- Email: [email protected]
Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity
FERTILITY AND STERILITY威 VOL. 72, NO. 3, SEPTEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity Armand Zini, M.D., Victor Mak, M.D., Donna Phang, M.Sc., and Keith Jarvi, M.D. Division of Urology, Department of Surgery, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
Objective: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. Design: Prospective, observational study. Setting: University-based infertility clinic. Patient(s): Twenty-five nonazoospermic men. Intervention(s): Semen samples (n ⫽ 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange–treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. Main Outcome Measure(s): Sperm motility and DNA integrity. Result(s): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). Conclusion(s): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles. (Fertil Steril威 1999;72:496 –9. ©1999 by American Society for Reproductive Medicine.) Key Words: Sperm DNA, sperm motility, semen processing, male infertility
Received January 25, 1999; revised and accepted April 20, 1999. Reprint requests: Armand Zini, M.D., Mount Sinai Hospital, 600 University Avenue, Suite 1525, Toronto, Ontario, Canada M5G 1X5 (FAX: 416-5868354; E-mail: azini@mtsinai .on.ca). 0015-0282/99/$20.00 PII S0015-0282(99)00295-2
496
Sperm washing is an integral part of assisted reproductive technologies such as IVF and IUI. It is important, however, to use a sperm processing technique that is gentle yet enables the recovery of a concentrated and highly functional sperm population. Many of the conventional centrifugation techniques commonly used for cell separation are toxic to spermatozoa. It is reported that serial centrifugation of semen induces significant reactive oxygen species (ROS) release by spermatozoa and semen leukocytes, resulting in sperm dysfunction (1). In contrast, gentler sperm separation techniques such as density-gradient centrifugation and swim-up allow for good sperm recovery with minimal sperm dysfunction (1–3).
objective of this study was to examine the effect of standard semen processing by Percoll density-gradient centrifugation on human sperm DNA integrity.
To date, little is known about the effect of sperm processing on sperm DNA integrity. The
Semen samples (n ⫽ 25) were obtained from consecutively seen nonazoospermic men
MATERIALS AND METHODS Materials Acridine orange (AO) was purchased from PolySciences (Warrington, PA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO) and were at least reagent grade.
Sperm Preparation, Treatments, and Motility Analysis
presenting for infertility evaluation at our institution, under ongoing internal review board approval. Samples were produced by masturbation after 3–5 days of sexual abstinence and allowed to liquefy at room temperature. After liquefaction of semen, standard semen parameters (volume, density, motility, and morphology) were obtained according to World Health Organization guidelines (4). All the semen samples had motile sperm and none had significant leukocytospermia as per World Health Organization guidelines (4). Patient information for this study remained confidential and within the institution. At our institution, institutional review board approval is not necessary for prospective observational studies; therefore, it was not obtained. Approximately 0.5 mL of semen was layered on discontinuous two-layer (45% and 90%) and four-layer (20%, 40%, 65%, and 90%) Percoll gradients and centrifuged at 600 ⫻ g for 20 minutes in 15-mL conical tubes (5). The medium used to dilute the Percoll was modified Ham’s F-10 medium (supplemented with 1% bovine serum albumin and 25 mM of N-2hydroxyethylpiperazine-N⬘-2-ethanesulphonic acid [HEPES], pH 8.0). Spermatozoa collected from the bottom layer (90% layer) of the two-layer and four-layer Percoll gradients were resuspended in modified Ham’s F-10 medium to a sperm concentration of approximately 10 –20 ⫻ 106/mL for assessment of motility. An aliquot of 1 ⫻ 106 spermatozoa (approximately 25–100 L) from each sample (the bottom layer of the two-layer and four-layer Percoll gradients) was resuspended in 1 mL of ice-cold phosphate-buffered saline (pH 7.4) and centrifuged at 600 ⫻ g for 5 minutes. The pellet was resuspended in 1 mL of ice-cold TNE (0.01 M of tris(hydroxymethyl)aminomethane hydrochloride, 0.15 M of NaCl, and 1 mM of ethylenediaminetetraacetic acid, pH 7.4) and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and stored at ⫺70°C until later assessment of sperm DNA integrity. These samples were labeled as processed two-layer and processed four-layer. An aliquot of unprocessed semen (approximately 25–100 L, containing 1 ⫻ 106 spermatozoa) was suspended in 1 mL of ice-cold phosphate-buffered saline (pH 7.4) and centrifuged at 600 ⫻ g for 5 minutes. The pellet was resuspended in ice-cold TNE and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and immediately stored at ⫺70°C until later assessment of sperm DNA denaturation. These samples were labeled as whole.
