Creatine kinase activity in human spermatozoa and seminal plasma lacks predictive value for male fertility in in vitro fertilization

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1 FERTILITY AND STERILITY VOL. 69, NO. 4, APRIL 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Creatine kinase activity in human spermatozoa and seminal plasma lacks predictive value for male fertility in in vitro fertilization Claus Rolf, M.D., Hermann M. Behre, M.D., Trevor G. Cooper, Ph.D., Birgit Koppers, Dr.Med., and Eberhard Nieschlag, M.D. Institute of Reproductive Medicine of the University, Münster, Germany Received October 14, 1997; revised and accepted December 2, Supported by the Deutsche Forschungsgemeinschaft Bonn, Germany, Confocal Research Group The Male Gamete: Production, Maturation, Function, grant Ni 130/15 and by the Federal Health Ministry, Bonn, Germany (C.R.). Reprint requests: Eberhard Nieschlag, M.D., Institute of Reproductive Medicine of the University, Domagkstraße 11, D Münster, Germany (FAX: ; MUENSTER.DE) /98/$19.00 PII S (97) Objective: To examine the predictive value of creatine kinase in human spermatozoa and seminal plasma from patients treated in an IVF program. Design: Prospective, blind clinical study. Setting: Male infertility clinic. Patient(s): Seventy-three patients and 32 fathers (age, 45 years). Intervention(s): Determination of creatine kinase activity in seminal plasma, washed spermatozoa, and swim-up purified spermatozoa from patients treated in an IVF program. Main Outcome Measure(s): Creatine kinase activity in seminal plasma, washed spermatozoa, and swim-up purified spermatozoa. Result(s): Creatine kinase activity in washed spermatozoa correlated significantly with normal sperm morphology. No significant correlations were found between creatine kinase activity or creatine kinase isoenzyme ratio in seminal plasma, washed spermatozoa, or swim-up purified spermatozoa and success in the IVF program. The percentage of spermatozoa with normal morphology was significantly lower in patients whose sperm did not fertilize than in patients whose sperm did fertilize oocytes and in the control group of proven fathers. Conclusion(s): Total creatine kinase activity and creatine kinase isoenzyme distribution are not sperm function markers for prediction of male fertility in IVF treatment. (Fertil Steril 1998;69: by American Society for Reproductive Medicine.) Key Words: Creatine kinase, isoenzymes, IVF, fertility, spermatozoal morphology Creatine kinase (EC ) catalyzes the reversible N-phosphorylation of creatine, thus maintaining an immediately accessible energy reservoir in the cell. Creatine phosphate Adenosine diphosphate Creatine kinase O Adenosine triphosphate Creatine Cells requiring high energy, such as spermatozoa, are characterized by high creatine kinase activity. The use of energy by spermatozoa is coupled strictly to its production, and in spermatozoa of some species, creatine kinase has been considered to be the key enzyme in the generation, transport, and use of energy (1). It is generally accepted that there are two creatine kinase isoenzymes in human spermatozoa. Although the presence of CK-B in human spermatozoa is not disputed, the identity of the second creatine kinase species is not fully established. According to Wallimann et al. (2), the second isoenzyme in human spermatozoa is of mitochondrial origin (CK-Mi), but Huszar (3) considers the second isoenzyme, CK-M, to be of cytosolic origin, based on its mobility in agarose gels, which corresponds to the mobility of creatine kinase isoenzymes in muscle. In this article, we have assumed that CK-Mi and not CK-M is present in human sperm, based on our results of agarose gel 727

