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Keywords:

  • Structure;
  • human sperm;
  • decondensation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

ABSTRACT: Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4′,6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.

Sperm DNA fragmentation is increasingly being recognized as an important cause of infertility. Recent clinical studies indicate that DNA fragmentation levels above 30%, as measured by the Sperm Chromatin Structure Assay (SCSA), are not compatible with the initiation and maintenance of a term pregnancy (Evenson et al, 1999; Larson et al, 2000).

A number of tests are currently available for the measurement of sperm DNA fragmentation (De Jonge, 2002). These include the TUNEL assay (Gorczyca et al, 1993a,b), the comet assay (Hughes et al, 1996), the chromomycin A3 test (Manicardi et al, 1995), the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) test (Fernández et al, 1998, 2002; Fernández and Gosálvez, 2002), and the SCSA test (Evenson et al, 1980, 1985, 1991, 1999; Evenson and Melamed, 1983; Evenson and Jost, 1994). Recent data indicate that SCSA test values, expressed as COMP αt (currently designated “DFI” [DNA fragmentation index]), are significantly correlated with pregnancy rate in vivo and in vitro. In a recent series that included more than 25 couples undergoing in vitro fertilization and intracytoplasmic sperm injection (ICSI) cycles, no term pregnancy occurred when COMP αt values were more than 27% in the semen samples utilized in these cycles (Larson et al, 2000). All pregnancies occurred when COMP αt values were less than 30%. Of the 9 patients in whom a biochemical pregnancy occurred when COMP αt values were greater than 30% (34%-37%), no term pregnancy was observed, and all pregnancies ended in first trimester abortion (Larson et al, 2000).

When somatic cells or spermatozoa with nonfragmented DNA are immersed in an agarose matrix and directly exposed to lysing solutions, the resulting deproteinized nuclei show extended halos of DNA dispersion, as monitored by fluorescence microscopy using specific DNA fluorochromes (Cook and Brazell, 1978; Ankem et al, 2002). The halos correspond to relaxed DNA loops attached to the residual nuclear structure. These deproteinized nuclei are called “nucleoids.” The presence of DNA breaks promotes the expansion of the halo of the nucleoid and is the basis for the halo test to detect DNA damage (Roti Roti and Wright, 1987; Smith and Sykes, 1992).

In this study, we introduce the Sperm Chromatin Dispersion (SCD) test as a novel test for the assessment of sperm DNA fragmentation. This assay is based on the halo test and on our observation 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. These results were confirmed by a subsequent DBD-FISH assay.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Preparation of Semen Samples

Semen samples were obtained from males attending a clinic for infertility screening (n = 20) and from normozoospermic healthy sperm donors (n = 10). Semen samples were classified as having normal or abnormal semen parameters according to World Health Organization (1999) criteria. Samples with abnormal semen parameters are defined as those having a sperm concentration less than or equal to 20 million/mL, a sperm motility less than 50%, and/or normal forms less than or equal to 14%. Samples with leukocytes were excluded from the study. In a separate set of experiments, semen samples obtained from a healthy donor with normal semen parameters and from an infertility patient with abnormal semen parameters were subjected to a discontinuous ISolate density gradient (Irvine Scientific, Santa Ana, Calif). The resulting fractions between the seminal plasma interface and the 90% ISolate interface (F1), between the F1 interface and the 90% pellet (F2), and the 90% pellet were aspirated and washed twice in modified human tubal fluid (mHTF) medium (Irvine). Aliquots of the original semen and each of the fractions were used to determine sperm concentration, motility, and morphology.