Controls To assess the possible influence of Ham’s F-10 medium or Percoll on sperm DNA integrity (independent of the centrifugation process), we examined DNA integrity in unFERTILITY & STERILITY威
processed semen (n ⫽ 11 consecutively seen patients) incubated in Ham’s F-10 medium alone or in Ham’s F-10 medium plus 90% Percoll. An aliquot of whole semen was diluted in Ham’s F-10 medium or Ham’s F-10 medium plus 90% Percoll (1/10 vol/vol) and incubated for 30 minutes at room temperature. Samples then were centrifuged at 600 ⫻ g for 5 minutes and the pellet was resuspended in ice-cold TNE and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and immediately stored at ⫺70°C until later assessment of sperm DNA denaturation.
Acridine Orange Sperm Staining Sperm DNA integrity was assessed with the sperm chromosome structure assay described by Evenson et al. (6). Stored sperm samples (processed and whole) were thawed on ice, fixed in 70% ethanol for 30 minutes, and rehydrated in TNE at room temperature. The fixed samples (200-L volume at a sperm concentration of 5 ⫻ 106/mL) were treated for 30 seconds with 400 L of a solution of 0.1% Triton X-100, 0.15 M of NaCl, and 0.08 N of HCl, pH 1.2. After 30 seconds, 1.2 mL of staining buffer (6 g/mL of AO, 37 mM of citric acid, 126 mM of Na2HPO4, 1 mM of disodium ethylenediaminetetraacetic acid, and 0.15 M of NaCl, pH 6.0) was admixed to the test tube and analyzed by flow cytometry.
Flow Cytometry After excitation by a 488-nm wavelength light source, AO bound to double-stranded DNA fluoresces green (515– 530 nm) and AO bound to single-stranded DNA fluoresces red (ⱖ630 nm). Three minutes after AO staining, the samples were analyzed in a fluorescence-activated cell sorter Calibur flow cytometer (Becton Dickinson, San Jose, CA). A minimum of 5,000 cells were analyzed by a fluorescenceactivated cell sorter interfaced with a data handler (CELLQUEST 3.1; Becton Dickinson, San Jose, CA) on a Power Macintosh 7600/132 computer (Macintosh, Cupertino, CA). The proportion of cells that exhibited abnormal emission of red fluorescence (reflecting the percentage of sperm with denatured DNA) was recorded. Interassay variability (⬍5%) was verified by repeated assessments of control semen samples (results not shown). Fresh and frozenthawed samples yielded similar results (⬍5% variability) (results not shown).
Statistical Analysis
Values are expressed as means ⫾ 1 SD. A one-way analysis of variance was used to determine differences in the percentage of sperm motility and in the percentage of sperm that exhibited DNA denaturation between groups. Pearson’s correlation was performed to examine the relation between the percentage of sperm with DNA denaturation and standard semen parameters using SAS software (SAS, Cary, NC). P⬍.05 was considered statistically significant. 497
TABLE 1 Sperm motility and sperm DNA denaturation before and after Percoll density-gradient centrifugation. Sperm motility Sperm DNA denaturation (%) (%)
Semen sample Whole semen Processed (two-layer Percoll) Processed (four-layer Percoll)
44 ⫾ 21 54 ⫾ 24* 57 ⫾ 25*
18 ⫾ 13 34 ⫾ 22* 32 ⫾ 21*
Note: Values are means ⫾ SD. * Statistically significant difference (versus whole semen). Zini. Adverse effect of semen processing. Fertil Steril 1999.