2 electrophoresis separation of sperm creatine kinase from CK-MM standards. In clinical studies, higher creatine kinase activity has been found in spermatozoa from oligozoospermic men than in spermatozoa from normozoospermic men, but no relationship between sperm creatine kinase and their motility or morphology has been found (4). In another study, spermatozoa displaying low total creatine kinase activity but a high proportion of mitochondrial creatine kinase were more likely to fertilize in IVF than spermatozoa with higher total creatine kinase activity (5). The investigators suggested that the proportion of spermatozoal creatine kinase activity present as the CK-M isoenzyme may be used to predict the fertility of men and to explain some cases of male infertility. Spermatozoa with a high total creatine kinase content were considered to be immature and deficient in various functions, including zona binding and fertilizing (6). To date, a high proportion of sperm function tests fail to predict fertilizing capacity of spermatozoa; thus, new methods for evaluating sperm function are warranted. The percentage of sperm with normal morphology, mean linearity, acrosome status, and sperm zona pellucida binding test were considered to be related to fertilization rates in IVF. Several other sperm function tests provided no additional information about fertilization rates (7). The present study was performed to evaluate the role of spermatozoal creatine kinase in male fertility by comparing the relationship between creatine kinase activity and fertilizing potential of spermatozoa from patients attending the clinic for IVF treatment. MATERIALS AND METHODS Patients and Samples Seventy-three men who, together with their female partners, were undergoing IVF treatment were enrolled in this study. In 56 cases the indication for IVF was bilateral tubal obstruction, in 7 cases the indication for IVF was unilateral tubal obstruction, in 7 cases three or more inseminations had been performed previously without a pregnancy being achieved, in 2 cases two inseminations each were performed, and in 1 case the indication for IVF was immunologic infertility. To clarify the source of enzyme measured, we measured creatine kinase activity in swim-up spermatozoa, sperm washed free of seminal plasma, and undiluted, sperm-free seminal plasma. Because creatine kinase activity was measured in samples remaining after IVF sample preparation, activity could not be measured in seminal plasma, washed spermatozoa, and swim-up purified spermatozoa in all samples. Creatine kinase activity was measured in swim-up purified spermatozoa from 63 patients and in washed spermatozoa from 45 patients. In 41 of these patients, creatine kinase activity was determined both in washed spermatozoa and in swim-up purified spermatozoa. In 36 patients, creatine kinase activity was determined in seminal plasma. Creatine kinase activity was measured in seminal plasma and in washed spermatozoa from a control group of 32 healthy male volunteers 45 years who had fathered at least one child within the last 5 years. Semen Analysis Human semen was obtained by masturbation at the Institute of Reproductive Medicine. Ejaculate analysis was performed according to World Health Organization guidelines (8) and included physical parameters (ejaculate volume, ph, color) as well as analysis of spermatozoal concentration, motility, and morphology. Papanicolaou staining was used for evaluating spermatozoal morphology. Internal quality control of semen parameters was performed according to the methods of Cooper et al. (9). Normospermia was diagnosed in 17 patients, teratospermia in 28 patients, asthenospermia in 8 patients, and oligozoospermia in 5 patients. Thirteen of the proven fathers had normospermia, 14 men had teratospermia, 2 men had asthenospermia and oligozoospermia, and 1 patient had asthenospermia. Fertilization in IVF In 11 of 73 cases no fertilization occurred, in 40 cases fertilization occurred but no pregnancy was achieved, and the wives of 22 patients achieved a pregnancy, leading to a pregnancy rate (PR) of 30.1%. Reagents All reagents for routine creatine kinase determination were obtained from Sigma Chemie, Deisenhofen, Germany, with the exception of DEAE-Sephadex (Pharmacia Biotech, Uppsala, Sweden). The selectivity of the modified creatine kinase assay was checked by electrophoretic separation on precast agarose gels (Helena Laboratories, Beaumont, TX). Processing of Spermatozoa for IVF Treatment All sperm preparations were performed using the swim-up method (10). One milliliter of ejaculate was mixed with 3 ml of Ham s F-10 medium (Flow Laboratories, Irvine, CA) supplemented with human serum albumin (0.3 mg/ml). The sample was centrifuged for 10 minutes at 2,000 g, and the supernatant was discarded. The pellet then was dispersed gently in 2 ml of culture medium and recentrifuged for 10 minutes at 2,000 g. After removal of the supernatant, 1 ml of the culture medium was layered over the pellet and spermatozoa were allowed to swim up for 60 minutes at 37 C in 5% CO 2 in air. Next, 500 L ofthe swim-up supernatant was removed and sperm concentration and motility were determined. 728 Rolf et al. Predictive value of creatine kinase Vol. 69, No. 4, April 1998