SCD Test

Aliquots of 0.2 mL of raw semen and of the different ISolate gradient fractions in mHTF medium were either analyzed directly or frozen in liquid nitrogen prior to analysis. Samples were thawed at room temperature and diluted in mHTF medium to obtain sperm concentrations that ranged between 5 and 10 million/mL. The suspensions were mixed with 1% low-melting-point aqueous agarose (to obtain a 0.7% final agarose concentration) at 37°C. Aliquots of 50 μL of the mixture were pipetted onto a glass slide precoated with 0.65% standard agarose dried at 80°C, covered with a coverslip (24 by 60 mm), and left to solidify at 4°C for 4 minutes. As in the halo test or the comet assay, the agarose matrix allows for work with unfixed sperm on a slide in a suspensionlike environment. Coverslips were carefully removed, and slides were immediately immersed horizontally in a tray with freshly prepared acid denaturation solution (0.08 N HCl) for 7 minutes at 22°C in the dark to generate restricted single-stranded DNA (ssDNA) motifs from DNA breaks. The denaturation was then stopped, and proteins were removed by a transfer of the slides to a tray with neutralizing and lysing solution 1 (0.4 M Tris, 0.8 M DTT, 1% SDS, and 50 mM EDTA, pH 7.5) for 10 minutes at room temperature, which was followed by incubation in neutralizing and lysing solution 2 (0.4 M Tris, 2 M NaCl, and 1% SDS, pH 7.5) for 5 minutes at room temperature. Slides were thoroughly washed in Tris-borate-EDTA buffer (0.09 M Tris-borate and 0.002 M EDTA, pH 7.5) for 2 minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each), and air dried. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) (2 μg/mL) (Roche Diagnostics, Barcelona, Spain) in Vectashield (Vector Laboratories, Burlingame, Calif) for fluorescence microscopy or with the Diff-Quik reagent (Baxter Healthcare Corporation Inc, McGaw, Ill) for brightfield microscopy.

DBD-FISH

A human whole genome probe (4.3 ng/μL in 50% formamide/2× standard saline citrate [SSC], 10% dextran sulfate, and 100 mM calcium phosphate, pH 7.0) (1× SSC is 0.015 M sodium citrate and 0.15 M sodium chloride [NaCl], pH 7.0), which had been biotin labeled by nick translation, was denatured and hybridized overnight at room temperature on dried slides processed for the SCD test; it was then washed twice in 50% formamide/2× SSC, pH 7.0, for 5 minutes, and twice in 2× SSC, pH 7, for 3 minutes, at room temperature. The hybridized probe was detected with streptavidin-indocarbocyamine (Cy3) (1:200) (Sigma Chemical Co, St Louis, Mo), and cells were counterstained with DAPI (Fernández et al, 1998, 2002; Fernández and Gosálvez, 2002).

Fluorescence Microscopy and Digital Image Analysis

Slides were viewed under a DMRB epifluorescence microscope (Leica, Wetzlar, Germany) equipped with a DMRD photoexposer, PL Fluotar 100× or 40× objectives, and appropriate fluorescence filters for DAPI and Cy3. Images were acquired using a high-sensitivity charge-coupled device camera (Ultrapix 1600, AstroCam, Perkin Elmer Optoelectronics, Santa Clara, Calif), which detects over 16000 gray levels and allows the subtraction of the current dark image and a correction for nonuniform sample illumination. Groups of 450 digital images, 2 per cell (DAPI stain and corresponding DBD-FISH signal), were taken for each experimental point under similar conditions; these were then stored in the file format of the camera (in files with an.apf extension) and thereafter converted to files with an.img format. Each experiment was repeated at least twice. Image analysis was performed using a semiautomatic routine designed with Visilog 5.1 software (Noesis, Courtaboeuf, France). This allows for thresholding, background subtraction, and measurement of the surface area in pixels from both the halo and the whole nucleoid observed under DAPI staining, as well as observation of mean fluorescence intensity (MFI; mean gray level) of the DBD-FISH signal. The halo size of each cell was evaluated by the relative parameter: surface of the halo ÷ surface of the whole nucleoid (Figure 1). Since some sperm cells may have different nuclear sizes, this relative parameter avoids the distortion that would result if absolute sizes were considered. DNA dispersion patterns were established in 450 sperm cells from semen samples obtained from patients with normal (n = 5) and abnormal (n = 5) semen parameters. Each individual spermatozoon was simultaneously scored by light microscopy and digital image analysis (DIA) by 2 different observers. Statistical analysis was carried out using the Student's t test and one- and two-way analyses of variance (P < .05).

image

Figure 1. . Demonstration of how the relative halo size is obtained by digital image analysis (DIA). The DAPI (4′,6-diamidino-2-phenylindole) stained sperm nucleoid (blue fluorescence, left) contains a central core and a peripheral halo of dispersed DNA loops. Using DIA software, the relative halo size is obtained from the halo surface (upper right) and the whole nucleoid surface (lower right). Dividing the halo surface by the respective whole nucleoid surface, we obtain the relative halo size as it relates to the nucleoid.

