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Sperm chromatin assessment

Ashok Agarwal, Tamer M Said

Introduction
Semen analysis is the basic and most commonly used test for predicting fertility; however, the standard measurements of sperm concentration, percentage motility, and morphology may not reveal subtle sperm defects. In this context, sperm chromatin abnormalities have been studied extensively in the past decade as a cause for male infertility.1 The focus on the genomic integrity of the male gamete has been further intensified by the growing concern of transmission of genetic diseases through assisted reproductive techniques (ART), specifically intracytoplasmic sperm injection (ICSI). Accumulating evidence indicates that a negative correlation exists between disturbances in the organization of the genomic material in sperm nuclei and the fertility potential of spermatozoa, whether in vivo or in vitro.2,3 This emphasizes that stable DNA, which is capable of decondensation at the appropriate time in the fertilization process, is one of the criteria needed to consider a spermatozoon fertile.4 Conventional semen analysis per se cannot cover the diverse array of biological properties that the spermatozoon expresses as a highly specialized cell.5,6 In addition, the results of semen analyses can be very subjective and prone to intra- and interobserver variability.7 At the present time, it is clear that a sperm chromatin structure of poor quality may be indicative of male subfertility, regardless of the number, motility, and morphology of spermatozoa. Sperm chromatin structure evaluation is an independent measure of sperm quality that provides good diagnostic and prognostic capabilities. Therefore, it may be considered a reliable predictor of a couples inability to become pregnant,8 and may also have an impact on the offspring, resulting in infertility.9 Many techniques have been described for evaluation of the chromatin status. In this chapter, we describe the normal sperm chromatin architecture and the causative factors leading to its aberrations. We also provide the rationale and the different
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methodologies employed for sperm chromatin assessment.

Human sperm chromatin structure

The nuclear status of sperm cells is determined by two major events that occur during spermiogenesis: acquisition of the final nuclear shape and the replacement of somatic-type histones by protamines (sperm-specific basic nuclear proteins) leading to highly packaged chromatin. Sperm DNA is organized in a specific manner to keep the chromatin in the nucleus compact and stable. It is packed into a tight, almost crystalline status that is at least six times more condensed than mitotic chromosomes. It occupies nearly the entire nucleus volume, whereas somatic cell DNA only partly fills the nucleus.10 This DNA organization not only permits the very tightly packaged genetic information to be transferred to the egg, but also ensures that the DNA is delivered in a physical and chemical form that allows the developing embryo to access the genetic information.11 Sperm nuclei do not have the volume required for the type of packaging present in somatic cells, because packing the DNA in even a single, closely packed nucleosome would require 9.9 m3, which is more than twice the volume of an average sperm nucleus. Thus, a completely different type of DNA packaging must be present in mammalian sperm nuclei.12 Organization of chromatin for packaging in the spermatozoon takes place at four different levels: chromosomal anchoring, which refers to the attachment of the DNA to the nuclear annulus; formation of DNA loop domains as the DNA attaches to the newly added nuclear matrix; replacement of histones by protamines, which condense the DNA into compact doughnuts; and chromosomal positioning.12 The histones are first displaced by transition proteins

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(TNPs), which are removed from the condensing chromatin at later stages and replaced by protamines. It is of interest to note that the condensation of chromatin begins in the posterior pole and proceeds apically, which is a unique feature in humans that is not present in other mammalian species.13 Sperm epididymal maturation implies a final stage of chromatin organization involving protamine cross-linking by disulfide bond formationa step that is supported by the fact that protamines contain a significant number of cysteine residues that participate in sperm chromatin compaction by forming multiple inter- and intraprotamine disulfide cross-links. All of these interactions make mammalian DNA the most condensed eukaryotic DNA.14

Indications of sperm chromatin assessment

Diagnosis of male infertility
The positive relationship between poor sperm parameters and DNA damage in spermatozoa points to inherent problems.20 In general, infertile patients have higher levels of DNA strand breaks than seen in fertile males.21 Moreover, spermatozoa from infertile patients are generally more susceptible to the effects of DNA-damaging agents such as H2O2 and radiographic exposure.22 Thus, the sperm DNA integrity may be considered an objective marker of sperm function that serves as a significant prognostic factor for male infertility. Recently, we discovered a significant increase in sperm chromatin structure assay (SCSA)-defined DNA damage in sperm from infertile men with normal semen parameters, leading us to speculate that sperm DNA damage analysis may reveal a hidden abnormality of sperm DNA in infertile men classified with idiopathic infertility based on apparently normal standard semen parameters.23

Origin of sperm chromatin abnormalities

Sperm nuclear chromatin abnormalities/DNA damage could occur at the time of, or be the result of, DNA packing at spermiogenesis.15 Environmental stress, gene mutations, and chromosomal abnormalities can disturb the highly refined biochemical events that occur during spermatogenesis. This can ultimately lead to an abnormal chromatin structure that is incompatible with fertility. However, the exact mechanism(s) by which chromatin abnormalities/ DNA damage arise in human spermatozoa are not precisely understood. Three main theories have been proposed: defective sperm chromatin packaging, apoptosis, and oxidative stress (OS).

