Androlog Mail:
A PREMATURE REQUIEM FOR THE SPERM CREATINE KINASE TEST
In the April 1998 issue of Fertility and Sterility (69:727, 1998) Rolf
et. al., published an article entitled: "Creatine kinase activity in human
spermatozoa and seminal plasma lacks predictive value for male fertility in
vitro fertilization", which concluded that the total creatine kinase
activity or creatine kinase isoform distribution did not predict male
fertility in IVF treatment. The study was based on data from 73 couples who
were treated with IVF. Although the title suggests that the utility of CK
parameters was evaluated in cases of male infertility, in actuality none of
the couples had male factor infertility. In fact, the 73 men had high mean
sperm concentrations and motilities of approximately 78 million sperm/mL and
54%. Therefore, the selection of subjects and the the semen characteristics
of husbands, prevented the authors from evaluating the utility of the CK
test, as it is intended, for male factor infertility.
In establishing sperm creatine kinase (CK) activity as an objective
biochemical marker of sperm maturity and fertility, at Yale we analyzed
almost 700 samples in four studies(1-4). As opposed to the Rolf study, in
which almost all samples were in the 26-165 million sperm/mL range, we
utilized samples in all sperm concentration ranges and the distribution
above and below 20 million sperm/mL was 1:1. Our data showed that there were
differences in sperm CK activity among men and that there was an inverse
correlation between sperm concentration and sperm CK activity.
With CK-immunocytochemistry of individual spermatozoa we showed that
increased CK activity was related to retained cytoplasm and CK content
confined in the cytoplasmic compartment (5). We hypothesised that increased
cytoplasmic retention signified an interruption of spermiogenesis and
diminished sperm maturity. In sperm that had completed cytoplasmic extrusion
and showed low CK activity, we found a new sperm CK isoform (in addition to
the CK-B) with electrophoretic properties similar to that of the muscle CK-M
isoform. The ratio of the CK-M and CK-B [%CK-M/ (CK-B + CK-M)] thus
predicted the proportions of mature and immature sperm in the samples (4).
We have not yet identified a function for the CK-M isoform but consider it
as a developmental marker, similar to that of the CK-M in muscle which is
expressed only upon commencement of contractile activity. Another example is
the developmental switch from fetal to adult hemoglobin. Three other
laboratories confirmed the value of sperm CK measurements in semen or in
sperm fractions prepared by swim up or gradient centrifugation (6-9).
In subsequent work, we demonstrated the clinical utility of sperm CK
parameters for prediction of male fertility. For instance, in couples with
oligospermic husbands treated with intrauterine insemination, a
subpopulation of 24 fertile and 18 infertile men in the 5-20 million
sperm/mL concentration range had identical semen characteristics (mean sperm
concentrations: 11.9 and 11.9 million/mL; mean sperm motility: 24% and 23%).
The only difference between the men who did or did not achieve pregnacy was
an approximately 100% elevation in sperm CK values in the infertile group,
indicating diminished sperm maturity (3).
The value of CK-M ratio in the assessment of male fertility was shown
in our blinded study of 84 couples treated with IVF at two institutions
(10). All 16 pregnancies occurred in couples with husbands prospectively
classified in the "CK-M fertile" range. If the men were in the CK-M fertile
range and the couple had at least one fertilized oocyte, indicating the lack
of oocyte factor for the infertility, the predictive rate of the CK-M ratio
was 30.4% per cycle. It has became evident that the primary beneficiaries of
the CK test are oligospermic men who by conventional semen parameters would
be considered of diminished fertility, but utilizing the sperm biochemical
parameters can be proven to have sperm with adequate maturity. For these
men, the infertility work up could be shifted to the wife s condition,
and/or treatment could commence thereby alleviating the consideration of
surgical or medical intervention to elevate sperm counts (11).
Why did the CK activity and CK-M ratio did not reflect male fertility
in the Rolf study?
First, In addition to the lack of couples with male infertility in the
study population, the authors were looking for CK activity differences in
the swim up fractions prepared from samples with high sperm concentrations
where we previously showed that none exists. Our first study of sperm CK on
150 samples (1) demonstrated an inverse relationship between the incidence
of immature sperm in semen and the sperm concentration in the samples.
Further, due to the lower incidence of immature sperm (thus higher
representation of mature sperm) in high sperm concentration samples, the
increase in the proportion of mature sperm in the swim up fractions vs. the
semen is narrow. Consequently, in the four sperm concentration groups: <10
million/mL; 10-20 million/mL; 20-30 million/mL and >30 million sperm/mL;
both the mean CK activities and the CK activity differences between the
initial semen and of the swim up fractions progressively declined (CK IU/108
sperm): 1.7 vs. 0.72; 0.69 vs. 0.30; 0.31 vs. 0.19 (P< 0.01-0.001) and 0.098
vs. 0.073 (not different). Thus, Rolf et al., using the high sperm
concentration samples essentially confirmed our previous results regarding
the lack of swim up mediated improvement in the >30 million sperm/mL group.
Second, there is extensive discussion in the Rolf paper how our
laboratory overestimates and reports higher CK activity because of
deficiencies in our sperm preparation methods. Yet, their CK data of 5-9
IU/107 sperm is 1000-fold higher than that of our values. It is either a
methodological inconsistency which needs to be followed up or a calculation
error. If indeed, the sperm CK activity measured in Munster is 5-9 mIU/107
sperm (or 0.05-0.09 IU/ 108 sperm) which is identical to our CK values of
0.07-0.1 in the >30 million sperm/mL group, than Rolf et.al., have also
confirmed that the CK activity data are not different in the Munster and
Yale laboratories thus our sperm preparation methods are competent.