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Brightfield Microscopy

Slides were also stained with the Diff-Quik solutions used for sperm morphology, and the degree of DNA dispersion was assessed by brightfield microscopy. A minimum of 300 spermatozoa were evaluated by 2 different observers.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Effect of Acid Pretreatment on Sperm DNA Dispersion

When somatic cells with fragmented DNA are exposed to a denaturing solution, either acid or alkaline, prior to deproteinization, the spreading of the DNA fragments is as large as or even larger than when omitting the denaturation step (Fernández et al, 2002). However, as shown in Figure 2a through c, when sperm with fragmented DNA are exposed to a denaturing acid solution prior to deproteinization, the halos of DNA dispersion are absent or extremely small compared to those observed in sperm nuclei with nonfragmented DNA, as confirmed by DBD-FISH. In sharp contrast, when sperm are not preexposed to a denaturing acid solution, it is difficult to distinguish differences in nuclear DNA dispersion between sperm with fragmented and nonfragmented DNA (Figure 2d). The results shown in Figure 2a through c were obtained with the same semen sample as used in Figure 2d, where all spermatozoa exhibited DNA dispersion halos.

image

Figure 2. . Spermatozoa embedded in an agarose microgel on a slide treated with acid solution prior to the lysis step. (a) DAPI (4′,6-diamidino-2-phenylindole) staining (blue fluorescence) showing spermatozoa with different patterns of DNA dispersion: 1) large-sized halo; 2) medium-sized halo; 3) very small-sized halo; and 4) no halo. (b) Sequential DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) labeling with a whole genome probe (red fluorescence), demonstrating extensive DNA breakage in those nuclei with very small halos or no halo at all in (a). (c) Same sperm cells stained with Diff-Quik staining solution for brightfield microscopy. (d) Spermatozoa from the same sample as that used in (a—c), embedded in an agarose microgel on a slide not treated with acid solution prior to the lysis step and then stained with DAPI (blue fluorescence). Note that in contrast to the sperm nuclei shown in (a—c), all sperm nuclei show halos of DNA dispersion. The sperm nucleoid in the lower left shows a higher halo and a clear nuclear disintegration, indicating extensive DNA breaks. (e) The Sperm Chromatin Dispersion (SCD) test—processed spermatozoa showing no DNA dispersion halo by DAPI staining (blue fluorescence) and (f) the same cells showing DNA fragmentation by DBD-FISH (red fluorescence). The cell in the upper left is of larger size than that of the sperm showed in the middle right of the figure, whereas the 2 cells located below appear to be partially disaggregated, loose material, so their DBD-FISH labeling of DNA breaks is not as intense as in the rest. (g) Immature germ cell, with a much larger size and a larger halo than that of the spermatozoa under DAPI staining and (h) the same cell processed for DBD-FISH. Note the extremely low signal intensity by DBD-FISH. Spermatozoa from the 90% pellet (i, j) and F1 fractions (k, l) were obtained by ISolate gradient centrifugation and processed for the SCD test (i, k) and DBD-FISH (j, l). The great majority of spermatozoa in the 90% pellet fraction show large halos of DNA dispersion under DAPI staining (blue fluorescence) and very faint labeling by DBD-FISH (red fluorescence). Occasionally, some spermatozoa are found with very small halos or none at all and intense labeling by DBD-FISH. In sharp contrast, approximately half of the spermatozoa in fraction 1 show very small halos or no halo at all by the SCD test and extensive DNA fragmentation by DBD-FISH.

