research article assessment of chromatin maturity in human...

Hindawi Publishing CorporationAdvances in UrologyVolume 2013 Article ID 578631 8 pageshttpdxdoiorg1011552013578631

Research ArticleAssessment of Chromatin Maturity inHuman Spermatozoa Useful Aniline Blue Assay forRoutine Diagnosis of Male Infertility

Afifa Sellami12 Nozha Chakroun12 Soumaya Ben Zarrouk12 Hanen Sellami1

Sahbi Kebaili3 Tarek Rebai12 and Leila Keskes1

1 Histology-Embryology-Biology of Reproduction Laboratory Medical School Sfax 3029 Tunisia2Histology Embryology Research Unit Faculty of Medicine Medical School Sfax 3029 Tunisia3 Gynaecology and Obstetrics Department Hedi Chaker Academic Hospital Sfax 3029 Tunisia

Correspondence should be addressed to Afifa Sellami sellamiafifayahoofr

Received 17 June 2013 Accepted 3 September 2013

Academic Editor Axel S Merseburger

Copyright copy 2013 Afifa Sellami 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

During spermatogenesis sperm chromatin undergoes structural changes and results in a high condensation This nuclearcompactionwould be useful as a predictor of sperm fertilization capacity and pregnancy outcomeWe purpose to evaluate firstly therelationship among chromatinmaturity assessed by aniline blue staining (AB) and the semen parameters in infertile men Secondlywe analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa Fifty-one ejaculates wereinvestigated by semen analysis and stained for chromatin condensationwithAB to distinguish between unstainedmature spermandstained immature sperm AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare therates of immature sperm before and after selection Neat semen were divided into two groups G1 (119899 = 31) immature sperm lt20and G2 (119899 = 20) immature sperm ge20 No significant differences were detected in sperm concentration motility and normalmorphology between G1 and G2 However the rates of some morphology abnormalities were higher in G2 head abnormalities(119875 = 001) and microcephalic sperm (119875 = 002) We founded significant correlation between sperm immaturity and acrosomeabnormalities (119903 = 0292119875 = 003) Sperm selection has significantly reduced the rates of immature sperm A better understandingof chromatin structure and its impact on the sperm potential is needed to explore male infertility

1 Introduction

Routine semen analysis is the basic analysis in the explorationof male infertility It provides useful data concerning spermcount sperm motility and viability sperm morphology per-formance of genital glands and ejaculation Although thecontribution of the semen analysis is limited on the assess-ment of some important criteria implicated in the spermfunctional potential In fact it is well established that thematurity of sperm chromatin is essential for fertilizing capac-ity of spermatozoa and embryonic development [1ndash3] Theproportion of sperm with abnormal chromatin condensationin the ejaculate could be a prognostic factor in assessing thechances of fertilization and pregnancy [4] Indeed duringspermiogenesis spermnuclear is completely reorganized and

undergoes characteristic rearrangements with an importantcompaction [4ndash6] This chromatin condensation involves areplacement of somatic and testismdashspecific nucleoproteinshistones by transition proteins then by more basic proteinsnamed protamines P1 and P2 [5 7] with a proper ratio ofprotamine 1 to protamine 2 (P1P2) [8] These protaminespack nuclear DNA tightly into highly condensed chromatinin fact the sperm nucleus will have an important mechanicaland chemical stability [9]Thenuclear compaction is involvedin the protection of paternal genome during the transit ofspermatozoa through the male and female genital tractsand during its interaction with oocyte Abnormalities inchromatin condensation can cause nuclear damages as DNAdenaturation or fragmentation often associated with maleinfertility [10]

2 Advances in Urology

The addition of the evaluation of sperm head maturity toroutine semen analysis improves the assessment of fertility inmen [11] Many analytical techniques have been proposed toassess nuclear compaction in ejaculated sperm Some cyto-chemical or fluorescent dyes were used acidic aniline blue todetect excessive presence of histones [11ndash15] chromomycinA3 applied for the evaluation of protamine deficiency [16 17]and acridine orange [17] and toluidine blue staining used forassessing sperm chromatin structure and packaging [7 18]These different dyes have the advantage of providing suitableslides for use on a light or fluorescent microscope Anilineblue (AB) test evaluates the degree of sperm chromatincompaction ormaturation and it is able to detect sperm chro-matin defects related to their nucleoprotein content [11 14]This method allows discriminating between the presence oflysine-rich histones and arginine- and cysteine-rich protami-nes in sperm nuclei [11 19] Consequently histone-rich nucleiof immature spermwill take up the blue stain whereasmatureprotamine-rich nuclei remain unstained Several questionsobviously arise including (i) can the (AB) test predict themaleprognostic fertility and (ii) can it be a useful complementassay to direct the treatment of couple infertility

Many studies have shown negative correlations betweenthe defects of sperm chromatin integrity and male fertil-ity potential [20] However there is a controversy on thecorrelation between sperm nucleus maturity evaluated withAB staining and some semen parameters firstly and spermfertilization capacity and embryo quality secondly Otherreports have found a negative predictive value of nuclearcompaction in pregnancy outcome during assisted reproduc-tion treatment [10] So the aim of sperm selection techniquesapplied in assisted procreation techniques is not only basedon the assessment of classic parameters as sperm motilitynumeration and morphology but also involves more othersperm characteristics such as nuclear integrity

The aims of the present study were to assess the spermchromatin maturity in infertile men using acidic aniline bluestaining and to evaluate the relationship among chromatinsperm status and the semen parameters in male infertilityMoreover we analyzed whether the sperm gradient densitycentrifugation technique is effective to select mature sperma-tozoa

