. (Color version available online.)
Comparing the CK-M values with the sperm concentration, a cut-off value for CK-M with 0.048 UI/108 sperm was calculated with 85.70% sensitivity and 72.73% specificity. AUC below ROC curve was 0.862 (P <.001; Fig. 1). Examining the number of pathologic forms of sperm cells whose presence is the marker of morphologic spermatogenesis disorder, which correlates with the established cut-off values for CK of 0.093, it has been established that patients with CK values >0.093 have a total number of pathologic forms higher than 0.40 (87.5% sensitivity, 77.3% specificity, AUC was 0.832, P <.001; Fig. 2). When the pathologic sperm forms are defined as head, neck, and tail disorders and compared with the cut-off CK values established for normal spermatogenesis with the help of the ROC curve, results are obtained that indicate that patients with CK values <0.09 U/L have normal spermatogenesis and pathologic disorder of the head in <15% (62.5% sensitivity, 52.5% specificity, AUC was 0.623, P <.05), neck <12% (67% sensitivity, 65% specificity, AUC was 0.701, P <.05), and tail <10% (75% sensitivity, 61% specificity, AUC was 0.671, P <.05; Table 2).
COMMENT Previous studies have shown an inverse correlation between CK activities in sperm cells and the number of sperm cells, pointing that there is a metabolic difference between sperm cells in normozoospermic and oligozoospermic group of patients.4,5,16 Such findings indicate a disorder related to sperm maturation, which is why CK 1298
is considered the predictor of sperm maturity.17 Mature spermatozoa show a higher concentration of the CK-M isoform that is expressed only during the last phase of spermiogenesis in elongated spermatids and in mature sperm.4,5,18 Studies suggest that the plasma membrane changes take place in spermiogenesis simultaneously with cytoplasmic extrusion and the expression of the new sperm-specific CK-M.4,18 On the basis of the results of previous studies, one can ask a theoretical question about which marker is better for predicting male fertility, CK, or determining sperm morphology. Measuring CK activities is an objective marker, whereas determining sperm morphology is a subjective marker, subject to major variations depending on the technician. Sperm cells that substantially completed the cytoplasmic extrusions could be subject, during spermatogenesis, to the interruption or halt of biochemical maturation in later development. Thus, biochemical sperm maturation and functional sperm ability can be reduced without any visible changes in sperm morphology, which in some cases explains the disharmony between CK values and the number of pathologic sample forms.17 The analysis of the relationship between the CK and sperm morphology indicates that there is a relationship between raised levels of CK and the increased retention of cytoplasm and morphologic disorders of the sperm head and tail.16,19 We compared the presence of pathologic changes in the head, neck, and tail with the CK and CK-M values in normozoospermic and oligozoospermic patients. The level of maturation is defined by CK and CK-M values, whose cut-off values were established previously. It was shown that CK and CK-M values were increased in patients who had >15% of morphologic changes to the head, >12% present cytoplasmic residue, and >12% of sperm tail disorder. Huszar et al3,9 have shown that there is a relationship between elevated cytoplasmic retention and head disorder, followed by increased number of big, round sperm heads or increased incidence of amorphous sperm heads. It must be taken into consideration that the cytoplasmic residue is sometimes not noticed in native and stained slides, so one gets the impression that the sperm heads are of changed dimensions. It has been shown that sperm tail, which arises during the genesis of immature sperm cells and which has not completed cytoplasmic extrusion, is shorter because of a standstill in sperm genetic development.18 Therefore, in these cases, a short tail and the failed remodeling of UROLOGY 82 (6), 2013
cytoplasm is a cause of reduced interaction with the zona pellucida. The process of sperm genesis was followed by cytoplasmic extrusion and the expression of CK-M isoforms by remodeling the sperm plasma membrane, which facilitates the formation of the space required for sperm and egg cell interaction,18 so that a possibility for a binding-zone site is considered a part of the remodeling process of the plasma membrane in mature sperm cells. If the morphologic changes to sperm cells are regarded in the light of related events in sperm genesis, the connection between sperm immaturity and the increasing number of changes in the sperm head, neck, and tail clearly suggest the connection of morphologic and biochemical parameters. Morphology alone, even following strict criteria does not have a major predictive significance, but morphology with biochemical markers can be used as predictive markers in the assessment of sperm maturity. The difference between cell maturation, which is a gene regulated process, and epididymal maturation, which includes modifications to improve motility, functional membrane integrity to increase resistance to premature acrosome reaction, and efficiency in the female reproductive tract, is important in the maturation process.
CONCLUSION In conclusion, the relation between sperm morphology and biochemical markers included in the maturation process is established during the sperm genesis process. If the results of these markers are used together with the morphology of the spermatozoa in the interpretation of infertility, it would lead us to better insight of the fertility potential of the each patient. References 1. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol. 1991;164:542-551. 2. Bourgeron T. Mitochondrial function and male infertility. Results and Problems in Cell Differentiation. 2000;28:187-210. 3. Huszar G, Vigue L. Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology. Mol Reprod Dev. 1993;34:292-298. 4. Huszar G, Patrizio P, Vigue L, et al. Cytoplasmic extrusion and the switch from creatine kinase B to M isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa. J Androl. 1998;19:11-20.
UROLOGY 82 (6), 2013
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