Assisted reproductive techniques

During ICSI, the sperm cell is injected directly into the cytoplasm of the mature oocyte. Because the sperm membraneoocyte interaction is no longer relevant, increased emphasis is placed on the quality of the sperm chromatin and the ability of the oocyte to initiate decondensation and pronuclear formation. In general, the ICSI fertilization rate does not exceed 6580%24 despite the mechanical injection of one sperm into a mature oocyte. The fertilization rate is lower than expected possibly because sperm that are selected from semen of patients with male factor infertility may have defects in their DNA. Therefore, although the most normal-appearing and motile spermatozoa are selected, there is always a small percentage of sperm used in in vitro fertilization (IVF)/ICSI that contain varying degrees of DNA damage.2 Semen samples characterized by increased DNA fragmentation levels are often associated with decreased fertilization rates and/or embryo cleavage following IVF and ICSI, and may be linked to an increase in early embryo death.25 In a recent study (Fig 7.1), we tested the correlation of sperm DNA damage with different ART outcomes. We reported that the percentage of DNA fragmentation index (DFI) was significantly higher in infertile men who did not achieve a clinical pregnancy with ART (38 (interquartile range 2843)) than in those who did (21 (1325); p = 0.001). In addition, we found no clinical pregnancy when the

Contributing factors
The most important factor contributing to sperm chromatin damage is leukocytospermia. It may result in reactive oxygen species (ROS)-induced crossdamage of sperm,16 and is associated with proinflammatory mediators such as cytokines, causing alterations in the regulation of spermiogenesis and subsequently chromatin aberrations. Similarly, OS may be the underlying reason why sperm DNA from smokers contains more strand breaks than that from non-smokers.17 High levels of sperm DNA damage can be seen following exposure to irradiation and chemotherapeutic agents and may persist for several months.18 Finally, sperm preparation techniques involving repeated high-speed centrifugation and the isolation of spermatozoa from the protective antioxidant environment provided by seminal plasma may contribute to increased sperm DNA damage via mechanisms that are mediated by the enhanced generation of ROS.19

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IUI n = 19 Semen analysis Clinical pregnancy % DNA fragmentation Normal n=4 (+) n=2 18 7 () n=2 44 9 Abnormal n = 15 (+) n=3 20 7 () n =12 35 12 Normal n=2 (+) n=1 13 0

IVF n = 10 Abnormal n=8 (+) n=2 21 5 () n=6 38 13 Normal n=0

ICSI n=4 Abnormal n=4 (+) n=1 28 0 () n=3 37 12

() n=1 28 0

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

Table 7.1 Various methods for assessing sperm chromatin abnormalities.

Assay Acidic aniline blue28 Toluidine blue stain29 Chromomycin A330 Sperm chromatin dispersion31 DNA breakage detectionfluorescent in situ hybridization32 In situ nick translation33 Acridine orange34 TUNEL35 Parameter Nuclear maturity (DNA protein composition) DNA fragmentation Nuclear maturity (DNA protein composition) DNA fragmentation DNA fragmentation (ssDNA) DNA fragmentation (ssDNA) DNA denaturation (acid) DNA fragmentation Method of analysis Optical microscopy Optical microscopy Fluorescent microscopy Fluorescent microscopy Fluorescent microscopy Fluorescent microscopy Flow cytometry Fluorescent microscopy Flow cytometry Optical microscopy Fluorescent microscopy Flow cytometry Fluorescent microscopy Flow cytometry High-performance liquid chromatography

Comet (neutral)36 (alkaline)37 Sperm chromatin structure38 8-OHdG measurement39

DNA fragmentation (dsDNA) DNA fragmentation (ssDNA /dsDNA) DNA denaturation (acid/heat) 8-OHdG

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

DFI was higher than 28%.26 Thus, assessment of sperm chromatin may help to predict the success rates of ART.

Cancer patients
Patients with cancer are often referred to sperm banks before chemotherapy, radiation therapy, or surgery is initiated. Although pregnancies and births have been reported using cryopreserved sperm from patients with cancer, these semen samples have decreased fertilization potential. The extent of DNA damage may help to determine how semen should be cryopreserved before therapy begins. Specimens with high

sperm concentration and motility and low levels of DNA damage should be preserved in relatively large aliquots that are suitable for intrauterine insemination (IUI). If a single specimen of good quality is available, then it should be preserved in multiple small aliquots suitable for IVF or ICSI.27

Evaluation of sperm nuclear DNA damage

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

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These assays include simple staining techniques such as the acidic aniline blue (AAB) and toluidine blue (TB) stains, fluorescent staining techniques such as the sperm chromatin dispersion (SCD) test, chromomycin A3 (CMA3), DNA breakage detection fluorescent in situ hybridization assay (DBDFISH), in situ nick translation (NT), and flow cytometricbased sperm chromatin structure assay (SCSA). Some assays employ more than one method for the analysis of their results. Examples of these assays include the acridine orange (AO) and terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate-nick end labeling (TUNEL) assays. Other methods less frequently used include high-performance liquid chromatography (HPLC).

Immature sperm chromatin may or may not correlate with asthenozoospermic samples and abnormal morphology patterns.28,40 Most important is the finding that chromatin condensation as visualized by aniline blue staining is a good predictor for IVF outcome, although it cannot determine the fertilization potential, cleavage, and pregnancy rate following ICSI.42

Toluidine blue stain Principle

Toluidine blue is a basic nuclear dye used for metachromatic and orthochromatic staining of chromatin. It becomes heavily incorporated in the damaged dense chromatin. This stain is a sensitive structural probe for DNA structure and packaging.