RESULTS The semen samples tested had a mean sperm density of 24 ⫻ 106/mL (range, 8 – 43 ⫻ 106/mL), a mean sperm motility of 43% (range, 13%– 86%), and a mean percentage of sperm that exhibited denatured DNA of 18% (range, 6%– 60%). Sperm motility increased in 72% and 80% of the samples, respectively, after two-layer and four-layer Percoll density-gradient centrifugation. As shown in Table 1, mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen. In contrast, after both two-layer and four-layer Percoll density-gradient centrifugation, the percentage of spermatozoa with denatured DNA increased in 84% of the samples tested. As seen in Table 1, the mean percentage of sperm with denatured DNA increased significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen. A significant negative correlation was observed between the percentage of ejaculated sperm with denatured DNA and the percentage of motile sperm (r ⫽ ⫺48, P⬍.05). A weak negative correlation was observed between the percentage of ejaculated sperm with denatured DNA and sperm density (r ⫽ ⫺38, P⬍.06). There was no significant correlation between the percentage of normal forms (World Health Organization sperm morphology criteria) and the percentage of ejaculated sperm with denatured DNA. Incubation of spermatozoa in Ham’s F-10 medium or Ham’s F-10 medium plus Percoll did not have a negative influence on sperm DNA integrity. The mean percentage of spermatozoa with denatured DNA in diluted semen (Ham’s F-10 medium and Ham’s F-10 medium plus Percoll) was not significantly different than that of whole undiluted semen (19.4% ⫾ 9.5% and 15.9% ⫾ 6.4% versus 20.4% ⫾ 12.1%, P⬎.05).
DISCUSSION Seminal plasma is an important source of antioxidants (7–9); therefore, separating spermatozoa from seminal plasma during semen processing results in a prooxidant state. 498
Zini et al.
Effect of semen processing on sperm DNA
This is reflected by the finding that repetitive washing of sperm by serial centrifugation increases ROS production and impairs sperm function dramatically, particularly in samples with excessive generation of semen ROS (1). This observation emphasizes the importance of semen processing techniques on the production of ROS and potential oxidative injury to spermatozoa. As a result, most infertility centers use gentler sperm separation techniques, such as densitygradient centrifugation and swim-up, to minimize sperm toxicity (1–3). Nonetheless, even these gentler techniques result in small but detectable levels of ROS production (1). Using Percoll density-gradient centrifugation, highly motile, morphologically normal spermatozoa are collected in the higher-density layer (5). Generally, the mild stress induced by this sperm separation technique is not sufficient to result in excessive sperm membrane lipid peroxidation, and consequently, the recovered spermatozoa demonstrate improved motility relative to the unprocessed sample (1). However, in samples that generate high ROS levels, a loss of sperm motility and the capacity for sperm-oocyte fusion (which also is dependent on sperm membrane characteristics) may occur, resulting in impaired fertilization in vivo (i.e., IUI) and in vitro (i.e., standard IVF) (10, 11). In this study, we observed a significant increase in mean sperm motility after Percoll preparation, although approximately 25% of the semen samples showed a decline in sperm motility after processing. In contrast, the DNA integrity of the recovered spermatozoa was significantly impaired compared with unprocessed semen. These observations imply that sperm DNA integrity is more sensitive to the stress induced by centrifugation than is sperm motility. Although it has not been proven, we suspect that as previously reported, sperm DNA was subjected to oxidative stress during the centrifugation process (1). In keeping with the observed differential sensitivity of sperm DNA and motility to centrifugal forces, Aitken et al. (12) recently showed that a 3-hour incubation of spermatozoa with up to 100 M of H2O2 or 10 mM of the reduced form of nicotinamide adenine dinucleotide phosphate had no significant effect on the percentage of motile spermatozoa (12). However, the same concentrations of oxidants were found to increase DNA fragmentation significantly. A more remote explanation for the observed increase in DNA denaturation after Percoll treatment is that Percoll separation may select spermatozoa with augmented DNA fragmentation. This latter possibility is unlikely if we consider the negative correlation between standard semen parameters (density and motility) and sperm DNA denaturation observed in this study. Although it was not formally assessed, it also is possible that the centrifugal process induces sperm capacitation and, as previously reported, this could result in increased sperm DNA denaturation (13). The medium used for sperm processing can influence the Vol. 72, No. 3, September 1999
