Infertility

The Correlation of Biochemical andMorphologic Parameters in the Assessmentof Sperm MaturityOtas Durutovic, Natasa Lalic, Dragica Milenkovic-Petronic, Nebojsa Bojanic,Dejan Djordjevic, Bogomir Milojevic, Nebojsa Ladjevic, Ana Mimic, Lidija Tulic,Zoran Dzamic, and Sava Micic

OBJECTIVE To examine the relationship between biochemical markers and morphologic sperm characteris-

Financial Disclosure: The authoFrom the Clinic of Urology, Cl

Medicine University of Belgrade,Belgrade, Serbia; the Centre of ASerbia, Belgrade, Serbia; and theof Serbia, Belgrade, SerbiaReprint requests: Bogomir Mil

Serbia, Resavska 51, 11000 BelgSubmitted: April 1, 2013, acc

1296 ª 2013 ElseAll Rights Re

tics, including head, neck, and tail changes.

METHODS 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, oli-gozoospermic, severe oligozoospermic, and asthenozoospermic, according to the sperm concen-tration and motility.

RESULTS 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 levelscorrelated negatively with sperm concentration and sperm motility, but correlated positively withthe pathologic sperm form. Patients with CK values >0.093 have a total number of pathologicforms 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 pathologicdisorder of the head <15%, neck <12%, and tail <10%.

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 usedtogether with the morphology of the spermatozoa in the interpretation of infertility, it would leadus to better insight of the fertility potential of the each patient. UROLOGY 82: 1296e1299, 2013.� 2013 Elsevier Inc.

he parameters of conventional semen analysis(sperm concentration, motility, and morphology)

Thave failed to show a consistent correlation with

pregnancy rates.1,2 Therefore, biochemical markers areincluded as the objective indicator of biologic sperm powers.

A number of independent studies have indicated thatdefective sperm function is associated with elevated levelsof certain key enzymes, such as creatine kinase (CK),3-5

lactate dehydrogenase,6 and glucose-6-phosphate dehy-drogenase.7 CK levels in human sperm are an objectivebiochemical marker of sperm maturity and fertilizingpotential.3,8,9 Immunocytochemical studies of CK levels inindividual spermatozoa have demonstrated that increasedCK concentrations reflect residual cytoplasm in sperm that

rs declare that they have no relevant financial interests.inical Center of Serbia, Belgrade, Serbia; the School ofBelgrade, Serbia; the Center for Medical Biochemistry,nesthesiology and Reanimatology, Clinical Centre ofClinic for Gynecology and Obstetrics, Clinical Centre

ojevic, Ph.D., Clinic of Urology, Clinical Center ofrade, Serbia. E-mail: em2bogomir@yahoo.comepted (with revisions): August 12, 2013

vier Inc.served

was not extruded during late spermiogenesis.10,11 Interestin CK has been stimulated by studies suggesting thatdefective sperm function is associated with defects inspermatogenesis that leads to the release of immaturespermatozoa 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 seminalplasma, and CK-M, the isoform of spermatozoa, which ismitochondrial origin.13 The biochemical parameters of CKactivity and CK-M isoform ratio were evaluated in severalstudies.7,8 In couples with oligozoospermic husbandstreated with intrauterine insemination, sperm CK activi-ties differed between fertile and infertile men, although thesperm concentration and motility values between the 2groups were similar or identical. A logistic regressionanalysis in fertile and infertile oligozospermic men furtherindicated that, whereas the occurrence of pregnanciescorrelated with CK activities, the sperm concentrationvalues did not contribute to this correlation.14

The purpose of this study was to examine the rela-tionship between biochemical CK and CK-M markersand morphologic sperm characteristics, including head,neck, and tail changes.

0090-4295/13/$36.00http://dx.doi.org/10.1016/j.urology.2013.08.031

Table 1. Correlation analysis of sperm characteristics withcreatine kinase (CK) and CK-M activity

CharacteristicsCorrelationwith CK (r*)

PValue

Correlationwith CK-M

(r*)P

Value

Sperm concentration(�106/mL)

�0.653 <.01 �0.528 <.01

Motility (%) �0.448 <.01 �0.405 <.01Morphology 0.628 <.01 0.636 <.01

CK, creatine kinase.* Pearson’s correlation coefficient.

Figure 1. The receiver operating characteristic test to deter-mine the sensitivity and the specificity of CK-M activities indetecting pathologic sperm maturation, which is defined bydisorder in sperm number. (Color version available online.)