Acidic aniline blue stain Principle

The AAB stain discriminates between lysine-rich histones and arginine/cysteine-rich protamines. This technique provides a specific positive reaction for lysine and reveals differences in the basic nuclear protein composition of ejaculated human spermatozoa. Histone-rich nuclei of immature spermatozoa are rich in lysine and will consequently take up the blue stain. On the other hand, protaminerich nuclei of mature spermatozoa are rich in arginine and cysteine and contain relatively low levels of lysine, which means they will not be stained by aniline blue.40

Technique
The protocol of the TB stain includes four steps. The smears are air-dried, fixed in freshly made 96% ethanolacetone (1 : 1) at 4C for 30 minutes, hydrolyzed in 0.1 N Hcl at 4C for 5 minutes, and rinsed three times in distilled water for 2 minutes each. Smears are stained with 0.05% TB (Merck, Poole, Dorset, UK) for 10 minutes. The staining buffer consists of 50% citrate phosphate (McIlvain buffer, pH 3.5). Permanent preparations are dehydrated in tertiary butanol twice for 3 minutes each at 37C, and in Histoclear (RA Lambs Labs, Apex, NC) twice for 3 minutes each. Afterwards, the preparations are embedded in DPX. Sperm heads with good chromatin integrity stain light blue, and those of diminished integrity stain violet (purple) (Fig 7.2a).29

Technique
Slides are prepared by smearing 5 l of either raw or washed semen sample. The slides are air-dried and fixed for 30 minutes in 3% glutaraldehyde in phosphate-buffered saline (PBS). The smear is dried and stained for 5 minutes in 5% aqueous aniline blue solution (pH 3.5). Sperm heads containing immature nuclear chromatin stain blue, and those with mature nuclei do not take up the stain. The percentage of spermatozoa stained with aniline blue is determined by counting 200 spermatozoa per slide under bright field microscopy.28

Clinical significance
Due to the cooperative nature of metachromatic staining, which indicates poor sperm integrity, only severe DNA damage is revealed. Nevertheless, TB staining may be considered a fairly reliable method for assessing the sperm chromatin. Abnormal nuclei (purple-violet sperm heads) have been shown to be correlated with counts of red-orange sperm heads as revealed by the AO method.43

Clinical significance
Results of AAB have shown a clear association between abnormal sperm chromatin and male infertility.41 However, the correlation between the percentage of aniline blue-stained spermatozoa and other sperm parameters remains controversial.

Advantages and limitations

In general, the AAB and TB methods are simple and inexpensive and have the advantage of providing permanent preparations for use on an ordinary microscope. The smears stained with the TB method can also be used for morphological assessment of the cells.

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

In this way, the TB stain method has the advantage, in comparison with the AAB method. However, these methods have the inherent limits of repeatability dictated by dye equilibrium variations and by a limited number of cells which can be reasonably scored.

Clinical significance
As a discriminator of IVF success (> 50% oocytes fertilized), CMA3 staining has a sensitivity of 73% and specificity of 75%. Therefore, it can distinguish between 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 fertilization entirely, and suggested that poor chromatin packaging contributes to a failure in the decondensation process and probably reduced fertility. It appears that semen samples with high CMA3 positivity (> 30%) may have significantly lower fertilization rates if used for ICSI.25

Chromomycin A3 assay Principle

Chromomycin A3 is a guaninecytosine-specific fluorochrome that reveals chromatin that is poorly packaged in human spermatozoa via indirect visualization of protamine-deficient DNA. Chromomycin A3 and protamines compete for the same binding sites in the DNA. Therefore, high CMA3 fluorescence is a strong indicator of the low protamination state of spermatozoa.30

Advantages and limitations

The CMA3 assay yields reliable results as it is strongly correlated with other assays used in the evaluation of sperm chromatin.30 In addition, the sensitivity and specificity of the CMA3 stain are comparable to those of the AAB stain (75% and 82%, 60% and 91%, respectively) if used in evaluation of the chromatin status in infertile men.46 However, it is important to note that all of these assays mentioned to this point are limited by observer subjectivity.

Technique
For CMA3 staining, semen smears are first fixed in methanolglacial acetic acid 3 : 1 at 4C for 20 minutes and are then allowed to air-dry at room temperature for 20 minutes. The slides are treated for 20 minutes with 100 l CMA3 solution. The CMA3 solution consists of 0.25 mg/ml CMA3 in McIlvains buffer (pH 7.0) supplemented with 10 mmol/l MgCl2. The slides are rinsed in buffer and mounted with 1 : 1 v/v PBSglycerol. The slides are then kept at 4C overnight after which evaluation of fluorescence is performed using a fluorescent microscope. A total of 200 spermatozoa are randomly evaluated on each slide. Evaluation of CMA3 staining is done by distinguishing spermatozoa that stain bright yellow (CMA3 positive) from those that stain a dull yellow (CMA3 negative).30

DNA breakage detectionfluorescent in situ hybridization assay Principle

Cells embedded within an agarose matrix on a slide are exposed to an alkaline unwinding solution, which transforms DNA-strand breaks into single-stranded DNA (ssDNA) motifs. After neutralizing and protein removal, ssDNA is accessible to hybridization with whole genome or specific DNA

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probes. The probe highlights the chromatin area to be analyzed. As DNA breaks increase, the more ssDNA is produced by the alkaline solution and the more the probe hybridizes, resulting in an increase in the fluorescence intensity and surface area of the FISH signal. Abnormal chromatin packaging in sperm cells greatly increases the accessibility of DNA ligands and the sensitivity of DNA to denaturation by alkali, and this relates to the presence of intense labeling (red fluorescence) by DBDFISH. Therefore, DBDFISH allows in situ detection and quantification of DNA breaks and reveals structural features in the sperm chromatin.32,47

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

In situ nick translation assay Principle

The NT assay quantifies the incorporation of biotinylated deoxyuridine triphosphate (dUTP) at singlestrand DNA breaks in a reaction that is catalyzed by the template-dependent enzyme DNA polymerase I. It specifically stains spermatozoa that contain appreciable and variable levels of endogenous DNA damage. The NT assay indicates anomalies that have occurred during remodeling of the nuclear DNA in spermatozoa, and in doing so is more likely to detect sperm anomalies that are not indicated by morphology.