3 All patients were treated with the same protocol for IVF-ET as described elsewhere (10). After aliquots of swim-up sperm suspensions were added to the oocytes, the remaining suspension was used for determination of creatine kinase activity. Assay Methods Electrophoretic Separation The ejaculates were processed and analyzed in the manner described by Huszar et al. (5). Preparation of Spermatozoa for Creatine Kinase Determination Creatine kinase activity was determined in seminal plasma, washed spermatozoa, and swim-up separated spermatozoa. Seminal plasma was centrifuged at 2,000 g for 10 minutes at 4 C. The activity in the sperm-free supernatant was measured immediately. For creatine kinase determination in spermatozoa, a proportion of 200 L of the ejaculate was diluted in 10 ml of washing buffer (0.05 M tris(hydroxymethyl)aminomethane [Tris; Sigma Chemical Co., Deisenhofen, Germany] buffer, 0.1 M NaCl, ph 8.0) and centrifuged for 10 minutes at 2,000 g at 4 C and the supernatant was discarded. The pellet then was dispersed gently in 10 ml of washing buffer and recentrifuged for 10 minutes at 2,000 g. After removal of the supernatant, the pellet was solubilized in 100 L of extraction media (ice-cold 1% v/v octylphenolpolyethylene glycolether [Triton X-100; Serva, Heidelberg, Germany] in washing buffer). The spermatozoal membranes were disrupted by vortexing at full speed for 5 minutes. An aliquot of 10 L was taken for determination of sperm count by hemocytometry. Thereafter, the samples were stored at 80 C for a maximum of 5 months until creatine kinase determination was performed. After thawing, the samples were centrifuged at 5,000 g for 10 minutes. The supernatant was transferred to an Eppendorf (Eppendorf, Hamburg, Germany) reaction tube (1.5 ml), and the pellet again was extracted in 100 L ofextraction media. After vortexing and centrifuging the sample at 5,000 g for 10 minutes, we added the supernatant to the supernatant from the first extraction step. This repeated detergent treatment with intermediate freezing-and-thawing procedure allowed determination of most of the spermatozoal creatine kinase activity. Cumulative creatine kinase activity in washed spermatozoa after three extractions was 70% 18, 25% 15, and 5% 2.8 for CK-Mi and 89% 6, 10% 5, and 1% 1.8 for CK-B, respectively (mean SD; n 6). Separation of Isoenzymes To separate CK-Mi from CK-B isoenzymes, an anion exchange batch assay was developed. DEAE-Sephadex A-50 ion-exchanger was prepared by allowing 1 g to swell in 250 ml of washing buffer at 25 C. The buffer was decanted and replaced three times to remove fines, and the slurry so formed was stored at 4 C (10). Fifty microliters of this anion exchange slurry was added to 100 L of spermatozoal extract. After vortexing the sample for 1 minute and incubating it for 5 minutes on ice, we centrifuged the sample at 2,500 g for 5 minutes at 4 C. Seventy-five microliters of purified supernatant was used directly for CK-Mi activity determination. We calculated CK-B activity by subtracting the CK-Mi activity from total creatine kinase activity (11). Creatine Kinase Activity Total creatine kinase activity was measured by spectrophotometry. Enzyme activity was assayed with a commercial kit (creatine kinase [CK] Procedure No. 47-UV; Sigma Chemie, Deisenhofen, Germany). We used a Pye Unicam PU 8610 spectrophotometer (Pye Unicam, Cambridge, United Kingdom) at a temperature of 37 C and a wavelength of 340 nm. The reproducibility was assessed from a pool of five semen samples from three volunteers. The intra-assay coefficient of variation for total creatine kinase was 9%, and for the