1 Department of Urology Graduate School of Medicine and Pharmaceutical Sciences for Research University of Toyama2630 Sugitani Toyama-shi Toyama 930-0194 Japan
2Department of Medical Biology Graduate School of Medicine and Pharmaceutical Sciences for Research University of Toyama2630 Sugitani Toyama-shi Toyama 930-0194 Japan
Correspondence should be addressed to Akira Komiya komiyamedu-toyamaacjp
Received 20 February 2014 Accepted 16 June 2014 Published 3 July 2014
Academic Editor Emilio A Martinez
Copyright copy 2014 Akira Komiya et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility Thirty malepatients who visited our Male Infertility Clinic were enrolled These patients underwent conventional semen analyses Acrobeadstests and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads testscoring after 24 hours of incubationThe presence of acrosome reactions was evaluated using the Acrobeads test The spermatozoawere classified into three groups (I) those bound to MH61-beads (II) motile spermatozoa that did not bind to MH61-beadsand (III) immotile spermatozoa that did not bind to MH61-beads The percentage of spermatozoa with large nuclear vacuoles(LNV) was compared between the three groups The degree of sperm nuclear vacuolation was evaluated in 17992 ejaculatedspermatozoa The mean LNVs were 24 in group I 58 in group II and 98 in group III These values were significantlydifferent from each other (119875 lt 0001 paired t-test) There were no correlations between the LNV values and the Acrobeadsscores In conclusion the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa andspermatozoa with maintained motility and higher in the immotile spermatozoa that did not bind to MH61-beads
1 Introduction
In the field of assisted reproductive technology (ART) par-ticularly that involving intracytoplasmic sperm injection(ICSI) one should select the single bestmotile spermatozoonalthough the ability to identify fertile spermatozoa under alight microscope without denaturation is limited [1ndash4] Inthis context Bartoovrsquos group reported that the ultramor-phological status of subcellular organelles in the head issignificantly associated with the outcomes of natural and IVFfertilization [5 6] This group further developed a methodto assess the detailed morphology of motile spermatozoain real time at a high magnification using a light micro-scope the motile sperm organelle morphology examination(MSOME) used in the field of ART [7] The examination isperformed in real time using an inverted light microscope
equipped with high-power Nomarski differential interfer-ence contrast (DIC) optics enhanced by digital imaging toachieve a magnification up to 6300times MSOME has yielded amore strict definition of normal spermatozoa than conven-tional semen analyses and new abnormalities particularlysperm head vacuoles have been described [8] The high-magnification observation technique has been adapted toselect the best spermatozoa for oocyte injection introducinga new technique named intracytoplasmic morphologicallyselected sperm injection (IMSI) Morphological normalcy ofthe spermnucleus as defined byMSOME is significantly andpositively associated with both the rate of fertilization and thepregnancy outcomes [8] In addition the rate of pregnancyafter IMSI is significantly higher than that observed followingroutine ICSI procedures (660 versus 300) [9] Morerecently the results of ICSI with sperm selected according to
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 178970 8 pageshttpdxdoiorg1011552014178970
2 The Scientific World Journal
conventional light microcopy or MSOME have been contro-versial and the technique is thought to be time-consumingHowever a recent meta-analysis showed a trend towards animprovement in IMSI outcomes versus that obtained withICSI [10] To date teratozoospermia has been proposed to bea preferential indication forMSOME and IMSI [11] while theonly confirmed indication for IMSI is recurrent implantationfailure following ICSI [12]
Numerous vacuoles have been identified inside the spermnucleus under a light microscope at high magnifications It isgenerally accepted that vacuolated spermatozoa are classifiedas having an abnormal morphology [13 14] AmongMSOMEparameters the presence of a sperm nuclear vacuole is one ofthe most important findings Recent results have shown thatthe presence of sperm nuclear vacuoles is negatively corre-lated with the rates of fertilization pregnancy and implan-tation [7 8 15ndash18] Sperm vacuolation has also been reportedto be negatively related to the parameters in conventionaland computer-assisted semen analyses (CASA) [14 19]Thesefindings suggest that the observation of sperm vacuolationcan be used to predict the sperm function
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of sperm functionTo date the results of investigations regarding the origins ofvacuoles have been controversial and while some authorsreport vacuoles to originate within the nucleus others reportan acrosomal origin It has been suggested that at leastsome nuclear vacuoles are of acrosomal origin [20 21] Inaddition sperm head vacuoles are thought to be produced atearlier stages of spermmaturation and that normal acrosomereactions are more likely to be induced in spermatozoa with-out large nuclear vacuoles [22] In this context the Acrob-eads test can be used to evaluate acrosome reactions insperm using amonoclonal antibody that binds to the anteriorportion of acrosome-reacted sperm [23 24]
The purpose of the present study was to further inves-tigate the relationship between sperm nuclear vacuolationand the sperm functions including acrosome reactions byconducting Acrobeads tests and assessments of the spermmotility status after 24 hours of incubation by a high-magnification microscope
2 Materials and Methods
The Institutional Review Board of the University of Toyamaapproved this study (number 23-128) Ethical consent for thework to be carried out was provided and signed informedconsent was obtained from each patient evaluated in thisstudy The study conformed to the principles outlined in theDeclaration of Helsinki
21 Sample Collection We enrolled 30 male patients whovisited the Male Infertility Clinic at Toyama UniversityHospital The patient ages ranged from 26 to 49 years witha mean of 36 years (plusmn57 standard deviation) The durationof infertility ranged from 7 to 105 months with a mean of 37months (plusmn27 standard deviation) The semen samples werecollected following masturbation from infertile male patients
who visited the Male Infertility Clinic at Toyama UniversityHospital The semen samples were collected after at least fivedays of abstinence allowed to liquefy at room temperatureand evaluated within one hour of collection using manualconventional semen analyses [25] which were performedas previously described [26] All manual assessments wereperformed by a single experienced laboratory technician(Y K) and the sperm concentrations were assessed usingan improved Neubauer hemocytometer The samples werediluted according to the instructions of the WHO laboratorymanual (1999) [25] To determine the degree of spermmotility a 10 120583L sample was loaded onto a clear slide glassand covered with a 22 times 22mm2 cover glass under a positivephase-contrast microscope at a total magnification of times400Male factors were generally screened based on medicalhistory physical examinations conventional semen analysesblood tests including assessments of sex hormones andmeasurements of the testicular volume using an orchidome-ter scrotal ultrasonography and transrectal ultrasonographyVaricocele was diagnosed during scrotal examinations withthe patient in a standing position and was graded as previ-ously described [23] Patients were excluded from this study iftheir final sperm concentration was lt40times106mL after swim-up selection
22 Acrobeads Test Liquefied semen samples were dilutedwith an equal volume of modified Biggers Whitten andWhittingham (mBWW)mediumat 37∘CThe compoundwascentrifuged for five minutes at 200timesg The supernatant fluidwas discarded and 12mL of mBWW medium was placedover the centrifuged sperm pellet The centrifuge tube waskept at an angle of 5∘ for one hour at 37∘C and motile spermwere collected from the medium using the swim-up methodOne milliliter of the upper pole of the medium was removedand centrifuged for five minutes at 300timesg The pellets werewashed twice in mBWW medium containing 03 humanserum albumin (HSA) and then resuspended in mBWWmedium containing 35 HSA at a sperm concentration of40 times 106mL
The Acrobeads test (FUSO Pharmaceutical IndustriesOsaka Japan) was performed according to the previouslydescribed method [23 27] using immunobeads coated withMH61 monoclonal antibodies (MH61-beads) [28] whichbinds to the anterior portion of acrosome-reacted spermOne hundred microliters of prepared sperm suspensionwas divided into a 96-well tissue culture plate (CorningCorning NY USA) Serial dilutions weremade withmBWWmedium35HSA at 1 2 1 4 and 1 8 and 10 120583L ofmBWWmedium35 HSA including 15 times 104MH61-beads wasadded to each well The plates were incubated at 37∘C in 5CO2 in humidified air Agglutinated sperm-bead complexeswere observed using an inverted phase-contrast microscopewith 100x magnification at 24 hours of incubationThe visualfield in each specimen was divided into five portions andeach field was considered to be positive when no beads freefrombinding to the sperm foundwere found If three ormoreof the fields were positive the well was judged to be positiveWhen less than three fields were positive in anywell the score
The Scientific World Journal 3
was 0 Therefore at least some spermatozoa were bound toMH61 even in the cases with a score of 0 When positiveagglutination was observed at dilution of 1 1 1 2 1 4 or1 8 the test results were scored as 1 2 3 or 4 respectivelyTo observe sperm vacuolation the sperm suspension wasplaced onto a glass bottom dish (WillCo-Dish WillCo WellsBV AmsterdamThe Netherlands) instead of a 96-well tissueculture plate
23 Observation of Spermatozoa Using a High-MagnificationMicroscope The spermatozoa placed on a glass bottomdish were analyzed at 3700times using an inverted microscopeequipped with Nomarski differential interference contrastoptics (IX71 Olympus Tokyo) and a video system (FX630Olympus Tokyo) A 60-times (142 numerical aperture) objectivelens was used with oil Images of the spermatozoa werecaptured and stored on a video system using an image-filingsoftware program FlvFs (Flovel Tokyo) We spent 30 to 60minutes capturing and analyzing the images of each ejaculateThe spermatozoa were classified into three groups (I) thosebound to MH61-beads after the acrosome reaction at 24hours of incubation regardless of the motility (II) motilespermatozoa that did not bind to MH61-beads and (III)immotile spermatozoa that did not bind to MH61-beads Atleast 500 spermatozoa per ejaculate and 100 spermatozoaper each group were evaluated using the high-magnificationmicroscope [14] A nuclear vacuole was defined as ldquolargerdquo ifthe maximum diameter of the vacuole was more than 50of the width of the sperm head [14] Using this system weevaluated large nuclear vacuoles (LNVs) not only in motilespermatozoa but also in immotile spermatozoa (Figure 1)
The percentage of spermatozoa with LNVswas calculatedfor each sample and compared between groups I II and III