2 Materials and Methods

21 Patients Our study was carried out in 51 semen samplesfrommale partner of infertile couple attending theHistology-Embryology Laboratory of Sfax Medical School (Tunisia) forsemen investigationsThe patients were aged between 28 and49 years old with a mean age (plusmnstandard deviation (SD)) of3528 plusmn 062 years

22 Collection of Semen Samples Semen samples were col-lected bymasturbation after 3ndash5 days of sexual abstinence andallowed to liquefy for 30 minutes at 37∘C

23 Semen Analysis Basic semen analysis consisted in themeasurement of semen volume sperm concentration (hemo-cytometer method) motility (total motility rapid progressive

(type a) slow progressive (type b) and nonprogressive (typec)) vitality andmorphology All parameters were carried outaccording to the World Health Organization (WHO) guide-lines [21]

24 Semen Preparation Among these 51 samples 12 wereproceeded by Sill select density gradient centrifugation (119899 =12) Sill select (Fertipro NV Belgium) is a silane-coated silicaparticles solution that allows for separation of spermatozoaaccording to their density A 2-layer gradient was preparedusing respectively solutions of 90 and 45 Sill select Byusing a sterile pipette 1mL of liquefied semen sample wasplaced on top of the upper layer in a conical 15mL centrifugetube The tube was centrifuged at 300 g for 20 minutes Thesupernatant was then removed and the sperm pellet wassuspended in a volume of 3mL of Ferticult medium (FertiproNV Belgium) It was again centrifuged at 500 g for 10minutesThe final sperm pellet was suspended in 03mL of Ferticultmedium and examined for the sperm concentrationmotilitymorphology and AB staining

25 Acidic Aniline Blue Staining For each neat semen sample(119899 = 51) we evaluated the sperm nuclei chromatin conden-sation using AB staining Among these samples we also eva-luated by AB staining the nuclear maturity of spermatozoarecovered after a Sill select density gradient centrifugation(119899 = 12)

AB staining was performed as previously described byHofmann and Hilscher [19] After liquefaction 1mL of neatsemen was washed twice in 02M phosphate buffered (pH =72) We spread 20120583L of sperm pellet on glass slides andsperm smears are allowed to dry in air For the 12 sampleson which we performed a Sill select gradient density smearswere made with 20120583L from the final sperm pellet after adouble washing in 02M phosphate buffered (pH = 72)

Smears were fixed with a solution of 3 buffered glu-taraldehyde in 02M phosphate buffer (pH = 72) for 30min-utes Slides were then stained with 5 aqueous aniline bluesolution mixed with 4 acetic acid (pH = 35) for 5 minutes

For each stained smear 200 spermatozoa were evaluatedwith light microscope in oil immersion magnification (100xobjective) Spermatozoawith unstained nuclei are considerednormal (mature chromatin) while those blue stained wereconsidered abnormal (immature chromatin) (Figure 1) Theresults were expressed as percentages of nuclear unstainedand stained sperm An ejaculate with a rate of blue-stainednucleus sperm less than 20 was considered normal [4 11]

26 Statistical Analysis A statistical analysis was performedusing SPSS 130 software Statistical tests including Studentrsquos119905-test Pearsonrsquos and Spearmanrsquos correlations and linearregression were used The statistical significance was consid-ered for 119875 values lt005

3 Results

The mean values (plusmnSD) and ranges of semen parametersunstained (mature) sperm head (Figure 1(a)) and blue-stained (immature) spermhead (Figure 1(b)) are summarizedin Table 1

Advances in Urology 3

lowast

lowast

lowast

lowast

lowast

(a)

lowast

lowast

lowast

lowast

lowast

(b)

Figure 1 Sperm chromatin condensation assessed by aniline blue (a) Sample showing mainly mature sperm with unstained nucleus (lowast)(b) Sample showing mainly immature sperm (⧫) with blue-stained nucleus

Table 1 Means and ranges of semen parameters and AB tingibilityof sperm nuclear in the study samples (119899 = 51)

Mean plusmn SD RangesVolume (mL) 342 plusmn 173 09ndash11Total motility () 4078 plusmn 1115 15ndash55

Rapid progressive motility ldquoardquo () 1372 plusmn 760 0ndash25Slowly progressive motility ldquobrdquo () 2137 plusmn 608 5ndash35No progressive motility ldquocrdquo () 578 plusmn 209 5ndash15

Sperm concentration (millionsmL) 7520 plusmn 6405 620ndash392Vitality () 7619 plusmn 1032 45ndash97Normal morphology () 909 plusmn 697 1ndash30Unstained (mature) sperm head () 7945 plusmn 1344 30ndash99Blue-stained (immature) sperm head () 2066 plusmn 1376 1ndash70

Our samples were divided into two groups group G1 (119899 =31) blue-stained nuclei (immature sperm) lt20 and groupG2 (119899 = 20) blue-stained nuclei (immature sperm) ge20We compared the means values of semen parameters in eachgroup and we have not found any significant differencesbetween G1 and G2 (Table 2)

The comparison of spermocytogram data showed manydifferences between G1 and G2 groups The average numbersof morphological abnormalities (100 spermatozoa analyzed)were higher in G2 concerning abnormalities of sperm head(119875 = 001) mainly microcephalic spermatozoa (119875 = 002)and acrosome abnormalities (119875 = 009) (Table 2)

The rate of spermatozoa with blue-stained nuclei wasnegatively correlated with the percentage of those withnormalmorphology (Figure 2)We also found significant andpositive correlations between chromatin maturity and spermhead abnormalities (Figure 3) and acrosome abnormalities(Figure 4)

The sperm selection by density gradient increased signif-icantly the sperm total motility and decreased significantlythe rates of sperm with abnormal morphology and immaturechromatin (Table 3 Figures 5 and 6)

30002500200015001000500000Normal morphology sperm ()