CK-Mi/creatine kinase total ratio it was 10.5%. The interassay (day to day) variation for creatine kinase activity was 12%, and for the CK-Mi/creatine kinase total ratio it was 13.5%. Statistical Evaluation Linear regression analysis was performed to examine the relationships between creatine kinase activity and creatine kinase ratios and conventional semen parameters. All variables were checked for normal distribution by applying the Kolmogorov-Smirnov one-sample test for goodness-of-fit. When appropriate, the variables were logarithmically or arcsine transformed before analysis to achieve normal distributions. Multifactor analysis of variance was applied for testing significant differences between the study groups. Two-sided P values of 0.05 were considered statistically significant. Differences in PRs in the different study groups were tested by the 2 test. Computations were performed using the statistical software package Statgraphics Plus 5.2 (STSC Inc., Rockville, MD). RESULTS Separation of Creatine Kinase Isoenzymes of Washed Spermatozoa by Electrophoresis We were unable to confirm the electrophoretic method of Huszar et al. (5) to measure creatine kinase isoenzymes in washed spermatozoa. When the method of Huszar et al. (5) was used, high CK-B activity was detectable in sperm pellets from oligozoospermic and azoospermic men. The top panel of Figure 1 shows the creatine kinase activity in the centrifuged pellet from the ejaculate of a nonobstructive azoosper- FERTILITY & STERILITY 729

4 FIGURE 1 Electrophoretic separation of ejaculates prepared according to Huszar et al. (5). The shaded areas indicate the location of the creatine kinase (CK) isoenzyme peaks of the patient s samples. The open areas indicate the location of the creatine kinase isoenzyme peaks of the commercial standard containing CK-MM, CK-MB, and CK-BB. Top panel: Creatine kinase activity in the washed pellet of an azoospermic patient. Major peak CK-BB. Total creatine kinase activity 133 U/L. Bottom panel: Creatine kinase activity in the washed pellet of a normospermic patient. Major peak CK-Mi Mi. Total creatine kinase activity 284 U/L U/L) and those who did not (mean SD, U/L) (Fig. 2). No correlation was found between creatine kinase activity in seminal plasma and conventional semen parameters (sperm concentration, progressive motility, and normal morphology). Creatine Kinase Activity in Washed Spermatozoa of Patients Undergoing IVF and Proven Fathers In the group of proven fathers, the mean creatine kinase activity of washed spermatozoa was U/10 7 spermatozoa (mean SD) and the mean CK-Mi/creatine kinase total ratio was In patients whose spouses were successful in IVF treatment, the mean creatine kinase activity was U/10 7 spermatozoa. In this group, the mean CK-Mi/creatine kinase total ratio was In patients who failed in IVF treatment, the mean creatine kinase activity was spermatozoa. In this group, the mean CK-Mi/creatine kinase total ratio was The differences in creatine kinase activity or CK-Mi/creatine kinase total ratios between the different groups were not significant (Fig. 3). A significant inverse correlation between creatine kinase activity in the washed spermatozoa and the percentage of spermatozoa with normal morphology was found (r 0.50; P 0.03). No significant correlation was found between creatine kinase activity and the sperm concentration or the percentage of progressive motile spermatozoa. No correlation was found between creatine kinase activity and CK-Mi ratios in washed FIGURE 2 mic patient to be mainly CK-B. This demonstrates that the enzyme activity is related not to the presence of spermatozoa but presumably to the contaminating seminal plasma. The influence of creatine kinase activity of seminal plasma originating from epithelial cells, leukocytes, immature germ cells, cell debris, and seminal plasma itself must be considered. In contrast, the bottom panel of Figure 1 shows the creatine kinase activity of an ejaculate from a normozoospermic patient to be CK-Mi because it is separable from the CK-M standard. Creatine Kinase Activity in Seminal Plasma of IVF Patients and Proven Fathers The creatine kinase activity in seminal plasma from fathers of the