- Email: [email protected]
Effect of long-term storage on deoxyribonucleic acid damage and motility of sperm bank donor specimens
Effect of long-term storage on deoxyribonucleic acid damage and motility of sperm bank donor specimens The percentage of sperm DNA damage in samples from sperm bank donors was not significantly different (P¼.17), whereas the percentage of motile cells was lower (P¼.009) after long-term (9–13 years) compared with short-term (1–5 years) storage. Density gradient isolation reduced the difference in sperm motility between the two groups. (Fertil Steril 2008;90:1327–30. 2008 by American Society for Reproductive Medicine.)
Cryopreservation is accompanied by damage to a variety of sperm cell organelles due to intracellular ice crystal formation, cellular dehydration and osmotic injury. Controversy still exists concerning the cryodamage to sperm DNA. One study indicated DNA quality deterioration (1). Another found that only the sperm DNA of infertile men was damaged, whereas that of fertile men was unaffected (2). However, another study was unable to show any DNA injury and attributed the sperm damage after cryopreservation mainly to membrane changes induced by thawing (3). In addition to the freezing itself, storage duration may also exert its impact. The currently accepted cryobiological view is that there is no functional loss during proper storage at 196 C in liquid nitrogen for indefinite periods (4). Nevertheless, there is a paucity of controlled studies designed to detect reproductive performance of cryopreserved sperm as a consequence of storage length (4). The process of DNA fragmentation in spermatozoa progresses even after ejaculation (5). However, the use of density gradient centrifugation enriches the unaffected sperm population by excluding those with nicked DNA and with poorly condensed chromatin (6). The objective of this study was to determine whether cryostorage duration in liquid nitrogen led to sperm deterioration, indicated by DNA fragmentation and motility percentage. The impact of density gradient selection was also tested. Freezing was processed as previously described (7). Each specimen was diluted by adding an equal volume of the freezing medium test yolk buffer (Irvine Scientific, Santa Ana, CA). After equilibration for 15–20 minutes at room temperature, the specimen was sealed in 0.5-mL straws Received May 2, 2007; revised and accepted July 16, 2007. This study was carried out under the auspices of the Alan and Ada Selwyn Chair in Clinical Infertility Research and Molecular Medicine (Melbourne, Australia) (grant recipient, G.P.). The research constitutes part of the M.Sc. thesis of A. Edelstein at the Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel. Reprint requests: Leah Yogev, Ph.D., The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: 972-3-6925696; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1343
(I.M.V., Paris, France) and cooled in a semi-programmable freezer (Nicool LM-10; Air Liquid, Paris, France). The straws were cooled gradually and then transferred to liquid nitrogen (196 C) for storage. Percentage of postthaw motility before commencement of storage was evaluated by thawing one straw from each frozen specimen after 30 minutes in liquid nitrogen. Thawing was conducted at 34 C for 5 minutes. Sperm criteria for recruitment of sperm bank donors, freezing procedure, equipment, and medium composition remained unchanged throughout the entire study period. Assessment of the possible effect of long-term cryostorage was performed on sperm of 30 sperm bank donors. The participants comprised young (22–28-year-old) unmarried Caucasian students, almost all of whom were born in Israel. The study was approved by the local institutional review board committee in accordance with the Helsinki Declaration of 1975. The only inclusion criterion for the two groups was storage length. Available donor samples were thawed after 9–13 years of storage (long-term group, n ¼ 16) and after 1–5 years (short-term group, n ¼ 14). The definition for longand short-term storage was decided arbitrarily, by splitting the accessible specimens into two groups with a comparable number of participants. The samples from the long-term group had been cryostored for women who already had a child and had required preservation from the same donor for future use. The women from this group later requested termination of the cryostorage. The short-term group comprised donors with enough available samples for routine inseminations. Both groups constitute a small section of the preserved donor population. No difference was found in motility percentage before freezing (55% 1.1% and 57% 0.8% [mean SE]; median 55% and 57%, respectively; P¼.08) and after freezing–thawing and before storage (41% 2.2% and 35% 2.0%; median 41% and 35%, respectively; P¼.07), between the long- and short-term groups. Sperm motility was observed using the Makler chamber (Sefi Medical Instruments, Haifa, Israel). The percentage of DNA-damaged spermatozoa was determined by the
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1327
terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The In Situ Cell Death Detection Kit (Roche Diagnostics, GmbH, Penzberg, Germany) was used for the TUNEL assay. This kit uses fluorescein-deoxyuridine triphosphate (red color) to label sites of DNA fragmentation according to the manufacturer’s instructions. Final suspensions of sperm were fixed with 4% paraformaldehyde and were permeabilized with 1% Triton X-100 in DDH2O. This was followed by incubation in the dark at 37 C for 1 hour in TUNEL reaction mixture containing enzyme solution (calf thymus terminal deoxynucleotidyl transferase) and label solution (nucleotides mixture). Negative (omitting the enzyme terminal transferase) and positive (using deoxyribonuclease I, 1 mg/mL for 10 minutes at room temperature) controls were performed in each experiment. A total of 200 cells per slide were randomly analyzed under an Olympus AX-70 microscope (Tokyo, Japan). Normal-shaped spermatozoa were identified initially, using phase contrast, and then analyzed by red fluorescent staining (detection range of 570–620 nm).