7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = minus0266

P = 005

Figure 2 Correlation between sperm nucleus maturity and spermmorphology

4 Discussion

According to some literature data [14 22 23] our studydemonstrated that there is no correlation between the degreeof sperm chromatin condensation assessed by AB stainingand some sperm parameters including motility vitality andsperm countThese results suggested that chromatin conden-sation constitutes a valuable parameter in the assessment ofmale fertility completely independent of conventional spermparameters [24] In contrast other studies reported signifi-cant correlations between sperm chromatin immaturity andthe decrease of sperm count and progressive motility [17 25]In fact protamination anomalies are probably related to ageneralized abnormal spermiogenesis with defective semenparameters in result [25] However the inherent difficultieswith the AB assay are the visualization and accurate counting


Table 2 Comparison of semen parameters between G1 and G2 groups

Spermogram data (Mean plusmn SE) G1 (119899 = 31) G2 (119899 = 20) P valueVolume (mL) 343 plusmn 196 341 plusmn 140 097Total motility () 42 plusmn 12 3904 plusmn 98 034

Rapid progressive motility ldquoardquo () 141 plusmn 825 1214 plusmn 643 019Slowly progressive motility ldquobrdquo () 2133 plusmn 628 2142 plusmn 594 095No progressive motility ldquocrdquo () 583 plusmn 230 571 plusmn 179 083

Sperm concentration (millionsmL) 8558 plusmn 7206 6037 plusmn 4831 014Vitality () 7693 plusmn 1048 7514 plusmn 1025 054Normal morphology () 1046 plusmn 715 714 plusmn 636 008

Abnormalities of sperm head (100 spz analyzed) 15535 plusmn 3218 17689 plusmn 2801 001Microcephalic spz () 84 plusmn 56 13 plusmn 77 002Acrosome abnormalities () 6456 plusmn 1434 7123 plusmn 1436 009Cytoplasmic droplets () 820 plusmn 603 1047 plusmn 808 02

24000210001800015000120009000Total number of sperm head abnormalities (100 sperm observed)

7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0425

P = 0003

Figure 3 Correlation between sperm nucleus maturity and spermhead abnormalities

Table 3 Comparison between total motility rates of normalmorphology and AB tingibility of sperm nuclear before and aftersperm selection (119899 = 12)

Before spermselection

After spermselection P value

Total motility () 3824 plusmn 906 5712 plusmn 1017 lt0001Normal morphology () 1016 plusmn 565 1741 plusmn 538 002Blue-stained head sperm () 1066 plusmn 967 350 plusmn 410 001

of the mature sperm that are clear colored particularly inoligozoospermic ejaculates with reduced sperm concentra-tion Several different counterstains were tested includingrose bengal stain janus green stain and eosin Y stain whichgave the best results and was therefore used to improve thevisualization of excessive histones in sperm [26 27]

100009000800070006000500040003000Acrosome abnormalities ()

7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0292

P = 003

Figure 4 Correlation between sperm nucleus maturity and acro-some abnormalities

Furthermore we founded a correlation between the nor-mal sperm morphology and the nuclear maturity We notedalso that the degree of sperm chromatin compaction was sig-nificantly correlated with the average number of sperm headabnormalities and the acrosome abnormalities These resultsconfirm earlier investigations [12 19 24 28ndash30] that demon-strated significant correlations between sperm morphologyand its nuclear condensation Similarly Kazerooni et al [17]showed a significantly higher percentage of AB-positive sper-matozoa amongmen with teratozoospermia when comparedwith normozoospermic group (316 versus 141 119875 lt0001) More recently Zini et al [31] showed significantcorrelation between morphological sperm head defects andnuclear condensation anomalies and suggested that thesesperm dysmorphisms may in part due to incomplete spermnuclear compaction These results were in accordance with


0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12

Normal morphology before sperm selection ()Normal morphology after sperm selection ()

Figure 5Normalmorphology before and after sperm selection (119899 =12)

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12

Blue-stained head before sperm selection ()Blue-stained head after sperm selection ()

Figure 6 Nuclear maturity before and after sperm selection (119899 =12)

those of Boitrelle et al [32] who used high-magnification(times10000) contrast microscopy to study sperm morphologyand demonstrated that the presence of sperm head vacuoleswas associated with a complete or partial failure of chromatincondensation However Adham et al [33] reported thatthe morphological changes of the acrosomal vesicle duringspermatogenesis are concomitant with chromatin conden-sation Therefore it could be suggested that the failure ofthe initial chromatin condensation during spermatogenesisleads to impairment of acrosome attachment to the nucleusinvolvement or to dehiscence of acrosome from the nucleus

[33] In the opposite no correlationwas foundbetween spermmorphology and sperm condensation evaluated by AB test inother studies [14]

In light of these results we suggest that some alterations inthe process of spermiogenesis may promote multiple defectsin remodeling and compaction of the sperm chromatin Onthe basis of some recent studies [8 34] it has been shown thatprotamine P2 precursors play a pivotal role in maintainingthe P1P2 ratioThus any defect in pre-P2 mRNA translationappears to cause abnormal sperm morphogenesis reducedsperm motility and subsequent male infertility [35 36]

Secondly the compaction of the nuclear chromatin andthe DNA integrity are two linked parameters [10 37ndash39]To explore simultaneously these important criteria acridineorange staining was used concomitantly to the AB test[17 40] and a significant correlation was found betweenthe two dyes Thereby the nuclei protamination requires aspatial change in DNA structure with the intervention ofendogenous nucleases such as topoisomerase II which playsan important role in the phenomena of breakage and repairsperm DNA [41 42] So when repair systems are notsufficiently effective the nuclear DNA fragmentation mayincrease [43]