functional status of the recovered spermatozoa. It has been shown that some of the media can be prooxidant. In particular, in Ham’s F-10 medium (the sperm suspension medium used in this study), the presence of vitamin C and trace metals can generate ROS (10). However, the use of antioxidants (albumin, taurine, hypotaurine, and glutathione) in sperm preparation techniques (albumin was used in this study) can protect spermatozoa from oxidative injury (14, 15). The differential sensitivity of sperm DNA and the sperm plasma membrane to oxidative stress is clinically relevant. Spermatozoa exposed to mild oxidative conditions can possess damaged DNA yet have intact or even improved capacity for sperm-oocyte fusion and sperm motility, and thus participate in normal fertilization (12, 16). Moreover, with IVF with intracytoplasmic sperm injection, it is possible for spermatozoa with impaired motility and potential DNA damage to participate in fertilization. These findings urge us to recognize the limitations of relying on sperm motility as an index of overall sperm function, particularly sperm DNA integrity. Although the implications of using DNA-damaged spermatozoa for assisted reproductive technologies are unknown, there is cause for concern (12). Sperm DNA damage may be repaired by endogenous endonucleases; however, incomplete repair may result in the transmission of genetic abnormalities to the offspring if these spermatozoa are used in assisted reproduction (17, 18). In this study, we monitored sperm DNA denaturation by flow cytometry and used this index as a measure of sperm DNA integrity. Sperm DNA denaturation previously has been shown to correlate with male fertility potential. Aravindan et al. (19) recently showed that sperm DNA denaturation correlates very highly with DNA fragmentation monitored by single sperm cell gel electrophoresis (“Comet” assay) and in situ nick translation. These data suggest that sperm DNA denaturation is a valid marker of DNA integrity. Our data demonstrate that the improvement in sperm motility after standard density-gradient centrifugation is not associated with a similar improvement in sperm DNA integrity. These data urge us to reexamine our current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles. The use of antioxidants in sperm preparation techniques may be one approach to reduce oxidative stress to spermatozoa.
FERTILITY & STERILITY威
Acknowledgment: The authors are indebted to Donald Evenson, Ph.D., for his thoughtful recommendations regarding the flow cytometry protocol.
References 1. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988;9:367–76. 2. Morales P, Vantman D, Barros C, Vigil P. Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction. Hum Reprod 1991;6:401– 4. 3. Ng FL, Liu DY, Baker HW. Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod 1992;7:261– 6. 4. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1993. 5. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl 1992;13:368 –78. 6. Evenson DP, Darzynkiewics Z, Melamed MR. Comparison of human and mouse sperm chromatin heterogeneity to fertility. Science 1980; 240:1131–3. 7. Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 1979;31:531–7. 8. Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers. Fertil Steril 1992;58:809 –16. 9. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in the semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl 1993;16:183– 8. 10. Aitken JR, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 1994;16:259 – 67. 11. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164:542–51. 12. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59:1037– 46. 13. Royere D, Hamamah S, Nicolle JC, Lansac J. Does in vitro capacitation alter chromatin stability in ejaculated spermatozoa? Cytochemical studies. Mol Reprod Dev 1991;28:177– 82. 14. Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240 –7. 15. Lopes S, Juriscova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896 –900. 16. 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;13:1864 –71. 17. Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 1989;57:717–23. 18. Maione B, Pittoggi C, Achene L, Lorenzini R, Spadafora C. Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol 1997;16:1087–97. 19. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. 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;236:231–7.