FIGURE 1 Box and whisker plot showing percentage of damaged DNA (upper panel) and motile spermatozoa (lower panel) thawed after long-term (9–13 years) and short-term (1–5 years) storage in liquid nitrogen. Specimens (washed and gradient isolated sperm) were observed immediately after thawing (0 time) and after 24 hours of incubation. The box encloses the middle half of the data (supplying first and third quartile) and is bisected by median value. The vertical lines indicate the range of ‘‘typical’’ data values. Density gradient ¼ after isolation on two-layer density gradient. aSignificant difference between long- and short-term storage of ‘‘washed’’ and ‘‘density gradient’’ samples at 0 time (P¼ .009 and P¼ .004, respectively) and after 24 hours of ‘‘washed’’ sample incubation (P¼ .023).
To compare frozen–thawed sperm DNA fragmentation after different sperm preparation methods (i.e., washed and treated with density gradient isolation), each thawed sperm sample was divided into two aliquots. One aliquot was washed twice with ‘‘medium’’ (human tubal fluid [Irvine Scientific] þ 1% human serum albumin [Kamapharm, Kibbutz Beit Kama, Israel]), and the pellet was suspended in 0.4 mL of medium. Washing out the freezing medium was essential for the TUNEL staining technique. The second aliquot was layered onto Isolate (Irvine Scientific) two-layer discontinuous gradient and centrifuged for 20 minutes at 400 g. The sperm pellet was resuspended in 1 mL of medium and washed twice. The effect of in vitro postthawed sperm specimen incubation on DNA fragmentation progress was monitored in all specimens after incubation of 24 hours at room temperature (8). Results are presented as mean SE. The statistical significance level was set to .05, and commercial software (SPSS 13.0 for Windows; SPSS, Chicago, IL) was used for the analysis. Comparisons between the two groups (short- and long-term cryostorage) were performed using the Mann-Whitney nonparametric test. Long-term cryopreservation of donor samples did not affect DNA integrity compared with the short-term group (37% 3.4% and 31% 3.2%, respectively; P¼.17; observed power: 0.248) (Fig. 1). After in vitro incubation a higher but nonsignificant percentage of sperm cells with damaged DNA was observed in the long-term group (54% 4.1%) compared with the short period of cryopreservation (45% 3.1%; P¼.08; observed power: 0.39). The minor difference between the two groups in terms of the percentage of sperm cells with damaged DNA was also not significant after isolation of a high-quality spermatozoa population from the samples (31% 2.9% and 26% 3.9% for long- and short-term storage, respectively). Conversely, 1328
Edelstein et al.
Correspondence
Edelstein. Long-Storage effect on Sperm Cells. Fertil Steril 2008.