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DNA Dispersion Patterns and Correlation Between DNA Dispersion and DNA Fragmentation

Four dispersion patterns were clearly observed when scoring SCD test—processed sperm nuclei by either fluorescence or brightfield microscopy: 1) nuclei with large DNA dispersion halos; 2) nuclei with medium-sized halos; 3) nuclei with very small-sized halos; and 4) nuclei with no halo (Figure 2a and c). Very occasionally, it may be difficult to discriminate between spermatozoa with medium- and large-sized halos. In these cases, if the halo width is similar to or larger than the minor diameter of the core of the nucleoid, it will be considered a sperm cell with a large halo. The presence of DNA breaks in spermatozoa was confirmed by subsequent hybridization with a whole genome probe using the DBD-FISH assay. As shown in Figure 2b, sperm with large DNA dispersion halos (pattern 1) show very faint to undetectable levels of DBD-FISH labeling in specific nuclear areas. These probably correspond to chromocenters resulting from the clustering of repetitive satellite DNA sequences that are very sensitive to the denaturation. Sperm with medium-sized halos (pattern 2) exhibit a slightly higher DBD-FISH signal (Figure 2b) than nuclei with large halos. In contrast, sperm nuclei with very small halos (pattern 3) or no halo at all (pattern 4) showed very strong DBD-FISH labeling, which corresponded to those sperm nuclei with extensive DNA fragmentation. A previous study had already reported this latter result (Fernández et al, 2000).

It is noteworthy that, in some samples, sperm with non-dispersed nuclei appeared with different levels of disintegration (Figure 2e and f). Another important observation is that spermatids and/or somatic cell nuclei are clearly distinguished from spermatozoa. These cells show a significantly larger diameter than those of sperm nuclei and have either a very low-intensity DBD-FISH signal or an undetectable DBD-FISH signal (Figure 2g and h). However, when these cells are apoptotic, they exhibit very highly dispersed DNA spots around a faint residual nuclear structure and strong DBD-FISH labeling (Fernández et al, 2002).

Digital vs Manual Analysis

Each individual spermatozoon was simultaneously scored manually and by DIA analysis by 2 different observers. Table 1 shows the Halo Surface-Whole Nucleoid Surface ratio and the corresponding MFI values in DBD-FISH for all 4 cell patterns from 5 healthy sperm donors. Differences were statistically significant (P < .05) among the 4 cell patterns, confirming the inverse correlation between halo size and DBD-FISH signal. Figure 3 visually represents the DIA analysis in 450 sperm cells, each sperm cell being characterized by its data pair: 1) the Halo Surface-Whole Nucleoid Surface ratio under DAPI staining, and 2) the MFI under DBD-FISH. Furthermore, the cell pattern, as scored manually, is also identified. There was an excellent correlation between the results obtained manually and those obtained by DIA analysis. When a sperm nucleus was manually assigned the pattern of a large halo, there was a 3.7% probability that this cell could correspond to a medium-sized halo, as determined by DIA analysis. Conversely, the probability that spermatozoa scored as medium-sized halos corresponded to a large-sized halo was only 2.2%. The probability that a sperm cell scored as a medium-sized halo could correspond to a very small-sized halo was also 2.2%. Conversely, the probability that a sperm cell scored as a very small-sized halo could correspond to a medium-sized halo was 3.1%. Finally, the probability that a sperm nucleus scored (by qualitative analysis) as a very small-sized halo (or as an undetectable halo) that did not have extensively fragmented DNA was 1.8%, and this error could increase to 3.1% in the hypothetical case that this population was exclusively composed of cells with very small halos. The probability of error was less than 3% in interslide scoring for all different patterns and in interindividual scoring among 3 different subjects. In conclusion, the manual scoring of SCD test—processed sperm samples appears to be an accurate method for the analysis of sperm DNA fragmentation.

Table 1. . Correlation between Halo Surface-Whole Nucleoid Surface (HS/WNS) ratio and mean fluorescence intensity (MFI) of the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) signal for the nuclei patterns identified by fluorescence and brightfield microscopy *
 Large HaloMedium HaloVery Small HaloNo Halo
  1. *Values represent the mean ± standard deviation of the HS/WNS ratio of sperm nuclei stained with DAPI (4′, 6-diamidino-2-phenylindole) and the MFI of the DBD-FISH signal for the 4 nuclei patterns manually established. A total of 450 sperm cells per sample were scored in samples from 5 healthy donors. The percentage of each of these 4 patterns is also indicated.