24 Statistical Analysis Thestatistical analysis of the datawascarried out using the JMP 801 statistical software package(SAS Institute Japan Tokyo) Paired and unpaired Studentrsquos 119905-testswere used to compare the values between the groupsThechi-square test was used to examine differences in categoricalvariables Spearmanrsquos rank correlation coefficient was usedto determine the correlations between the proportion ofspermatozoa with large nuclear vacuoles (LNVs) and theconventional semen parameters A value of 119875 lt 005 wasdefined as being statistically significant
3 Results
31 Conventional Semen Parameters and Acrobeads TestResults The semen volume was 33 plusmn 17mL (mean plusmn stan-dard deviation) the sperm count was 535 plusmn 334 (times106mL)the proportion of sperm exhibiting motility was 437 plusmn 115and the proportion of sperm with a normal morphology was50 plusmn 44 according to the conventional semen analysisConventional semen parameters were normal in 11 cases(including six patients with palpable varicocele) and abnor-mal in 19 cases (13 patients with palpable varicocele andsix patients with idiopathic male infertility) The Acrobeadsscore was 0 in two cases 1 in one case 2 in 11 cases 3
M
M
M
M
M
10120583m
10120583m
10120583m10120583m10120583m
lowast
lowast
lowastlowast
lowastlowast
Figure 1 High-magnification observation of a sperm head (times600)M MH61-bead lowastspermatozoa bound to an MH61-bead withoutlarge nuclear vacuoles lowastlowastspermatozoa bound to an MH61-beadwith large nuclear vacuoles spermatozoa not bound to MH61-beads without large nuclear vacuoles spermatozoa not bound toMH61-beads with large nuclear vacuolesThe arrows indicate spermlarge nuclear vacuoles
in 6 cases and 4 in no cases The scores in the patientswith normozoospermia tended to be higher than those withteratozoospermia andor asthenozoospermia however thedifference was not significant (Table 1)
32 Observation of Spermatozoa Using a High-MagnificationMicroscope High-magnification observation of spermato-zoa was performed on a glass bottom plate at a dilution of 1 2in 27 cases a dilution of 1 4 in one case and a dilution of 1 8in two cases according to the final sperm count in the spermsuspension following the use of the swim-up methods andAcrobeads tests The LNVs (average plusmn standard deviationminimum-maximum) were 24 plusmn 21 (0ndash81) in group I58 plusmn 39 (09ndash192) in group II and 98 plusmn 43 (43ndash186) in group III These values were significantly differentfrom each other (I versus II 119875 lt 0001 I versus III 119875 lt0001 II versus III 119875 lt 0001 paired 119905-test) There were nocorrelations between the LNVs values and the Acrobeadsscores (Table 2) or conventional semen parameters (data notshown) in this cohort
4 Discussion
In the present study we investigated the relationship betweensperm nuclear vacuolation and the sperm functions includ-ing acrosome reactions by conducting Acrobeads tests andassessments of the sperm motility status after 24 hours ofincubation by a high-magnification microscope MSOME isa method used to evaluate motile spermatozoa however weapplied a high-magnification microscope not only to motilespermatozoa but also to immotile ones The LNV variedaccording to the status of MH61-binding and motility after24 hours of incubation at 37∘C The LNV values in thespermatozoa that bound to MH61-beads after the acrosomereactions were significantly lower than those of the motile
4 The Scientific World Journal
Table 1 Acrobeads scores and the results of the semen analysis
Total number of observed spermatozoa 6474 5129 6389Number of observed spermatozoa foreach patient (mean plusmn SD) 2158 plusmn 94 1710 plusmn 443 2130 plusmn 200
LNV proportion of spermatozoa with large nuclear vacuoles SD standard deviation lowastStatistically significant
and immotile spermatozoa that did not bind to MH61-beadsafter 24 hours of incubation Spermatozoa that bind toMH61-beads are thought to do so due to acrosome reactions TheLNV values in the motile spermatozoa were significantlylower than those observed in the immotile spermatozoaafter 24 hours of incubation These results indicate thatspermatozoa with LNVs are less likely to undergo acrosomereactions and maintain motility up to 24 hours at 37∘C Therelationship between sperm nuclear vacuolation and spermmotility observed in the present study is consistent withthe findings of our previous report [14] In contrast theLNV values observed in this cohort were smaller thanthose noted in our previous report This may be due to thedifferences in the patientsrsquo backgrounds the semen qualityandor methods used for sperm preparation In the presentstudy patients with normozoospermia were included andspermatozoa selection was performed using the swim-upmethod On the other hand the cohort evaluated in ourprevious study did not include normozoospermic patientsand the semen samples were processed using density gradientcentrifugation In this context Monqaut et al reported thatthe use of sperm processing methods including swim-upmethod and density gradient centrifugation allows for theselection of sperm with a lower level of nuclear vacuolizationand a higher level of sperm motility [29] In that study theswim-up method produced samples with less vacuolizationIn a report by Watanabe et al the LNV was 46 afterboth of the density gradient centrifugation and the swim-up method in high-quality semen samples in which themean values of sperm concentration and motility were 419
millionmL and 533 respectively [30] The definitions ofLNV may also account for the LNV Our definition ofvacuolated spermatozoa is different from and stricter thanothers [8 13] Therefore the LNV values observed in thepresent study are consistent with the findings of other reports
As shown in Table 2 there were no differences in theLNV values between the ejaculates with low and highAcrobeads scores The spermatozoa bound to MH61-beadsexhibited lower LNV values while those not bound toMH61-beads demonstrated higher LNV values regardlessof the Acrobeads scores The spermatozoa that lost motilityat 24 hours of incubation also showed higher LNV valuesregardless of the Acrobeads scores Spermatozoa with anormal function may be present in semen with abnormalAcrobeads scores In contrast the semen with abnormalAcrobeads scores may include the spermatozoa with bothnormal and abnormal functions Based on these resultswe speculate that the Acrobeads test reflects the quality ofsemen as a whole whereas the LNV reflects the degreeof normality of the individual sperm functions Thereforeit is sensible to select sperm according to the LNV whenperforming ICSI
Sperm vacuolation has been reported to be negativelyrelated to parameters of conventional and computer-assistedsemen analyses The ratio of the vacuole area to the spermhead area is negatively correlated with a poor sperm mor-phology [19] The proportion of large nuclear vacuoles inprocessed motile spermatozoa demonstrates significant cor-relations with decreased sperm count sperm motility totalsperm count motile sperm count and total motile sperm
The Scientific World Journal 5
count on conventional semen analyses [14] In addition theproportion of sperm with large nuclear vacuoles exhibitssignificant correlations with objective parameters of spermmotility such as linearity and the beatcross frequencymeasured using SMAS a CASA system [14] Varicocelerepair reduces the proportion of large nuclear vacuoles inmotile spermatozoa [31]Therefore the observation of spermvacuolation can be used to predict the sperm function andevaluate the therapeutic effects
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of the sperm func-tion Montjean et al demonstrated that induced acrosomereactions are not correlated with significant modificationof sperm nuclear condensation or morphology (Bartoovrsquoscriteria) [20] The authors simultaneously observed a highlysignificant decrease in the presence of vacuoles followingacrosome reaction induction Kacem et al evaluated the acro-some status using sperm organellar morphological examina-tions [21] In that study vacuoles were present in 61 of thespermatozoa when acrosomal material or intact acrosomeswere observed in comparison with the 29 observed whenthe spermatozoa were acrosome-reacted (119875 lt 00001) Inone study the induction of the acrosomal reactions usingionophore A23587 from 174 to 361 significantly increasedthe percentage of vacuole-free spermatozoa from 412 to638 (119875 lt 0001) [21] These data suggest that some nuclearvacuoles are of acrosomal origin Peer et al investigated theeffects of incubation at 37∘C on the morphological normalcyof the sperm nucleus [32] Their study showed that aftertwo hours of incubation at 37∘C there was a significantincrease in the frequency of vacuolated nuclei No significantmorphological changes in sperm nuclei were observed afterprolonged incubation at 21∘C Next after two hours ofincubation the incidence of spermatozoa with vacuolatednuclei was significantly higher at 37∘C than at 21∘C Morerecently however Neyer et al reported that incubationtemperatures (20 or 37∘C) andor the induction of oxidativestress do not stimulate the formation of new vacuoles [22]In that study after inducing the acrosome reactions nomodifications were detected in the vacuolated spermatozoaThese results suggest that some sperm head vacuoles areproduced at earlier stages of sperm maturation and thatnormal acrosome reactions are more likely to be induced inspermatozoa without large nuclear vacuoles In this contexttheAcrobeads test can be used to evaluate acrosome reactionsin the sperm using a monoclonal antibody that binds to theanterior portion of acrosome-reacted sperm [23 24] Theresults of tests using Acrobeads show good reproducibilityand are correlated with the results of sperm penetrationassays using zona-free hamster eggs and IVF [24 33 34]Therefore the acrosome status determined according tothe Acrobeads test is a valuable parameter for estimatingthe capacity for fertilization in males with infertility Inour previous report the Acrobeads score was found to berelated to the sperm concentration and sperm motility in 81ejaculates [27] Komori et al reported that spermmotility andthe percentage of sperm with an abnormal morphology hadan effect on the Acrobeads test results in 114 ejaculates [33]In the present study a similar trend was observed however it
was not statistically significantThismay be due to the smallersample size of 30 semen samples used in this study
It is not clear how sperm vacuolation affects acrosomereactions and the maintenance of motility The etiology ofsperm nuclear vacuoles also remains unclear Human spermhead vacuoles are physiological structures that are formedduring the process of sperm development and maturationprocess [35] Nuclear vacuoles may be the remnants ofunnecessary cytoplasm and organelles that should have beeneliminated during spermiogenesis [36 37] More recentlyPerdrix et al showed that vacuoles are located inside thenucleus using transmission electron microscopy [38] Excessresidual membrane constituents can be a source of reac-tive oxygen species (ROS) ROS expose sperm to excessiveoxidative stress resulting in DNA damage [39ndash44] DNAdamage is thought to reduce male fertility and cause-specific treatments in patients with a high level of spermDNA damage result in significant DNA improvement [45ndash53] Many studies have indicated that there is a positiverelationship between sperm DNA fragmentation and thepresence of large nuclear vacuoles in the sperm nuclear area[53ndash55] Furthermore several studies have reported that largevacuoles are associated with chromatin condensation failure[38 56ndash60] DNA damage alters the special cellular functionsof human spermatozoa resulting in diminished acrosomereactions with reduced rates of fertilization Ozmen et alreported that negative correlations were identified betweenincreased DNA damage and acrosome reactions andor theviability of human spermatozoa especially in cases involvingreduced fertilization rates [61] In addition Morakinyo et alreported that oxidative stress induced by calcium antagonistsdecreases the percentage value of acrosomal-reacted spermin rats [62] Therefore the results in the present study can beexplained by oxidative stress in spermatozoa andor spermDNA damage associated with LNVs