motility percentage was significantly lower in the long-term storage group than in the short-term storage group immediately after washing (28% 2.0% and 36% 2.2%, respectively; P¼.009), gradient isolation (49% 1.9% and 58% Vol. 90, No. 4, October 2008
2.2%, respectively; P¼.004), and after incubation of washed specimens (15% 2.1% and 22% 1.8%, respectively; P¼.023). This difference proved to be not significant after incubation of the gradient isolated fraction (37% 2.3% and 42% 3.0%, respectively) (Fig. 1). In the present study sperm DNA was not damaged after a very long-term storage period, compared with shortterm storage. The stability of sperm DNA during extended storage may encourage a long period of cryopreservation for fertility rescue when needed. Nonetheless, the above findings were demonstrated in a relatively small group. Pregnancy rates after IUI, IVF, and intracytoplasmic sperm injection were inversely related to sperm DNA damage (9, 10). Nonetheless, there is still some controversy concerning the importance of DNA integrity for pregnancy rate (11, 12). However, it may be concluded that as long as enough total progressive–motile sperm cells survive, the use of IUI is adequate. In cases of decreased motility, pooling of samples is recommended to achieve the amount of progressive–motile cells that is needed for IUI (7). It has been shown that cryopreservation causes deterioration of sperm chromatin quality (1, 13). The high rate of damaged sperm DNA may contribute to the difference between the number of IUI cycles needed for pregnancy achievement when using frozen–thawed instead of fresh donor sperm (3, 14, 15). However, a different observation was reported concerning the potential damage of the freezing– thawing process: whereas freezing and thawing of normal specimens triggered the apoptotic machinery, no significant changes were observed in DNA fragmentation (16). These diverse findings may be a result of the disparate methods used, as well as different inclusion criteria for patients recruited to the study groups. Cryopreservation of human semen induces damage at different cellular levels. Moreover, it was recently found that some cryoinjuries occurred shortly after the freezing process, whereas others may occur later (17). In this recent research, samples cryostored for more than 15 weeks showed a significant increase in a-tubulin immunodetection compared with samples thawed after a shorter period (17). The TUNEL assay labels DNA strand breaks from any insult, in both apoptotic and nonapoptotic cells. It has been reported that sperm DNA fragmentation activity continued after ejaculation during the 24-hour in vitro incubation of the fresh samples (6). The involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation was suggested. Reactive oxygen species may explain the deterioration of the frozen–thawed semen during incubation as well, given that freezing and thawing procedures are known to release oxygen species (18). Concerning fertilization, to date only one report has indicated a small decrease in bull conception after a storage Fertility and Sterility
period of >5 years (19), whereas no impact was shown by others (5, 20). The relatively minor effects on fertility of long-term semen storage in liquid nitrogen may escape its detection. Density gradient centrifugation techniques may enrich the normal sperm population by separating those with nicked DNA from poorly condensed chromatin cells. Having enough progressive motile sperm cells in the cryopreserved donor specimens may ensure successful IUI treatments.To the best of our knowledge, the present study demonstrates for the first time that human sperm DNA integrity can be successfully preserved during very long-term cryostorage in liquid nitrogen, whereas in sperm motility of the same samples a limited deterioration was observed. The capability of the density gradient isolation technique to amend this damage is emphasized.
Acknowledgments: The authors thank Mrs. Bella Gore of The Institute for the Study of Fertility for her skillful and excellent technical assistance; Mr. Doron Comaheshter of The Statistics Service, Tel Aviv Medical Center, for statistical assistance; and Mrs. Sigalit Siso of The Graphic Unit, Tel Aviv Medical Center, for the skilled graphic illustration.
Avital Edelstein, M.Sc. Haim Yavetz, M.D. Sandra E. Kleiman, Ph.D. Ron Hauser, M.D. Amnon Botchan, M.D. Gedalia Paz, Ph.D. Leah Yogev, Ph.D. The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel REFERENCES 1. Gandini L, Lombardo F, Lenzi A, Spano M, Dondero F. Cryopreservation and sperm DNA integrity. Cell Tissue Bank 2006;7:91–8. 2. Donnelly ET, Steele EK, McClure N, Lewis EM. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001;16:1191–9. 3. Duru NK, Morshedi MS, Schuffner A, Oehninger S. Cryopreservationthawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation. J Androl 2001;22:646–51. 4. Clarke GN, Liu DY, Baker HWG. Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen. Fertil Steril 2006;86: 721–2. 5. Murtori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 2003;24:253–62. 6. Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, et al. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod 2000;15:1112–6. 7. Yogev L, Kleiman S, Shabtai E, Botchan A, Gamzu R, Paz G, et al. Seasonal variations in pre- and post-thaw donor sperm quality. Hum Reprod 2004;19:880–5.
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8. Petrella C, Hsieh J, Thrift K, Jarow JP, Zacur H, Zhao Y. Optimizing incubation conditions for the preservation of sperm motility in processed semen samples. Fertil Steril 2005;84:513–5. 9. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001;10: 2160–5. 10. O’brien J, Zini A. Sperm DNA integrity and male infertility. Urology 2005;65:16–22. 11. Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 2004;19:1409–17. 12. Payne JF, Raburn DJ, Couchman GM, Price TM, Jamison MG, Walmer DK. Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques. Fertil Steril 2005;84:356–64. 13. De Paula TS, Bertolla RP, Spaine DM, Cunha MA, Schor N, Cedenho AP. Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia. Fertil Steril 2006;86:597–600.