control group was slightly but not significantly higher than the creatine kinase activity in seminal plasma from the men of the infertile couples (mean SD, U/L vs U/L). No differences in creatine kinase activity in seminal plasma were found between the patients who achieved a pregnancy in IVF (mean SD, Creatine kinase (CK) activity in seminal plasma from fertile and infertile men. Lower and upper margins of the boxes indicate 25th and 75th percentiles, horizontal bars on the whiskers mark 10th and 90th percentiles. In addition, all values are plotted individually. The median is indicated by a line within the box. 730 Rolf et al. Predictive value of creatine kinase Vol. 69, No. 4, April 1998

5 FIGURE 3 Total creatine kinase (CK) activity in washed spermatozoa from fertile and infertile men (A) and distribution of creatine kinase isoenzymes (CK-Mi/creatine kinase total ratio) in these groups (B). Lower and upper margins of the boxes indicate 25th and 75th percentiles, and horizontal bars on the whiskers mark 10th and 90th percentiles. In addition, all values are plotted individually. The median is indicated by a line within the box. spermatozoa. No significant correlation between CK-Mi ratios and any semen parameter was found. Creatine Kinase Activity in Swim-Up Spermatozoa of Patients Undergoing IVF The differences in creatine kinase activity of spermatozoa between the different groups of infertile patients were not significant. In patients whose spouses achieved pregnancy after IVF treatment (n 17), the mean creatine kinase activity was U/10 7 spermatozoa and the mean CK-Mi/creatine kinase total ratio was In patients whose spermatozoa fertilized oocytes but who failed to achieve a pregnancy (n 37), the mean creatine kinase activity was U/10 7 spermatozoa and the mean CK-Mi/creatine kinase total ratio was In patients whose spermatozoa did not fertilize the oocytes (n 9), the mean creatine kinase activity was U/10 7 spermatozoa and the mean CK-Mi/creatine kinase total ratio was (Fig. 4). A significant inverse correlation was observed between creatine kinase activity and CK-Mi ratios in swim-up spermatozoa (r 0.34). In the swim-up purified spermatozoa, no significant correlation between creatine kinase activity or creatine kinase ratio and semen parameters was found. Creatine kinase activity was slightly but not significantly higher in washed spermatozoa than in the swim-up purified spermatozoa (Figs. 3 and 4). The CK-Mi ratio was in the same range in both samples. Morphology of Sperm and IVF Results The percentage of spermatozoa with normal morphology was significantly lower in patients whose spermatozoa did not fertilize eggs in vitro than in patients whose spermatozoa did and in the control group (Table 1). The difference in sperm morphology between patients whose spermatozoa did not fertilize and patients who achieved a pregnancy was not significant, probably because of the relatively small number of patients. Sperm concentration and motility were not correlated with the outcome of IVF treatment. DISCUSSION The dimeric creatine kinase molecule consists of two distinct polypeptide subunits, designated B (brain type) and M (muscle type). Creatine kinase molecules thus are composed of either two identical subunits (CK-MM or CK-BB) or a pair of different subunits (CK-MB). In mitochondria, another CK dimer (CK-Mi-Mi) occurs (12). In human spermatozoa, CK-B is localized in the sperm tail by indirect immunofluorescence staining and CK-Mi is localized in the mitochondria of the midpiece region by direct immunogold staining (2). This compartmentalization of isoenzymes is consistent with the postulated function of creatine kinase of providing energy for sperm motility. The B isoenzyme generates adenosine triphosphate (ATP) from creatine phosphate and adenosine diphosphate close to the site of use by flagellar dynein ATPases. For this system to be effective, another isoenzyme of creatine kinase is required in the sperm midpiece, one that generates creatine phosphate from mitochondrial ATP. This is the mitochondrial isoenzyme CK-Mi. This high-energy shuttle has been shown to operate in human spermatozoa with lactate as the sole energy source (13). FERTILITY & STERILITY 731