There are some limitations to the present study Thenumber of ejaculates was relatively small The results mayhave been different if we had obtained more samples espe-cially with respect to the relationships between theAcrobeadsscores and semen quality or LNV However our cohortwas adequately large to analyze the differences in LNVaccording to the presence of acrosome reactions and themaintenance of sperm motility Acrobeads tests can beperformed only in relatively high-quality semen processedusing the swim-up method Therefore if the presence ofacrosome reactions is evaluated using other methods withlower quality semen samples the results will be differentNo relationships in the sperm vacuolation or Acrobeadsscores between pregnancy or birth rates were found althoughonly four of 30 couples achieved pregnancy including twonatural conceptions and two pregnancies via ICSI over amedian follow-up of five months (data not shown) Suchinformation would be beneficial in clinical practice Nolifestyle factors including smoking body mass index andalcohol consumption were found to be correlatedwith spermvacuolation or the Acrobeads scores although these factorsmay have had a potential negative impact on sperm vacuoledevelopment (data not shown) However these factors havenot been previously discussed in the literature
6 The Scientific World Journal
5 Conclusions
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
conventional light microcopy or MSOME have been contro-versial and the technique is thought to be time-consumingHowever a recent meta-analysis showed a trend towards animprovement in IMSI outcomes versus that obtained withICSI [10] To date teratozoospermia has been proposed to bea preferential indication forMSOME and IMSI [11] while theonly confirmed indication for IMSI is recurrent implantationfailure following ICSI [12]
Numerous vacuoles have been identified inside the spermnucleus under a light microscope at high magnifications It isgenerally accepted that vacuolated spermatozoa are classifiedas having an abnormal morphology [13 14] AmongMSOMEparameters the presence of a sperm nuclear vacuole is one ofthe most important findings Recent results have shown thatthe presence of sperm nuclear vacuoles is negatively corre-lated with the rates of fertilization pregnancy and implan-tation [7 8 15ndash18] Sperm vacuolation has also been reportedto be negatively related to the parameters in conventionaland computer-assisted semen analyses (CASA) [14 19]Thesefindings suggest that the observation of sperm vacuolationcan be used to predict the sperm function
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of sperm functionTo date the results of investigations regarding the origins ofvacuoles have been controversial and while some authorsreport vacuoles to originate within the nucleus others reportan acrosomal origin It has been suggested that at leastsome nuclear vacuoles are of acrosomal origin [20 21] Inaddition sperm head vacuoles are thought to be produced atearlier stages of spermmaturation and that normal acrosomereactions are more likely to be induced in spermatozoa with-out large nuclear vacuoles [22] In this context the Acrob-eads test can be used to evaluate acrosome reactions insperm using amonoclonal antibody that binds to the anteriorportion of acrosome-reacted sperm [23 24]
The purpose of the present study was to further inves-tigate the relationship between sperm nuclear vacuolationand the sperm functions including acrosome reactions byconducting Acrobeads tests and assessments of the spermmotility status after 24 hours of incubation by a high-magnification microscope
2 Materials and Methods
The Institutional Review Board of the University of Toyamaapproved this study (number 23-128) Ethical consent for thework to be carried out was provided and signed informedconsent was obtained from each patient evaluated in thisstudy The study conformed to the principles outlined in theDeclaration of Helsinki
21 Sample Collection We enrolled 30 male patients whovisited the Male Infertility Clinic at Toyama UniversityHospital The patient ages ranged from 26 to 49 years witha mean of 36 years (plusmn57 standard deviation) The durationof infertility ranged from 7 to 105 months with a mean of 37months (plusmn27 standard deviation) The semen samples werecollected following masturbation from infertile male patients
who visited the Male Infertility Clinic at Toyama UniversityHospital The semen samples were collected after at least fivedays of abstinence allowed to liquefy at room temperatureand evaluated within one hour of collection using manualconventional semen analyses [25] which were performedas previously described [26] All manual assessments wereperformed by a single experienced laboratory technician(Y K) and the sperm concentrations were assessed usingan improved Neubauer hemocytometer The samples werediluted according to the instructions of the WHO laboratorymanual (1999) [25] To determine the degree of spermmotility a 10 120583L sample was loaded onto a clear slide glassand covered with a 22 times 22mm2 cover glass under a positivephase-contrast microscope at a total magnification of times400Male factors were generally screened based on medicalhistory physical examinations conventional semen analysesblood tests including assessments of sex hormones andmeasurements of the testicular volume using an orchidome-ter scrotal ultrasonography and transrectal ultrasonographyVaricocele was diagnosed during scrotal examinations withthe patient in a standing position and was graded as previ-ously described [23] Patients were excluded from this study iftheir final sperm concentration was lt40times106mL after swim-up selection
22 Acrobeads Test Liquefied semen samples were dilutedwith an equal volume of modified Biggers Whitten andWhittingham (mBWW)mediumat 37∘CThe compoundwascentrifuged for five minutes at 200timesg The supernatant fluidwas discarded and 12mL of mBWW medium was placedover the centrifuged sperm pellet The centrifuge tube waskept at an angle of 5∘ for one hour at 37∘C and motile spermwere collected from the medium using the swim-up methodOne milliliter of the upper pole of the medium was removedand centrifuged for five minutes at 300timesg The pellets werewashed twice in mBWW medium containing 03 humanserum albumin (HSA) and then resuspended in mBWWmedium containing 35 HSA at a sperm concentration of40 times 106mL
The Acrobeads test (FUSO Pharmaceutical IndustriesOsaka Japan) was performed according to the previouslydescribed method [23 27] using immunobeads coated withMH61 monoclonal antibodies (MH61-beads) [28] whichbinds to the anterior portion of acrosome-reacted spermOne hundred microliters of prepared sperm suspensionwas divided into a 96-well tissue culture plate (CorningCorning NY USA) Serial dilutions weremade withmBWWmedium35HSA at 1 2 1 4 and 1 8 and 10 120583L ofmBWWmedium35 HSA including 15 times 104MH61-beads wasadded to each well The plates were incubated at 37∘C in 5CO2 in humidified air Agglutinated sperm-bead complexeswere observed using an inverted phase-contrast microscopewith 100x magnification at 24 hours of incubationThe visualfield in each specimen was divided into five portions andeach field was considered to be positive when no beads freefrombinding to the sperm foundwere found If three ormoreof the fields were positive the well was judged to be positiveWhen less than three fields were positive in anywell the score
The Scientific World Journal 3
was 0 Therefore at least some spermatozoa were bound toMH61 even in the cases with a score of 0 When positiveagglutination was observed at dilution of 1 1 1 2 1 4 or1 8 the test results were scored as 1 2 3 or 4 respectivelyTo observe sperm vacuolation the sperm suspension wasplaced onto a glass bottom dish (WillCo-Dish WillCo WellsBV AmsterdamThe Netherlands) instead of a 96-well tissueculture plate
23 Observation of Spermatozoa Using a High-MagnificationMicroscope The spermatozoa placed on a glass bottomdish were analyzed at 3700times using an inverted microscopeequipped with Nomarski differential interference contrastoptics (IX71 Olympus Tokyo) and a video system (FX630Olympus Tokyo) A 60-times (142 numerical aperture) objectivelens was used with oil Images of the spermatozoa werecaptured and stored on a video system using an image-filingsoftware program FlvFs (Flovel Tokyo) We spent 30 to 60minutes capturing and analyzing the images of each ejaculateThe spermatozoa were classified into three groups (I) thosebound to MH61-beads after the acrosome reaction at 24hours of incubation regardless of the motility (II) motilespermatozoa that did not bind to MH61-beads and (III)immotile spermatozoa that did not bind to MH61-beads Atleast 500 spermatozoa per ejaculate and 100 spermatozoaper each group were evaluated using the high-magnificationmicroscope [14] A nuclear vacuole was defined as ldquolargerdquo ifthe maximum diameter of the vacuole was more than 50of the width of the sperm head [14] Using this system weevaluated large nuclear vacuoles (LNVs) not only in motilespermatozoa but also in immotile spermatozoa (Figure 1)
The percentage of spermatozoa with LNVswas calculatedfor each sample and compared between groups I II and III
24 Statistical Analysis Thestatistical analysis of the datawascarried out using the JMP 801 statistical software package(SAS Institute Japan Tokyo) Paired and unpaired Studentrsquos 119905-testswere used to compare the values between the groupsThechi-square test was used to examine differences in categoricalvariables Spearmanrsquos rank correlation coefficient was usedto determine the correlations between the proportion ofspermatozoa with large nuclear vacuoles (LNVs) and theconventional semen parameters A value of 119875 lt 005 wasdefined as being statistically significant
3 Results
31 Conventional Semen Parameters and Acrobeads TestResults The semen volume was 33 plusmn 17mL (mean plusmn stan-dard deviation) the sperm count was 535 plusmn 334 (times106mL)the proportion of sperm exhibiting motility was 437 plusmn 115and the proportion of sperm with a normal morphology was50 plusmn 44 according to the conventional semen analysisConventional semen parameters were normal in 11 cases(including six patients with palpable varicocele) and abnor-mal in 19 cases (13 patients with palpable varicocele andsix patients with idiopathic male infertility) The Acrobeadsscore was 0 in two cases 1 in one case 2 in 11 cases 3
M
M
M
M
M
10120583m
10120583m
10120583m10120583m10120583m
lowast
lowast
lowastlowast
lowastlowast
Figure 1 High-magnification observation of a sperm head (times600)M MH61-bead lowastspermatozoa bound to an MH61-bead withoutlarge nuclear vacuoles lowastlowastspermatozoa bound to an MH61-beadwith large nuclear vacuoles spermatozoa not bound to MH61-beads without large nuclear vacuoles spermatozoa not bound toMH61-beads with large nuclear vacuolesThe arrows indicate spermlarge nuclear vacuoles
in 6 cases and 4 in no cases The scores in the patientswith normozoospermia tended to be higher than those withteratozoospermia andor asthenozoospermia however thedifference was not significant (Table 1)
