Effect of MennInfertility on Serum Creatine Kinase Activity

mThe present study was carried out on 60 patientsm with male factor infertility comparedmwith 60 healthy controls, their ages ranged betweenn20-56 years in Maternity and Childhood Teaching Hilla Hospital and in a biochemistry laboratory of Pharmacy college of Babylon university, between Junem2014 – Junuary 2015. The purpose of this study is to investigate CK activity, serum creatine,mcreatinine levels and semen parametersmin infertile men only ,infertile with DM, smoking ifertile,nand hypertention with infertile patients. The present resultsbshowed a statistically significant deferences (p<0.05) in creatine kinasehactivity, creatine and seminal characteristics while nonmsignificant in creatinine and in Abnormal sperm morphology % . There were statistically significant deferences betweenkthe biochemical and seminal parameters inodiabetes , hypertension and smoking patients and tomcontrol group at a p valuem(P<0.05). There were no observed significantkdifferences in creatinine and Abnormal sperm morphology % of smokerskcompared to control group (P>0.05). The same resultskwere obtained in diabetic and hypertensionkwhen compared tojcontrol group (P<0.05). Our resultsjindicated that thejdiabetes , hypertension and smoking reducelserum CK creatinine and semen parameters inkmale infertility . Enzymetic activity of CK inkserum is a biochemical marker in determining infertility and this biochemicalkmarker will represents an important diagnostic feature withmseminal parameters in


Introduction
Infertility is a medical condition characterized by ankinability men to cause pregnancy in akfertile women.Approximately 50% ofiinfertile couples are related to male factor.Infertility iskcommon among couples with childbearinguage (Sidhu et al., 1998).Infertility is a growingiproblem in the world.In 2010, an estimatedo48.5 million couples worldwideuwere infertile (Mascarenhas et al., 2012).Theietiology of male factoroinfertility is poorly understood.Maleofactor infertility is a commonocondition with unknownpetiology in most of the cases .One of the reasonspthat lead to infertility in men is theoabnormality of Sperm , as well as a number of diseasespand lifestyle-related and the effect onofertility are linked to and the effect onpfertility in men, such as obesity, diabetesland smoking (Hirsh , 2003;SANDRO et al., 2012;Gaur et al., 2007).Creatine kinase (CK) is a mitochondrialpand cytosolic enzyme.This enzymeocatalyzes the conversion of creatine toiphosphocreatine and consumes adenosineotriphosphate (ATP) and adenosine diphosphateo(ADP) as follows: (Oda et al.,2010)The CK enzyme is distributed in variousporgans and cell types such as: brain, spermatozoa, mskeletal, heart muscle, retina , hair cellkof the inner ear,osmooth muscle, and nervouspsystems (Maysoon, 2012).CK is an important enzyme in tissueocell that consumespATP rapidly.This enzyme suppliesoATP to theosperm.Its biological role is to provide an ATPpbuffering system for tissues that requireplarge amounts of energy (Ghassan and Hedef, 2009).Many Studies show that the phosphoryl creatine and ATPpshuttle are important energyisources for sperm (Miyaji et al., 2001).Thus CK is an important enzyme inpsperm.Serum creatinine is an important indicator of renalphealth.Creatinine is producedpvia a biological system involvingocreatine, phosphocreatine , and adenosine triphosphate.It is removed from the blood by thepkidneys (Allen et al., 2012).Creatine is synthesized primarily in the liver and thenotransported through blood to the other organs,pmuscle, and brain, where, throughpphosphorylation, it becomes the high-energy compound phosphocreatine (Taylor, 1989).Creatine conversion toiphosphocreatine is catalyzed by creatineikinase; spontaneous formation of creatinineioccurs during the reaction (Mcleish and Kenyon, 2005).
A centralocompound in the energy metabolism of cells in tissuespwith a highly fluctuating energy demand isuCreatine.The non-enzymaticoconversion of creatine toucreatinine, which is finallyoexcreted in urine, the creatine bodyopool must be maintained by de novo synthesispand nutritional intake.The de novopsynthesis is mainly localized topliver, kidney, and pancreas (Wyss and Kaddurah-Daouk, 2000).The creatine/phosphocreatine systempis an essential part forjcellular phosphate coupled energypstorage and production, especially in tissuesosubject to high metabolickdemands.This system is important to transfer energyofrom mitochondria to thejiflagellum, which is essentially for the swimming ofpsperms.Therefore, we propose that CK has anpimportant role in spermomovement.The aim of this study was topdetermine the mean concentration of serum CKoand semen parameters of infertilelmales and to compare the result of serum CKoconcentration with semen parameterslbetween infertile males and healthy normal fertile volunteersl(control group).This research was trying tokexamine differences inoserum CK , creatine and creatinine levelsk between normal healthy donors and infertileppatients with various diagnoses , to determinelthe link between these levels and theoquality of sperm in DM , smoking and hypertentionpinfertile males.

