Introduction

Semen analysis is the basic and most commonlyused test for predicting fertility; however, the stan-dard measurements of sperm concentration, percent-age motility, and morphology may not reveal subtlesperm defects. In this context, sperm chromatinabnormalities have been studied extensively in thepast decade as a cause for male infertility.1 The focuson the genomic integrity of the male gamete hasbeen further intensified by the growing concern oftransmission of genetic diseases through assistedreproductive techniques (ART), specifically intracy-toplasmic sperm injection (ICSI). Accumulating evi-dence indicates that a negative correlation existsbetween disturbances in the organization of thegenomic material in sperm nuclei and the fertilitypotential of spermatozoa, whether in vivo orin vitro.2,3 This emphasizes that stable DNA, which iscapable of decondensation at the appropriate time inthe fertilization process, is one of the criteria neededto consider a spermatozoon fertile.4 Conventionalsemen analysis per se cannot cover the diverse arrayof biological properties that the spermatozoonexpresses as a highly specialized cell.5,6 In addition,the results of semen analyses can be very subjectiveand prone to intra- and interobserver variability.7

At the present time, it is clear that a sperm chro-matin structure of poor quality may be indicative ofmale subfertility, regardless of the number, motility,and morphology of spermatozoa. Sperm chromatinstructure evaluation is an independent measure ofsperm quality that provides good diagnostic andprognostic capabilities. Therefore, it may be consid-ered a reliable predictor of a couple’s inability tobecome pregnant,8 and may also have an impact onthe offspring, resulting in infertility.9

Many techniques have been described for evalua-tion of the chromatin status. In this chapter, wedescribe the normal sperm chromatin architectureand the causative factors leading to its aberrations.We also provide the rationale and the different

methodologies employed for sperm chromatinassessment.

Human spermchromatin structure

The nuclear status of sperm cells is determined bytwo major events that occur during spermiogenesis:acquisition of the final nuclear shape and thereplacement of somatic-type histones by protamines(sperm-specific basic nuclear proteins) leading tohighly packaged chromatin. Sperm DNA is orga-nized in a specific manner to keep the chromatin inthe nucleus compact and stable. It is packed into atight, almost crystalline status that is at least sixtimes more condensed than mitotic chromosomes. Itoccupies nearly the entire nucleus volume, whereassomatic cell DNA only partly fills the nucleus.10

This DNA organization not only permits the verytightly packaged genetic information to be trans-ferred to the egg, but also ensures that the DNA isdelivered in a physical and chemical form thatallows the developing embryo to access the geneticinformation.11

Sperm nuclei do not have the volume required forthe type of packaging present in somatic cells,because packing the DNA in even a single, closelypacked nucleosome would require 9.9 µm3, which ismore than twice the volume of an average spermnucleus. Thus, a completely different type of DNApackaging must be present in mammalian spermnuclei.12 Organization of chromatin for packaging inthe spermatozoon takes place at four different levels:chromosomal anchoring, which refers to the attach-ment of the DNA to the nuclear annulus; formationof DNA loop domains as the DNA attaches to thenewly added nuclear matrix; replacement of histonesby protamines, which condense the DNA into com-pact doughnuts; and chromosomal positioning.12 Thehistones are first displaced by transition proteins

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(TNPs), which are removed from the condensingchromatin at later stages and replaced by protamines.It is of interest to note that the condensation of chro-matin begins in the posterior pole and proceedsapically, which is a unique feature in humans thatis not present in other mammalian species.13 Spermepididymal maturation implies a final stage of chro-matin organization involving protamine cross-linkingby disulfide bond formation—a step that is supportedby the fact that protamines contain a significantnumber of cysteine residues that participate insperm chromatin compaction by forming multipleinter- and intraprotamine disulfide cross-links. All ofthese interactions make mammalian DNA the mostcondensed eukaryotic DNA.14

Origin of spermchromatin abnormalities

Sperm nuclear chromatin abnormalities/DNA dam-age could occur at the time of, or be the result of,DNA packing at spermiogenesis.15 Environmentalstress, gene mutations, and chromosomal abnormali-ties can disturb the highly refined biochemicalevents that occur during spermatogenesis. This canultimately lead to an abnormal chromatin structurethat is incompatible with fertility. However, the exactmechanism(s) by which chromatin abnormalities/DNA damage arise in human spermatozoa are notprecisely understood. Three main theories have beenproposed: defective sperm chromatin packaging,apoptosis, and oxidative stress (OS).

Contributing factors

The most important factor contributing to spermchromatin damage is leukocytospermia. It may resultin reactive oxygen species (ROS)-induced cross-damage of sperm,16 and is associated with proin-flammatory mediators such as cytokines, causingalterations in the regulation of spermiogenesis andsubsequently chromatin aberrations. Similarly, OSmay be the underlying reason why sperm DNA fromsmokers contains more strand breaks than that fromnon-smokers.17 High levels of sperm DNA damagecan be seen following exposure to irradiation andchemotherapeutic agents and may persist for severalmonths.18 Finally, sperm preparation techniquesinvolving repeated high-speed centrifugation andthe isolation of spermatozoa from the protectiveantioxidant environment provided by seminalplasma may contribute to increased sperm DNAdamage via mechanisms that are mediated by theenhanced generation of ROS.19