32 Observation of Spermatozoa Using a High-MagnificationMicroscope High-magnification observation of spermato-zoa was performed on a glass bottom plate at a dilution of 1 2in 27 cases a dilution of 1 4 in one case and a dilution of 1 8in two cases according to the final sperm count in the spermsuspension following the use of the swim-up methods andAcrobeads tests The LNVs (average plusmn standard deviationminimum-maximum) were 24 plusmn 21 (0ndash81) in group I58 plusmn 39 (09ndash192) in group II and 98 plusmn 43 (43ndash186) in group III These values were significantly differentfrom each other (I versus II 119875 lt 0001 I versus III 119875 lt0001 II versus III 119875 lt 0001 paired 119905-test) There were nocorrelations between the LNVs values and the Acrobeadsscores (Table 2) or conventional semen parameters (data notshown) in this cohort
4 Discussion
In the present study we investigated the relationship betweensperm nuclear vacuolation and the sperm functions includ-ing acrosome reactions by conducting Acrobeads tests andassessments of the sperm motility status after 24 hours ofincubation by a high-magnification microscope MSOME isa method used to evaluate motile spermatozoa however weapplied a high-magnification microscope not only to motilespermatozoa but also to immotile ones The LNV variedaccording to the status of MH61-binding and motility after24 hours of incubation at 37∘C The LNV values in thespermatozoa that bound to MH61-beads after the acrosomereactions were significantly lower than those of the motile
4 The Scientific World Journal
Table 1 Acrobeads scores and the results of the semen analysis
Total number of observed spermatozoa 6474 5129 6389Number of observed spermatozoa foreach patient (mean plusmn SD) 2158 plusmn 94 1710 plusmn 443 2130 plusmn 200
LNV proportion of spermatozoa with large nuclear vacuoles SD standard deviation lowastStatistically significant
and immotile spermatozoa that did not bind to MH61-beadsafter 24 hours of incubation Spermatozoa that bind toMH61-beads are thought to do so due to acrosome reactions TheLNV values in the motile spermatozoa were significantlylower than those observed in the immotile spermatozoaafter 24 hours of incubation These results indicate thatspermatozoa with LNVs are less likely to undergo acrosomereactions and maintain motility up to 24 hours at 37∘C Therelationship between sperm nuclear vacuolation and spermmotility observed in the present study is consistent withthe findings of our previous report [14] In contrast theLNV values observed in this cohort were smaller thanthose noted in our previous report This may be due to thedifferences in the patientsrsquo backgrounds the semen qualityandor methods used for sperm preparation In the presentstudy patients with normozoospermia were included andspermatozoa selection was performed using the swim-upmethod On the other hand the cohort evaluated in ourprevious study did not include normozoospermic patientsand the semen samples were processed using density gradientcentrifugation In this context Monqaut et al reported thatthe use of sperm processing methods including swim-upmethod and density gradient centrifugation allows for theselection of sperm with a lower level of nuclear vacuolizationand a higher level of sperm motility [29] In that study theswim-up method produced samples with less vacuolizationIn a report by Watanabe et al the LNV was 46 afterboth of the density gradient centrifugation and the swim-up method in high-quality semen samples in which themean values of sperm concentration and motility were 419
millionmL and 533 respectively [30] The definitions ofLNV may also account for the LNV Our definition ofvacuolated spermatozoa is different from and stricter thanothers [8 13] Therefore the LNV values observed in thepresent study are consistent with the findings of other reports
As shown in Table 2 there were no differences in theLNV values between the ejaculates with low and highAcrobeads scores The spermatozoa bound to MH61-beadsexhibited lower LNV values while those not bound toMH61-beads demonstrated higher LNV values regardlessof the Acrobeads scores The spermatozoa that lost motilityat 24 hours of incubation also showed higher LNV valuesregardless of the Acrobeads scores Spermatozoa with anormal function may be present in semen with abnormalAcrobeads scores In contrast the semen with abnormalAcrobeads scores may include the spermatozoa with bothnormal and abnormal functions Based on these resultswe speculate that the Acrobeads test reflects the quality ofsemen as a whole whereas the LNV reflects the degreeof normality of the individual sperm functions Thereforeit is sensible to select sperm according to the LNV whenperforming ICSI
Sperm vacuolation has been reported to be negativelyrelated to parameters of conventional and computer-assistedsemen analyses The ratio of the vacuole area to the spermhead area is negatively correlated with a poor sperm mor-phology [19] The proportion of large nuclear vacuoles inprocessed motile spermatozoa demonstrates significant cor-relations with decreased sperm count sperm motility totalsperm count motile sperm count and total motile sperm
The Scientific World Journal 5
count on conventional semen analyses [14] In addition theproportion of sperm with large nuclear vacuoles exhibitssignificant correlations with objective parameters of spermmotility such as linearity and the beatcross frequencymeasured using SMAS a CASA system [14] Varicocelerepair reduces the proportion of large nuclear vacuoles inmotile spermatozoa [31]Therefore the observation of spermvacuolation can be used to predict the sperm function andevaluate the therapeutic effects
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of the sperm func-tion Montjean et al demonstrated that induced acrosomereactions are not correlated with significant modificationof sperm nuclear condensation or morphology (Bartoovrsquoscriteria) [20] The authors simultaneously observed a highlysignificant decrease in the presence of vacuoles followingacrosome reaction induction Kacem et al evaluated the acro-some status using sperm organellar morphological examina-tions [21] In that study vacuoles were present in 61 of thespermatozoa when acrosomal material or intact acrosomeswere observed in comparison with the 29 observed whenthe spermatozoa were acrosome-reacted (119875 lt 00001) Inone study the induction of the acrosomal reactions usingionophore A23587 from 174 to 361 significantly increasedthe percentage of vacuole-free spermatozoa from 412 to638 (119875 lt 0001) [21] These data suggest that some nuclearvacuoles are of acrosomal origin Peer et al investigated theeffects of incubation at 37∘C on the morphological normalcyof the sperm nucleus [32] Their study showed that aftertwo hours of incubation at 37∘C there was a significantincrease in the frequency of vacuolated nuclei No significantmorphological changes in sperm nuclei were observed afterprolonged incubation at 21∘C Next after two hours ofincubation the incidence of spermatozoa with vacuolatednuclei was significantly higher at 37∘C than at 21∘C Morerecently however Neyer et al reported that incubationtemperatures (20 or 37∘C) andor the induction of oxidativestress do not stimulate the formation of new vacuoles [22]In that study after inducing the acrosome reactions nomodifications were detected in the vacuolated spermatozoaThese results suggest that some sperm head vacuoles areproduced at earlier stages of sperm maturation and thatnormal acrosome reactions are more likely to be induced inspermatozoa without large nuclear vacuoles In this contexttheAcrobeads test can be used to evaluate acrosome reactionsin the sperm using a monoclonal antibody that binds to theanterior portion of acrosome-reacted sperm [23 24] Theresults of tests using Acrobeads show good reproducibilityand are correlated with the results of sperm penetrationassays using zona-free hamster eggs and IVF [24 33 34]Therefore the acrosome status determined according tothe Acrobeads test is a valuable parameter for estimatingthe capacity for fertilization in males with infertility Inour previous report the Acrobeads score was found to berelated to the sperm concentration and sperm motility in 81ejaculates [27] Komori et al reported that spermmotility andthe percentage of sperm with an abnormal morphology hadan effect on the Acrobeads test results in 114 ejaculates [33]In the present study a similar trend was observed however it
was not statistically significantThismay be due to the smallersample size of 30 semen samples used in this study
It is not clear how sperm vacuolation affects acrosomereactions and the maintenance of motility The etiology ofsperm nuclear vacuoles also remains unclear Human spermhead vacuoles are physiological structures that are formedduring the process of sperm development and maturationprocess [35] Nuclear vacuoles may be the remnants ofunnecessary cytoplasm and organelles that should have beeneliminated during spermiogenesis [36 37] More recentlyPerdrix et al showed that vacuoles are located inside thenucleus using transmission electron microscopy [38] Excessresidual membrane constituents can be a source of reac-tive oxygen species (ROS) ROS expose sperm to excessiveoxidative stress resulting in DNA damage [39ndash44] DNAdamage is thought to reduce male fertility and cause-specific treatments in patients with a high level of spermDNA damage result in significant DNA improvement [45ndash53] Many studies have indicated that there is a positiverelationship between sperm DNA fragmentation and thepresence of large nuclear vacuoles in the sperm nuclear area[53ndash55] Furthermore several studies have reported that largevacuoles are associated with chromatin condensation failure[38 56ndash60] DNA damage alters the special cellular functionsof human spermatozoa resulting in diminished acrosomereactions with reduced rates of fertilization Ozmen et alreported that negative correlations were identified betweenincreased DNA damage and acrosome reactions andor theviability of human spermatozoa especially in cases involvingreduced fertilization rates [61] In addition Morakinyo et alreported that oxidative stress induced by calcium antagonistsdecreases the percentage value of acrosomal-reacted spermin rats [62] Therefore the results in the present study can beexplained by oxidative stress in spermatozoa andor spermDNA damage associated with LNVs
There are some limitations to the present study Thenumber of ejaculates was relatively small The results mayhave been different if we had obtained more samples espe-cially with respect to the relationships between theAcrobeadsscores and semen quality or LNV However our cohortwas adequately large to analyze the differences in LNVaccording to the presence of acrosome reactions and themaintenance of sperm motility Acrobeads tests can beperformed only in relatively high-quality semen processedusing the swim-up method Therefore if the presence ofacrosome reactions is evaluated using other methods withlower quality semen samples the results will be differentNo relationships in the sperm vacuolation or Acrobeadsscores between pregnancy or birth rates were found althoughonly four of 30 couples achieved pregnancy including twonatural conceptions and two pregnancies via ICSI over amedian follow-up of five months (data not shown) Suchinformation would be beneficial in clinical practice Nolifestyle factors including smoking body mass index andalcohol consumption were found to be correlatedwith spermvacuolation or the Acrobeads scores although these factorsmay have had a potential negative impact on sperm vacuoledevelopment (data not shown) However these factors havenot been previously discussed in the literature