Technique
To perform this assay, sperm cells are mixed with lowmelting-point agarose to a final concentration of 0.7% at 37C. A volume of 300 l of the mixture is pipetted onto polystyrene slides and allowed to solidify at 4C. The slides are immersed into a freshly prepared alkaline denaturation solution (0.03 mol/l NaOH, 1 mol/l NaCl) for 5 minutes at 22C in the dark, to generate ssDNA from DNA breaks. The denaturation is then stopped, and proteins are removed by transferring the slides 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 ethylenediaminetetra-acetic acid (EDTA), pH 7.5) for 10 minutes at room temperature, which is followed by incubation in neutralizing and lysing solution 2 (0.4 mol/l Tris, 2 mol/l NaCl, and 1% SDS, pH 7.5) for 20 minutes at room temperature. The slides are thoroughly washed in Trisborate EDTA buffer (0.09 mol/l Trisborate and 0.002 mol/l EDTA, pH 7.5) for 15 minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each), and air-dried. A human whole genome probe is hybridized overnight (4.3 ng/l in 50% formamide/2 standard saline citrate (SSC), 10% dextran sulfate, and 100 mmol/l calcium phosphate, pH 7.0) (1 SSC is 0.015 mol/l sodium citrate and 0.15 mol/l sodium chloride, pH 7.0). It is then washed twice in 50% formamide/2 SSC, pH 7.0, for 5 minutes, and twice in 2 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 with 4,6-diamidino-2-phenylindole (DAPI) (1 g/ml) and visualized using fluorescent microscopy (Fig 7.2b).32

Technique
To perform the assay, smears containing 500 sperm each should be prepared. The fluorescent staining solution is prepared by mixing 10 l streptavidin fluoresceinisothiocyanate, 90 l Tris buffer, and 900 l double-distilled water. One hundred microliters of this solution is added to the slides. The incubation is carried out in a moist chamber at 37C for 30 minutes. After incubation, the slides are rinsed in PBS twice, washed with distilled water, and finally mounted with a 1 : 1 mixture of PBS and glycerol. The slides are examined using fluorescent microscopy. A total of 100200 spermatozoa should be counted, and those fluorescing and hence incorporating the dye are classified as having endogenous nicks.33

Clinical significance
Sperm nuclear integrity as assessed by the NT assay demonstrates a very clear relationship with sperm motility and morphology and, to a lesser extent, sperm concentration.48,49 Results of the assay are supported by the strong positive correlations detected with the sensitivity of CMA3 and TUNEL assays (r = 0.86; p < 0.05 and r = 0.87; p < 0.05, respectively).30 The NT assay is also able to indicate if there is damage arising from factors such as heat exposure50 or the generation of ROS following exposure to leukocytes within the male reproductive tract.51

Advantages and limitations Advantages and limitations

Although the assay reveals chromatin structural features, it is expensive, time-consuming, and involves sophisticated procedures. The assay is of less clinical The advantage of the NT assay is that the reaction is based on direct labeling of termini of DNA breaks,

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and thus the lesions that are measured are identifiable at the molecular level. In addition, if flow cytometry is used to analyze the results, it may be performed on fixed cells, as the time of cell storage in ethanol may vary.33

Clinical significance
Staining with AO shows a significant difference between fertile males and those who are infertile with different andrologic pathologies. The cut-off value set to differentiate between fertile and infertile men varies between 20 and 50%.8,34,53 Studies show that ssDNA that is detected by a low incidence (< 50%) of green AO fluorescence negatively affects the fertilization process in a classical IVF program.52,54,55 However, no correlation was found with pregnancy rate and live births achieved by ICSI, except in patients having 0% of spermatozoa with ssDNA, in whom the pregnancy rate was significantly high.54

Acridine orange assay Principle

The AO assay measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ by quantifying the metachromatic shift of AO fluorescence from green (native DNA) to red (denatured DNA). The fluorochrome AO intercalates into double-stranded DNA as a monomer and binds to single-stranded DNA as an aggregate. The monomeric AO bound to native DNA fluoresces green, whereas the aggregated AO on denatured DNA fluoresces red (Fig 7.2c).52

Advantages and limitations

The AO assay is a biologically stable measure of sperm quality. The interassay variability is less than 5%, rendering the technique highly reproducible.5 A strong positive correlation exists between the AO assay and other techniques used to evaluate ssDNA, e.g. TUNEL assay.56 The AO assay still requires expensive instrumentation if flow cytometry is used to interpret the results. Also, observer subjectivity may hinder the results if fluorescent microscopy is used.

Technique
The AO assay may be used for either fluorescence or flow cytometry. To perform this assay for fluorescent microscopy, thick smears are fixed in Carnoys fixative (methanol : acetic acid 1 : 3) for at least 2 hours. The slides are stained for 5 minutes and gently rinsed with deionized water. At least 200 cells should be counted so that the estimate of the numbers of sperm with green and red fluorescence is accurate. For flow cytometry, aliquots of semen (about 25100 l, containing 1 million spermatozoa) are suspended in 1 ml of ice-cold PBS (pH 7.4) and centrifuged at 600 g for 5 minutes. The pellet is resuspended in ice-cold TNE (0.01 mol/l Tris-HCl, 0.15 mol/l NaCl, and 1 mmol/l ethylenediaminetetraacetic acid (EDTA), pH 7.4) and again centrifuged at 600 g for 5 minutes. The pellet is then resuspended in 200 l of ice-cold TNE with 10% glycerol and immediately fixed in 70% ethanol for 30 minutes. The fixed samples are treated for 30 seconds with 400 l of a solution of 0.1% Triton X-100, 0.15 mol/l NaCl, and 0.08N HCl, pH 1.2. After 30 seconds, 1.2 ml of staining buffer (6 g/ml AO, 37 mmol/l citric acid, 126 mmol/l Na2HPO4, 1 mmol/l disodium EDTA, 0.15 mol/l NaCl, pH 6.0) is admixed to the test-tube and analyzed by flow cytometry. After excitation by a 488-nm wavelength light source, AO bound to double-stranded DNA fluoresces green (515530 nm) and AO bound to single-stranded DNA fluoresces red (630 nm or greater). A minimum of 5000 cells are analyzed by fluorescent activated cell sorting (FACS).34