499
Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity Armand Zini, M.D., Victor Mak, M.D., Donna Phang, M.Sc., and Keith Jarvi, M.D. Division of Urology, Department of Surgery, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
Objective: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. Design: Prospective, observational study. Setting: University-based infertility clinic. Patient(s): Twenty-five nonazoospermic men. Intervention(s): Semen samples (n ⫽ 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange–treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. Main Outcome Measure(s): Sperm motility and DNA integrity. Result(s): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). Conclusion(s): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles. (Fertil Steril威 1999;72:496 –9. ©1999 by American Society for Reproductive Medicine.) Key Words: Sperm DNA, sperm motility, semen processing, male infertility
Received January 25, 1999; revised and accepted April 20, 1999. Reprint requests: Armand Zini, M.D., Mount Sinai Hospital, 600 University Avenue, Suite 1525, Toronto, Ontario, Canada M5G 1X5 (FAX: 416-5868354; E-mail: azini@mtsinai .on.ca). 0015-0282/99/$20.00 PII S0015-0282(99)00295-2
496
Sperm washing is an integral part of assisted reproductive technologies such as IVF and IUI. It is important, however, to use a sperm processing technique that is gentle yet enables the recovery of a concentrated and highly functional sperm population. Many of the conventional centrifugation techniques commonly used for cell separation are toxic to spermatozoa. It is reported that serial centrifugation of semen induces significant reactive oxygen species (ROS) release by spermatozoa and semen leukocytes, resulting in sperm dysfunction (1). In contrast, gentler sperm separation techniques such as density-gradient centrifugation and swim-up allow for good sperm recovery with minimal sperm dysfunction (1–3).
objective of this study was to examine the effect of standard semen processing by Percoll density-gradient centrifugation on human sperm DNA integrity.
To date, little is known about the effect of sperm processing on sperm DNA integrity. The
Semen samples (n ⫽ 25) were obtained from consecutively seen nonazoospermic men
MATERIALS AND METHODS Materials Acridine orange (AO) was purchased from PolySciences (Warrington, PA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO) and were at least reagent grade.
Sperm Preparation, Treatments, and Motility Analysis
presenting for infertility evaluation at our institution, under ongoing internal review board approval. Samples were produced by masturbation after 3–5 days of sexual abstinence and allowed to liquefy at room temperature. After liquefaction of semen, standard semen parameters (volume, density, motility, and morphology) were obtained according to World Health Organization guidelines (4). All the semen samples had motile sperm and none had significant leukocytospermia as per World Health Organization guidelines (4). Patient information for this study remained confidential and within the institution. At our institution, institutional review board approval is not necessary for prospective observational studies; therefore, it was not obtained. Approximately 0.5 mL of semen was layered on discontinuous two-layer (45% and 90%) and four-layer (20%, 40%, 65%, and 90%) Percoll gradients and centrifuged at 600 ⫻ g for 20 minutes in 15-mL conical tubes (5). The medium used to dilute the Percoll was modified Ham’s F-10 medium (supplemented with 1% bovine serum albumin and 25 mM of N-2hydroxyethylpiperazine-N⬘-2-ethanesulphonic acid [HEPES], pH 8.0). Spermatozoa collected from the bottom layer (90% layer) of the two-layer and four-layer Percoll gradients were resuspended in modified Ham’s F-10 medium to a sperm concentration of approximately 10 –20 ⫻ 106/mL for assessment of motility. An aliquot of 1 ⫻ 106 spermatozoa (approximately 25–100 L) from each sample (the bottom layer of the two-layer and four-layer Percoll gradients) was resuspended in 1 mL of ice-cold phosphate-buffered saline (pH 7.4) and centrifuged at 600 ⫻ g for 5 minutes. The pellet was resuspended in 1 mL of ice-cold TNE (0.01 M of tris(hydroxymethyl)aminomethane hydrochloride, 0.15 M of NaCl, and 1 mM of ethylenediaminetetraacetic acid, pH 7.4) and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and stored at ⫺70°C until later assessment of sperm DNA integrity. These samples were labeled as processed two-layer and processed four-layer. An aliquot of unprocessed semen (approximately 25–100 L, containing 1 ⫻ 106 spermatozoa) was suspended in 1 mL of ice-cold phosphate-buffered saline (pH 7.4) and centrifuged at 600 ⫻ g for 5 minutes. The pellet was resuspended in ice-cold TNE and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and immediately stored at ⫺70°C until later assessment of sperm DNA denaturation. These samples were labeled as whole.