6 The Scientific World Journal
5 Conclusions
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
was 0 Therefore at least some spermatozoa were bound toMH61 even in the cases with a score of 0 When positiveagglutination was observed at dilution of 1 1 1 2 1 4 or1 8 the test results were scored as 1 2 3 or 4 respectivelyTo observe sperm vacuolation the sperm suspension wasplaced onto a glass bottom dish (WillCo-Dish WillCo WellsBV AmsterdamThe Netherlands) instead of a 96-well tissueculture plate
23 Observation of Spermatozoa Using a High-MagnificationMicroscope The spermatozoa placed on a glass bottomdish were analyzed at 3700times using an inverted microscopeequipped with Nomarski differential interference contrastoptics (IX71 Olympus Tokyo) and a video system (FX630Olympus Tokyo) A 60-times (142 numerical aperture) objectivelens was used with oil Images of the spermatozoa werecaptured and stored on a video system using an image-filingsoftware program FlvFs (Flovel Tokyo) We spent 30 to 60minutes capturing and analyzing the images of each ejaculateThe spermatozoa were classified into three groups (I) thosebound to MH61-beads after the acrosome reaction at 24hours of incubation regardless of the motility (II) motilespermatozoa that did not bind to MH61-beads and (III)immotile spermatozoa that did not bind to MH61-beads Atleast 500 spermatozoa per ejaculate and 100 spermatozoaper each group were evaluated using the high-magnificationmicroscope [14] A nuclear vacuole was defined as ldquolargerdquo ifthe maximum diameter of the vacuole was more than 50of the width of the sperm head [14] Using this system weevaluated large nuclear vacuoles (LNVs) not only in motilespermatozoa but also in immotile spermatozoa (Figure 1)
The percentage of spermatozoa with LNVswas calculatedfor each sample and compared between groups I II and III
24 Statistical Analysis Thestatistical analysis of the datawascarried out using the JMP 801 statistical software package(SAS Institute Japan Tokyo) Paired and unpaired Studentrsquos 119905-testswere used to compare the values between the groupsThechi-square test was used to examine differences in categoricalvariables Spearmanrsquos rank correlation coefficient was usedto determine the correlations between the proportion ofspermatozoa with large nuclear vacuoles (LNVs) and theconventional semen parameters A value of 119875 lt 005 wasdefined as being statistically significant
3 Results
31 Conventional Semen Parameters and Acrobeads TestResults The semen volume was 33 plusmn 17mL (mean plusmn stan-dard deviation) the sperm count was 535 plusmn 334 (times106mL)the proportion of sperm exhibiting motility was 437 plusmn 115and the proportion of sperm with a normal morphology was50 plusmn 44 according to the conventional semen analysisConventional semen parameters were normal in 11 cases(including six patients with palpable varicocele) and abnor-mal in 19 cases (13 patients with palpable varicocele andsix patients with idiopathic male infertility) The Acrobeadsscore was 0 in two cases 1 in one case 2 in 11 cases 3
M
M
M
M
M
10120583m
10120583m
10120583m10120583m10120583m
lowast
lowast
lowastlowast
lowastlowast
Figure 1 High-magnification observation of a sperm head (times600)M MH61-bead lowastspermatozoa bound to an MH61-bead withoutlarge nuclear vacuoles lowastlowastspermatozoa bound to an MH61-beadwith large nuclear vacuoles spermatozoa not bound to MH61-beads without large nuclear vacuoles spermatozoa not bound toMH61-beads with large nuclear vacuolesThe arrows indicate spermlarge nuclear vacuoles
in 6 cases and 4 in no cases The scores in the patientswith normozoospermia tended to be higher than those withteratozoospermia andor asthenozoospermia however thedifference was not significant (Table 1)
32 Observation of Spermatozoa Using a High-MagnificationMicroscope High-magnification observation of spermato-zoa was performed on a glass bottom plate at a dilution of 1 2in 27 cases a dilution of 1 4 in one case and a dilution of 1 8in two cases according to the final sperm count in the spermsuspension following the use of the swim-up methods andAcrobeads tests The LNVs (average plusmn standard deviationminimum-maximum) were 24 plusmn 21 (0ndash81) in group I58 plusmn 39 (09ndash192) in group II and 98 plusmn 43 (43ndash186) in group III These values were significantly differentfrom each other (I versus II 119875 lt 0001 I versus III 119875 lt0001 II versus III 119875 lt 0001 paired 119905-test) There were nocorrelations between the LNVs values and the Acrobeadsscores (Table 2) or conventional semen parameters (data notshown) in this cohort
4 Discussion
In the present study we investigated the relationship betweensperm nuclear vacuolation and the sperm functions includ-ing acrosome reactions by conducting Acrobeads tests andassessments of the sperm motility status after 24 hours ofincubation by a high-magnification microscope MSOME isa method used to evaluate motile spermatozoa however weapplied a high-magnification microscope not only to motilespermatozoa but also to immotile ones The LNV variedaccording to the status of MH61-binding and motility after24 hours of incubation at 37∘C The LNV values in thespermatozoa that bound to MH61-beads after the acrosomereactions were significantly lower than those of the motile
4 The Scientific World Journal
Table 1 Acrobeads scores and the results of the semen analysis
Total number of observed spermatozoa 6474 5129 6389Number of observed spermatozoa foreach patient (mean plusmn SD) 2158 plusmn 94 1710 plusmn 443 2130 plusmn 200
LNV proportion of spermatozoa with large nuclear vacuoles SD standard deviation lowastStatistically significant
and immotile spermatozoa that did not bind to MH61-beadsafter 24 hours of incubation Spermatozoa that bind toMH61-beads are thought to do so due to acrosome reactions TheLNV values in the motile spermatozoa were significantlylower than those observed in the immotile spermatozoaafter 24 hours of incubation These results indicate thatspermatozoa with LNVs are less likely to undergo acrosomereactions and maintain motility up to 24 hours at 37∘C Therelationship between sperm nuclear vacuolation and spermmotility observed in the present study is consistent withthe findings of our previous report [14] In contrast theLNV values observed in this cohort were smaller thanthose noted in our previous report This may be due to thedifferences in the patientsrsquo backgrounds the semen qualityandor methods used for sperm preparation In the presentstudy patients with normozoospermia were included andspermatozoa selection was performed using the swim-upmethod On the other hand the cohort evaluated in ourprevious study did not include normozoospermic patientsand the semen samples were processed using density gradientcentrifugation In this context Monqaut et al reported thatthe use of sperm processing methods including swim-upmethod and density gradient centrifugation allows for theselection of sperm with a lower level of nuclear vacuolizationand a higher level of sperm motility [29] In that study theswim-up method produced samples with less vacuolizationIn a report by Watanabe et al the LNV was 46 afterboth of the density gradient centrifugation and the swim-up method in high-quality semen samples in which themean values of sperm concentration and motility were 419
millionmL and 533 respectively [30] The definitions ofLNV may also account for the LNV Our definition ofvacuolated spermatozoa is different from and stricter thanothers [8 13] Therefore the LNV values observed in thepresent study are consistent with the findings of other reports
As shown in Table 2 there were no differences in theLNV values between the ejaculates with low and highAcrobeads scores The spermatozoa bound to MH61-beadsexhibited lower LNV values while those not bound toMH61-beads demonstrated higher LNV values regardlessof the Acrobeads scores The spermatozoa that lost motilityat 24 hours of incubation also showed higher LNV valuesregardless of the Acrobeads scores Spermatozoa with anormal function may be present in semen with abnormalAcrobeads scores In contrast the semen with abnormalAcrobeads scores may include the spermatozoa with bothnormal and abnormal functions Based on these resultswe speculate that the Acrobeads test reflects the quality ofsemen as a whole whereas the LNV reflects the degreeof normality of the individual sperm functions Thereforeit is sensible to select sperm according to the LNV whenperforming ICSI
Sperm vacuolation has been reported to be negativelyrelated to parameters of conventional and computer-assistedsemen analyses The ratio of the vacuole area to the spermhead area is negatively correlated with a poor sperm mor-phology [19] The proportion of large nuclear vacuoles inprocessed motile spermatozoa demonstrates significant cor-relations with decreased sperm count sperm motility totalsperm count motile sperm count and total motile sperm
The Scientific World Journal 5
count on conventional semen analyses [14] In addition theproportion of sperm with large nuclear vacuoles exhibitssignificant correlations with objective parameters of spermmotility such as linearity and the beatcross frequencymeasured using SMAS a CASA system [14] Varicocelerepair reduces the proportion of large nuclear vacuoles inmotile spermatozoa [31]Therefore the observation of spermvacuolation can be used to predict the sperm function andevaluate the therapeutic effects
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of the sperm func-tion Montjean et al demonstrated that induced acrosomereactions are not correlated with significant modificationof sperm nuclear condensation or morphology (Bartoovrsquoscriteria) [20] The authors simultaneously observed a highlysignificant decrease in the presence of vacuoles followingacrosome reaction induction Kacem et al evaluated the acro-some status using sperm organellar morphological examina-tions [21] In that study vacuoles were present in 61 of thespermatozoa when acrosomal material or intact acrosomeswere observed in comparison with the 29 observed whenthe spermatozoa were acrosome-reacted (119875 lt 00001) Inone study the induction of the acrosomal reactions usingionophore A23587 from 174 to 361 significantly increasedthe percentage of vacuole-free spermatozoa from 412 to638 (119875 lt 0001) [21] These data suggest that some nuclearvacuoles are of acrosomal origin Peer et al investigated theeffects of incubation at 37∘C on the morphological normalcyof the sperm nucleus [32] Their study showed that aftertwo hours of incubation at 37∘C there was a significantincrease in the frequency of vacuolated nuclei No significantmorphological changes in sperm nuclei were observed afterprolonged incubation at 21∘C Next after two hours ofincubation the incidence of spermatozoa with vacuolatednuclei was significantly higher at 37∘C than at 21∘C Morerecently however Neyer et al reported that incubationtemperatures (20 or 37∘C) andor the induction of oxidativestress do not stimulate the formation of new vacuoles [22]In that study after inducing the acrosome reactions nomodifications were detected in the vacuolated spermatozoaThese results suggest that some sperm head vacuoles areproduced at earlier stages of sperm maturation and thatnormal acrosome reactions are more likely to be induced inspermatozoa without large nuclear vacuoles In this contexttheAcrobeads test can be used to evaluate acrosome reactionsin the sperm using a monoclonal antibody that binds to theanterior portion of acrosome-reacted sperm [23 24] Theresults of tests using Acrobeads show good reproducibilityand are correlated with the results of sperm penetrationassays using zona-free hamster eggs and IVF [24 33 34]Therefore the acrosome status determined according tothe Acrobeads test is a valuable parameter for estimatingthe capacity for fertilization in males with infertility Inour previous report the Acrobeads score was found to berelated to the sperm concentration and sperm motility in 81ejaculates [27] Komori et al reported that spermmotility andthe percentage of sperm with an abnormal morphology hadan effect on the Acrobeads test results in 114 ejaculates [33]In the present study a similar trend was observed however it