HS/WNS0.87 ± 0.030.73 ± 0.060.45 ± 0.090
MFI12.32 ± 6.6829.87 ± 18.22154.91 ± 87.46386.91 ± 258.31
% patterns74.7710.207.287.73
image

Figure 3. . Comparison of digital and manual scoring in 450 sperm cells. Using digital image analysis (DIA), each sperm cell was defined by its data pair: 1) the Halo Surface-Whole Nucleoid Surface ratio under DAPI (4′,6-diamidino-2-phenylindole) staining, and 2) the mean fluorescence intensity (MFI) under DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH). Its characterization by manual analysis is also indicated. Note that a few cells without halos show disintegrated nuclei. Although their DBD-FISH labeling is complete, the MFI is faint because the DBD-FISH signal is dispersed.

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SCD Test Values in Semen Samples From Healthy Sperm Donors and Infertility Patients

In order to determine DNA fragmentation levels in semen samples obtained from healthy sperm donors and infertility patients as determined by the SCD test, semen samples from healthy sperm donors and from patients attending a clinic for infertility screening were evaluated. As shown in Table 2, the percentage of nondispersed nuclei (ie, nuclei with very small halos or none at all) in semen samples obtained from infertility patients ranged from 10% to 81%. In addition, 40% of the samples from patients with normal semen parameters and 60% of the samples from patients with abnormal semen parameters had nondispersed nuclei values above 30%. In contrast, only 10% of the samples from healthy sperm donors had non-dispersed nuclei values in semen above 30%, with values ranging from 3.6% to 35% (Table 2). The SCD test values of healthy sperm donors were significantly different from those of infertility patients (mean ± SD) (16.7 ± 9.9 vs 35.4 ± 18.3, P < .05). No statistically significant differences were found between samples from infertility patients with normal or abnormal semen parameters (32.1 ± 20.4 vs 38.7 ± 16.3, P > .05). It is worth mentioning that semen samples from healthy sperm donors were used in intrauterine insemination cycles and that, with the exception of sample 2, which had a nondispersed nuclei value of 35%, all resulted in a term pregnancy.

Table 2. . Sperm Chromatin Dispersion (SCD) test values in semen samples from healthy donors and infertility patients
Sample Group% Dispersed Nuclei% Nondispersed Nuclei
  1. *All semen samples from sperm donors were used in donor insemination cycles and, with the exception of sample 2, all resulted in a term pregnancy. A minimum of 300 spermatozoa per sample were scored.

Sperm donors  
    Sample 1*96.43.6
    Sample 265.035.0
    Sample 389.011.0
    Sample 486.113.9
    Sample 594.55.5
    Sample 682.317.7
    Sample 772.028.0
    Sample 887.013.0
    Sample 985.214.8
    Sample 1075.025.0
Patients  
    Normal semen parameters  
        Sample 1181.018.0
        Sample 1228.471.6
        Sample 1340.659.4
        Sample 1483.216.8
        Sample 1569.031.0
        Sample 1673.426.6
        Sample 1754.745.3
        Sample 1890.010.0
        Sample 1972.827.2
        Sample 2085.214.8
    Abnormal semen parameters  
        Sample 2119.081.0
        Sample 2268.631.4
        Sample 2368.431.6
        Sample 2471.029.0
        Sample 2560.139.9
        Sample 2667.532.5
        Sample 2773.027.0
        Sample 2849.850.2
        Sample 2971.428.6
        Sample 3064.036.0

DNA Dispersion in Sperm Subsets Isolated by Gradient Centrifugation

In a different set of experiments, the SCD test was applied to the assessment of DNA fragmentation in sperm subsets isolated by density gradient centrifugation. Previous studies have shown that the level of DNA fragmentation, as measured by the SCSA test, was highest in the low-density fractions and lowest in the 90% sperm pellet (Ollero et al, 2001). In our study, 300 sperm cells were scored from raw semen and from each of the 3 ISolate gradient fractions by the SCD test and DBD-FISH. The results show that the level of DNA fragmentation was also highest in the low-density fraction, F1, and lowest in the 90% pellet, which is in excellent agreement with previous studies (Figure 2i through l; Table 3).