Indications of spermchromatin assessment

Diagnosis of male infertility

The positive relationship between poor sperm para-meters and DNA damage in spermatozoa points toinherent problems.20 In general, infertile patientshave higher levels of DNA strand breaks than seen infertile males.21 Moreover, spermatozoa from infertilepatients are generally more susceptible to the effectsof DNA-damaging agents such as H2O2 and radi-ographic exposure.22 Thus, the sperm DNA integritymay be considered an objective marker of spermfunction that serves as a significant prognostic factorfor male infertility. Recently, we discovered a signif-icant increase in sperm chromatin structure assay(SCSA)-defined DNA damage in sperm from infertilemen with normal semen parameters, leading us tospeculate that sperm DNA damage analysis mayreveal a hidden abnormality of sperm DNA in infer-tile men classified with idiopathic infertility basedon apparently normal standard semen parameters.23

Assisted reproductive techniques

During ICSI, the sperm cell is injected directly intothe cytoplasm of the mature oocyte. Because thesperm membrane–oocyte interaction is no longerrelevant, increased emphasis is placed on the qualityof the sperm chromatin and the ability of the oocyteto initiate decondensation and pronuclear forma-tion. In general, the ICSI fertilization rate does notexceed 65–80%24 despite the mechanical injection ofone sperm into a mature oocyte. The fertilizationrate is lower than expected possibly because spermthat are selected from semen of patients with malefactor infertility may have defects in their DNA.Therefore, although the most normal-appearing andmotile spermatozoa are selected, there is always asmall percentage of sperm used in in vitro fertiliza-tion (IVF)/ICSI that contain varying degrees of DNAdamage.2 Semen samples characterized by increasedDNA fragmentation levels are often associated withdecreased fertilization rates and/or embryo cleavagefollowing IVF and ICSI, and may be linked to anincrease in early embryo death.25

In a recent study (Fig 7.1), we tested the correla-tion of sperm DNA damage with different ART out-comes. We reported that the percentage of DNAfragmentation index (DFI) was significantly higherin infertile men who did not achieve a clinical preg-nancy with ART (38 (interquartile range 28–43))than in those who did (21 (13–25); p = 0.001). Inaddition, we found no clinical pregnancy when the

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DFI was higher than 28%.26 Thus, assessment ofsperm chromatin may help to predict the successrates of ART.

Cancer patients

Patients with cancer are often referred to sperm banksbefore chemotherapy, radiation therapy, or surgery isinitiated. Although pregnancies and births have beenreported using cryopreserved sperm from patientswith cancer, these semen samples have decreased fer-tilization potential. The extent of DNA damage mayhelp to determine how semen should be cryopre-served before therapy begins. Specimens with high

sperm concentration and motility and low levels ofDNA damage should be preserved in relatively largealiquots that are suitable for intrauterine insemina-tion (IUI). If a single specimen of good quality isavailable, then it should be preserved in multiplesmall aliquots suitable for IVF or ICSI.27

Evaluation of spermnuclear DNA damage

Different methods may be used to evaluate thestatus of the sperm chromatin for the presence ofabnormalities or simply immaturity (Table 7.1).

95Sperm chromatin assessment

Assay Parameter Method of analysis

Acidic aniline blue28 Nuclear maturity (DNA Optical microscopyprotein composition)

Toluidine blue stain29 DNA fragmentation Optical microscopyChromomycin A3

30 Nuclear maturity (DNA Fluorescent microscopyprotein composition)

Sperm chromatin dispersion31 DNA fragmentation Fluorescent microscopyDNA breakage detection–fluorescent DNA fragmentation (ssDNA) Fluorescent microscopy

in situ hybridization32

In situ nick translation33 DNA fragmentation (ssDNA) Fluorescent microscopyFlow cytometry

Acridine orange34 DNA denaturation (acid) Fluorescent microscopyFlow cytometry

TUNEL35 DNA fragmentation Optical microscopyFluorescent microscopyFlow cytometry

Comet (neutral)36 DNA fragmentation (dsDNA) Fluorescent microscopy(alkaline)37 DNA fragmentation (ssDNA /dsDNA)

Sperm chromatin structure38 DNA denaturation (acid/heat) Flow cytometry8-OHdG measurement39 8-OHdG High-performance liquid

chromatography

8-OHdG, 8-hydroxy-2-deoxyguanosine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA

Table 7.1 Various methods for assessing sperm chromatin abnormalities.

% DNAfragmentation

Clinicalpregnancy

18 ± 7

(+)n = 2

44 ± 9

(−)n = 2

20 ± 7

(+)n = 3

35 ± 12

(−)n =12

13 ± 0

(+)n = 1

28 ± 0

(−)n = 1

21 ± 5

(+)n = 2

38 ± 13

(−)n = 6

⎯

⎯

⎯

⎯

28 ± 0

(+)n = 1

37 ± 12

(−)n = 3

IUIn = 19

IVFn = 10

ICSIn = 4

Normaln = 4

Abnormaln = 15

Semenanalysis

Normaln = 2

Abnormaln = 8

Normaln = 0

Abnormaln = 4

Fig 7.1 Flow diagram of the study showing the number of patients in each group and their clinical pregnancy outcome. Results ofpercentage DNA fragmentation are expressed as mean ± standard deviation. IUI, intrauterine insemination; IVF, in vitro fertilization;ICSI, intracytoplasmic sperm injection.