was not statistically significantThismay be due to the smallersample size of 30 semen samples used in this study
It is not clear how sperm vacuolation affects acrosomereactions and the maintenance of motility The etiology ofsperm nuclear vacuoles also remains unclear Human spermhead vacuoles are physiological structures that are formedduring the process of sperm development and maturationprocess [35] Nuclear vacuoles may be the remnants ofunnecessary cytoplasm and organelles that should have beeneliminated during spermiogenesis [36 37] More recentlyPerdrix et al showed that vacuoles are located inside thenucleus using transmission electron microscopy [38] Excessresidual membrane constituents can be a source of reac-tive oxygen species (ROS) ROS expose sperm to excessiveoxidative stress resulting in DNA damage [39ndash44] DNAdamage is thought to reduce male fertility and cause-specific treatments in patients with a high level of spermDNA damage result in significant DNA improvement [45ndash53] Many studies have indicated that there is a positiverelationship between sperm DNA fragmentation and thepresence of large nuclear vacuoles in the sperm nuclear area[53ndash55] Furthermore several studies have reported that largevacuoles are associated with chromatin condensation failure[38 56ndash60] DNA damage alters the special cellular functionsof human spermatozoa resulting in diminished acrosomereactions with reduced rates of fertilization Ozmen et alreported that negative correlations were identified betweenincreased DNA damage and acrosome reactions andor theviability of human spermatozoa especially in cases involvingreduced fertilization rates [61] In addition Morakinyo et alreported that oxidative stress induced by calcium antagonistsdecreases the percentage value of acrosomal-reacted spermin rats [62] Therefore the results in the present study can beexplained by oxidative stress in spermatozoa andor spermDNA damage associated with LNVs
There are some limitations to the present study Thenumber of ejaculates was relatively small The results mayhave been different if we had obtained more samples espe-cially with respect to the relationships between theAcrobeadsscores and semen quality or LNV However our cohortwas adequately large to analyze the differences in LNVaccording to the presence of acrosome reactions and themaintenance of sperm motility Acrobeads tests can beperformed only in relatively high-quality semen processedusing the swim-up method Therefore if the presence ofacrosome reactions is evaluated using other methods withlower quality semen samples the results will be differentNo relationships in the sperm vacuolation or Acrobeadsscores between pregnancy or birth rates were found althoughonly four of 30 couples achieved pregnancy including twonatural conceptions and two pregnancies via ICSI over amedian follow-up of five months (data not shown) Suchinformation would be beneficial in clinical practice Nolifestyle factors including smoking body mass index andalcohol consumption were found to be correlatedwith spermvacuolation or the Acrobeads scores although these factorsmay have had a potential negative impact on sperm vacuoledevelopment (data not shown) However these factors havenot been previously discussed in the literature
6 The Scientific World Journal
5 Conclusions
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
Total number of observed spermatozoa 6474 5129 6389Number of observed spermatozoa foreach patient (mean plusmn SD) 2158 plusmn 94 1710 plusmn 443 2130 plusmn 200
LNV proportion of spermatozoa with large nuclear vacuoles SD standard deviation lowastStatistically significant
and immotile spermatozoa that did not bind to MH61-beadsafter 24 hours of incubation Spermatozoa that bind toMH61-beads are thought to do so due to acrosome reactions TheLNV values in the motile spermatozoa were significantlylower than those observed in the immotile spermatozoaafter 24 hours of incubation These results indicate thatspermatozoa with LNVs are less likely to undergo acrosomereactions and maintain motility up to 24 hours at 37∘C Therelationship between sperm nuclear vacuolation and spermmotility observed in the present study is consistent withthe findings of our previous report [14] In contrast theLNV values observed in this cohort were smaller thanthose noted in our previous report This may be due to thedifferences in the patientsrsquo backgrounds the semen qualityandor methods used for sperm preparation In the presentstudy patients with normozoospermia were included andspermatozoa selection was performed using the swim-upmethod On the other hand the cohort evaluated in ourprevious study did not include normozoospermic patientsand the semen samples were processed using density gradientcentrifugation In this context Monqaut et al reported thatthe use of sperm processing methods including swim-upmethod and density gradient centrifugation allows for theselection of sperm with a lower level of nuclear vacuolizationand a higher level of sperm motility [29] In that study theswim-up method produced samples with less vacuolizationIn a report by Watanabe et al the LNV was 46 afterboth of the density gradient centrifugation and the swim-up method in high-quality semen samples in which themean values of sperm concentration and motility were 419
millionmL and 533 respectively [30] The definitions ofLNV may also account for the LNV Our definition ofvacuolated spermatozoa is different from and stricter thanothers [8 13] Therefore the LNV values observed in thepresent study are consistent with the findings of other reports
As shown in Table 2 there were no differences in theLNV values between the ejaculates with low and highAcrobeads scores The spermatozoa bound to MH61-beadsexhibited lower LNV values while those not bound toMH61-beads demonstrated higher LNV values regardlessof the Acrobeads scores The spermatozoa that lost motilityat 24 hours of incubation also showed higher LNV valuesregardless of the Acrobeads scores Spermatozoa with anormal function may be present in semen with abnormalAcrobeads scores In contrast the semen with abnormalAcrobeads scores may include the spermatozoa with bothnormal and abnormal functions Based on these resultswe speculate that the Acrobeads test reflects the quality ofsemen as a whole whereas the LNV reflects the degreeof normality of the individual sperm functions Thereforeit is sensible to select sperm according to the LNV whenperforming ICSI
Sperm vacuolation has been reported to be negativelyrelated to parameters of conventional and computer-assistedsemen analyses The ratio of the vacuole area to the spermhead area is negatively correlated with a poor sperm mor-phology [19] The proportion of large nuclear vacuoles inprocessed motile spermatozoa demonstrates significant cor-relations with decreased sperm count sperm motility totalsperm count motile sperm count and total motile sperm
The Scientific World Journal 5
count on conventional semen analyses [14] In addition theproportion of sperm with large nuclear vacuoles exhibitssignificant correlations with objective parameters of spermmotility such as linearity and the beatcross frequencymeasured using SMAS a CASA system [14] Varicocelerepair reduces the proportion of large nuclear vacuoles inmotile spermatozoa [31]Therefore the observation of spermvacuolation can be used to predict the sperm function andevaluate the therapeutic effects
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of the sperm func-tion Montjean et al demonstrated that induced acrosomereactions are not correlated with significant modificationof sperm nuclear condensation or morphology (Bartoovrsquoscriteria) [20] The authors simultaneously observed a highlysignificant decrease in the presence of vacuoles followingacrosome reaction induction Kacem et al evaluated the acro-some status using sperm organellar morphological examina-tions [21] In that study vacuoles were present in 61 of thespermatozoa when acrosomal material or intact acrosomeswere observed in comparison with the 29 observed whenthe spermatozoa were acrosome-reacted (119875 lt 00001) Inone study the induction of the acrosomal reactions usingionophore A23587 from 174 to 361 significantly increasedthe percentage of vacuole-free spermatozoa from 412 to638 (119875 lt 0001) [21] These data suggest that some nuclearvacuoles are of acrosomal origin Peer et al investigated theeffects of incubation at 37∘C on the morphological normalcyof the sperm nucleus [32] Their study showed that aftertwo hours of incubation at 37∘C there was a significantincrease in the frequency of vacuolated nuclei No significantmorphological changes in sperm nuclei were observed afterprolonged incubation at 21∘C Next after two hours ofincubation the incidence of spermatozoa with vacuolatednuclei was significantly higher at 37∘C than at 21∘C Morerecently however Neyer et al reported that incubationtemperatures (20 or 37∘C) andor the induction of oxidativestress do not stimulate the formation of new vacuoles [22]In that study after inducing the acrosome reactions nomodifications were detected in the vacuolated spermatozoaThese results suggest that some sperm head vacuoles areproduced at earlier stages of sperm maturation and thatnormal acrosome reactions are more likely to be induced inspermatozoa without large nuclear vacuoles In this contexttheAcrobeads test can be used to evaluate acrosome reactionsin the sperm using a monoclonal antibody that binds to theanterior portion of acrosome-reacted sperm [23 24] Theresults of tests using Acrobeads show good reproducibilityand are correlated with the results of sperm penetrationassays using zona-free hamster eggs and IVF [24 33 34]Therefore the acrosome status determined according tothe Acrobeads test is a valuable parameter for estimatingthe capacity for fertilization in males with infertility Inour previous report the Acrobeads score was found to berelated to the sperm concentration and sperm motility in 81ejaculates [27] Komori et al reported that spermmotility andthe percentage of sperm with an abnormal morphology hadan effect on the Acrobeads test results in 114 ejaculates [33]In the present study a similar trend was observed however it
was not statistically significantThismay be due to the smallersample size of 30 semen samples used in this study