MATERIALS AND METHODS

Patient SelectionSemen samples of 154 men referred for semen analysis to theAndrology Laboratory of the Clinic of Urology, Clinical Centerof Serbia were studied. Semen specimen was collected aftera requested abstinence of 3-7 days. Median time of abstinencefor normozoospermic group was 3.5 � 0.9 days, for oligozoo-spermic group was 3.8 � 0.8 days, for severe oligozoospermicgroup was 3.6 � 0.9 days, and for asthenozoospermic group was3.6 � 0.5 days.

The specimens were allowed to liquefy at 37�C for 30 minutesbefore evaluation of the sperm characteristics (concentration,motility, and morphology). Semen analysis was performedmanually according to the World Health Organization guide-lines, and morphology was examined using strict criteria (WorldHealth Organization, 1999).15

According to the sperm concentration and motility, patientswere divided into 4 groups: normozoospermic, oligozoospermic,severe oligozoospermic, and asthenozoospermic.

CK EstimationThe CK values were defined by kinetic Olympus assay (CK testOSR 6279). The sperm cells were prepared using 250 mLejaculated seminal plasmas extracted 3 times by the repeatedflushing of sediment sperm cells with an imidazole (0.15 MNaCl and 0.03 M imidazole at pH 7.0). The residue is rinsedafter the last buffer flushing, drained dry, and resuspended in0.1% Triton X-100 solution with intense vortex stirring forapproximately 20 seconds. The sample was centrifuged again,and the supernatant was analyzed for CK activity. The CKactivity was expressed as international units/108 spermatozoa.

The separation of CK-M isoenzymes from CK-B isoenzymessupernatant was obtained from the previously described proce-dure, which was performed by passing through a DEAESephadex A-50 column. The separation of CK isoenzyme withthe help of an ion exchange column: the column is prepared byswelling 1-g Sephadex A-50 in 250 mL buffers at roomtemperature. The swelling Sephadex is than rinsed 3 times andthe result is stored in the fridge at 4�C. A 100 mL of preparedsperm supernatant is added to the Eppendorff centrifuge tube of50 mL Sephardexes. Seventy-five microliters of supernatants isused to determine the CK activity that originates from the CK-M forms. CK-B activity is obtained by subtracting the CK-Mactivity from the total CK sperm activity. The obtainedactivity was expressed as international units/108 spermatozoa.

Statistical AnalysisStatistical analysis was performed using the SPSS Windowsstatistical packet. The difference between patient groups wascalculated using the Mann-WhitneyU test. Correlation betweenCK and also CK-M with sperm concentration, motility, andmorphology was done. Statistical significance was obtained usinganalysis of variance. Significant differences were accepted withvalues ofP<.05. Receiver operating characteristic (ROC) test wasused to determine predictive values of CK activities. As the CKdistribution and sperm morphology are not uniform, a log trans-formation of these parameters was done to reduce the skew level.

RESULTSThe differences in CK and CK-M levels between nor-mozoospermic and the 2 groups of oligozoospermic

UROLOGY 82 (6), 2013

patients were significantly different (P <.01). Patientswith normozoospermia had CK values of 0.097 � 0.026UI/108 sperm cells and CK-M 0.041 � 0.015 UI/108

sperm cells, whereas patients with oligozoospermia hadCK values of 0.46 � 0.30 UI/108 sperm cells and CK-M0.11 � 0.07 UI/108 sperm cells. Patients with severeoligozoospermia 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 withsperm concentration and sperm motility. The CK andCK-M levels correlated positively with the pathologicsperm forms (Table 1).

Using the ROC curves, cut-off values were obtained forthe CK and CK-M that significantly correlate with thesperm concentration. The calculated cut-off value for CKfrom 0.093 UI/108 sperm had a sensitivity of 93.75% anda specificity of 86.36%. The area under the curve (AUC)was 0.957 (P <.001).

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Figure 2. The receiver operating characteristic test todetermine the sensitivity and the specificity of morphology indetecting pathologic sperm maturation, which is defined bydisorder in CK activities. (Color version available online.)

Table 2. Cut-off values, specificities, and sensitivities andthe area under the curve for defining head, neck, and tailsperm pathology

PathologicSperm Forms

Cut-off

Sensitivity(%)

Specificity(%) AUC

PValue

Head 0.15 62.5 52.5 0.623 <.05Neck 0.12 67 65 0.701 <.05Tail 0.12 75 61 0.671 <.05

AUC, area under the curve.