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These assays include simple staining techniquessuch as the acidic aniline blue (AAB) and toluidineblue (TB) stains, fluorescent staining techniquessuch as the sperm chromatin dispersion (SCD) test,chromomycin A3 (CMA3), DNA breakage detection–fluorescent in situ hybridization assay (DBD–FISH),in situ nick translation (NT), and flow cytometric-based sperm chromatin structure assay (SCSA).Some assays employ more than one method forthe analysis of their results. Examples of theseassays include the acridine orange (AO) and termi-nal deoxynucleotidyl transferase-mediated fluores-cein-deoxyuridine triphosphate-nick end labeling(TUNEL) assays. Other methods less frequentlyused include high-performance liquid chromato-graphy (HPLC).

Acidic aniline blue stain

Principle

The AAB stain discriminates between lysine-richhistones and arginine/cysteine-rich protamines.This technique provides a specific positive reactionfor lysine and reveals differences in the basicnuclear protein composition of ejaculated humanspermatozoa. Histone-rich nuclei of immature sper-matozoa are rich in lysine and will consequentlytake up the blue stain. On the other hand, protamine-rich nuclei of mature spermatozoa are rich in argi-nine and cysteine and contain relatively low levelsof lysine, which means they will not be stained byaniline blue.40

Technique

Slides are prepared by smearing 5 µl of either raw orwashed semen sample. The slides are air-dried andfixed for 30 minutes in 3% glutaraldehyde in phos-phate-buffered saline (PBS). The smear is dried andstained for 5 minutes in 5% aqueous aniline bluesolution (pH 3.5). Sperm heads containing immaturenuclear chromatin stain blue, and those with maturenuclei do not take up the stain. The percentage ofspermatozoa stained with aniline blue is determinedby counting 200 spermatozoa per slide under brightfield microscopy.28

Clinical significance

Results of AAB have shown a clear associationbetween abnormal sperm chromatin and male infer-tility.41 However, the correlation between the per-centage of aniline blue-stained spermatozoa andother sperm parameters remains controversial.

Immature sperm chromatin may or may not correlatewith asthenozoospermic samples and abnormalmorphology patterns.28,40 Most important is thefinding that chromatin condensation as visualizedby aniline blue staining is a good predictor forIVF outcome, although it cannot determine thefertilization potential, cleavage, and pregnancy ratefollowing ICSI.42

Toluidine blue stain

Principle

Toluidine blue is a basic nuclear dye used formetachromatic and orthochromatic staining ofchromatin. It becomes heavily incorporated in thedamaged dense chromatin. This stain is a sensitivestructural probe for DNA structure and packaging.

Technique

The protocol of the TB stain includes four steps. Thesmears are air-dried, fixed in freshly made 96%ethanol–acetone (1 : 1) at 4°C for 30 minutes,hydrolyzed in 0.1 N Hcl at 4°C for 5 minutes, andrinsed three times in distilled water for 2 minuteseach. Smears are stained with 0.05% TB (Merck,Poole, Dorset, UK) for 10 minutes. The staining bufferconsists of 50% citrate phosphate (McIlvain buffer,pH 3.5). Permanent preparations are dehydrated intertiary butanol twice for 3 minutes each at 37°C, andin Histoclear (RA Lambs Labs, Apex, NC) twice for3 minutes each. Afterwards, the preparations areembedded in DPX. Sperm heads with good chro-matin integrity stain light blue, and those of dimin-ished integrity stain violet (purple) (Fig 7.2a).29

Clinical significance

Due to the cooperative nature of metachromaticstaining, which indicates poor sperm integrity, onlysevere DNA damage is revealed. Nevertheless, TBstaining may be considered a fairly reliable methodfor assessing the sperm chromatin. Abnormal nuclei(purple-violet sperm heads) have been shown to becorrelated with counts of red-orange sperm heads asrevealed by the AO method.43

Advantages and limitations

In general, the AAB and TB methods are simple andinexpensive and have the advantage of providing per-manent preparations for use on an ordinary micro-scope. The smears stained with the TB method canalso be used for morphological assessment of the cells.

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In this way, the TB stain method has the advantage, incomparison with the AAB method. However, thesemethods have the inherent limits of repeatability dic-tated by dye equilibrium variations and by a limitednumber of cells which can be reasonably scored.

Chromomycin A3 assay

Principle

Chromomycin A3 is a guanine–cytosine-specific flu-orochrome that reveals chromatin that is poorlypackaged in human spermatozoa via indirect visual-ization of protamine-deficient DNA. ChromomycinA3 and protamines compete for the same bindingsites in the DNA. Therefore, high CMA3 fluorescenceis a strong indicator of the low protamination state ofspermatozoa.30