Sperm chromatin dispersion test Principle

If spermatozoa with nonfragmented DNA are immersed in an agarose matrix and directly exposed to lysing solutions, the resulting deproteinized nuclei (nucleoids) show extended halos of DNA dispersion as monitored by fluorescent microscopy. The presence of DNA breaks promotes the expansion of the halo of the nucleoid.57 The SCD test is based on the principle that when sperm are treated with an acid solution prior to lysis buffer, the DNA dispersion halos that are observed in sperm nuclei with nonfragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all in sperm nuclei with fragmented DNA.

Technique
Aliquots of either raw or washed semen samples should be adjusted to concentrations ranging between 5 and 10 million/ml. The suspensions are mixed with 1% low-melting-point aqueous agarose (to obtain a 0.7% final agarose concentration) at 37C. Aliquots of 50 l of the mixture should be pipetted onto a glass slide precoated with 0.65% standard agarose dried at 80C, covered with

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a coverslip, and left to solidify at 4C for 4 minutes. The coverslips are then carefully removed, and the slides are immediately immersed horizontally in a tray of freshly prepared acid denaturation solution (0.08N HCl) for 7 minutes at 22C in the dark, which generates restricted single-stranded DNA (ssDNA) motifs from DNA breaks. Denaturation is then stopped, and the proteins are removed by transferring the slides to a tray with neutralizing and lysing solution 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 room temperature. The slides are then incubated in neutralizing and lysing solution 2 (0.4 mol/l Tris, 2 mol/l NaCl, and 1% SDS, pH 7.5) for 5 minutes at room temperature. The slides are thoroughly washed in Tris-borateEDTA buffer (0.09 mol/l Tris-borate and 0.002 mol/l EDTA, pH 7.5) for 2 minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each), and air-dried. Cells are stained with DAPI (4,6-diamidino-2-phenylindole) (2 g/ml) for fluorescence microscopy (Fig 7.3a).31

nucleus comet head and the resulting tail. The tail lengths are used as an index for the damage. Also, the tail moment, which is the product of the tail length and intensity (fraction of total DNA in the tails), has been used as a measuring parameter. The tail moment can be more precisely defined as being equivalent to the torsional moment of the tail.59

Technique
In this assay, sperm cells are cast into miniature agarose gels on microscope slides and lysed in situ to remove DNA-associated proteins and to allow the compacted DNA in the sperm to relax. The lysis buffer (Tris 10 mmol/l, 0.5 mol/l EDTA, and 2.5 mol/l NaCl, pH 10) contains 1% Triton X-100, 40 mmol/l dithiothreitol, and 100 g/ml proteinase K). Microgels are then electrophoresed (20 minutes at 25 V/ 0.01 A) in neutral buffer (Tris 10 mmol/l containing 0.08 mol/l boric acid and 0.5 mol/l EDTA, pH 8.2), during which the damaged DNA migrates from the nucleus towards the anode. The DNA is visualized by staining the slides with the fluorescent DNA binding dye SYBR Green I. Comet measurements are performed using fluorescent microscopy. These measurements can be done either manually or with computerized image analysis (Fig 7.3b).36

Advantages and limitations

The major advantage of the SCD test is that it does not require the determination of color or fluorescence intensity. Rather, the percentage of spermatozoa with nondispersed (very small halos or none at all) or dispersed nuclei is determined, which can be easily and reliably accomplished by the naked eye. Furthermore, the test is simple, fast, and reproducible, and its results are comparable to those of the SCSA.31 Because the SCD test was recently introduced, little is known about its limitations and its clinical significance.

Clinical significance
The assay has been successfully used in the evaluation of DNA damage after cryopreservation.60 It may also predict embryo development after IVF and ICSI, especially in couples with unexplained infertility.61,62

Comet assay Principle

The comet assay, also known as single-cell gel electrophoresis for analysis of DNA damage in an individual cell, was first introduced by Ostling and Johanson in 1984.58 Neutral electrophoresis buffer conditions were used to show that the migration of double-stranded DNA loops from a damaged cell in the form of a tail unwinding from the relaxed supercoiled nucleus was proportional to the extent of damage inflicted on the cell. This finding took on the appearance of a comet with a tail when viewed using the fluorescent microscope and DNA stains. Singh et al. modified the comet assay in 198836 by using alkaline electrophoresis buffers to expose alkali-labile sites on the DNA; this modification increased the sensitivity of the assay to detect both single- and double-stranded DNA breaks.37 The damage is quantified by measuring the displacement between the genetic material of the

Advantages and limitations

The comet is a well-standardized assay that correlates significantly with TUNEL and SCSA assays.63 It is simple to perform, has a low intra-assay coefficient of variation, and a low performance cost.64 Because it is based on fluorescent microscopy, the assay requires an experienced observer to analyze the slides and interpret the results.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling assay Principle

The TUNEL assay quantifies the incorporation of dUTP at single- and double-strand DNA breaks in a reaction catalyzed by the template-independent