It is not clear how sperm vacuolation affects acrosomereactions and the maintenance of motility The etiology ofsperm nuclear vacuoles also remains unclear Human spermhead vacuoles are physiological structures that are formedduring the process of sperm development and maturationprocess [35] Nuclear vacuoles may be the remnants ofunnecessary cytoplasm and organelles that should have beeneliminated during spermiogenesis [36 37] More recentlyPerdrix et al showed that vacuoles are located inside thenucleus using transmission electron microscopy [38] Excessresidual membrane constituents can be a source of reac-tive oxygen species (ROS) ROS expose sperm to excessiveoxidative stress resulting in DNA damage [39ndash44] DNAdamage is thought to reduce male fertility and cause-specific treatments in patients with a high level of spermDNA damage result in significant DNA improvement [45ndash53] Many studies have indicated that there is a positiverelationship between sperm DNA fragmentation and thepresence of large nuclear vacuoles in the sperm nuclear area[53ndash55] Furthermore several studies have reported that largevacuoles are associated with chromatin condensation failure[38 56ndash60] DNA damage alters the special cellular functionsof human spermatozoa resulting in diminished acrosomereactions with reduced rates of fertilization Ozmen et alreported that negative correlations were identified betweenincreased DNA damage and acrosome reactions andor theviability of human spermatozoa especially in cases involvingreduced fertilization rates [61] In addition Morakinyo et alreported that oxidative stress induced by calcium antagonistsdecreases the percentage value of acrosomal-reacted spermin rats [62] Therefore the results in the present study can beexplained by oxidative stress in spermatozoa andor spermDNA damage associated with LNVs
There are some limitations to the present study Thenumber of ejaculates was relatively small The results mayhave been different if we had obtained more samples espe-cially with respect to the relationships between theAcrobeadsscores and semen quality or LNV However our cohortwas adequately large to analyze the differences in LNVaccording to the presence of acrosome reactions and themaintenance of sperm motility Acrobeads tests can beperformed only in relatively high-quality semen processedusing the swim-up method Therefore if the presence ofacrosome reactions is evaluated using other methods withlower quality semen samples the results will be differentNo relationships in the sperm vacuolation or Acrobeadsscores between pregnancy or birth rates were found althoughonly four of 30 couples achieved pregnancy including twonatural conceptions and two pregnancies via ICSI over amedian follow-up of five months (data not shown) Suchinformation would be beneficial in clinical practice Nolifestyle factors including smoking body mass index andalcohol consumption were found to be correlatedwith spermvacuolation or the Acrobeads scores although these factorsmay have had a potential negative impact on sperm vacuoledevelopment (data not shown) However these factors havenot been previously discussed in the literature
6 The Scientific World Journal
5 Conclusions
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
count on conventional semen analyses [14] In addition theproportion of sperm with large nuclear vacuoles exhibitssignificant correlations with objective parameters of spermmotility such as linearity and the beatcross frequencymeasured using SMAS a CASA system [14] Varicocelerepair reduces the proportion of large nuclear vacuoles inmotile spermatozoa [31]Therefore the observation of spermvacuolation can be used to predict the sperm function andevaluate the therapeutic effects
The status of nuclear vacuoles related to acrosome reac-tions has been investigated as a parameter of the sperm func-tion Montjean et al demonstrated that induced acrosomereactions are not correlated with significant modificationof sperm nuclear condensation or morphology (Bartoovrsquoscriteria) [20] The authors simultaneously observed a highlysignificant decrease in the presence of vacuoles followingacrosome reaction induction Kacem et al evaluated the acro-some status using sperm organellar morphological examina-tions [21] In that study vacuoles were present in 61 of thespermatozoa when acrosomal material or intact acrosomeswere observed in comparison with the 29 observed whenthe spermatozoa were acrosome-reacted (119875 lt 00001) Inone study the induction of the acrosomal reactions usingionophore A23587 from 174 to 361 significantly increasedthe percentage of vacuole-free spermatozoa from 412 to638 (119875 lt 0001) [21] These data suggest that some nuclearvacuoles are of acrosomal origin Peer et al investigated theeffects of incubation at 37∘C on the morphological normalcyof the sperm nucleus [32] Their study showed that aftertwo hours of incubation at 37∘C there was a significantincrease in the frequency of vacuolated nuclei No significantmorphological changes in sperm nuclei were observed afterprolonged incubation at 21∘C Next after two hours ofincubation the incidence of spermatozoa with vacuolatednuclei was significantly higher at 37∘C than at 21∘C Morerecently however Neyer et al reported that incubationtemperatures (20 or 37∘C) andor the induction of oxidativestress do not stimulate the formation of new vacuoles [22]In that study after inducing the acrosome reactions nomodifications were detected in the vacuolated spermatozoaThese results suggest that some sperm head vacuoles areproduced at earlier stages of sperm maturation and thatnormal acrosome reactions are more likely to be induced inspermatozoa without large nuclear vacuoles In this contexttheAcrobeads test can be used to evaluate acrosome reactionsin the sperm using a monoclonal antibody that binds to theanterior portion of acrosome-reacted sperm [23 24] Theresults of tests using Acrobeads show good reproducibilityand are correlated with the results of sperm penetrationassays using zona-free hamster eggs and IVF [24 33 34]Therefore the acrosome status determined according tothe Acrobeads test is a valuable parameter for estimatingthe capacity for fertilization in males with infertility Inour previous report the Acrobeads score was found to berelated to the sperm concentration and sperm motility in 81ejaculates [27] Komori et al reported that spermmotility andthe percentage of sperm with an abnormal morphology hadan effect on the Acrobeads test results in 114 ejaculates [33]In the present study a similar trend was observed however it
was not statistically significantThismay be due to the smallersample size of 30 semen samples used in this study
It is not clear how sperm vacuolation affects acrosomereactions and the maintenance of motility The etiology ofsperm nuclear vacuoles also remains unclear Human spermhead vacuoles are physiological structures that are formedduring the process of sperm development and maturationprocess [35] Nuclear vacuoles may be the remnants ofunnecessary cytoplasm and organelles that should have beeneliminated during spermiogenesis [36 37] More recentlyPerdrix et al showed that vacuoles are located inside thenucleus using transmission electron microscopy [38] Excessresidual membrane constituents can be a source of reac-tive oxygen species (ROS) ROS expose sperm to excessiveoxidative stress resulting in DNA damage [39ndash44] DNAdamage is thought to reduce male fertility and cause-specific treatments in patients with a high level of spermDNA damage result in significant DNA improvement [45ndash53] Many studies have indicated that there is a positiverelationship between sperm DNA fragmentation and thepresence of large nuclear vacuoles in the sperm nuclear area[53ndash55] Furthermore several studies have reported that largevacuoles are associated with chromatin condensation failure[38 56ndash60] DNA damage alters the special cellular functionsof human spermatozoa resulting in diminished acrosomereactions with reduced rates of fertilization Ozmen et alreported that negative correlations were identified betweenincreased DNA damage and acrosome reactions andor theviability of human spermatozoa especially in cases involvingreduced fertilization rates [61] In addition Morakinyo et alreported that oxidative stress induced by calcium antagonistsdecreases the percentage value of acrosomal-reacted spermin rats [62] Therefore the results in the present study can beexplained by oxidative stress in spermatozoa andor spermDNA damage associated with LNVs
There are some limitations to the present study Thenumber of ejaculates was relatively small The results mayhave been different if we had obtained more samples espe-cially with respect to the relationships between theAcrobeadsscores and semen quality or LNV However our cohortwas adequately large to analyze the differences in LNVaccording to the presence of acrosome reactions and themaintenance of sperm motility Acrobeads tests can beperformed only in relatively high-quality semen processedusing the swim-up method Therefore if the presence ofacrosome reactions is evaluated using other methods withlower quality semen samples the results will be differentNo relationships in the sperm vacuolation or Acrobeadsscores between pregnancy or birth rates were found althoughonly four of 30 couples achieved pregnancy including twonatural conceptions and two pregnancies via ICSI over amedian follow-up of five months (data not shown) Suchinformation would be beneficial in clinical practice Nolifestyle factors including smoking body mass index andalcohol consumption were found to be correlatedwith spermvacuolation or the Acrobeads scores although these factorsmay have had a potential negative impact on sperm vacuoledevelopment (data not shown) However these factors havenot been previously discussed in the literature
6 The Scientific World Journal
5 Conclusions
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
In this study the degree of sperm nuclear vacuolation wassignificantly lower in the acrosome-reacted spermatozoa andspermatozoa that maintained motility up to 24 hours ofincubation and higher in the immotile spermatozoa that didnot bind to MH61-beads These results support the conceptthat the degree of sperm nuclear vacuolation evaluated usinga high-magnification microscope reflects some of spermfunctions
Disclosure
The authors alone are responsible for the contents of thispaper
Conflict of Interests
The authors declare no conflict of interests regarding thepublication of this paper
References
[1] G Palermo H Joris P Devroey and A C Van SteirteghemldquoPregnancies after intracytoplasmic injection of single sperma-tozoon into an oocyterdquoThe Lancet vol 340 no 8810 pp 17ndash181992
[2] K CWorrilow S Eid DWoodhouse et al ldquoUse of hyaluronanin the selection of sperm for intracytoplasmic sperm injec-tion (ICSI) significant improvement in clinical outcomesmdashmulticenter double-blinded and randomized controlled trialrdquoHuman Reproduction vol 28 no 2 pp 306ndash314 2013
[3] A Jakab D Sakkas E Delpiano et al ldquoIntracytoplasmic sperminjection a novel selection method for sperm with normalfrequency of chromosomal aneuploidiesrdquo Fertility and Sterilityvol 84 no 6 pp 1665ndash1673 2005
[4] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertility and Sterility vol 99 no 4 pp 1023ndash1029 2013
[5] R Mashiach B Fisch F Eltes Y Tadir J Ovadia and B Bar-toov ldquoThe relationship between sperm ultrastructural featuresand fertilizing capacity in vitrordquo Fertility and Sterility vol 57no 5 pp 1052ndash1057 1992
[6] B Bartoov F Eltes M Pansky J Langzam M Reichart andY Soffer ldquoImproved diagnosis of male fertility potential viaa combination of quantitative ultramorphology and routinesemen analysesrdquo Human Reproduction vol 9 no 11 pp 2069ndash2075 1994
[7] B Bartoov A Berkovitz and F Eltes ldquoSelection of sperma-tozoa with normal nuclei to improve the pregnancy rate withintracytoplasmic sperm injectionrdquoThe New England Journal ofMedicine vol 345 no 14 pp 1067ndash1068 2001
[8] B Bartoov A Berkovitz F Eltes A Kogosowski Y Menezoand Y Barak ldquoReal-time fine morphology of motile humansperm cells is associated with IVF-ICSI outcomerdquo Journal ofAndrology vol 23 no 1 pp 1ndash8 2001