Technique

For CMA3 staining, semen smears are first fixed inmethanol–glacial acetic acid 3 : 1 at 4°C for 20 min-utes and are then allowed to air-dry at room temper-ature for 20 minutes. The slides are treated for20 minutes with 100 µl CMA3 solution. The CMA3

solution consists of 0.25 mg/ml CMA3 in McIlvain’sbuffer (pH 7.0) supplemented with 10 mmol/l MgCl2.The slides are rinsed in buffer and mounted with1 : 1 v/v PBS–glycerol. The slides are then kept at4°C overnight after which evaluation of fluorescenceis performed using a fluorescent microscope. A totalof 200 spermatozoa are randomly evaluated on eachslide. Evaluation of CMA3 staining is done by distin-guishing spermatozoa that stain bright yellow (CMA3

positive) from those that stain a dull yellow (CMA3

negative).30

Clinical significance

As a discriminator of IVF success (> 50% oocytesfertilized), CMA3 staining has a sensitivity of 73% andspecificity of 75%. Therefore, it can distinguishbetween IVF success and failure.44 In cases of ICSI,Sakkas et al.45 reported that the percentage of CMA3

positivity does not indicate failure of fertilizationentirely, and suggested that poor chromatin packagingcontributes to a failure in the decondensation processand probably reduced fertility. It appears that semensamples with high CMA3 positivity (> 30%) may havesignificantly lower fertilization rates if used for ICSI.25

Advantages and limitations

The CMA3 assay yields reliable results as it isstrongly correlated with other assays used in theevaluation of sperm chromatin.30 In addition, the sen-sitivity and specificity of the CMA3 stain are compa-rable to those of the AAB stain (75% and 82%, 60%and 91%, respectively) if used in evaluation of thechromatin status in infertile men.46 However, it isimportant to note that all of these assays mentionedto this point are limited by observer subjectivity.

DNA breakage detection—fluorescentin situ hybridization assay

Principle

Cells embedded within an agarose matrix on aslide are exposed to an alkaline unwinding solu-tion, which transforms DNA-strand breaks intosingle-stranded DNA (ssDNA) motifs. After neutral-izing and protein removal, ssDNA is accessible tohybridization with whole genome or specific DNA

97Sperm chromatin assessment

Fig 7.2 (a) Human ejaculate stained with toluidine blue: (1) mature sperm heads are light blue; (2) immature are violet. (b) DNAbreakage detection–fluorescence in situ hybridization (DBD–FISH) labeling with a whole genome probe (red fluorescence), demon-strating extensive DNA breakage in those nuclei that are intensely labeled. (c) Acridine orange (AO) stain to native DNA fluorescesgreen (1); whereas denatured DNA fluoresces red (2).

a

1 1

2

1

22

b c

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probes. The probe highlights the chromatin area tobe analyzed. As DNA breaks increase, the moressDNA is produced by the alkaline solution and themore the probe hybridizes, resulting in an increasein the fluorescence intensity and surface area of theFISH signal. Abnormal chromatin packaging insperm cells greatly increases the accessibility ofDNA ligands and the sensitivity of DNA to denatura-tion by alkali, and this relates to the presence ofintense labeling (red fluorescence) by DBD–FISH.Therefore, DBD–FISH allows in situ detection andquantification of DNA breaks and reveals structuralfeatures in the sperm chromatin.32,47

Technique

To perform this assay, sperm cells are mixed with low-melting-point agarose to a final concentration of 0.7%at 37°C. A volume of 300 µl of the mixture is pipettedonto polystyrene slides and allowed to solidify at 4°C.The slides are immersed into a freshly prepared alka-line denaturation solution (0.03 mol/l NaOH, 1 mol/lNaCl) for 5 minutes at 22°C in the dark, to generatessDNA from DNA breaks. The denaturation is thenstopped, and proteins are removed by transferring theslides to a tray with neutralizing and lysing solution 1(0.4 mol/l Tris, 0.8 mol/l dithiothreitol (DTT), 1%sodium dodecylsulfate (SDS), and 50 mmol/l ethyl-enediaminetetra-acetic acid (EDTA), pH 7.5) for10 minutes at room temperature, which is followedby incubation in neutralizing and lysing solution2 (0.4 mol/l Tris, 2 mol/l NaCl, and 1% SDS, pH 7.5)for 20 minutes at room temperature. The slides arethoroughly washed in Tris–borate– EDTA buffer (0.09mol/l Tris–borate and 0.002 mol/l EDTA, pH 7.5) for15 minutes, dehydrated in sequential 70%, 90%, and100% ethanol baths (2 minutes each), and air-dried. Ahuman whole genome probe is hybridized overnight(4.3 ng/µl in 50% formamide/2 × standard salinecitrate (SSC), 10% dextran sulfate, and 100 mmol/lcalcium phosphate, pH 7.0) (1 × SSC is 0.015 mol/lsodium citrate and 0.15 mol/l sodium chloride,pH 7.0). It is then washed twice in 50% forma-mide/2 × SSC, pH 7.0, for 5 minutes, and twice in2 × SSC (pH 7.0) for 3 minutes at room temperature.The hybridized probe is detected with streptavidin–indocarbocyamine (1 : 200) (Sigma Chemical Co.,St Louis, MO), and cells are counterstained with4′,6-diamidino-2-phenylindole (DAPI) (1 µg/ml) andvisualized using fluorescent microscopy (Fig 7.2b).32

Advantages and limitations

Although the assay reveals chromatin structural fea-tures, it is expensive, time-consuming, and involvessophisticated procedures. The assay is of less clinical

value as the results yielded are not superior to thoseof other, less cumbersome assays.32

In situ nick translation assay

Principle

The NT assay quantifies the incorporation of biotiny-lated deoxyuridine triphosphate (dUTP) at single-strand DNA breaks in a reaction that is catalyzed bythe template-dependent enzyme DNA polymerase I. Itspecifically stains spermatozoa that contain apprecia-ble and variable levels of endogenous DNA damage.The NT assay indicates anomalies that have occurredduring remodeling of the nuclear DNA in spermato-zoa, and in doing so is more likely to detect spermanomalies that are not indicated by morphology.