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a 4

1 3 2 1

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

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

interpreted by assessing 100500 sperm cells under fluorescent microscopy or by using FACS flow cytometry (Fig 7.4a and b).35

Clinical significance
The TUNEL assay has been widely used in male infertility research related to sperm chromatin/DNA abnormalities. It provides useful information in many cases of male infertility. A negative correlation was found between the percentage of DNAfragmented sperm and the motility, morphology, and concentration in the ejaculate. It also appears to be potentially useful as a predictor for IUI pregnancy rate, IVF embryo cleavage rate, and ICSI fertilization rate. In addition, it provides an explanation for recurrent pregnancy loss.2,6669

Technique
Identification of strand breaks can be quantified by flow cytometry or fluorescent microscopy in which DNA-damaged sperm fluoresce intensely.65 To assess the DNA fragmentation by TUNEL, about 2 106 sperm are fixed with 1% formaldehyde for 10 minutes at room temperature. The sample is centrifuged at 10 000 g for 4 minutes. After the sperm are washed in PBS (pH 7.4), they are resuspended in 100 l prewash buffer containing single-strength One-Phor-All buffer (100 mmol/l Tris-acetate, 100 mmol/l magnesium acetate, 500 mmol/l potassium acetate; and 0.1% Triton X-100) for 10 minutes at room temperature. Fixed sperm are spun out of the buffer and resuspended in 50 l of TdT buffer containing 3 mol/l biotin-16-dUTP, 12 mol/l deoxyadenosine triphosphate (dATP), 0.1% Triton X-100, and 10 U of TdT enzyme and incubated at 37C for 60 minutes. After two washes in PBS, the fixed, permeabilized sperm are resuspended in 100 l of staining buffer consisting of 0.1% Triton X-100 and 1% streptavidin/Texas red antibiotin and incubated at 4C in the dark for 30 minutes. The stained cells are washed in PBS/0.1% Triton X-100. To create negative controls, the enzyme terminal transferase may be omitted from the reaction mixture. To create positive controls, the samples are pretreated with 0.1 IU DNAase I for 30 minutes at room temperature and then labeled. Results may be

Advantages and limitations

We believe that although the flow cytometric method of assessment is generally more accurate and reliable, it is also sophisticated and expensive. On the other hand, the fluorescent TUNEL assay has demonstrated fairly good quality control parameters. The intraobserver variability was found to be < 8% and the interobserver variability was < 7% (Spearman rank correlation between observers was r = 0.54, p = 0.01).35

Sperm chromatin structure assay Principle

The SCSA relies on the fact that abnormal sperm chromatin has a greater susceptibility to the physical induction of partial DNA denaturation in situ. The extent of DNA denaturation following heat or acid treatment is determined by measuring the

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(a) 80

(b) 80

Relative cell courts

60 FITC-dUTP

60

40 M1 20

40 M1 20

0 100 101 102 103 104

0 100 101 102 103 104

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

metachromatic shift from green fluorescence (AO intercalated into double-stranded nucleic acid) to red fluorescence (AO associated with singlestranded DNA).70 Apparently, acid conditions partially denature protamine-complexed somatic cell DNA. This protocol has been divided into SCSAacid and SCSAheat to distinguish the physical means of inducing DNA denaturation. The two methods give essentially the same results, but the SCSAacid method is much easier to use. DNA damage that is SCSA-defined is manifested by the DFI.

used effectively in epidemiological studies of male infertility.71 In clinical applications, the SCSA parameters not only distinguish fertile and infertile men but also are able to classify men according to the level of in vivo fertility (pregnancy initiated in less than 3 months), moderate fertility (pregnancy initiated within 412 months), and no proven fertility (no pregnancy by 12 months). In addition, a DFI threshold was established that identifies samples compatible with pregnancy (< 30%).6 To the best of our knowledge, SCSA is the most successful assay in predicting the various outcomes of ART including the fertilization and implantation rates.7274

Technique
To perform the assay, semen samples are placed on crushed liquid ice; all succeeding steps are performed at 4C. 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-l aliquot is removed and admixed with 400 l 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 g/ml AO, chromatographically purified in 0.2 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 placed into the flow cytometer sample chamber.38

Advantages and limitations

The SCSA accurately estimates the percentage of DNA-damaged sperm and has a cut-off point (30% DFI) to differentiate between fertile and infertile samples. However, it requires the presence of expensive instrumentation (flow cytometer) and highly skilled technicians.

High-performance liquid chromatography Principle

This assay entails measurement of the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), which is a byproduct of oxidative DNA damage in the spermatozoa. It is the most commonly studied biomarker for oxidative DNA damage. Among various oxidative DNA adducts, 8-OHdG has been selected as a representative of oxidative DNA damage owing to its

Clinical significance
Because the SCSA is more constant over prolonged periods of time than routine World Health Organization (WHO) semen parameters, it may be

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high specificity, potent mutagenicity, and relative abundance in DNA.39

during the analysis, which can lead to inaccurate results. A fixed number of sperm cells should be analyzed as a precaution. However, the DNA yield cannot be excluded as a potential confounder.