6 FIGURE 4 Total creatine kinase (CK) activity in swim-up purified spermatozoa from patients with different IVF outcome (A) and the specific distribution of creatine kinase isoenzymes (CK-Mi/creatine kinase total ratio) in these groups (B). Lower and upper margins of the boxes indicate 25th and 75th percentiles, and horizontal bars on the whiskers mark 10th and 90th percentiles. In addition, all values are plotted individually. The median is indicated by a line within the box. In contrast to Wallimann et al. (2), Huszar (3) considers creatine kinase in spermatozoa to be cytosolic and not of mitochondrial origin, based on its mobility in agarose gels, corresponding to mobility of the creatine kinase isoenzymes in muscle. In 1988, Huszar et al. (4) found an inverse correlation between creatine kinase activity in spermatozoa and sperm concentration; no relationship between sperm creatine kinase and motility or morphology was found. In 1994, the same group (6) reported an inverse correlation between creatine kinase activity and morphology. In 1994, Huszar et al. (6) explained this new finding as being due to incomplete shedding of cytoplasm before release of the spermatozoa from the Sertoli cells, and a correlation between creatine kinase activity and sperm midpiece area was reported. This was supported by the lower level of creatine kinase activity of mature spermatozoa after separation on a Percoll gradient found by other groups (14 16). The role of CK-MM in sperm function has not been demonstrated, and a cytoplasmic location for the isoenzyme is at variance with a role in sperm motility. CK-B has been identified immunocytochemically in the cytoplasmic droplet of immature spermatozoa; these sperm are characterized by poor motility; in contrast, mature spermatozoa have shed the droplet and have very little cytoplasma yet are highly motile. In contrast, in our study no difference in creatine kinase activity was found between washed spermatozoa and swim-up purified spermatozoa. These observations can be explained in part by the different spermatozoal separation TABLE 1 Ejaculate parameters of patients undergoing IVF and proven fathers. Group Ejaculate volume (ml) Sperm concentration (10 6 /ml) Sperm motility (%) Normal morphology (%) Proven fathers (n 32) Patients and spouses achieving fertilization and pregnancy in IVF program (n 22) Patients achieving fertilization in IVF program (n 40) Patients achieving no fertilization in IVF program (n 11) * Note: Values are means SD. * P 0.05 (vs. groups of proven fathers and vs. couples with ET after IVF). 732 Rolf et al. Predictive value of creatine kinase Vol. 69, No. 4, April 1998

7 techniques (swim-up vs. Percoll), but the fact remains that different sperm subpopulations were separated. Only the CK-B isoenzyme is found in seminal plasma (17, 18), and this isoenzyme is several times higher than that in serum, suggesting a local production in the male genital system and a multiglandular origin (17). When creatine kinase activity in spermatozoa is being determined, contamination with creatine kinase activity from seminal plasma must be eliminated. In this study, the extraction method of Huszar et al. (5) was not adequate to separate seminal CK-B from CK-B of spermatozoa. Therefore, a revised extraction method was developed to eliminate such contamination and increase the yield of creatine kinase extracted from spermatozoa. Seminal debris from azoospermic ejaculates still contained CK-B. In normozoospermic ejaculates, the greatest proportion of CK isoenzymes was separable from CK-M and was considered to be CK-Mi, for which no electrophoretic standards are available. Asseo et al. (17) found statistically significant differences in creatine kinase activity in the seminal plasma of men with normospermia and oligoasthenospermia, yet with wide overlapping of the individual values. In our study, we did not find statistically significant differences in creatine kinase activity between the groups. In the present study, a significant inverse correlation was demonstrated between creatine kinase activity in washed spermatozoa and the percentage of spermatozoa with normal morphology, an observation in agreement with