Third, with respect to the CK-M ratios, Rolf et. al., assumed that we
were measuring mitochondrial CK-M despite the fact that we demonstrated that
our "CK-M" is different from that of conventional CK families. These
conclusions were drawn based on the lack of heterodimer formation with the
B isoform, molecular weight and lack of immunological crossreactivity (12).
Moreover, Rolf et.al., determined the mitochondrial CK isoform by a batch
chromatography procedure non-validated with sperm extracts. The bound
activity was considered to be mitochondrial CK but the identity of the
enzyme was not confirmed. This CK activity could have originated from
another isoform or from a mixture of CK isoforms. Further, unlike in our
laboratory where we directly measure both the CK-B and CK-M isoforms for the
CK-M ratio, in Munster the CK-B activity was not actually measured. It was
only a derived value by subtracting the CK activity removed by the
ion-exchange beads from an initial activity. No reproducibility or the rate
of recoveries by this method were reported. Because the Munster group did
measure a CK isoform different from our CK-M, and the methods were not
validated, the CK-M ratio data of the two laboratories are not comparable.
Fourth, there are methodological variations between the Yale and
Munster procedures in the sperm preparation and in the long-term freezing of
the samples. For instance, we expect that the omission of DTT or similar
agents which protect the SH-groups in CK-B, would result in the loss and
underestimation of the CK-B activity and conversely in artificially higher
CK-M/CK-B ratios (whatever CK isoform was actually measured).
We can conclude that the study by Rolf et. al., failed to test the
utility of the CK parameters in the evaluation of male fertility because:
I. there were no couples with male factor infertility among the IVF patient
population;
II. almost exclusive use of sperm samples in the > 30 million sperm/mL
range;
III. failure to consider published data which demonstrated that the
proportion of mature sperm does not improve in the swim-up fractions from
samples with high sperm concentrations;
IV. potential errors in the reported sperm CK activity values, and
measurements of a CK isoform different from the CK-M with methods not
validated in sperm extracts;
V. variances between Munster and Yale in the handling and long-term freezing
of the sperm samples.
We welcome other clinical studies directed to sperm CK parameters and
are available for discussion with colleagues about all aspects of design and
methods. Because we developed an antiserum specific for the CK-M, methods
utilizing immunological approaches will be available.
REFERENCES
1. Huszar G., Corrales M., Vigue L. (1988) Correlation between sperm
creatine phosphokinase activity and sperm concentrations in normospermic and
oligospermic men.
Gamete Research, 19:67-75.
2. Huszar, G., Vigue, L., Corrales, M. (1988) Sperm creatine phosphokinase
activity as a measure of sperm quality in normospermic, variablespermic and
oligospermic men.
Biology of Reproduction, 38:1061-1066.
3. Huszar, G., Vigue, L., Corrales, M. (1990) Sperm creatine kinase activity
in fertile and infertile oligospermic men.
J. of Andrology, 11:40-46.
4. Huszar, G. and Vigue, L. (1990) Spermatogenesis related change in the
synthesis of the creatine kinase B-type and M-type isoforms in human
spermatozoa.
Molecular Reproduction and Development, 25:258-262.
5. Huszar, G. and Vigue, L. (1993) Incomplete development of human
spermatozoa is associated with increased creatine phosphokinase
concentrations and abnormal head morphology.
Molecular Reproduction and Development, 34:292-298.
6. Orlando, C., Krausz, C., Forti G., Casano R. (1994) Simultaneous
measurement of sperm LDH, LDHx, CPK activities and ATP content in
normospermic and oligospermic men.
Int J. of Andrology, 17:13-18.
7. Aitken, J., Krausz, C., Buckingham, D. (1994) Relationship between
biochemical markers for residual sperm cytoplasm, reactive oxygen species
generation, and the presence of leukocytes and precursor germ cells in human
sperm suspensions.
Molecular Reproduction and Development, 39:268-279.
8. Gomez, E., Buckingham, DW., Brindle, J., Lanzafame, F., Irvine, DS.,
Aitken, RJ. (1996) 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. of Andrology, 17:276-287.
9. Sidhu, RS., Yongjin, W., Agarwal, A. (1997) Creatine kinase level and
lipid peroxidation rate in human spermatozoa from patients with cancer.
J. of Assisted Reproduction and Genetics, 14:538-542.
10. Huszar, G., Vigue, L. and Morshedi, M. (1992) Sperm creatine
phosphokinase M-isoform ratios and fertilizing potential of men: A blinded
study of 84 couples treated with in vitro fertilization.
Fertility and Sterility, 57: 882-888.
11. Huszar, G. (1994) The role of sperm creatine kinase in the assessment of
male fertility. Reproductive Medicine Review, 3:179-187.
12. Huszar, G., and Vigue, L. (1997) Creatine kinase (CK-M) isoform in human
sperm: isolation of CK-M and development of a CK-M specific antibody.
Abstract, 1997 Ann. Meeting American Society of Andrology, Baltimore, MD.
Gabor Huszar, M.D.
E-Mail: HuszarGB@MASPO3.MAS.YALE.edu
Fax: (203)-737-1200
This archive was generated by hypermail 2.1.2 : Mon Jun 11 2001 - 22:44:39 CDT