Technique

To perform the assay, smears containing 500 spermeach should be prepared. The fluorescent stainingsolution is prepared by mixing 10 µl streptavidin–fluorescein–isothiocyanate, 90 µl Tris buffer, and900 µl double-distilled water. One hundred micro-liters of this solution is added to the slides. The incu-bation is carried out in a moist chamber at 37°C for30 minutes. After incubation, the slides are rinsed inPBS twice, washed with distilled water, and finallymounted with a 1 : 1 mixture of PBS and glycerol.The slides are examined using fluorescent microscopy.A total of 100–200 spermatozoa should be counted,and those fluorescing and hence incorporating thedye are classified as having endogenous nicks.33

Clinical significance

Sperm nuclear integrity as assessed by the NT assaydemonstrates a very clear relationship with spermmotility and morphology and, to a lesser extent,sperm concentration.48,49 Results of the assay aresupported by the strong positive correlations detec-ted with the sensitivity of CMA3 and TUNEL assays(r = 0.86; p < 0.05 and r = 0.87; p < 0.05, res-pectively).30 The NT assay is also able to indicateif there is damage arising from factors such asheat exposure50 or the generation of ROS followingexposure to leukocytes within the male reproduc-tive tract.51

Advantages and limitations

The advantage of the NT assay is that the reaction isbased on direct labeling of termini of DNA breaks,

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and thus the lesions that are measured areidentifiable at the molecular level. In addition, ifflow cytometry is used to analyze the results, it maybe performed on fixed cells, as the time of cellstorage in ethanol may vary.33

Acridine orange assay

Principle

The AO assay measures the susceptibility of spermnuclear DNA to acid-induced denaturation in situ byquantifying the metachromatic shift of AO fluores-cence from green (native DNA) to red (denaturedDNA). The fluorochrome AO intercalates intodouble-stranded DNA as a monomer and bindsto single-stranded DNA as an aggregate. Themonomeric AO bound to native DNA fluorescesgreen, whereas the aggregated AO on denaturedDNA fluoresces red (Fig 7.2c).52

Technique

The AO assay may be used for either fluorescence orflow cytometry. To perform this assay for fluorescentmicroscopy, thick smears are fixed in Carnoy’s fixa-tive (methanol : acetic acid 1 : 3) for at least 2 hours.The slides are stained for 5 minutes and gentlyrinsed with deionized water. At least 200 cellsshould be counted so that the estimate of thenumbers of sperm with green and red fluorescenceis accurate.

For flow cytometry, aliquots of semen (about25–100 µl, containing 1 million spermatozoa) aresuspended in 1 ml of ice-cold PBS (pH 7.4) and cen-trifuged at 600 g for 5 minutes. The pellet is resus-pended in ice-cold TNE (0.01 mol/l Tris-HCl,0.15 mol/l NaCl, and 1 mmol/l ethylenediaminetetra-acetic acid (EDTA), pH 7.4) and again centrifuged at600 g for 5 minutes. The pellet is then resuspendedin 200 µl of ice-cold TNE with 10% glycerol andimmediately fixed in 70% ethanol for 30 minutes.The fixed samples are treated for 30 seconds with400 µl of a solution of 0.1% Triton X-100, 0.15 mol/lNaCl, and 0.08N HCl, pH 1.2. After 30 seconds,1.2 ml of staining buffer (6 µg/ml AO, 37 mmol/l cit-ric acid, 126 mmol/l Na2HPO4, 1 mmol/l disodiumEDTA, 0.15 mol/l NaCl, pH 6.0) is admixed to thetest-tube and analyzed by flow cytometry. After exci-tation by a 488-nm wavelength light source, AObound to double-stranded DNA fluoresces green(515–530 nm) and AO bound to single-strandedDNA fluoresces red (630 nm or greater). A minimumof 5000 cells are analyzed by fluorescent activatedcell sorting (FACS).34

Clinical significance

Staining with AO shows a significant differencebetween fertile males and those who are infertile withdifferent andrologic pathologies. The “cut-off” valueset to differentiate between fertile and infertile menvaries between 20 and 50%.8,34,53 Studies show thatssDNA that is detected by a low incidence (< 50%) ofgreen AO fluorescence negatively affects the ferti-lization process in a classical IVF program.52,54,55

However, no correlation was found with pregnancyrate and live births achieved by ICSI, except inpatients having 0% of spermatozoa with ssDNA, inwhom the pregnancy rate was significantly high.54

Advantages and limitations

The AO assay is a biologically stable measure ofsperm quality. The interassay variability is less than5%, rendering the technique highly reproducible.5 Astrong positive correlation exists between the AOassay and other techniques used to evaluate ssDNA,e.g. TUNEL assay.56 The AO assay still requires expen-sive instrumentation if flow cytometry is used tointerpret the results. Also, observer subjectivity mayhinder the results if fluorescent microscopy is used.