Table 3. . Percentage of dispersed and nondispersed (with no halo or minimal-sized halo) nuclei in raw semen and the corresponding ISolate gradient fractions
 Concentration (million/mL)Dispersed Nuclei*Nondispersed Nuclei*Φn × Cn
  1. *Percentage of dispersed and nondispersed nuclei in raw semen and in subsets of human spermatozoa isolated by ISolate gradient centrifugation of 2 semen samples with normal and abnormal semen parameters, respectively. Semen 1: normal World Health Organization (WHO) parameters; semen 2: abnormal WHO parameters. Φn × Cn: percentage of nondispersed nuclei in the ISolate fraction (Φn) multiplied by the corresponding fractional sperm concentration (Cn). Sperm concentration values are expressed in million per milliliter.

Semen 113776.723.3
    F17062.137.118.95
    F23087.912.03.24
        90% pellet3794.44.01.08
    Total23.27
Semen 212.857.142.9
    F19.562.850.537.48
    F22.175.024.03.93
        90% pellet1.280.719.51.83
    Total43.24

In order to rule out the production of fragmentation artifacts during the manipulation of sperm cells during the SCD test procedure, the level of nondispersed and dispersed nuclei was assessed in raw semen and the corresponding ISolate fractions of 2 semen samples with normal and abnormal semen parameters, respectively. The results indicate that the sum of the percentage of spermatozoa with nondispersed DNA in the different gradient fractions (Φn), as measured by the SCD test, multiplied by the fractional sperm concentration of each fraction (Cn) corresponded to the percentage of nondispersed nuclei found in raw semen (ΦS) to within 1% (Table 3). Note that ΦS = (ΦF1 × CF1 + ΦF2 × CF2 + ΦP × CP), where ΦS is the percentage of sperm nuclei with nondispersed nuclei in raw semen, and ΦF1 × CF1, ΦF2 × CF2, and ΦP × CP are the percentages of nondispersed nuclei in ISolate fractions F1 and F2 and the 90% pellet multiplied by their corresponding fractional sperm concentration, respectively.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

It has been previously established that DNA breaks increase ssDNA production after treatment with denaturing agents (eg, heat, acid, or alkali) (Ahnström, 1988). The unwinding assay currently used in radiobiology to study radiation-induced DNA breaks is based on the fact that denaturing solutions, generally alkaline, produce ssDNA areas starting from the end of the DNA breaks. As DNA breaks increase, more ssDNA is generated by the denaturing solution. The acid solution is weaker than the alkali. When sperm nuclei contain fragmented DNA, the denaturing solution transforms the regions with extensive DNA breaks into ssDNA motifs (Gorzcyca et al, 1993a,b). These motifs are susceptible to hybridization with a fluorescent whole genome probe, and this is the rationale behind DNA breakage detection by DBD-FISH. The mechanism responsible for the suppression of the production of DNA halos in sperm nuclei with extensive DNA fragmentation remains unknown. The DBD-FISH test results obtained in this study demonstrate that, in addition to the presence of extensive DNA breaks, the generation of high amounts of ssDNA is a necessary condition for suppressing the generation of DNA dispersion halos in sperm cells with fragmented DNA. The results suggest that ssDNA interacts within the sperm head in such a way that the removal of most nuclear proteins by lysing solutions does not result in dispersion of the DNA fragments. However, this is not observed in apoptotic somatic cell nuclei, suggesting that it may be the result of the peculiar structure and organization of sperm chromatin. The mild acid solution does not produce ssDNA in sperm nuclei without fragmented DNA, except in small chromocenters containing DNA sequences highly sensitive to denaturation. Therefore, the DNA loops may spread in large dispersion halos. It should be pointed that when using an alkaline solution (0.03 M NaOH and 1 M NaCl)—a much stronger denaturant than the mild acid solution—the background DBD-FISH signal increases in sperm with nonfragmented DNA. Accordingly, the DNA dispersion halos appear smaller than when using the acid solution. On the other hand, the halos observed when sperm cells are lysed without acid pretreatment (Figure 2d) are larger than when the acid pretreatment is used. This supports the inverse correlation that is observed between the yield of ssDNA and the extent of the DNA dispersion halo in spermatozoa. This is consistent with the recent report by Ankem et al (2002). In addition, our results suggest a correlation between defective chromatin packaging and DNA dispersion in mature spermatozoa, as shown by the presence of nondispersed nuclei and DNA fragmentation. This correlation had been suggested indirectly by the studies of chromomycin A3 fluorochrome accessibility to sperm nucleus and in situ nick translation sensitivity (Sakkas et al, 1996).