Technique
Step I DNA extraction is performed with chloroform isoamyl alcohol (12 : 1 v/v) after the sperm cells are washed with sperm wash buffer (10 mmol/l TrisHCl, 10 mmol/l EDTA, 1 mol/l NaCl, pH 7.0) and lysed at 55C for 1 hour with 0.9% SDS, 0.5 mg/ml proteinase K, and 0.04 mol/l dithiothreitol (DTT). After ribonuclease A treatment to remove RNA residue, the extracted DNA is dissolved in 10 mmol/l Tris-HCl (pH 7.0) for DNA digestion. Step II Enzymatic DNA digestion is performed with three enzymes: DNAase. I, nuclease P1, and alkaline phosphatase. The final solution is dried under reduced temperature and pressure and is redissolved in distilled and deionized water for HPLC. Step III The third step is HPLC analysis. The HPLC system used for 8-OHdG measurements consists of a pump, a partisphere 5 C18 column, an electrochemical detector, an ultraviolet detector, an autosampler, and an integrator. The mobile phase consists of 20 mmol/l NH4H2PO4, 1 mmol/l EDTA, and 4% methanol (pH 4.7). The calibration curves for 8-OHdG are established with standard 8-OHdG, and the results are expressed as 8-OHdG/104 dG.39

Conclusion
In summary, we emphasize the importance of assessing sperm for chromatin abnormalities as it may provide useful information in cases of male idiopathic infertility and in men pursuing assisted reproduction. Sperm chromatin assessment is an independent measure of sperm quality that provides better diagnostic and prognostic capabilities than standard sperm parameters for male fertility potential. There are multiple assays that may be used for evaluation of the sperm chromatin status. Most of these assays have many advantages as well as limitations. The choice of which assay to be performed depends on many factors such as the expense, the available laboratory facilities, and the presence of experienced technicians. The establishment of a cutoff point between normal levels in the average fertile population and the minimal levels of sperm DNA integrity required for achieving pregnancy still remains to be investigated. Such an average range or value is still lacking for most of these assays except for the SCSA.

Clinical significance
The assay provides the most direct evidence suggesting the involvement of oxidative sperm DNA damage in male infertility, based on the finding that levels of 8-OHdG in sperm are significantly higher in infertile patients than in fertile controls and have an inverse relationship with sperm concentration.75 Levels of 8-OHdG in sperm DNA have been reported to be increased in smokers, and they inversely correlate with the intake and seminal plasma concentration of vitamin C, the most important antioxidant in sperm. If not repaired, 8-OHdG modifications in DNA are mutagenic and may cause embryo loss, malformations, or childhood cancer. Moreover, this modification could be a marker of OS in sperm, which may have negative effects on sperm function.76

References
1. Sakkas D, Mariethoz E, Manicardi G, et al. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4: 317. 2. Sun J, Jurisicova A, Casper R. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997; 56: 6027. 3. Spano M, Bonde J, Hollund H, et al. Sperm chromatin damage impairs human fertility. Fertil Steril 2000; 73: 4350. 4. Amann R. Can fertility potential of a seminal sample be predicted accurately? J Androl 1989; 16: 8998. 5. Zini A, Kamal K, Phang D, et al. Biologic variability of sperm DNA denaturation in infertile men. Urology 2001; 58: 25861. 6. Evenson D, Larson K, Jost L. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23: 2543. 7. Keel B, Webster B. The standard semen analysis. In Webster B (ed.) CRC Handbook of the Laboratory Diagnosis and Treatment of Infertility. Boca Raton, FL: CRC Press, 1990: 2769.

Advantages and limitations

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

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8. Evenson D, Jost L, Marshall D, et al. Utility of the sperm chromatin structure assay as a diagnostic tool in the human fertility clinic. Hum Reprod 1999; 14: 103949. 9. Aitken, R. The Amoroso lecture. The human spermatozoaa cell in crisis. J Reprod Fertil 1999; 115: 17. 10. Fuentes-Mascorro G, Serrano H, Rosado A. Sperm chromatin. Arch Androl 2000; 45: 21525. 11. Poccia D. Remodeling of nucleoproteins during spermatogenesis fertilization and early development. Int Rev Cytol 1986; 105: 165. 12. Ward W, Coffey D. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 1991; 44: 56974. 13. Dadoune, J. Nuclear status of human sperm cells. Micron 1995; 26: 32345. 14. Ward W, Coffey D. Specific organization of genes in relation to the sperm nuclear matrix. Biochem Biophys Res Commun 1994; 173: 205. 15. Sailer B, Jost L, Evenson, D. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl 1995; 16: 807. 16. Comhaire F, Mahmoud A, Depuydt C, et al. Mechanism and effects of male genital tract infection on sperm quality and fertilizing potential: the andrologists viewpoint. Hum Reprod Update 1999; 5: 3938. 17. Potts R, Newbury C, Smith G, et al. Sperm chromatin damage associated with male smoking. Mutat Res 1999; 423: 10311. 18. Arnon J, Meirow D, Lewis-Roness H, et al. Genetic and teratogenic effects of cancer treatments on gametes and embryos. Hum Reprod Update 2001; 7: 394403. 19. Zalata A, Hafez T, Comhaire F. Evaluation of the role of reactive oxygen species in male infertility. Hum Reprod 1995; 10: 144451. 20. Lopes S, Sun J, Jurisicova A, et al. Sperm deoxyribonucleic acid fragmentation is increased in poorquality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998; 69: 52832. 21. Host E, Lindenberg S, Kahn J, et al. DNA strand beaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynecol Scand 1999; 78: 3369. 22. McKelvey-Martin V, Melia N, Walsh I, et al. Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1). Human bladder washings and transitional cell carcinoma of the bladder; and (2). Human sperm and male infertility. Mutat Res 1997; 375: 93104. 23. Saleh R, Agarwal A, Nelson D, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002; 78: 31318. 24. Shen S, Khabani A, Klein N, et al. Statistical analysis of factors affecting fertilization rates and clinical outcome associated with intracytoplasmic sperm injection. Fertil Steril 2003; 79: 35560. 25. Sakkas D, Urner F, Bizzaro D, et al. Sperm nuclear DNA damage and altered chromatin structure: effect