Controls To assess the possible influence of Ham’s F-10 medium or Percoll on sperm DNA integrity (independent of the centrifugation process), we examined DNA integrity in unFERTILITY & STERILITY威
processed semen (n ⫽ 11 consecutively seen patients) incubated in Ham’s F-10 medium alone or in Ham’s F-10 medium plus 90% Percoll. An aliquot of whole semen was diluted in Ham’s F-10 medium or Ham’s F-10 medium plus 90% Percoll (1/10 vol/vol) and incubated for 30 minutes at room temperature. Samples then were centrifuged at 600 ⫻ g for 5 minutes and the pellet was resuspended in ice-cold TNE and again centrifuged at 600 ⫻ g for 5 minutes. The pellet then was resuspended in 200 L of ice-cold TNE with 10% glycerol (final sperm concentration, 5 ⫻ 106/mL) and immediately stored at ⫺70°C until later assessment of sperm DNA denaturation.
Acridine Orange Sperm Staining Sperm DNA integrity was assessed with the sperm chromosome structure assay described by Evenson et al. (6). Stored sperm samples (processed and whole) were thawed on ice, fixed in 70% ethanol for 30 minutes, and rehydrated in TNE at room temperature. The fixed samples (200-L volume at a sperm concentration of 5 ⫻ 106/mL) were treated for 30 seconds with 400 L of a solution of 0.1% Triton X-100, 0.15 M of NaCl, and 0.08 N of HCl, pH 1.2. After 30 seconds, 1.2 mL of staining buffer (6 g/mL of AO, 37 mM of citric acid, 126 mM of Na2HPO4, 1 mM of disodium ethylenediaminetetraacetic acid, and 0.15 M of NaCl, pH 6.0) was admixed to the test tube and analyzed by flow cytometry.
Flow Cytometry After excitation by a 488-nm wavelength light source, AO bound to double-stranded DNA fluoresces green (515– 530 nm) and AO bound to single-stranded DNA fluoresces red (ⱖ630 nm). Three minutes after AO staining, the samples were analyzed in a fluorescence-activated cell sorter Calibur flow cytometer (Becton Dickinson, San Jose, CA). A minimum of 5,000 cells were analyzed by a fluorescenceactivated cell sorter interfaced with a data handler (CELLQUEST 3.1; Becton Dickinson, San Jose, CA) on a Power Macintosh 7600/132 computer (Macintosh, Cupertino, CA). The proportion of cells that exhibited abnormal emission of red fluorescence (reflecting the percentage of sperm with denatured DNA) was recorded. Interassay variability (⬍5%) was verified by repeated assessments of control semen samples (results not shown). Fresh and frozenthawed samples yielded similar results (⬍5% variability) (results not shown).
Statistical Analysis
Values are expressed as means ⫾ 1 SD. A one-way analysis of variance was used to determine differences in the percentage of sperm motility and in the percentage of sperm that exhibited DNA denaturation between groups. Pearson’s correlation was performed to examine the relation between the percentage of sperm with DNA denaturation and standard semen parameters using SAS software (SAS, Cary, NC). P⬍.05 was considered statistically significant. 497
TABLE 1 Sperm motility and sperm DNA denaturation before and after Percoll density-gradient centrifugation. Sperm motility Sperm DNA denaturation (%) (%)
Semen sample Whole semen Processed (two-layer Percoll) Processed (four-layer Percoll)
44 ⫾ 21 54 ⫾ 24* 57 ⫾ 25*
18 ⫾ 13 34 ⫾ 22* 32 ⫾ 21*
Note: Values are means ⫾ SD. * Statistically significant difference (versus whole semen). Zini. Adverse effect of semen processing. Fertil Steril 1999.