Subjuctoselection
The studyywas done during the period fromoJune 2014 to Junuary 2015.All mesurmentspwere done in a clinical biochemistry laboratory of college Pharmacy, university of Babylon.The study sample includep60 infertile patients agedofrom 20 to 56 years , and 60 apperantly healthyovolunteers in the same ages range as control group.This study involved patientsiand healthy subjects werekinvistigated for the enzymes activity of CK , creatine, creatinine kand semen analysis .
CK was determinedkaccording to Biolabo manufacturekkit [CK-NAC].The principle of kit is enzymeticimethod described by Oliver andjmodified by Rosalki and later by Szasz (Szasz et al., 1976).Creatinine wasudetermined according to Biolabojmanufacture kit (Jaffe's reaction , colorimetricureaction) and involving thejalkaline sodium picrate method, is the widelyhaccepted for creatinineomeasurement (Tietz, 1999).Creatine was determined by The nonenzymaticlmethod genemlly employed innbiological samples lack specificity, because ofointerference from sever& compounds onormally present.The determination of creatinine andocreatine in serum is based on theiJaffh reaction after conversionoof creatine into creatinine.The mathmaticall relationobetween creatine and creatinine is described byoratio of molecular weight of creatine to molecularuweight of creatinine.

Semen collectionoand preparation
Samples of semenoejaculate were collected from allpmarried patients and volunteers in laboratorypof Maternity and Childhood Teaching Hilla Hospital, and brought within 20 minutsointo a clean aseptic vails.Afteriejaculation the specimens were allowed tooliquefy at 37°C for 30 minutes before theosperm characteristics (concentration, motility,oand morphology) were evaluated.seminalofluid analysis was performed to measure spermlconcentration, sperm morphology,osperm motility in accordance with the recommendationspof the World HealthpOrganization (WHO) (World Health Organization, 1999).Seminal plasmapwas separated by centrifugation atp2000 x g for 10 minutes at roomptemperature.The supernatantowas removed immediately andpkept in 20ºC.The specimen and allkmicroscopic andumacroscopic examinationsjwere examined according to WHO criteria.
The semen samples were liquefiedjafter 30 minuts at room temperature.Semen samples werepplaced at 37°C for liquefaction,pfollowed by routine semen analysis, and theoremaining semen samples wereocentrifuged at 500xgofor 30 minutes.oTheupper layer seminalpplasma was collected for theodeterminations of biochemical markers.Theosupernatant was measured by apcentrifuge test tube and it was used foroenzymatic measurements (Dandekar and Parker ,1999).

Statistical analysis.
All data collectedpfrom patients and control groupspwere analysed using SPSS programoversion 15.The data were analysed as mean andpstandard deviation (SD), also the significantovalue was examined as p<0.05.

Results and Discussion:
The analysed datapfor serum of infertile patients showp a decrease in both CK activity and creatinepconcentration with respect topcontrol group, while there is no changepin creatinine conc., as shownoin table (1).It seems that theoCK activity decreaseofrom 98.45+.20.595 in control group to 70.5 + 6.128 IU/L in patients group, at the same timepcreatine conc.wasdecreasedpfrom 0.908+0.123to 0.76 + 0 .157 in both control and patientogroups respectively ,The resultsoin table(1) indicate significantodifference at (P<0.05) .No availableidata are present to compare ouroresults with other studies.The activity of creatine kinaseo(CK) in serum has been observed in akvariety of clinical conditions.These results may beifound as a consequence of diminishedoefflux of the muscleienzyme in serum from reducedophysical activity causedpby illness or advanced agehor may result from reducedomuscle mass accompanyingomuscle wasting orocachectic states (Sidney ,1998).

