1 Division of Reproductive Sciences, The Toronto General Hospital, The Samuel Lunenfeld Research Institute, and 2 Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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Key words: DNA fragmentation/semen analysis/spermatozoa/swim-up
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Introduction |
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In an attempt to prevent damage by centrifugation and generation of ROS, other methods of sperm preparation have been developed using density gradient media or direct swim-up from the original sperm sample (Mortimer, 1991). The latter method has recently been recommended for sperm preparation (World Health Organization, 1999
). Some studies have found that Percoll did not improve sperm quality as measured by motility, velocity, percentage normal morphology and intact acrosomes (Ng et al., 1992
) whereas others have found improved performance as judged by the hamster egg penetration assay (Chan and Tucker, 1992
). Assessment of DNA integrity following any preparation method has been limited to date. It has recently been reported that Percoll preparation of spermatozoa can lead to a 2-fold increase in denatured spermatozoa (Zini et al., 1999
). A double wash centrifugation followed by a swim-up from the pellet actually improved sperm chromatin structure properties (Spano et al., 1999
). It was therefore of interest to determine whether the normal swim-up technique involving centrifugation, as used in our IVF programme, could lead to sperm DNA damage, and to compare this with one that does not use a centrifugation step.
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Materials and methods |
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Semen samples were collected after 48 h of abstinence. A small aliquot (~50 µl) of the original semen sample was fixed in 4% paraformaldehyde to determine the percentage DNA fragmentation. Semen analysis was performed according to published methods (World Health Organization, 1987). Motility was assessed qualitatively since most of the samples were used for insemination in IVF.
Normal swim-up technique
Sperm preparation using a double wash and centrifugation procedure was carried out as previously described (Lopes et al., 1998a) and at the time this was the normal method used in our laboratories. Briefly, one of the two portions of semen was diluted with 2xvolume of Ham's F10 (Gibco, Life Technologies, Grand Island, NY, USA) containing 10% synthetic serum substitute (SSS) (Irvine Scientific, Irvine, CA, USA) and then centrifuged at 220 g for 10 min. The supernatant was transferred to another tube. The pellet was resuspended in 2 ml of Ham's F10 with 10% SSS. Both supernatant and pellet were centrifuged at 220 g for 10 min and the supernatants discarded. The pellets were combined and resuspended in 0.5 ml of Ham's F10 with 10% SSS. This suspension was layered gently under 1 ml of Ham's F10 with 10% SSS. The tube was slanted and incubated for 1 h at 37°C in a 5% CO2 incubator. The swim-up sperm fraction was then fixed in 4% paraformaldehyde for later determination of DNA fragmentation.
Direct swim-up technique
The second portion of each semen sample was used for the direct semen swim-up procedure without centrifugation. The semen sample, 0.100.15 ml, was layered gently under 0.3 ml of Ham's F10 with 10% SSS and incubated for 1 h at 37°C in a 5% CO2 incubator. The swim-up portion was then fixed in 4% paraformaldehyde as above.
Sperm DNA fragmentation
DNA fragmentation in the spermatozoa was measured using a modification of the method of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin end-labelling (TUNEL) as we previously described (Lopes et al., 1998a). The air-dried slides were washed in phosphate-buffered saline (PBS, pH 7.4 prepared in house), then spermatozoa were permeabilized with Triton X-100 (Caledon Laboratories Ltd, ON, Canada). A buffer containing 10 U of TdT enzyme (Pharmacia LKB Biotech, Piscataway, NJ, USA), 3 µmol/l biotin-16-dUTP (Boehringer Mannheim, Laval, PQ, Canada), 12 µmol/l dATP Pharmacia LKB Biotech), and 0.1% Triton X-100 was added to the slide and allowed to incubate at 37°C for 60 min. Following TdT exposure, spermatozoa were treated with a staining buffer containing 1% streptavidin/Texas red anti-biotin (Calbiochem-Novabiochem Corporation, La Jolla, CA, USA) and incubated at 4°C in the dark for 30 min. The stained cells were washed in PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO, USA), which stains all chromatin, prior to analysis. Percentage of spermatozoa with DNA damage was determined by dividing TUNEL positive spermatozoa by total spermatozoa analysed as determined by DAPI staining. At least 200 cells were counted to determine the percentage.
Statistical analysis
Statistical analyses were done using the SPSS statistical package version 10 for windows. One-way analysis of variance with Tukey's post hoc testing and, where appropriate, linear regression were applied to determine the correlation between the DNA fragmentation in the original semen sample and semen parameters. The Wilcoxon signed ranks test was also used to compare differences between groups for DNA fragmentation.
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Results |
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Discussion |
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Another study (Zini et al., 1999), using the same chromatin structure assay of Evenson (Evenson et al., 1980
), found in 25 non-azoospermic men that with two- or four-layered Percoll density-gradient gradient centrifugation, there was a 2-fold increase in denatured sperm DNA when compared to whole semen. They did not compare any other sperm preparation method. These latter results call into question whether Percoll density preparation of spermatozoa actually improves sperm quality. After our study was completed, a new edition of the Laboratory Manual (World Health Organization, 1999
) appeared in which it was recommended that the standard method for sperm preparation should avoid centrifugation. The results of our study, however, suggests that there is little to be gained in terms of DNA fragmentation but much is lost in terms of recovery when centrifugation is avoided.
Given the speed at which ROS can cause sperm damage (Aitken and Clarkson, 1988; Lopes et al., 1998a
) one would have expected that DNA fragmentation would be increased after the normal (centrifugation) swim-up, and especially at 24 h after insemination. In fact, the normal swim-up procedure for preparing spermatozoa appears to result in very low rates of sperm DNA damage and appears to be free of any detrimental effect on the fertilizing ability of the spermatozoa as noted in the DNA data from samples used for IVF. Even the two samples with high DNA fragmentation in the original semen improved after the normal swim-up procedure and were effective in fertilizing oocytes. It would have been interesting to compare the fertilizing ability of sperm prepared by the two methods but this was not the original objective of the study.