Comparing the CK-M values with the sperm concen-tration, a cut-off value for CK-M with 0.048 UI/108 spermwas calculated with 85.70% sensitivity and 72.73% speci-ficity. AUC below ROC curve was 0.862 (P <.001; Fig. 1).

Examining the number of pathologic forms of spermcells whose presence is the marker of morphologic sper-matogenesis disorder, which correlates with the estab-lished cut-off values for CK of 0.093, it has beenestablished that patients with CK values >0.093 havea 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-offCK values established for normal spermatogenesis withthe help of the ROC curve, results are obtained thatindicate that patients with CK values <0.09 U/L havenormal spermatogenesis and pathologic disorder of thehead 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).

COMMENTPrevious studies have shown an inverse correlationbetween CK activities in sperm cells and the number ofsperm cells, pointing that there is a metabolic differencebetween sperm cells in normozoospermic and oligozoo-spermic group of patients.4,5,16 Such findings indicatea disorder related to sperm maturation, which is why CK

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is considered the predictor of sperm maturity.17 Maturespermatozoa show a higher concentration of the CK-Misoform that is expressed only during the last phase ofspermiogenesis in elongated spermatids and in maturesperm.4,5,18 Studies suggest that the plasma membranechanges take place in spermiogenesis simultaneously withcytoplasmic extrusion and the expression of the newsperm-specific CK-M.4,18

On the basis of the results of previous studies, one canask a theoretical question about which marker is better forpredicting male fertility, CK, or determining spermmorphology. Measuring CK activities is an objectivemarker, whereas determining sperm morphology isa subjective marker, subject to major variations depend-ing on the technician. Sperm cells that substantiallycompleted the cytoplasmic extrusions could be subject,during spermatogenesis, to the interruption or halt ofbiochemical maturation in later development. Thus,biochemical sperm maturation and functional spermability can be reduced without any visible changes insperm morphology, which in some cases explains thedisharmony between CK values and the number ofpathologic sample forms.17 The analysis of the relation-ship between the CK and sperm morphology indicatesthat there is a relationship between raised levels of CKand the increased retention of cytoplasm and morpho-logic disorders of the sperm head and tail.16,19

We compared the presence of pathologic changes inthe head, neck, and tail with the CK and CK-M values innormozoospermic and oligozoospermic patients. The levelof maturation is defined by CK and CK-M values, whosecut-off values were established previously. It was shownthat CK and CK-M values were increased in patients whohad >15% of morphologic changes to the head, >12%present cytoplasmic residue, and >12% of sperm taildisorder. Huszar et al3,9 have shown that there is a rela-tionship between elevated cytoplasmic retention andhead disorder, followed by increased number of big, roundsperm heads or increased incidence of amorphous spermheads. It must be taken into consideration that thecytoplasmic residue is sometimes not noticed in nativeand stained slides, so one gets the impression that thesperm heads are of changed dimensions.

It has been shown that sperm tail, which arises duringthe genesis of immature sperm cells and which has notcompleted cytoplasmic extrusion, is shorter because ofa standstill in sperm genetic development.18 Therefore, inthese cases, a short tail and the failed remodeling of

UROLOGY 82 (6), 2013

cytoplasm is a cause of reduced interaction with the zonapellucida. The process of sperm genesis was followed bycytoplasmic extrusion and the expression of CK-M iso-forms by remodeling the sperm plasma membrane, whichfacilitates the formation of the space required for spermand egg cell interaction,18 so that a possibility fora binding-zone site is considered a part of the remodelingprocess of the plasma membrane in mature sperm cells.

If the morphologic changes to sperm cells are regardedin the light of related events in sperm genesis, theconnection between sperm immaturity and the increasingnumber of changes in the sperm head, neck, and tailclearly suggest the connection of morphologic andbiochemical parameters. Morphology alone, evenfollowing strict criteria does not have a major predictivesignificance, but morphology with biochemical markerscan be used as predictive markers in the assessment ofsperm maturity. The difference between cell maturation,which is a gene regulated process, and epididymal matu-ration, which includes modifications to improve motility,functional membrane integrity to increase resistance topremature acrosome reaction, and efficiency in the femalereproductive tract, is important in the maturation process.

CONCLUSIONIn conclusion, the relation between sperm morphologyand biochemical markers included in the maturationprocess is established during the sperm genesis process. Ifthe results of these markers are used together with themorphology of the spermatozoa in the interpretation ofinfertility, it would lead us to better insight of the fertilitypotential of the each patient.

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