Table (1):
The present resultspin Table (2) showed a comparison betweenoseminal characteristics inoinfertile male and control groups .The presentodata revealed that there was a statisticallyosignificant differences (P<0.05) of sperm cellocount per 1 ml of seminal fluidoand seminal fluid volumepwhen compared with controlpgroup.Also differences are seenoin the other seminal parameters aspwell: Sperm active motility, Sperm sluggishomotility, Normal sperm morphologyp% and sperm concentration per 1 ml whenpcompared with control group.Therepare no significant differencespin Abnormal sperm morphologyopercentage when compared withocontrol group.semen analysis is anoessential of the laboratory evalutionpof the infertile men and it still provides thepfundamental information on which cliniciansobase their initial diagnosis , so it is imperativekthat it is performed asoaccurately as possible.
The results in the table (2) showed aocomparison between seminal characteristicsoin infertile male and control groups .Sperm concentration,psperm cell count, percentage ofpSperm active motility , Spermosluggish motility and seminal fluidovolume in sperm decreased differences at (P<0.05), and Normalosperm morphology percentagepwas significantly higer than contrologroup.There are noosignificant differences in abnormal spermomorphology percentage when compared withocontrol group.The seminal analysis andu that CK activity in serum have help toodefine the severity of the maleofactor.A low spermocount and the decreasedoin thepsperm motility (movement)owhich indicates the Sperm abnormalities.ouroresults agreed with otherostudies (Sallmen et al., 2006;Magnusdottir et al., 2005;Ahmed et al., 2012).Thereois no significant effect aboutothe sperm abnormality, so we suggestofor more future study using otheroparameters .They are a critical factor inomale infertility.
More thano90% of male infertility cases are due toolow sperm counts, poor spermoquality, or both.The remaining casesoof male infertility can bepcaused by many conditions, including anatomicalpproblems, hormonal imbalances, andpgenetic defects.Aging also canoreduce sperm counts andpmotility.If less than 40%pof sperm are able to move inpa straight line, the condition ispconsidered abnormal.Sperm that movepsluggishly, these results may be due topgenetic or other defects that render thempincapable of fertilizing theoegg.Poor sperm motilitypmay be associated with DNApfragmentation and may increase theprisk of passing on geneticpdiseases.(3) showed there wereosignificant deferences observedpbetween the biochemical andoseminal parameters in diabetic , hypertentionoand smoking patients with compared to controlpgroup at p value (P < 0.05).There werepno observed significant differences inkcreatinine and abnormal spermomorphology % of smokers, diabetic and hypertentionkwith compared to control group (P < 0.05).The main cause of male infertilityhis low semen quality.In men who have infertilitykcan be caused by low sperm countkdue to endocrine problems, drugs, jradiation, or infection.There may be testicularkmalformations, hormoneoimbalance, or blockage of theoman's duct systemo (Mishail et al., 2009).
Ourpresults showedo that diabetes can leadk to reduced sperm quality due toodeficiencies in the semen bquality is used as an himportant measure of male infertility.Our jfindings were agree with Garcia-Diez et al.was fstated that type 1 diabetes mellitusu(insulin-dependent) lowers seminal fluid volume,rthe concentration, motility, and the rproportion of normal shape spermatozoay (Garcia-Diez and Corrales-Hernandes, 1991).Sexual dysfunction in all its fforms (reduced erection, fimpotence, and other libido dissociations) fis an accompanying phenomenon dof the diabetic disease.Testicular fdysfunction, impotence, ddecreased fertility potentialfand retrograde fejaculations are conditions thatjhave been described in diabetichmales.Diabetes is also the mostucommon cause of erectileudysfunction in men.