Notwithstanding the fact that DNA repair is feasible, it is possible that the damage noted (Mortimer, 1991) may be due to the three centrifugation steps normally carried out by some laboratories (Aitken and Clarkson, 1988
). Centrifugation removes the antioxidant properties (Aitken, 1999
) of seminal plasma and induces iatrogenic DNA damage in spermatozoa (Twigg et al., 1998c
). This is more pronounced with poor quality sperm samples with numerous leukocytes. In fact, 55% of oligozoospermic patients were found to have defective sperm function as assessed by the hamster egg penetration assay, and elevated production of ROS (Aitken et al., 1989
). Semen samples with poor semen analysis parameters and poor fertilization and cleavage rates in IVF, were found to have increased proportion of spermatozoa with DNA fragmentation (Sun et al., 1997
). Such sperm samples with high DNA fragmentation, when used for ICSI, were correlated with failed fertilization (Lopes et al., 1998b
). In the current study >90% of the original samples were normal as judged by the semen parameters. This fact may have precluded the observation of higher percentage of sperm DNA damage.
Linear regression analysis of the data in this study indicated that there was a significant relationship between DNA fragmentation and sperm morphology. This is in contrast to other data (Irvine et al., 2000) showing that sperm concentration most accurately reflected the incidence of sperm DNA damage and (Hughes et al., 1996
) that spermatozoa from normospermic men, in contrast to that from infertile men, were more susceptible to DNA damage by irradiation. A significant negative correlation between semen quality as assessed by motility, morphology and concentration and DNA damage has been demonstrated (Sun et al., 1997
). Using a TUNEL-coupled flow cytometry method, it was shown that the extent of sperm DNA fragmentation in unselected and swim-up spermatozoa was positively related to abnormal morphology and associated with defects of the sperm tail (Muratori et al., 2000
). A negative correlation was found between DNA breakage and progressive motility. In a similar study, using a TUNEL assay, with spermatozoa from infertile men (Barroso et al., 2000
), low sperm motility was associated with high DNA damage. These variations in correlations among the different studies may be a reflection of the variations in the methods used for semen analyses. The morphological analysis employed in our study can be criticized in view of the stricter criteria being employed in many laboratories. However, there is a common observation among all the studies that DNA damage is related to sperm parameters.
In summary, our results suggest that swim-up separation of motile spermatozoa from normal semen, either with or without centrifugation, does not increase the level of DNA damage. In addition, double centrifugation of normal spermatozoa does not impair the fertilizing ability of the recovered spermatozoa.
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Notes |
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References |
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Aitken, R. J. and Clarkson, J. S. (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl., 9, 367376.[Abstract]
Aitken, R.J., Clarkson, J.S., Hargreave, T.B. et al. (1989) Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J. Androl., 10, 214220.
Aitken, R.J., Gordon, E., Harkiss, D. et al. (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol. Reprod., 59, 10371046.
Barroso, G., Morshedi, M. and Oehninger, S. (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum. Reprod., 15, 13381344.
Chan, S.Y.W. and Tucker, M.J. (1992) Differential sperm performance as judged by the zona-free hamster egg penetration test relative to differing sperm penetration techniques. Hum. Reprod., 7, 255260.[Abstract]
Evenson, D.P., Darzynkiewics, Z. and Melamed, M.R. (1980) Comparison of human and mouse sperm chromatin heterogeneity to fertility. Science, 240, 11311133.
Hughes, C.M., Lewsi, S.E.M., McKelvey-martin, V.J. et al. (1996) A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol. Hum. Reprod., 2, 613619.[Abstract]
Irvine, D.S., Twigg, J.P., Gordon, E.L. et al. (2000) DNA integrity in human spermatozoa: relationships with semen quality. J. Androl., 21, 3344.
Lopes, S., Jurisicova, A., Sun, J-G. and Casper R.F. (1998a) Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod., 13, 896900.[Abstract]
Lopes, S., Sun, J-G., Jurisicova, A., et al. (1998b) Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril., 69, 528532.[ISI][Medline]
Mortimer, D. (1991) Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum. Reprod., 6, 173176.[ISI][Medline]
Muratori, M., Piomboni, P., Baldi, E. et al. (2000) Functional and ultrastructural features of DNA-fragmented human sperm. J. Androl., 21, 903912.
Ng, F.L.H., Liu, D.Y. and Baker, H.W.G. (1992) Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum. Reprod., 7, 261266.[Abstract]
Spano, M., Cordelli, E., Leter, G. et al. (1999) Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. Mol. Hum. Reprod., 5, 2937.
Sun, J-G., Jurisicova, A. and Casper, R.F. (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod., 56, 602607.[Abstract]
Twigg, J.P., Irvine, D.S. and Aitken, R.J. (1998a) Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod., 13, 18641871.[Abstract]
Twigg, J., Fulton, N., Gomez, E. et al. (1998b) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod., 13, 14291436.[Abstract]
Twigg, J., Irvine, D.S., Houston, P. et al. (1998c) Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol. Hum. Reprod., 4, 439445.[Abstract]
World Health Organization (1987) Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction, 2nd edn. Cambridge University Press, Cambridge.
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Zini, A., Mak, V., Phang, D. and Jarvi, K. (1999) Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity. Fertil. Steril., 72, 496499.[ISI][Medline]
Submitted on February 23, 2001; accepted on May 22, 2001.