What are the advantages and disadvantages of the SCD test compared to other existing methodologies? Unlike currently available semiquantitative tests for the determination of sperm DNA fragmentation (eg, the TUNEL assay, the comet assay, and the chromomycin A3 test), the SCD test does not rely on the determination of either color or fluorescence intensity. Rather, the endpoint measured by the SCD test consists of determining the percentage of spermatozoa with nondispersed (very small halos or none at all) or dispersed nuclei, which can be easily and reliably accomplished by the naked eye. As the results of this study indicate, the use of DIA did not significantly improve the accuracy of the test results compared to brightfield microscopy, implying that the scoring of these patterns by brightfield microscopy provides an accurate means for the determination of DNA dispersion and, therefore, DNA fragmentation in spermatozoa.

The current gold standard for the quantitative determination of sperm DNA fragmentation is the SCSA test. This test relies on the measurement by flow cytometry of green and red fluorescence intensity emitted by spermatozoa with double-stranded DNA (dsDNA) and ssDNA, respectively, following acid denaturation and acridine orange staining (Evenson et al, 1999). Spermatozoa with dsDNA reflect spermatozoa with intact DNA, and spermatozoa with ssDNA are indicative of spermatozoa with fragmented DNA. The DNA fragmentation levels obtained in our study in samples from infertile males and healthy donors are consistent with the results reported by Evenson (1999) and Ollero et al (2001). In addition, the DNA fragmentation values obtained in sperm subsets isolated by ISolate gradient centrifugation are also consistent with the results recently reported using the SCSA test (Ollero et al, 2001; Alvarez et al, 2002). These results indicate that the DNA fragmentation values obtained with the SCD test are comparable to those obtained with the SCSA test. Studies are currently under way to correlate the SCD test values with those of the SCSA test and with fertilization and pregnancy outcome.

What are the implications of DNA fragmentation in the outcome of in vivo and in vitro fertilization? In vitro fertilization of metaphase II oocytes with spermatozoa that have damaged DNA could potentially lead to failed fertilization, defective embryo development, implantation failure, or early abortion (Genesca et al, 1992; Parinaud et al, 1993; Twigg et al, 1998; Evenson et al, 1999). One could speculate that those samples with high DNA fragmentation values should produce lower fertilization rates after ICSI than the samples with low DNA fragmentation values. This hypothesis is currently being tested in our laboratory.

In conclusion, the results of this study show that the SCD test is a simple, fast, accurate, and highly reproducible method for the analysis of sperm DNA fragmentation in semen and processed sperm. Moreover, it has a turn-around time of less than 1 hour (scoring included) and reagent costs per sample of about $0.5, allowing the simultaneous processing of several samples per slide. Finally, the SCD test does not require the use of complex instrumentation: it can be carried out with equipment normally available in andrology laboratories (ie, light microscopes), and the test endpoints (nondispersed and dispersed nuclei) can be easily assessed by laboratory technicians. Therefore, the SCD test could potentially be used for the routine screening of sperm DNA fragmentation in the andrology laboratory.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