Similarly many studies reported an association betweensperm nuclei maturity evaluated by AB test and someDNA abnormalities in ejaculated human sperm [32 44ndash46] Indeed the common factor underlying sperm imma-turity and aneuploidies is the diminished expression ofHspA2 whose lack may cause diminished cellular transportof proteins such as DNA-repair enzymes essential duringspermiogenesis [47] Kovanci et al [47] demonstrated a closecorrelation between the incidence of immature spermatozoaand disomies (119903 = 07119875 lt 0001)These results suggested thepresence of a link between chromosomal meiotic segregationand the dynamic process of nucleoproteins during malegametogenesis [48]

Currently sperm selection techniques during medicalassisted procreation are designed to obtain sperm withldquoidealrdquomotility andmorphology Otherwise fertilization andpregnancy rate might be affected by sperm nucleus subtleanomalies that remain undetected by the biologist duringthe routine selection procedure [49] For this we used theAB staining test to evaluate the status of sperm nuclearmaturity after selection and we compared rates of spermwith immature chromatin before and after gradient densityselection We noted a significant improvement of spermchromatin maturity after selection concomitantly with theincrease of the sperm motility and the decrease of the abnor-mal spermmorphology rates Our results are similar to thosereported by Sanchez et al [50] and le Lannou and Blanchard[51] studies which showed that sperm selection techniqueslike swim-up migration or density gradient centrifugationincrease the proportion of sperm with normal chromatinstructure Sperm populations prepared by these techniquescontained more homogeneous subpopulations with a higherdegree of nuclearmaturity but the density gradient techniquewould be the best procedure [51 52] These results are inaccordance with those of Sakkas et al [53] who indicatedthat both PureSperm and Percoll density gradient assays can


enrich the sperm population by separating those with poorlycondensed chromatin It seems that the sperm glass-woolfiltration method increases significantly the percentage ofnormal chromatin condensed spermatozoa in comparisonwith traditional swim-up or gradient density [52 54]

In several previous reports the AB tingibility of sperma-tozoa was indicated to correlate well with infertility [2 20]Talebi et al [55] reported significant lower rates of maturespermatozoa in the group of patients with unexplained recur-rent spontaneous abortion Indeed the clinical significance ofthe assessment of sperm chromatin maturity lies in its asso-ciation not only with natural conception rates but also withassisted reproduction success rates [10 56] Sadeghi et al [16]believed that the fertilization rate following intracytoplasmicsperm injection (ICSI) was significantly increased in thegroup with less than 10 abnormal chromatin using AB testThese results were in agreement with the study of Esterhuizenet al [57] who showed that fertilization rate in ICSI is lowerwhen semen samples with high rates of nuclear condensationare used Other studies reported that sperm with high ratesof blue-stained nucleus can affect fertilization rate followingin vitro fertilization procedure and pregnancy rates [58 59]Also the risk of spontaneous abortion would be significantlyhigher after artificial insemination using ejaculates with highrates of immature nuclear sperm [60 61]

So it has been shown that a sperm with protaminedeficiency and increased histone remnants leads to prematurechromatin condensation that is the cause of failures infertilization and embryo development [1 62] Moreover thechromatin of spermwith reduced amounts of protamines wasobserved to be susceptible to chemical disruption [63]

In contrast Hammadeh et al [64] found no differencein fertilization rate in patients with ejaculated sperms orsperms obtained from testicular extracts although there wasa significant difference in the percentage of AB stainingbetween the two groups (ejaculated spermatozoa 331 plusmn 189and spermatozoa extracted from testis biopsy 700 plusmn 177with 119875 lt 00001) However unlike aniline blue staining theyfound a significant difference in the percentage of CMA3-positive sperm between the two groups This differencebetween AB and CMA3 staining could be due to the fact thatCMA3 staining is amore sensitive and specific test andCMA3reveals not only the presence of excessive histones but alsoprotamine deficiency [59] Similarly Razavi et al [49] did notfind any correlation between excessive histones evaluated byAB staining and fertilization rate following ICSI This couldbe explained by the presence of confounding factors such asICSI procedure operatorrsquos selection of spermatozoa accordingto normal morphology that may influence the effect of spermchromatin status on ICSI outcomes [16]

5 Conclusions

To assess the sperm nuclear maturity in the exploration ofmale infertility we can use simple techniques such as ABstaining in addition to classic sperm exploration methods asspermogram and sperm selection assays Our study providesadditional data on the correlation between the quality ofsperm nuclear material and morphological defects primarily

the sperm head anomalies Sperm chromatin integrity isessential for successful fertilization embryo developmentand normal pregnancy and a protamine deficiency appearedto effect fertilization rate and embryo qualityTherefore thereare still controversies over the effect of sperm chromatinstatus on fertilization rate embryo quality and pregnancyoutcomes The use of more sophisticated techniques as chro-momycin A3 assay (CAM3) [59] and transmission electronmicroscopy (TEM) image cytometry [65] provides moreaccurate and specific results in the evaluation of the spermnucleus maturity but has many technical constraints to usein routine investigations We must continue and deepen thestudy of sperm chromatin quality in infertile men to betterunderstand their relationshipwith the fertilizing capacity andbetter assess their impact on the results of in vitro fertilizationand embryo development

Conflict of Interests

Theauthors declare that the studywas conductedwithout anycompeting interests

Acknowledgment

The authors would like to thank the technicians of theLaboratory of Sfax Medical School for their help in semenprocessing and their technical assistance

References

[1] D Sakkas F Urner D Bizzaro et al ldquoSperm nuclear DNAdamage and altered chromatin structure effect on fertilizationand embryo developmentrdquo Human Reproduction vol 13 no 4supplement pp 11ndash19 1998

[2] M E Hammadeh M Stieber G Haidl andW Schmidt ldquoAsso-ciation between sperm cell chromatin condensation morphol-ogy based on strict criteria and fertilization cleavage andpregnancy rates in an IVF programrdquo Andrologia vol 30 no 1pp 29ndash35 1998