Poor semen qualityuhas also been reported in udiabetic men, including decreased sperm motilityhand total count.Becauseosexuality and fertility are important aspects inothe lives of individuals andpcouples, andmconsideringpthatnoverm177mmillionoindividualsmworldwidepsuffermfromoDia betes (Agbajemetmal., 2007).Thismstudyphighlighted the diabetes causepimplications for sexualpproblems.Diabetes is a uwell-recognized cause of male sexual hdysfunction , which in itself may contribute to ysubfertility.The results of this hstudy showed a decrease in semenhvolume, sperm count, and spermkmotility in smokers infertilityopatients compared with non-smokers.pSmokinghas been caused ofodeath in our society andjthe most important public health issueoof our time and Tobaccoosmoking is killing 1pin 10 adults in worldwide (Ng et al., 2014).
Lifestyleofactors such as smokingjand substance abusejcan lead to problems withufertility in men.Chemicals such as : hnicotine, cyanide, and ncarbon monoxide, in ncigarette smoke effect on the rate of sperm.Maleusmokers can sufferpdecreased sperm qualityowith lower counts (numbers ofksperm) and motility (sperm'skability to move) .Smokingjmight also decrease theksperm's ability to fertilizekeggs that's mean may causeiinfertility (Dai et al., 2015).
A number of studiesnhave shown that theuharmful products in tobaccokdamage the testiclesiand kill sperm (Thompson and Bannigan, 2008 ;Agarwal et al., 2005;Robbins et al., 2005).Manyjstudies were similar touour study in that smoking reducesjsemen quality (Zhang et al., 2000;Gaur et al., 2007).The findings of thiskstudy shown that CK activity inkserum , sperm cells andktotal semen significantlyjdecreased with smoking.iThepresent study has beenisuggested that harmful componentsiof tobacco smoke areiable to pass through the blood-otestis barrier andmdamage the sperm.
These resulse indicated that exposure tojsmoke can diminish spermjmotility via inhibitionoof CK activity andicreatine.As sperm motilityudepends on intact mitochondrialjfunction and energy levels.Thus reducediintracellular creatine stores may contribute toidecreased sperm motility leading tokmale infertility.Theifindings in our study showed thatkthe sinificant deferencesiof CK activity in serum, sperm counticells, volume ,creatine, concentration iand normal morphology in patient with hypertensionu(high blood pressure) and DM , butiabnormal sperm morphology %uand creatinine showed noudeferences in those patients.jSeveralstudieshdemonstrated that the hypertension in menjcould be associated withjimpaired reproductiveupotential (Fogari et al., 2002).Finallyj, from thisuviewpoint , the presentustudy suggests that it isunecessary tofocus on thekpossible effects of DM , smokingkand hypertention as anketiology of maleuinfertility in men and the cause of reducedifertility may accompanied in a fact of existinguto the decrease concentration ofkserum CK and semenkquality .

Conclusion
Enzymatic activity of CK in serum is anjimportant biochemicaljmarker in determiningjinfertility and this biochemical marker isjrepresents an important diagnostic feature.hWefound from the biochemicaljand seminal parameters in the diabetic ,jhypertention and smoking causejimpaired sperm quality and CKklevels in male.As ajconsequence, this effect mayjbe one of the severalkimportant factors that possibly causekinfertility in male.This research waskperformed to discuss theirelation between diabetes,ihypertension and smoking with malekinfertility.In thisistudy we found that thereuwas a decrease in the sperm qualityjand CK levels inkserum.

value less than P < 0.05
Abnormal SpermoMorphology refers to shape andpstructure.Abnormally shaped sperm cannotofertilize an egg.In our findings ,othere are nopsignificant differences in Abnormalpsperm morphology % when compared withocontrol group, that's meanjno Abnormally shaped sperm .Lower amountspof volume and concentration canpbe a sign of prostate problems, blockage,nor retrograde ejaculation.Abnormal results may suggestpprostate gland problems orplack of sperm.The volume of the semen sample,papproximate number of total spermpcells, sperm motility, and % of sperm withpnormal morphology are measured.Thisois the most common type ofpfertility testing