the results of other groups (14, 15). Immature sperm with high-intensity creatine kinase immunostaining are deficient in oocyte recognition and binding in vitro (6). In 1992, Huszar et al. (5) suggested that the CK-MM ratio of human spermatozoa is predictive of fertilizing potential, independent of sperm concentrations. In a blind study with 84 infertile couples, pregnancies occurred only in patients with CK-MM ratios 10% and a significantly lower oocyte fertilization rate was observed in the group of the infertile patients (patients with 10% CK-M). From these observations, the investigators concluded that creatine kinase is an objective biochemical marker for sperm function. Our study did not confirm these results. We found a considerably higher proportion of CK-M (CK-Mi) than did Huszar et al. (5). In all samples of washed, as well as swim-up, spermatozoa, the proportion of CK-Mi exceeded 10%, although this was set as the threshold value for high likelihood for fertilization by Huszar et al. (5). The difference can be explained on methodological grounds, because in our study, contamination from creatine kinase in seminal plasma was eliminated by repeated washing and the extraction of CK-Mi was improved by repeated extraction with higher detergent concentration. In previous studies (10), we demonstrated that female partners of patients with reduced sperm counts, and especially reduced sperm motility, were not likely to become pregnant after IVF treatment, and therefore intracytoplasmic sperm injection was performed. Consequently, we could not evaluate the influence of severely reduced motility or sperm count on fertility. We cannot exclude the possibility that in patients with poorer conventional semen quality, significant differences between swim-up purified spermatozoa and washed spermatozoa may exist. However, determining creatine kinase activity or CK-Mi ratios provides no further information for patients with good conventional semen parameters and good chances for success in IVF. In 1996, Aziz et al. (19) confirmed that a detailed morphology assessment of sperm (average number of deformities per sperm) is a reliable predictor of the outcome of fertilization in vitro. A so-called creatine phosphate shuttle has been demonstrated in sea urchin spermatozoa (20). Given that the spermatozoon s mitochondria and tail are the sites of ATP production and consumption, respectively, and creatine kinase inactivation inhibits respiration and affects the pattern of sea urchin spermatozoa motility (20), creatine kinase activity may reflect sperm function. On the basis of our previous in vitro experiments, creatine kinase was not considered essential for motility of human spermatozoa, because energy can be supplied by glycolysis when glucose is available, the production of ATP by glycolysis and respiration being independent and complementary (13). Thus, the theory of creatine kinase as a sperm maturity marker is acceptable only when a mitochondrial origin of the second creatine kinase isoenzyme is assumed. In summary, our study could not confirm a relationship between creatine kinase activity of sperm and in vitro fertility. In addition, there was no correlation between fertility potential (percentage of oocytes fertilized or PRs) and the creatine kinase activity of seminal plasma, washed spermatozoa, or swim-up purified spermatozoa and between fertility potential and CK-Mi/creatine kinase total ratio. Therefore, we conclude that creatine kinase cannot be used as a sperm function marker for predicting male fertility in IVF treatment. Acknowledgments: The authors thank Ms. Barbara Hellenkemper and Ms. Heidi Beering for technical assistance in routine semen analysis. References 1. Wallimann T, Hemmer W. Creatine kinase in non muscle tissues and cells. Mol Cell Biochem 1994;133/134: Wallimann T, Moser H, Zurbriggen B, Wegmann G, Eppenberger HM. Creatine kinase isoenzymes in spermatozoa. J Muscle Res Cell Motil 1986;7: Huszar G. The role of sperm creatine kinase in the assessment of male fertility. Reprod Med Rev 1994;3: Huszar G, Corrales M, Vigue L. Correlation between sperm creatine FERTILITY & STERILITY 733

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