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

on fertilization and embryo development. Hum Reprod 1998; 13: 1119. Saleh R, Agarwal A, Nada E, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003; 79 (Suppl 3): 7987. Kobayashi H, Larson K, Sharma R, et al. DNA damage in cancer patients before treatment as measured by the sperm chromatin structure assay. Fertil Steril 2001; 75: 46975. Baker H, Liu D. Assessment of nuclear maturity. In: Acosta A, Kruger T, eds. Human Spermatozoa in Assisted Reproduction. London: CRC Press, 1996: 193203. Erenpreisa J, Freivalds T, Slaidina M, et al. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry 2003; 52: 1927. Manicardi G, Bianchi P, Pantano S, et al. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod 1995; 52: 8647. Fernandez J, Muriel L, Rivero M, et al. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003; 24: 5966. Fernandez J, Vazquez-Gundin F, Delgado A, et al. DNA breakage detectionFISH (DBDFISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000; 253: 7782. Gorczyza W, Gong J, Darzynkiewics Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993; 53: 194551. Zini A, Fischer M, Sharir S, et al. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology 2002; 60: 106972. Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000; 15: 133844. Singh N, McCoy M, Tice R, et al. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 18491. Singh N, Danner D, Tice R, et al. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989; 184: 46170. Evenson D, Jost L, Baer R, et al. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod Toxicol 1991; 5: 11525. Shen H, Ong C. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000; 28: 52936. Hammadeh M, Zeginiadov T, Rosenbaum P. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl 2001; 46: 99104. Foresta C, Zorzi M, Rossato M, et al. Sperm nuclear instability and staining with aniline blue: abnormal

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Sperm chromatin assessment 105

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

persistence of histones in spermatozoa in infertile men. Int J Androl 1992; 15: 3307. Hammadeh M, Stieber M, Haidl G, et al. Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia 1998; 30: 2935. Erenpreiss J, Bars J, Lipatnikova V, et al. Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 2001; 22: 4553. Esterhuizen A, Franken D, Lourens J, et al. Sperm chromatin packaging as an indicator of in vitro fertilization rates. Hum Reprod 2000; 15: 65761. Sakkas D, Urner F, Bianchi P, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996; 11: 83743. Franken D, Franken C, Guerre HDL, et al. Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining. Andrologia 1999; 31: 3616. Fernndez J, Goyanes V, Ramiro-Daz J, et al. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998; 82: 2516. Irvine D, Twigg J, Gordon E, et al. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000; 21: 3344. Tomlinson M, Moffatt O, Manicardi G, et al. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001; 16: 21605. Setchell B, Ekpe G, Zupp J, et al. Transient retardation in embryo growth in normal female mice made pregnant by males whose testes had been heated. Hum Reprod 1998; 13: 3427. Aitken R, Irvine D, Wu F. Prospective analysis of spermoocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991; 164: 54251. Hoshi K, Katayose H, Yanagida K, et al. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996; 66: 6349. Gopalkrishnan K, Hurkadli K, Padwal V, et al. Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andrologia 1999; 31: 27782. Variant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSIderived embryos. J Assist Reprod Genet 2002; 19: 31928. Katayose H, Yanagida K, Hashimoto S, et al. Use of diamideacridine orange fluorescence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79: 6706. Zini A, Bielecki R, Phang D, et al. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001; 75: 6747. Ankem M, Mayer E, Ward W, et al. Novel assay for determining DNA organization in human

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

spermatozoa: implication for male factor infertility. Urology 2002; 59: 5758. Ostling O, Johanson K. Microelectrophoretic study of radiation induced DNA damages on individual cells. Biochem Biophys Res Commun 1984; 123: 2918. Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995; 326: 12331. Duty S, Singh N, Ryan L, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17: 127480. Morris I, Ilott S, Dixon L, et al. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (comet assay) and its relationship to fertilization. Hum Reprod 2002; 17: 9908. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002; 17: 185662. Aravindan G, Bjordahl J, Jost L, et al. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single cell electrophoresis. Exp Cell Res 1997; 236: 2317. Chan P, Corselli J, Patton W, et al. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster ocytes. Fertil Steril 2001; 75: 18692. Lopes S, Jurisicova A, Sun J, et al. Reactive oxygen species: a potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998; 13: 896900. Duran E, Morshedi M, Taylor S, et al. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 2002; 17: 31228. Carrell D, Liu L, Peterson C, et al. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003; 49: 4955. Muratori M, Maggi M, Spinelli S, et al. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 2003; 24: 25362. Benchaib M, Braun V, Lornage J, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003; 18: 10238. Drazynkiewicz Z, Traganos F, Sharpless T, et al. Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 1975; 90: 41128. Spano M, Kolstad A, Larsen S, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998; 13: 2495505. Larson K, DeJonge C, Barnes A, et al. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000; 15: 171722. Januskauska A, Johannisson A, Rodriguez-Martinez H. Assessment of sperm quality through fluorometry and

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sperm chromatin structure assay in relation to field fertility of frozenthawed semen from Swedish AI bulls. Theriogenology 2001; 55: 94761. 74. Perera D, Pizzey A, Campbell A, et al. Sperm DNA damage in potentially fertile homozygous -thalassaemia patients with iron overload. Hum Reprod 2002; 17: 18205.

75. Kodama H, Yamaguchi R, Fukuda J, et al. Increased deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril 1997; 65: 51924. 76. Loft S, Kold-Jensen T, Hjollund N, et al. Oxidative DNA damage in human sperm influences time to pregnancy. Hum Reprod 2003; 18: 126572.