Sperm chromatin dispersion test

Principle

If spermatozoa with nonfragmented DNA areimmersed in an agarose matrix and directly exposedto lysing solutions, the resulting deproteinizednuclei (nucleoids) show extended halos of DNA dis-persion as monitored by fluorescent microscopy.The presence of DNA breaks promotes the expansionof the halo of the nucleoid.57 The SCD test is basedon the principle that when sperm are treated with anacid solution prior to lysis buffer, the DNA disper-sion halos that are observed in sperm nuclei withnonfragmented DNA after the removal of nuclearproteins are either minimally present or not pro-duced at all in sperm nuclei with fragmented DNA.

Technique

Aliquots of either raw or washed semen samplesshould be adjusted to concentrations rangingbetween 5 and 10 million/ml. The suspensions aremixed with 1% low-melting-point aqueous agarose(to obtain a 0.7% final agarose concentration) at37°C. Aliquots of 50 µl of the mixture shouldbe pipetted onto a glass slide precoated with0.65% standard agarose dried at 80°C, covered with

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a coverslip, and left to solidify at 4°C for 4 minutes.The coverslips are then carefully removed, and theslides are immediately immersed horizontally in atray of freshly prepared acid denaturation solution(0.08N HCl) for 7 minutes at 22°C in the dark, whichgenerates restricted single-stranded DNA (ssDNA)motifs from DNA breaks. Denaturation is thenstopped, and the proteins are removed by transfer-ring the slides to a tray with neutralizing and lysingsolution 1 (0.4 mol/l Tris, 0.8 mol/l DTT, 1% SDS,and 50 mmol/l EDTA, pH 7.5) for 10 minutes at roomtemperature. The slides are then incubated in neu-tralizing and lysing solution 2 (0.4 mol/l Tris,2 mol/l NaCl, and 1% SDS, pH 7.5) for 5 minutes atroom temperature. The slides are thoroughly washedin Tris-borate–EDTA buffer (0.09 mol/l Tris-borateand 0.002 mol/l EDTA, pH 7.5) for 2 minutes, dehy-drated in sequential 70%, 90%, and 100% ethanolbaths (2 minutes each), and air-dried. Cells arestained with DAPI (4′,6-diamidino-2-phenylindole)(2 µg/ml) for fluorescence microscopy (Fig 7.3a).31

Advantages and limitations

The major advantage of the SCD test is that it does notrequire the determination of color or fluorescenceintensity. Rather, the percentage of spermatozoa withnondispersed (very small halos or none at all) or dis-persed nuclei is determined, which can be easily andreliably accomplished by the naked eye. Furthermore,the test is simple, fast, and reproducible, and itsresults are comparable to those of the SCSA.31 Becausethe SCD test was recently introduced, little is knownabout its limitations and its clinical significance.

Comet assay

Principle

The comet assay, also known as single-cell gel elec-trophoresis for analysis of DNA damage in an indi-vidual cell, was first introduced by Ostling andJohanson in 1984.58 Neutral electrophoresis bufferconditions were used to show that the migration ofdouble-stranded DNA loops from a damaged cell inthe form of a tail unwinding from the relaxed super-coiled nucleus was proportional to the extent ofdamage inflicted on the cell. This finding took on theappearance of a comet with a tail when viewedusing the fluorescent microscope and DNA stains.Singh et al. modified the comet assay in 198836 byusing alkaline electrophoresis buffers to exposealkali-labile sites on the DNA; this modificationincreased the sensitivity of the assay to detect bothsingle- and double-stranded DNA breaks.37

The damage is quantified by measuring thedisplacement between the genetic material of the

nucleus “comet head” and the resulting tail. The taillengths are used as an index for the damage. Also,the “tail moment,” which is the product of the taillength and intensity (fraction of total DNA in thetails), has been used as a measuring parameter. Thetail moment can be more precisely defined as beingequivalent to the torsional moment of the tail.59

Technique

In this assay, sperm cells are cast into miniatureagarose gels on microscope slides and lysed in situ toremove DNA-associated proteins and to allow thecompacted DNA in the sperm to relax. The lysisbuffer (Tris 10 mmol/l, 0.5 mol/l EDTA, and 2.5 mol/lNaCl, pH 10) contains 1% Triton X-100, 40 mmol/ldithiothreitol, and 100 µg/ml proteinase K). Micro-gels are then electrophoresed (20 minutes at 25 V/0.01 A) in neutral buffer (Tris 10 mmol/l containing0.08 mol/l boric acid and 0.5 mol/l EDTA, pH 8.2),during which the damaged DNA migrates from thenucleus towards the anode. The DNA is visualized bystaining the slides with the fluorescent DNA bindingdye SYBR Green I. Comet measurements are per-formed using fluorescent microscopy. These mea-surements can be done either manually or withcomputerized image analysis (Fig 7.3b).36

Clinical significance

The assay has been successfully used in the evalua-tion of DNA damage after cryopreservation.60 It mayalso predict embryo development after IVF and ICSI,especially in couples with unexplained infertility.61,62

Advantages and limitations

The comet is a well-standardized assay that corre-lates significantly with TUNEL and SCSA assays.63 Itis simple to perform, has a low intra-assay coeffi-cient of variation, and a low performance cost.64

Because it is based on fluorescent microscopy, theassay requires an experienced observer to analyzethe slides and interpret the results.

