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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.
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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.