We would like to thank Dr Christopher de Jonge and Dr Donald Evenson for their critical review of this manuscript. We would also like to thank Dr Enrique Segrelles, Dr José Bayo, Dr Braulio Peramo, and Dr José Manuel Gallardo for their support.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  • Ahnström G.. Techniques to measure DNA strand breaks in cells: a review. Int J Radiat Biol. 1988;54: 695707.
  • Alvarez JG, Ollero M., Gil-Guzman E., et al. Increased DNA damage in leukocytospermic semen samples. Fertil Steril. 2002;78: 319329.
  • Ankem MK, Mayer E., Ward WS, Cummings KB, Barone JG. Novel assay for determining DNA organization in human spermatozoa: implications for male factor infertility. Urology. 2002;59: 575578.
  • Cook PP, Brazell IA. Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei. Eur J Biochem. 1978;84: 465477.
  • de Jonge C.. The clinical value of sperm nuclear DNA assessment. Hum Fertil. 2002;5: 5153.
  • Evenson DP. Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects. Reprod Fertil Dev. 1999;11: 115.
  • Evenson DP, Darzynkiewicz Z., Melamed MR. Relation of mammalian sperm heterogeneity to fertility. Science. 1980;210: 11311133.
  • Evenson DP, Higgins PJ, Grueneberg D., Ballachey BE. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry. 1985;6: 238253.
  • Evenson DP, Jost LK. Sperm chromatin structure assay: DNA denaturability. In: Darzynkiewicz Z., Robinson JP, Crissman HA, eds. Methods in Cell Biology. Vol 42. Flow Cytometry. 2nd ed. Orlando, Fla: Academic Press; 1994; 159176.
  • Evenson DP, Jost LK, Baer RK, Turner TW, Schrader SM. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod Toxicol. 1991;5: 115125.
  • Evenson DP, Jost LK, Marshall D., Zinaman MJ, Clegg E., Purvis K., de Angelis P., Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 1999;14: 10391049.
  • Evenson DP, Larson KJ, Jost LK. 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.
  • Evenson DP, Melamed MR. Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem. 1983;31: 248253.
  • Fernández JL, Gosálvez J.. Application of FISH to detect DNA damage: DNA breakage detection-FISH (DBD-FISH). Methods Mol Biol. 2002;203: 203216.
  • Fernández JL, Goyanes V., Gosálvez J.. DNA Breakage Detection-FISH (DBD-FISH). In: Rautenstrauss B., Liehr T., eds. FISH Technology—Springer Lab Manual. Heidelberg: Springer-Verlag; 2002; 282290.
  • Fernández JL, Goyanes VJ, Ramiro-Díaz J., Gosálvez J.. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet. 1998;82: 251256.
  • Fernández JL, Vázquez-Gundín F., Delgado A., Goyanes VJ, Ramiro-Díaz J., de la Torre J., Gosálvez J.. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res. 2000;453: 7782.
  • Genesca A., Caballin MR, Miro R., Benet J., Germa J.R., Egozcue J.. Repair of human sperm chromosome aberrations in the hamster egg. Hum Genet. 1992;82: 181186.
  • Gorczyca W., Gong J., Darzynkiewicz Z.. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 1993a;53: 945951.
  • Gorczyca W., Traganos F., Jesionowska H., Darzynkiewicz Z.. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res. 1993b;207: 202205.
  • Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W.. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men using a modified comet assay. Mol Hum Reprod. 1996;2: 613619.
  • Larson KL, DeJonge C., Barnes A., Jost L., Evenson DP. Relationship between assisted reproductive techniques (ART) outcome and status of chromatin integrity as measured by the Sperm Chromatin Structure Assay (SCSA). Hum Reprod. 2000;15: 17171722.
  • Manicardi GC, Bianchi PG, Pantano S., Azzoni P., Bizzaro D., Bianchi U., Sakkas D.. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod. 1995;52: 864867.
  • Ollero M., Gil-Guzmán E., Sharma RK, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16: 19121921.
  • Parinaud J., Mieusset R., Vieitez G., Labal B., Richoilley G.. Influence of sperm parameters on embryo quality. Fertil Steril. 1993;60: 888892.
  • Roti JL, Wright WD. Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair. Cytometry. 1987;8: 461467.
  • Sakkas D., Urner F., Bianchi PG, Bizarro D., Wagner I., Jaquenoud N., Manicardi G., Campana A.. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod. 1996;11: 837843.
  • Smith PJ, Sykes HR. Simultaneous measurement of cell cycle phase position and ionizing radiation induced DNA strand breakage in single human tumour cells using laser scanning confocal imaging. Int J Radiat Biol. 1992;61: 553560.
  • Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod. 1998;13: 18641871.
  • Organization World Health. WHO Laboratory Manual for the Examination of Human Semen and Semen—Cervical Mucus Interactions. 2nd ed. Cambridge, United Kingdom: Cambridge University Press; 1999.
Footnotes
  1. Supported by the Fondo de Investigaciones Sanitarias (FIS 01–3113), Plan Gallego de Investigación y Desarrollo Tecnológico de la Xunta de Galicia (PGIDT-01BIO02E) and PGIDIT 02PXIC91603PN, and the Consejo de Seguridad Nuclear from Spain.