[3] A D Esterhuizen D R Franken J G H Lourens E Prinslooand L H Van Rooyen ldquoSperm chromatin packaging as anindicator of in-vitro fertilization ratesrdquo Human Reproductionvol 15 no 3 pp 657ndash661 2000

[4] C Roux C Tripogney C Joanne et al ldquoSperm chromatin pack-aging as an indicator of in-vitro fertilization ratesrdquo GynecologieObstetrique et Fertilite vol 32 no 9 pp 792ndash798 2004

[5] J-P Dadoune ldquoExpression of mammalian spermatozoal nucle-oproteinsrdquoMicroscopy Research and Technique vol 61 no 1 pp56ndash75 2003

[6] J P Dadoune ldquoThe nuclear status of human sperm cellsrdquo Mic-ron vol 26 no 4 pp 323ndash345 1995

[7] A Hekmatdoost N Lakpour and M R Sadeghi ldquoSperm chro-matin integrity etiologies and mechanisms of abnormalityassays clinical importance preventing and repairing damagerdquoAvicenna Journal of Medical Biotechnology vol 1 no 3 pp 147ndash160 2009

[8] D T Carrell B R Emery and S Hammoud ldquoThe aetiology ofsperm protamine abnormalities and their potential impact onthe sperm epigenomerdquo International Journal of Andrology vol31 no 6 pp 537ndash545 2008


[9] J W Hou D Chen and R S Jeyendran ldquoSperm nuclear matu-rity in spinal cord-injuredmen evaluation by acidic aniline bluestainrdquo Archives of Physical Medicine and Rehabilitation vol 76no 5 pp 444ndash445 1995

[10] AAgarwal andTM Said ldquoRole of sperm chromatin abnormal-ities andDNA damage inmale infertilityrdquoHuman ReproductionUpdate vol 9 no 4 pp 331ndash345 2003

[11] J Auger M Mesbah C Huber and J P Dadoune ldquoAniline bluestaining as amarker of sperm chromatin defects associated withdifferent semen characteristics discriminates between provenfertile and suspected infertile menrdquo International Journal ofAndrology vol 13 no 6 pp 452ndash462 1990

[12] J P Dadoune M J Mayaux andM L Guihard-Moscato ldquoCor-relation between defects in chromatin condensation of humanspermatozoa stained by aniline blue and semen characteristicsrdquoAndrologia vol 20 no 3 pp 211ndash217 1988

[13] O Hingst S Blottner and C Franz ldquoChromatin condensationin cat spermatozoa during epididymal transit as studied byaniline blue and acridine orange stainingrdquo Andrologia vol 27no 5 pp 275ndash279 1995

[14] M E Hammadeh T Zeginiadov P Rosenbaum T Georg WSchmidt and E Strehler ldquoPredictive value of sperm chromatincondensation (aniline blue staining) in the assessment of malefertilityrdquo Archives of Andrology vol 46 no 2 pp 99ndash104 2001

[15] A R Talebi M R Moein N Tabibnejad and J GhasemzadehldquoEffect of varicocele on chromatin condensation and DNAintegrity of ejaculated spermatozoa using cytochemical testsrdquoAndrologia vol 40 no 4 pp 245ndash251 2008

[16] M R Sadeghi M Hodjat N Lakpour et al ldquoEffects of spermchromatin integrity on fertilization rate and embryo qualityfollowing intracytoplasmic sperm injectionrdquo Avicenna Journalof Medical Biotechnology vol 1 no 3 pp 173ndash180 2009

[17] T Kazerooni N Asadi L Jadid et al ldquoEvaluation of spermrsquoschromatin quality with acridine orange test chromomycin A3and aniline blue staining in couples with unexplained recurrentabortionrdquo Journal of Assisted Reproduction and Genetics vol 26no 11-12 pp 591ndash596 2009

[18] A M Andreetta J C Stockert and C Barrera ldquoA simplemethod to detect sperm chromatin abnormalities cytochemicalmechanism and possible value in predicting semen qualityin assisted reproductive proceduresrdquo International Journal ofAndrology vol 18 no 1 supplement pp 23ndash28 1995

[19] NHofmann and BHilscher ldquoUse of aniline blue to assess chro-matin condensation inmorphologically normal spermatozoa innormal and infertile menrdquo Human Reproduction vol 6 no 7pp 979ndash982 1991

[20] C Foresta M Zorzi M Rossato and A Varotto ldquoSperm nuc-lear instability and staining with aniline blue abnormal persis-tance of histones in spermatozoa in infertile menrdquo InternationalJournal of Andrology vol 15 no 4 pp 330ndash337 1992

[21] WHO World Health Organization Laboratory Manual forthe Examination of Human Semen and Sperm-Cervical MucusInteraction Cambridge University Press 4th edition 1999

[22] N Salsabili A Mehrsai B Jalalizadeh et al ldquoCorrelation ofsperm nuclear chromatin condensation staining method withsemen parameters and sperm functional tests in patients withspinal cord injury varicocele and idiopathic infertilityrdquoUrologyJournal vol 3 no 1 pp 32ndash37 2006

[23] A Sadek A S A Almohamdy A Zaki M Aref S M Ibrahimand T Mostafa ldquoSperm chromatin condensation in infertilemen with varicocele before and after surgical repairrdquo Fertilityand Sterility vol 95 no 5 pp 1705ndash1708 2011

[24] H S KimM J Kang S A Kim et al ldquoTheutility of spermDNAdamage assay using toluidine blue and aniline blue staining inroutine semen analysisrdquoClinical and Experimental ReproductiveMedicine vol 40 no 1 pp 23ndash28 2013