Terminal deoxynucleotidyltransferase-mediated deoxyuridinetriphosphate-nick end labeling assay

Principle

The TUNEL assay quantifies the incorporation ofdUTP at single- and double-strand DNA breaks in areaction catalyzed by the template-independent

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enzyme terminal deoxynucleotidyl transferase (TdT).This enzyme incorporates biotinlyated deoxyuridineto 3′-OH of DNA to create a signal, which increaseswith the number of DNA breaks. Sperm with normalDNA therefore have only background staining/fluorescence, while those with fragmented DNA(multiple chromatin 3′-OH ends) stain/fluorescebrightly.33

Technique

Identification of strand breaks can be quantified byflow cytometry or fluorescent microscopy in whichDNA-damaged sperm fluoresce intensely.65 To assessthe DNA fragmentation by TUNEL, about 2 × 106

sperm are fixed with 1% formaldehyde for 10 min-utes at room temperature. The sample is centrifugedat 10 000 g for 4 minutes. After the sperm arewashed in PBS (pH 7.4), they are resuspended in100 µl prewash buffer containing single-strengthOne-Phor-All® buffer (100 mmol/l Tris-acetate,100 mmol/l magnesium acetate, 500 mmol/l potas-sium acetate; and 0.1% Triton X-100) for 10 minutesat room temperature. Fixed sperm are spun out ofthe buffer and resuspended in 50 µl of TdT buffercontaining 3 µmol/l biotin-16-dUTP, 12 µmol/ldeoxyadenosine triphosphate (dATP), 0.1% TritonX-100, and 10 U of TdT enzyme and incubated at37°C for 60 minutes. After two washes in PBS, thefixed, permeabilized sperm are resuspended in100 µl of staining buffer consisting of 0.1% TritonX-100 and 1% streptavidin/Texas red antibiotin andincubated at 4°C in the dark for 30 minutes. Thestained cells are washed in PBS/0.1% Triton X-100.To create negative controls, the enzyme terminaltransferase may be omitted from the reaction mix-ture. To create positive controls, the samples arepretreated with 0.1 IU DNAase I for 30 minutes atroom temperature and then labeled. Results may be

interpreted by assessing 100–500 sperm cells underfluorescent microscopy or by using FACS flowcytometry (Fig 7.4a and b).35

Clinical significance

The TUNEL assay has been widely used in maleinfertility research related to sperm chromatin/DNAabnormalities. It provides useful information inmany cases of male infertility. A negative correlationwas found between the percentage of DNA-fragmented sperm and the motility, morphology, andconcentration in the ejaculate. It also appears to bepotentially useful as a predictor for IUI pregnancyrate, IVF embryo cleavage rate, and ICSI fertilizationrate. In addition, it provides an explanation forrecurrent pregnancy loss.2,66–69

Advantages and limitations

We believe that although the flow cytometric methodof assessment is generally more accurate and reliable,it is also sophisticated and expensive. On the otherhand, the fluorescent TUNEL assay has demonstratedfairly good quality control parameters. The intra-observer variability was found to be < 8% and theinterobserver variability was < 7% (Spearman rankcorrelation between observers was r = 0.54, p = 0.01).35

Sperm chromatin structure assay

Principle

The SCSA relies on the fact that abnormal spermchromatin has a greater susceptibility to the physicalinduction of partial DNA denaturation in situ. Theextent of DNA denaturation following heat oracid treatment is determined by measuring the

101Sperm chromatin assessment

Fig 7.3 (a) Spermatozoa embedded in an agarose microgel stained with DAPI (4’, 6-diamidino-2-phenylindole) staining (blue fluores-cence) and showing spermatozoa with different patterns of DNA dispersion: large-sized halo (1); medium-sized halo (2); very small-sized halo (3); and no halo (4). (b) Comet images showing damaged (1) and undamaged DNA (2).

ba

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metachromatic shift from green fluorescence (AOintercalated into double-stranded nucleic acid)to red fluorescence (AO associated with single-stranded DNA).70 Apparently, acid conditionspartially denature protamine-complexed somaticcell DNA. This protocol has been divided intoSCSAacid and SCSAheat to distinguish the physicalmeans of inducing DNA denaturation. The twomethods give essentially the same results, but theSCSAacid method is much easier to use. DNA damagethat is SCSA-defined is manifested by the DFI.