RESULTS The semen samples tested had a mean sperm density of 24 ⫻ 106/mL (range, 8 – 43 ⫻ 106/mL), a mean sperm motility of 43% (range, 13%– 86%), and a mean percentage of sperm that exhibited denatured DNA of 18% (range, 6%– 60%). Sperm motility increased in 72% and 80% of the samples, respectively, after two-layer and four-layer Percoll density-gradient centrifugation. As shown in Table 1, mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen. In contrast, after both two-layer and four-layer Percoll density-gradient centrifugation, the percentage of spermatozoa with denatured DNA increased in 84% of the samples tested. As seen in Table 1, the mean percentage of sperm with denatured DNA increased significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen. A significant negative correlation was observed between the percentage of ejaculated sperm with denatured DNA and the percentage of motile sperm (r ⫽ ⫺48, P⬍.05). A weak negative correlation was observed between the percentage of ejaculated sperm with denatured DNA and sperm density (r ⫽ ⫺38, P⬍.06). There was no significant correlation between the percentage of normal forms (World Health Organization sperm morphology criteria) and the percentage of ejaculated sperm with denatured DNA. Incubation of spermatozoa in Ham’s F-10 medium or Ham’s F-10 medium plus Percoll did not have a negative influence on sperm DNA integrity. The mean percentage of spermatozoa with denatured DNA in diluted semen (Ham’s F-10 medium and Ham’s F-10 medium plus Percoll) was not significantly different than that of whole undiluted semen (19.4% ⫾ 9.5% and 15.9% ⫾ 6.4% versus 20.4% ⫾ 12.1%, P⬎.05).
DISCUSSION Seminal plasma is an important source of antioxidants (7–9); therefore, separating spermatozoa from seminal plasma during semen processing results in a prooxidant state. 498
Zini et al.
Effect of semen processing on sperm DNA
This is reflected by the finding that repetitive washing of sperm by serial centrifugation increases ROS production and impairs sperm function dramatically, particularly in samples with excessive generation of semen ROS (1). This observation emphasizes the importance of semen processing techniques on the production of ROS and potential oxidative injury to spermatozoa. As a result, most infertility centers use gentler sperm separation techniques, such as densitygradient centrifugation and swim-up, to minimize sperm toxicity (1–3). Nonetheless, even these gentler techniques result in small but detectable levels of ROS production (1). Using Percoll density-gradient centrifugation, highly motile, morphologically normal spermatozoa are collected in the higher-density layer (5). Generally, the mild stress induced by this sperm separation technique is not sufficient to result in excessive sperm membrane lipid peroxidation, and consequently, the recovered spermatozoa demonstrate improved motility relative to the unprocessed sample (1). However, in samples that generate high ROS levels, a loss of sperm motility and the capacity for sperm-oocyte fusion (which also is dependent on sperm membrane characteristics) may occur, resulting in impaired fertilization in vivo (i.e., IUI) and in vitro (i.e., standard IVF) (10, 11). In this study, we observed a significant increase in mean sperm motility after Percoll preparation, although approximately 25% of the semen samples showed a decline in sperm motility after processing. In contrast, the DNA integrity of the recovered spermatozoa was significantly impaired compared with unprocessed semen. These observations imply that sperm DNA integrity is more sensitive to the stress induced by centrifugation than is sperm motility. Although it has not been proven, we suspect that as previously reported, sperm DNA was subjected to oxidative stress during the centrifugation process (1). In keeping with the observed differential sensitivity of sperm DNA and motility to centrifugal forces, Aitken et al. (12) recently showed that a 3-hour incubation of spermatozoa with up to 100 M of H2O2 or 10 mM of the reduced form of nicotinamide adenine dinucleotide phosphate had no significant effect on the percentage of motile spermatozoa (12). However, the same concentrations of oxidants were found to increase DNA fragmentation significantly. A more remote explanation for the observed increase in DNA denaturation after Percoll treatment is that Percoll separation may select spermatozoa with augmented DNA fragmentation. This latter possibility is unlikely if we consider the negative correlation between standard semen parameters (density and motility) and sperm DNA denaturation observed in this study. Although it was not formally assessed, it also is possible that the centrifugal process induces sperm capacitation and, as previously reported, this could result in increased sperm DNA denaturation (13). The medium used for sperm processing can influence the Vol. 72, No. 3, September 1999