[25] V W Aoki L Liu and D T Carrell ldquoIdentification and evalua-tion of a novel sperm protamine abnormality in a population ofinfertile malesrdquo Human Reproduction vol 20 no 5 pp 1298ndash1306 2005

[26] A Wong S S Chuan W C Patton J D Jacobson J Corselliand P J Chan ldquoAddition of eosin to the aniline blue assay toenhance detection of immature sperm histonesrdquo Fertility andSterility vol 90 no 5 pp 1999ndash2002 2008

[27] Y S Park M K Kim S H Lee et al ldquoEfficacy of testicularsperm chromatin condensation assay using aniline blue-eosinstaining in the IVF-ET cyclerdquo Clinical and Experimental Repro-ductive Medicine vol 38 no 3 pp 142ndash147 2011

[28] S Leiva M Loyola A M Agar and E Bustos-ObregonldquoEvaluation of DNAprotein status and nuclear maturity ofhuman spermrdquo Cytobios vol 78 no 312 pp 7ndash18 1994

[29] D R Franken C J Franken H de la Guerre and A de VilliersldquoNormal sperm morphology and chromatin packaging com-parison between aniline blue and chromomycin A3 stainingrdquoAndrologia vol 31 no 6 pp 361ndash366 1999

[30] L A Lazaros G A Vartholomatos E G Hatzi et al ldquoAssess-ment of sperm chromatin condensation and ploidy status usingflow cytometry correlates to fertilization embryo quality andpregnancy following in vitro fertilizationrdquo Journal of AssistedReproduction and Genetics vol 28 no 10 pp 885ndash891 2011

[31] A Zini S Phillips A Courchesne et al ldquoSperm headmorphol-ogy is related to high deoxyribonucleic acid stainability assessedby sperm chromatin structure assayrdquo Fertility and Sterility vol91 no 6 pp 2495ndash2500 2009

[32] 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 chromatincondensationrdquo Human Reproduction vol 26 no 7 pp 1650ndash1658 2011

[33] I M Adham K Nayernia E Burkhardt-Gottges et al ldquoTera-tozoospermia in mice lacking the transition protein 2 (Tnp2)rdquoMolecular Human Reproduction vol 7 no 6 pp 513ndash520 2001

[34] N Torregrosa D Domınguez-Fandos M I Camejo et alldquoProtamine 2 precursors protamine 1protamine 2 ratio DNAintegrity and other sperm parameters in infertile patientsrdquoHuman Reproduction vol 21 no 8 pp 2084ndash2089 2006

[35] K Tseden O Topaloglu A Meinhardt et al ldquoPremature trans-lation of transition protein 2mRNAcauses spermabnormalitiesand male infertilityrdquoMolecular Reproduction and Developmentvol 74 no 3 pp 273ndash279 2007

[36] L Nanassy L Liu J Griffin and D T Carrell ldquoThe clinicalutility of the protamine 1protamine 2 ratio in spermrdquo Proteinand Peptide Letters vol 18 no 8 pp 772ndash777 2011

[37] G N de Iuliis L KThomson L A Mitchell et al ldquoDNA dam-age in human spermatozoa is highly correlated with theefficiency of chromatin remodeling and the formation of 8-hydroxy-21015840-deoxyguanosine a marker of oxidative stressrdquo Biol-ogy of Reproduction vol 81 no 3 pp 517ndash524 2009

[38] R T Schulte D A Ohl M Sigman and G D Smith ldquoSpermDNA damage in male infertility etiologies assays and out-comesrdquo Journal of Assisted Reproduction and Genetics vol 27no 1 pp 3ndash12 2010


[39] R J Aitken andG N De Iuliis ldquoOn the possible origins of DNAdamage in human spermatozoardquo Molecular Human Repro-duction vol 16 no 1 Article ID gap059 pp 3ndash13 2009

[40] J Erenpreiss J Bars V Lipatnikova J Erenpreisa and J Zal-kalns ldquoComparative study of cytochemical tests for sperm chro-matin integrityrdquo Journal of Andrology vol 22 no 1 pp 45ndash532001

[41] D Sakkas EMariethoz GManicardi D Bizzaro P G Bianchiand U Bianchi ldquoOrigin of DNA damage in ejaculated humanspermatozoardquo Reviews of Reproduction vol 4 no 1 pp 31ndash371999

[42] R Henkel M Hajimohammad T Stalf et al ldquoInfluence ofdeoxyribonucleic acid damage on fertilization and pregnancyrdquoFertility and Sterility vol 81 no 4 pp 965ndash972 2004

[43] D Sakkas G Manicardi P G Bianchi D Bizzaro and UBianchi ldquoRelationship between the presence of endogenousnicks and sperm chromatin packaging in maturing and fertil-izing mouse spermatozoardquo Biology of Reproduction vol 52 no5 pp 1149ndash1155 1995

[44] F Morel S Mercier C Roux T Elmrini M C Clavequinand J L Bresson ldquoInterindividual variations in the disomyfrequencies of human spermatozoa and their correlation withnuclearmaturity as evaluated aniline blue stainingrdquoFertility andSterility vol 69 no 6 pp 1122ndash1127 1998

[45] L Sati L Ovari D Bennett S D Simon R Demir and GHuszar ldquoDouble probing of human spermatozoa for persistenthistones surplus cytoplasm apoptosis and DNA fragmenta-tionrdquo Reproductive BioMedicine Online vol 16 no 4 pp 570ndash579 2008

[46] L Ovari L Sati J Stronk A Borsos D C Ward and GHuszar ldquoDouble probing individual human spermatozoa ani-line blue staining for persistent histones and fluorescence in situhybridization for aneuploidiesrdquoFertility and Sterility vol 93 no7 pp 2255ndash2261 2010