Technique

To perform the assay, semen samples are placed oncrushed liquid ice; all succeeding steps are per-formed at 4°C. Samples are diluted with TNE buffer(0.15 mol/l NaCl, 0.01 mol/l Tris, 0.001 mol/l EDTA,pH 7.4) to obtain a sperm concentration of ≤ 2 × 106

sperm/ml. A 200-µl aliquot is removed and admixedwith 400 µl of a low-pH detergent solution(0.15 mol/l NaCl, 0.08N HCl, 0.01% Triton X-100,pH 1.4). After 30 seconds, 1.2 ml staining solution(6 µg/ml AO, chromatographically purified in0.2 mol/l Na2HPO4, 1 mmol/l disodium EDTA,0.15 mol/l NaCl, 0.1 mol/l citric acid monohydrate,pH 6.0) is added, and the stained sample is placedinto the flow cytometer sample chamber.38

Clinical significance

Because the SCSA is more constant over prolongedperiods of time than routine World HealthOrganization (WHO) semen parameters, it may be

used effectively in epidemiological studies of maleinfertility.71 In clinical applications, the SCSAparameters not only distinguish fertile and infertilemen but also are able to classify men according tothe level of in vivo fertility (pregnancy initiated inless than 3 months), moderate fertility (pregnancyinitiated within 4–12 months), and no proven fertil-ity (no pregnancy by 12 months). In addition, a DFIthreshold was established that identifies samplescompatible with pregnancy (< 30%).6 To the best ofour knowledge, SCSA is the most successful assay inpredicting the various outcomes of ART includingthe fertilization and implantation rates.72–74

Advantages and limitations

The SCSA accurately estimates the percentage ofDNA-damaged sperm and has a cut-off point (30%DFI) to differentiate between fertile and infertilesamples. However, it requires the presence of expen-sive instrumentation (flow cytometer) and highlyskilled technicians.

High-performance liquid chromatography

Principle

This assay entails measurement of the levels of8-hydroxy-2-deoxyguanosine (8-OHdG), which is abyproduct of oxidative DNA damage in the sperma-tozoa. It is the most commonly studied biomarkerfor oxidative DNA damage. Among various oxida-tive DNA adducts, 8-OHdG has been selected as arepresentative of oxidative DNA damage owing to its

Textbook of Assisted Reproductive Techniques102

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Fig 7.4 (a) Terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate-nick end labeling (TUNEL) assayfluorescent activated cell sorting (FACS) histograms with markers (M1) for detection of fluorescence set at 650 nm semen sample withlow percentage of sperm DNA fragmentation; (b) TUNEL assay FACS histograms with markers (M1) for a semen sample with highpercentage of sperm DNA fragmentation.

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high specificity, potent mutagenicity, and relativeabundance in DNA.39

Technique

Step I DNA extraction is performed with chloroform–isoamyl alcohol (12 : 1 v/v) after the sperm cells arewashed with sperm wash buffer (10 mmol/l Tris-HCl, 10 mmol/l EDTA, 1 mol/l NaCl, pH 7.0) andlysed at 55°C for 1 hour with 0.9% SDS, 0.5 mg/mlproteinase K, and 0.04 mol/l dithiothreitol (DTT).After ribonuclease A treatment to remove RNAresidue, the extracted DNA is dissolved in10 mmol/l Tris-HCl (pH 7.0) for DNA digestion.

Step II Enzymatic DNA digestion is performedwith three enzymes: DNAase. I, nuclease P1, andalkaline phosphatase. The final solution is driedunder reduced temperature and pressure and isredissolved in distilled and deionized water forHPLC.

Step III The third step is HPLC analysis. The HPLCsystem used for 8-OHdG measurements consists of apump, a partisphere 5 C18 column, an electrochemi-cal detector, an ultraviolet detector, an autosampler,and an integrator. The mobile phase consists of20 mmol/l NH4H2PO4, 1 mmol/l EDTA, and 4%methanol (pH 4.7). The calibration curves for8-OHdG are established with standard 8-OHdG,and the results are expressed as 8-OHdG/104 dG.39

Clinical significance

The assay provides the most direct evidence sug-gesting the involvement of oxidative sperm DNAdamage in male infertility, based on the finding thatlevels of 8-OHdG in sperm are significantly higher ininfertile patients than in fertile controls and have aninverse relationship with sperm concentration.75

Levels of 8-OHdG in sperm DNA have been reportedto be increased in smokers, and they inverselycorrelate with the intake and seminal plasmaconcentration of vitamin C, the most importantantioxidant in sperm. If not repaired, 8-OHdG modifi-cations in DNA are mutagenic and may cause embryoloss, malformations, or childhood cancer. Moreover,this modification could be a marker of OS in sperm,which may have negative effects on sperm function.76

Advantages and limitations

Although 8-OHdG is a potential marker for oxidativeDNA damage, artifactual oxidation of dG can occur

during the analysis, which can lead to inaccurateresults. A fixed number of sperm cells should beanalyzed as a precaution. However, the DNA yieldcannot be excluded as a potential confounder.

Conclusion

In summary, we emphasize the importance of assess-ing sperm for chromatin abnormalities as it may pro-vide useful information in cases of male idiopathicinfertility and in men pursuing assisted reproduc-tion. Sperm chromatin assessment is an independentmeasure of sperm quality that provides better diag-nostic and prognostic capabilities than standardsperm parameters for male fertility potential.

There are multiple assays that may be used forevaluation of the sperm chromatin status. Most ofthese assays have many advantages as well as limita-tions. The choice of which assay to be performeddepends on many factors such as the expense, theavailable laboratory facilities, and the presence ofexperienced technicians. The establishment of a cut-off point between normal levels in the average fertilepopulation and the minimal levels of sperm DNAintegrity required for achieving pregnancy stillremains to be investigated. Such an average range orvalue is still lacking for most of these assays exceptfor the SCSA.

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