functional status of the recovered spermatozoa. It has been shown that some of the media can be prooxidant. In particular, in Ham’s F-10 medium (the sperm suspension medium used in this study), the presence of vitamin C and trace metals can generate ROS (10). However, the use of antioxidants (albumin, taurine, hypotaurine, and glutathione) in sperm preparation techniques (albumin was used in this study) can protect spermatozoa from oxidative injury (14, 15). The differential sensitivity of sperm DNA and the sperm plasma membrane to oxidative stress is clinically relevant. Spermatozoa exposed to mild oxidative conditions can possess damaged DNA yet have intact or even improved capacity for sperm-oocyte fusion and sperm motility, and thus participate in normal fertilization (12, 16). Moreover, with IVF with intracytoplasmic sperm injection, it is possible for spermatozoa with impaired motility and potential DNA damage to participate in fertilization. These findings urge us to recognize the limitations of relying on sperm motility as an index of overall sperm function, particularly sperm DNA integrity. Although the implications of using DNA-damaged spermatozoa for assisted reproductive technologies are unknown, there is cause for concern (12). Sperm DNA damage may be repaired by endogenous endonucleases; however, incomplete repair may result in the transmission of genetic abnormalities to the offspring if these spermatozoa are used in assisted reproduction (17, 18). In this study, we monitored sperm DNA denaturation by flow cytometry and used this index as a measure of sperm DNA integrity. Sperm DNA denaturation previously has been shown to correlate with male fertility potential. Aravindan et al. (19) recently showed that sperm DNA denaturation correlates very highly with DNA fragmentation monitored by single sperm cell gel electrophoresis (“Comet” assay) and in situ nick translation. These data suggest that sperm DNA denaturation is a valid marker of DNA integrity. Our data demonstrate that the improvement in sperm motility after standard density-gradient centrifugation is not associated with a similar improvement in sperm DNA integrity. These data urge us to reexamine our current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles. The use of antioxidants in sperm preparation techniques may be one approach to reduce oxidative stress to spermatozoa.
FERTILITY & STERILITY威
Acknowledgment: The authors are indebted to Donald Evenson, Ph.D., for his thoughtful recommendations regarding the flow cytometry protocol.
References 1. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 1988;9:367–76. 2. Morales P, Vantman D, Barros C, Vigil P. Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction. Hum Reprod 1991;6:401– 4. 3. Ng FL, Liu DY, Baker HW. Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod 1992;7:261– 6. 4. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1993. 5. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl 1992;13:368 –78. 6. Evenson DP, Darzynkiewics Z, Melamed MR. Comparison of human and mouse sperm chromatin heterogeneity to fertility. Science 1980; 240:1131–3. 7. Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 1979;31:531–7. 8. Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers. Fertil Steril 1992;58:809 –16. 9. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in the semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. Int J Androl 1993;16:183– 8. 10. Aitken JR, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 1994;16:259 – 67. 11. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164:542–51. 12. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59:1037– 46. 13. Royere D, Hamamah S, Nicolle JC, Lansac J. Does in vitro capacitation alter chromatin stability in ejaculated spermatozoa? Cytochemical studies. Mol Reprod Dev 1991;28:177– 82. 14. Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998;13:1240 –7. 15. Lopes S, Juriscova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896 –900. 16. 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;13:1864 –71. 17. Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 1989;57:717–23. 18. Maione B, Pittoggi C, Achene L, Lorenzini R, Spadafora C. Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol 1997;16:1087–97. 19. Aravindan GR, Bjordahl J, Jost LK, Evenson DP. 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;236:231–7.
499