[47] E Kovanci T Kovacs E Moretti et al ldquoFISH assessment ofaneuploidy frequencies in mature and immature human sper-matozoa classified by the absence or presence of cytoplasmicretentionrdquo Human Reproduction vol 16 no 6 pp 1209ndash12172001

[48] F Morel C Roux and J L Bresson ldquoDisomy frequency esti-mated by multicolour fluorescence in situ hybridization degreeof nuclear maturity and teratozoospermia in human spermato-zoardquo Reproduction vol 121 no 5 pp 783ndash789 2001

[49] S Razavi M H Nasr-Esfahani M Mardani A Mafi and AMoghdam ldquoEffect of human sperm chromatin anomalies onfertilization outcome post-ICSIrdquo Andrologia vol 35 no 4 pp238ndash243 2003

[50] R Sanchez E Villagran J Risopatron and R Celis ldquoEvaluationof nuclear maturity in human spermatozoa obtained by sperm-preparation methodsrdquo Andrologia vol 26 no 3 pp 173ndash1761994

[51] D le Lannou andY Blanchard ldquoNuclearmaturity andmorphol-ogy of human spermatozoa selected by Percoll density gradientcentrifugation or swim-up procedurerdquo Journal of Reproductionand Fertility vol 84 no 2 pp 551ndash556 1988

[52] M E Hammadeh A Kuhnen A S Amer P Rosenbaum andW Schmidt ldquoComparison of sperm preparation methodseffect on chromatin and morphology recovery rates and theirconsequences on the clinical outcome after in vitro fertilizationembryo transferrdquo International Journal of Andrology vol 24 no6 pp 360ndash368 2001

[53] D Sakkas G C Manicardi M Tomlinson et al ldquoThe use oftwo density gradient centrifugation techniques and the swim-upmethod to separate spermatozoawith chromatin andnuclearDNA anomaliesrdquo Human Reproduction vol 15 no 5 pp 1112ndash1116 2000

[54] R R Henkel D R Franken C J Lombard and W-B SchillldquoSelective capacity of glass-wool filtration for the separationof human spermatozoa with condensed chromatin a possibletherapeutic modality for male-factor casesrdquo Journal of AssistedReproduction and Genetics vol 11 no 8 pp 395ndash400 1995

[55] A R Talebi S Vahidi A Aflatoonian et al ldquoCytochemical eval-uation of sperm chromatin and DNA integrity in couples withunexplained recurrent spontaneous abortionsrdquoAndrologia vol44 supplement 1 pp 462ndash470 2012

[56] C Jeulin D Feneux and C Serres ldquoSperm factors related tofailure of human in-vitro fertilizationrdquo Journal of Reproductionand Fertility vol 76 no 2 pp 735ndash744 1986

[57] A D Esterhuizen D R Franken J G H Lourens C van ZylI I Muller and L H Van Rooyen ldquoChromatin packaging asan indicator of human sperm dysfunctionrdquo Journal of AssistedReproduction and Genetics vol 17 no 9 pp 508ndash514 2000

[58] D Y Liu and H W G Baker ldquoSperm nuclear chromatinnormality relationship with sperm morphology sperm-zonapellucida binding and fertilization rates in vitrordquo Fertility andSterility vol 58 no 6 pp 1178ndash1184 1992

[59] M H Nasr-Esfahani S Razavi and M Mardani ldquoRelationbetween different human sperm nuclear maturity tests andin vitro fertilizationrdquo Journal of Assisted Reproduction andGenetics vol 18 no 4 pp 219ndash225 2001

[60] E E Mohamed and M A Mohamed ldquoEffect of sperm chro-matin condensation on the outcome of intrauterine insem-ination in patients with male factor infertilityrdquo Journal ofReproductive Medicine vol 57 no 9-10 pp 421ndash426 2012

[61] M-H Lin R Kuo-Kuang Lee S-H Li C-H Lu F-J Sunand Y-M Hwu ldquoSperm chromatin structure assay parametersare not related to fertilization rates embryo quality and preg-nancy rates in in vitro fertilization and intracytoplasmic sperminjection but might be related to spontaneous abortion ratesrdquoFertility and Sterility vol 90 no 2 pp 352ndash359 2008

[62] R Oliva ldquoProtamines and male infertilityrdquo Human Reproduc-tion Update vol 12 no 4 pp 417ndash435 2006

[63] C Cho W D Willis E H Goulding et al ldquoHaploinsufficiencyof protamine-1 or -2 causes infertility in micerdquoNature Geneticsvol 28 no 1 pp 82ndash86 2001

[64] M E Hammadeh S Al-Hasani S Doerr et al ldquoComparisonbetween chromatin condensation and morphology from testisbiopsy extracted and ejaculated spermatozoa and their relation-ship to ICSI outcomerdquo Human Reproduction vol 14 no 2 pp363ndash367 1999

[65] J Auger D Schoevaert I Negulesco and J Dadoune ldquoThenuclear status of human sperm cells by TEM image cytometrynuclear shape and chromatin texture in semen samples fromfertile and infertile menrdquo Journal of Andrology vol 14 no 6pp 456ndash463 1993

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The addition of the evaluation of sperm head maturity toroutine semen analysis improves the assessment of fertility inmen [11] Many analytical techniques have been proposed toassess nuclear compaction in ejaculated sperm Some cyto-chemical or fluorescent dyes were used acidic aniline blue todetect excessive presence of histones [11ndash15] chromomycinA3 applied for the evaluation of protamine deficiency [16 17]and acridine orange [17] and toluidine blue staining used forassessing sperm chromatin structure and packaging [7 18]These different dyes have the advantage of providing suitableslides for use on a light or fluorescent microscope Anilineblue (AB) test evaluates the degree of sperm chromatincompaction ormaturation and it is able to detect sperm chro-matin defects related to their nucleoprotein content [11 14]This method allows discriminating between the presence oflysine-rich histones and arginine- and cysteine-rich protami-nes in sperm nuclei [11 19] Consequently histone-rich nucleiof immature spermwill take up the blue stain whereasmatureprotamine-rich nuclei remain unstained Several questionsobviously arise including (i) can the (AB) test predict themaleprognostic fertility and (ii) can it be a useful complementassay to direct the treatment of couple infertility