[9] B Bartoov A Berkovitz F Eltes et al ldquoPregnancy rates arehigher with intracytoplasmic morphologically selected sperminjection than with conventional intracytoplasmic injectionrdquoFertility and Sterility vol 80 no 6 pp 1413ndash1419 2003
[10] A Souza Setti R C Ferreira D Paes deAlmeida Ferreira BragaR de Cassia Savio Figueira A Iaconelli Jr and E Borges JrldquoIntracytoplasmic sperm injection outcome versus intracyto-plasmic morphologically selected sperm injection outcome ameta-analysisrdquo Reproductive BioMedicine Online vol 21 no 4pp 450ndash455 2010
[11] A Perdrix and N Rives ldquoMotile sperm organelle morphologyexamination (MSOME) and sperm head vacuoles state of theart in 2013rdquoHuman Reproduction Update vol 19 no 5 pp 527ndash541 2013
[12] F Boitrelle B Guthauser L Alter et al ldquoHigh-magnificationselection of spermatozoa prior to oocyte injection confirmedand potential indicationsrdquo Reproductive BioMedicine Onlinevol 28 no 1 pp 6ndash13 2014
[13] WHO ldquoSemen analysisrdquo in WHO Laboratory Manual for theExamination and Processing of Human Semen pp 7ndash11 WorldHealth Organization Geneva Switzerland 2010
[14] A Komiya A Watanabe Y Kawauchi and H Fuse ldquoSpermwith large nuclear vacuoles and semen quality in the evaluationof male infertilityrdquo Systems Biology in Reproductive Medicinevol 59 no 1 pp 13ndash20 2013
[15] A Berkovitz F Eltes S Yaari et al ldquoThemorphological normal-cy of the sperm nucleus and pregnancy rate of intracytoplasmicinjection with morphologically selected spermrdquoHuman Repro-duction vol 20 no 1 pp 185ndash190 2005
[16] A Berkovitz F Eltes A Ellenbogen S Peer D Feldberg andB Bartoov ldquoDoes the presence of nuclear vacuoles in humansperm selected for ICSI affect pregnancy outcomerdquo HumanReproduction vol 21 no 7 pp 1787ndash1790 2006
[17] D P de Almeida Ferreira Braga A S Setti R C Figueira etal ldquoSperm organelle morphologic abnormalities contributingfactors and effects on intracytoplasmic sperm injection cyclesoutcomesrdquo Urology vol 78 no 4 pp 786ndash791 2011
[18] D Falagario A M Brucculeri R Depalo P Trerotoli E Citta-dini and G Ruvolo ldquoSperm head vacuolization affects clinicaloutcome in ICSI cycle A proposal of a cut-off valuerdquo Journal ofAssisted Reproduction and Genetics vol 29 no 11 pp 1281ndash12872012
[19] A Perdrix R Saıdi J F Menard et al ldquoRelationship betweenconventional sperm parameters and motile sperm organellemorphology examination (MSOME)rdquo International Journal ofAndrology vol 35 no 4 pp 491ndash498 2012
[20] D Montjean S Belloc M Benkhalifa A Dalleac and YMenezo ldquoSperm vacuoles are linked to capacitation and acroso-mal statusrdquoHuman Reproduction vol 27 no 10 pp 2927ndash29322012
[21] O Kacem C Sifer V Barraud-Lange et al ldquoSperm nuclear vac-uoles as assessed by motile sperm organellar morphologicalexamination are mostly of acrosomal originrdquo Reproductive Bio-Medicine Online vol 20 no 1 pp 132ndash137 2010
[22] A Neyer P Vanderzwalmen M Bach A Stecher D Spitzerand N Zech ldquoSperm head vacuoles are not affected by in-vitro conditions as analysed by a systemof sperm-microcapturechannelsrdquo Reproductive BioMedicine Online vol 26 no 4 pp368ndash377 2013
[23] H Fuse M Iwasaki I Mizuno and Y Ikehara-KawauchildquoEvaluation of acrosome reactivity using the Acrobeads testin varicocele patients findings before and after treatmentrdquoArchives of Andrology vol 49 no 1 pp 1ndash6 2003
[24] K Ohashi F Saji M Kato T Tsutsui T Tomiyama and OTanizawa ldquoAcrobeads test a new diagnostic test for assessment
The Scientific World Journal 7
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
of the fertilizing capacity of human spermatozoardquo Fertility andSterility vol 63 no 3 pp 625ndash630 1995
[25] WHO World Health Organization Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press New York NY USA1999
[26] T Akashi AWatanabe A Komiya andH Fuse ldquoEvaluation ofthe SpermMotility Analyzer System (SMAS) for the assessmentof sperm quality in infertile menrdquo Systems Biology in Reproduc-tive Medicine vol 56 no 6 pp 473ndash477 2010
[27] H Fuse M Sakamoto T Takamine T Kazama and TKatayama ldquoAcrobeads test using monoclonal antibody-coatedimmunobeads clinical applicationrdquo Archives of Andrology vol34 no 1 pp 1ndash7 1995
[28] M Okabe M Nagira Y Kawai S Matzno T Mimura andT Mayumi ldquoA human sperm antigen possibly involved inbinding andor fusion with zona-free hamster eggsrdquo Fertilityand Sterility vol 54 no 6 pp 1121ndash1126 1990
[29] A L Monqaut C Zavaleta G Lopez R Lafuente andM Brassesco ldquoUse of high-magnification microscopy for theassessment of sperm recovered after two different sperm pro-cessing methodsrdquo Fertility and Sterility vol 95 no 1 pp 277ndash280 2011
[30] S Watanabe A Tanaka S Fujii et al ldquoAn investigation of thepotential effect of vacuoles in human sperm on DNA damageusing a chromosome assay and the TUNEL assayrdquo HumanReproduction vol 26 no 5 pp 978ndash986 2011
[31] A Komiya AWatanabe Y Kawauchi andH Fuse ldquoAnalysis ofinter-examination differences in spermnuclear vacuoles amongmale patients with infertilityrdquo Systems Biology in ReproductiveMedicine vol 60 pp 35ndash42 2014
[32] S Peer F Eltes A Berkovitz R Yehuda P Itsykson andB Bartoov ldquoIs fine morphology of the human sperm nucleiaffected by in vitro incubation at 37∘Crdquo Fertility and Sterilityvol 88 no 6 pp 1589ndash1594 2007
[33] K Komori A Tsujimura Y Okamoto et al ldquoRelationshipbetween substances in seminal plasma and Acrobeads Testresultsrdquo Fertility and Sterility vol 91 no 1 pp 179ndash184 2009
[34] K Ohashi F Saji M Kato M Okabe T Mimura and OTanizawa ldquoEvaluation of acrosomal status using MH61-beadstest and its clinical applicationrdquo Fertility and Sterility vol 58no 4 pp 803ndash808 1992
[35] A Tanaka M Nagayoshi I Tanaka and H Kusunoki ldquoHumansperm head vacuoles are physiological structures formed dur-ing the sperm development and maturation processrdquo Fertilityand Sterility vol 98 no 2 pp 315ndash320 2012
[36] K Toshimori ldquoDynamics of the mammalian sperm headmodifications and maturation events from spermatogenesis toegg activationrdquo Advances in Anatomy Embryology and CellBiology vol 204 pp 5ndash94 2009
[37] K Toshimori and H C Ito ldquoHuman sperm ultrastructures andfertilityrdquo Journal of Mammalian Ova Research vol 25 pp 232ndash239 2008
[38] A Perdrix A Travers M H Chelli et al ldquoAssessment ofacrosome and nuclear abnormalities in human spermatozoawith large vacuolesrdquoHuman Reproduction vol 26 no 1 pp 47ndash58 2011
[39] S Chen W J Huang L S Chang and Y Wei ldquo8-Hydroxy-21015840-deoxyguanosine in leukocyte DNA of spermatic vein asa biomarker of oxidative stress in patients with varicocelerdquoJournal of Urology vol 172 pp 1418ndash1421 2004
[40] A Agarwal and T M Said ldquoOxidative stress DNA damageand apoptosis in male infertility a clinical approachrdquo BJUInternational vol 95 no 4 pp 503ndash507 2005
[41] R Smith H Kaune D Parodi et al ldquoIncreased sperm DNAdamage in patients with varicocele relationship with seminaloxidative stressrdquo Human Reproduction vol 21 no 4 pp 986ndash993 2006
[42] R J Aitken J K Wingate G N de Iuliis A J Koppersand E A McLaughlin ldquoCis-unsaturated fatty acids stimulatereactive oxygen species generation and lipid peroxidation inhuman spermatozoardquo The Journal of Clinical Endocrinology ampMetabolism vol 91 no 10 pp 4154ndash4163 2006
[43] T Ishikawa H Fujioka T Ishimura A Takenaka and MFujisawa ldquoIncreased testicular 8-hydroxy-21015840-deoxyguanosinein patients with varicocelerdquo BJU International vol 100 no 4pp 863ndash866 2007
[44] R DadaM B Shamsi S Venkatesh N P Gupta and R KumarldquoAttenuation of oxidative stress amp DNA damage in varicocelec-tomy implications in infertility managementrdquo Indian Journal ofMedical Research vol 132 no 12 pp 728ndash730 2010
[45] J OrsquoBrien and A Zini ldquoSperm DNA integrity and maleinfertilityrdquo Urology vol 65 no 1 pp 16ndash22 2005
[46] A Zini A Blumenfeld J Libman and J Willis ldquoBeneficialeffect of microsurgical varicocelectomy on human sperm DNAintegrityrdquo Human Reproduction vol 20 no 4 pp 1018ndash10212005
[47] A Zini and J Libman ldquoSperm DNA damage clinical signif-icance in the era of assisted reproductionrdquo Canadian MedicalAssociation Journal vol 175 no 5 pp 495ndash500 2006
[48] Practice Committee of the American Society for ReproductiveMedicine ldquoThe clinical utility of sperm DNA integrity testingrdquoFertility and Sterility vol 90 pp S178ndash180 2008
[49] F Papachristou M Simopoulou S Touloupidis C TsalikidisN Sofikitis and T Lialiaris ldquoDNA damage and chromosomalaberrations in various types of male factor infertilityrdquo Fertilityand Sterility vol 90 no 5 pp 1774ndash1781 2008
[50] S I Moskovtsev I Lecker J B MMullen et al ldquoCause-specifictreatment in patients with high spermDNA damage resulted insignificant DNA improvementrdquo Systems Biology in ReproductiveMedicine vol 55 no 2-3 pp 109ndash115 2009
[51] S I Moskovtsev J B M Mullen I Lecker et al ldquoFrequencyand severity of sperm DNA damage in patients with confirmedcases of male infertility of different aetiologiesrdquo ReproductiveBioMedicine Online vol 20 no 6 pp 759ndash763 2010
[52] Y Menezo B Dale and M Cohen ldquoDNA damage and repairin human oocytes and embryos a reviewrdquo Zygote vol 18 no 4pp 357ndash365 2010
[53] D Sakkas and J G Alvarez ldquoSperm DNA fragmentationmechanisms of origin impact on reproductive outcome andanalysisrdquo Fertility and Sterility vol 93 no 4 pp 1027ndash10362010
[54] J B AOliveira F CMassaro R L R Baruffi et al ldquoCorrelationbetween semen analysis bymotile sperm organelle morphologyexamination and sperm DNA damagerdquo Fertility and Sterilityvol 94 no 5 pp 1937ndash1940 2010
[55] F Skowronek G Casanova J Alciaturi et al ldquoDNA spermdamage correlates with nuclear ultrastructural sperm defectsinteratozoospermic menrdquo Andrologia vol 44 no 1 pp 59ndash652012
[56] F Boitrelle F Ferfouri J M Petit et al ldquoLarge human spermvacuoles observed in motile spermatozoa under high magni-fication nuclear thumbprints linked to failure of chromatin
8 The Scientific World Journal
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
condensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011
[57] KGopalkrishnanV Padwal P KMeherji J S Gokral R Shahand H S Juneja ldquoPoor quality of sperm as it affects repeatedearly pregnancy lossrdquo Archives of Andrology vol 45 no 2 pp111ndash117 2000
[58] J G Franco Jr A L Mauri C G Petersen et al ldquoLarge nuclearvacuoles are indicative of abnormal chromatin packaging inhuman spermatozoardquo International Journal of Andrology vol35 no 1 pp 46ndash51 2012
[59] N G Cassuto A Hazout I Hammoud et al ldquoCorrelationbetween DNA defect and sperm-head morphologyrdquo Reproduc-tive BioMedicine Online vol 24 no 2 pp 211ndash218 2012
[60] H Utsuno K Oka A Yamamoto and T Shiozawa ldquoEvaluationof sperm head shape at high magnification revealed correlationof spermDNA fragmentation with aberrant head ellipticity andangularityrdquo Fertility and Sterility vol 99 no 6 pp 1573ndash15802013
[61] B Ozmen G S Caglar F Koster B Schopper K Diedrichand S Al-Hasani ldquoRelationship between sperm DNA damageinduced acrosome reaction and viability in ICSI patientsrdquoReproductive BioMedicine Online vol 15 no 2 pp 208ndash2142007
[62] A Morakinyo B Iranloye and O Adegoke ldquoCalcium antag-onists modulate oxidative stress and acrosomal reaction in ratspermatozoardquo Archives of Medical Science vol 7 no 4 pp 613ndash618 2011