Many studies have shown negative correlations betweenthe defects of sperm chromatin integrity and male fertil-ity potential [20] However there is a controversy on thecorrelation between sperm nucleus maturity evaluated withAB staining and some semen parameters firstly and spermfertilization capacity and embryo quality secondly Otherreports have found a negative predictive value of nuclearcompaction in pregnancy outcome during assisted reproduc-tion treatment [10] So the aim of sperm selection techniquesapplied in assisted procreation techniques is not only basedon the assessment of classic parameters as sperm motilitynumeration and morphology but also involves more othersperm characteristics such as nuclear integrity

The aims of the present study were to assess the spermchromatin maturity in infertile men using acidic aniline bluestaining and to evaluate the relationship among chromatinsperm status and the semen parameters in male infertilityMoreover we analyzed whether the sperm gradient densitycentrifugation technique is effective to select mature sperma-tozoa

2 Materials and Methods

21 Patients Our study was carried out in 51 semen samplesfrommale partner of infertile couple attending theHistology-Embryology Laboratory of Sfax Medical School (Tunisia) forsemen investigationsThe patients were aged between 28 and49 years old with a mean age (plusmnstandard deviation (SD)) of3528 plusmn 062 years

22 Collection of Semen Samples Semen samples were col-lected bymasturbation after 3ndash5 days of sexual abstinence andallowed to liquefy for 30 minutes at 37∘C

23 Semen Analysis Basic semen analysis consisted in themeasurement of semen volume sperm concentration (hemo-cytometer method) motility (total motility rapid progressive

(type a) slow progressive (type b) and nonprogressive (typec)) vitality andmorphology All parameters were carried outaccording to the World Health Organization (WHO) guide-lines [21]

24 Semen Preparation Among these 51 samples 12 wereproceeded by Sill select density gradient centrifugation (119899 =12) Sill select (Fertipro NV Belgium) is a silane-coated silicaparticles solution that allows for separation of spermatozoaaccording to their density A 2-layer gradient was preparedusing respectively solutions of 90 and 45 Sill select Byusing a sterile pipette 1mL of liquefied semen sample wasplaced on top of the upper layer in a conical 15mL centrifugetube The tube was centrifuged at 300 g for 20 minutes Thesupernatant was then removed and the sperm pellet wassuspended in a volume of 3mL of Ferticult medium (FertiproNV Belgium) It was again centrifuged at 500 g for 10minutesThe final sperm pellet was suspended in 03mL of Ferticultmedium and examined for the sperm concentrationmotilitymorphology and AB staining

25 Acidic Aniline Blue Staining For each neat semen sample(119899 = 51) we evaluated the sperm nuclei chromatin conden-sation using AB staining Among these samples we also eva-luated by AB staining the nuclear maturity of spermatozoarecovered after a Sill select density gradient centrifugation(119899 = 12)

AB staining was performed as previously described byHofmann and Hilscher [19] After liquefaction 1mL of neatsemen was washed twice in 02M phosphate buffered (pH =72) We spread 20120583L of sperm pellet on glass slides andsperm smears are allowed to dry in air For the 12 sampleson which we performed a Sill select gradient density smearswere made with 20120583L from the final sperm pellet after adouble washing in 02M phosphate buffered (pH = 72)

Smears were fixed with a solution of 3 buffered glu-taraldehyde in 02M phosphate buffer (pH = 72) for 30min-utes Slides were then stained with 5 aqueous aniline bluesolution mixed with 4 acetic acid (pH = 35) for 5 minutes

For each stained smear 200 spermatozoa were evaluatedwith light microscope in oil immersion magnification (100xobjective) Spermatozoawith unstained nuclei are considerednormal (mature chromatin) while those blue stained wereconsidered abnormal (immature chromatin) (Figure 1) Theresults were expressed as percentages of nuclear unstainedand stained sperm An ejaculate with a rate of blue-stainednucleus sperm less than 20 was considered normal [4 11]

26 Statistical Analysis A statistical analysis was performedusing SPSS 130 software Statistical tests including Studentrsquos119905-test Pearsonrsquos and Spearmanrsquos correlations and linearregression were used The statistical significance was consid-ered for 119875 values lt005

3 Results

The mean values (plusmnSD) and ranges of semen parametersunstained (mature) sperm head (Figure 1(a)) and blue-stained (immature) spermhead (Figure 1(b)) are summarizedin Table 1


lowast

lowast

lowast

lowast

lowast

(a)

lowast

lowast

lowast

lowast

lowast

(b)











7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = minus0266

P = 005


4 Discussion









7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0425

P = 0003








7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0292

P = 003




0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12



0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12














5 Conclusions





Acknowledgment


References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

lowast

lowast

lowast

(a)

lowast

lowast

lowast

lowast

lowast

(b)











7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = minus0266

P = 005


4 Discussion









7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0425

P = 0003








7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0292

P = 003




0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12



0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12














5 Conclusions





Acknowledgment


References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0425

P = 0003








7000

6000

5000

4000

3000

2000

1000

000

Blue

-sta

ined

hea

d sp

erm

()

R = 0292

P = 003




0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12



0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12














5 Conclusions





Acknowledgment


References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10 11 12



0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12














5 Conclusions





Acknowledgment


References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions





Acknowledgment


References









































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article assessment of chromatin maturity in human...

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