1330
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Correspondence
14. Botchan A, Hauser R, Gamzu R, Yogev L, Paz G, Yavetz H. Results of 6139 artificial insemination cycles with donor spermatozoa. Hum Reprod 2001;16:2298–304. 15. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006;21:3876–81. 16. Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJ, et al. Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod 2004;71:1828–37. 17. Desrosier P, Legare C, Leclerc P, Sullivan R. Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertil Steril 2006;85:1744–52. 18. Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev 2001;59:451–8. 19. Haugan T, Gro¨hn YT, Kommisurd E, Ropstad E, Reksen O. Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception. Anim Reprod Sci 2007;97:1–11. 20. Rofeim O, Gilbert BR. Long-term effect of cryopreservation on human spermatozoa. Fertil Steril 2005;84:536–7.
Vol. 90, No. 4, October 2008
Cryopreservation is accompanied by damage to a variety of sperm cell organelles due to intracellular ice crystal formation, cellular dehydration and osmotic injury. Controversy still exists concerning the cryodamage to sperm DNA. One study indicated DNA quality deterioration (1). Another found that only the sperm DNA of infertile men was damaged, whereas that of fertile men was unaffected (2). However, another study was unable to show any DNA injury and attributed the sperm damage after cryopreservation mainly to membrane changes induced by thawing (3). In addition to the freezing itself, storage duration may also exert its impact. The currently accepted cryobiological view is that there is no functional loss during proper storage at 196 C in liquid nitrogen for indefinite periods (4). Nevertheless, there is a paucity of controlled studies designed to detect reproductive performance of cryopreserved sperm as a consequence of storage length (4). The process of DNA fragmentation in spermatozoa progresses even after ejaculation (5). However, the use of density gradient centrifugation enriches the unaffected sperm population by excluding those with nicked DNA and with poorly condensed chromatin (6). The objective of this study was to determine whether cryostorage duration in liquid nitrogen led to sperm deterioration, indicated by DNA fragmentation and motility percentage. The impact of density gradient selection was also tested. Freezing was processed as previously described (7). Each specimen was diluted by adding an equal volume of the freezing medium test yolk buffer (Irvine Scientific, Santa Ana, CA). After equilibration for 15–20 minutes at room temperature, the specimen was sealed in 0.5-mL straws Received May 2, 2007; revised and accepted July 16, 2007. This study was carried out under the auspices of the Alan and Ada Selwyn Chair in Clinical Infertility Research and Molecular Medicine (Melbourne, Australia) (grant recipient, G.P.). The research constitutes part of the M.Sc. thesis of A. Edelstein at the Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel. Reprint requests: Leah Yogev, Ph.D., The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: 972-3-6925696; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1343
(I.M.V., Paris, France) and cooled in a semi-programmable freezer (Nicool LM-10; Air Liquid, Paris, France). The straws were cooled gradually and then transferred to liquid nitrogen (196 C) for storage. Percentage of postthaw motility before commencement of storage was evaluated by thawing one straw from each frozen specimen after 30 minutes in liquid nitrogen. Thawing was conducted at 34 C for 5 minutes. Sperm criteria for recruitment of sperm bank donors, freezing procedure, equipment, and medium composition remained unchanged throughout the entire study period. Assessment of the possible effect of long-term cryostorage was performed on sperm of 30 sperm bank donors. The participants comprised young (22–28-year-old) unmarried Caucasian students, almost all of whom were born in Israel. The study was approved by the local institutional review board committee in accordance with the Helsinki Declaration of 1975. The only inclusion criterion for the two groups was storage length. Available donor samples were thawed after 9–13 years of storage (long-term group, n ¼ 16) and after 1–5 years (short-term group, n ¼ 14). The definition for longand short-term storage was decided arbitrarily, by splitting the accessible specimens into two groups with a comparable number of participants. The samples from the long-term group had been cryostored for women who already had a child and had required preservation from the same donor for future use. The women from this group later requested termination of the cryostorage. The short-term group comprised donors with enough available samples for routine inseminations. Both groups constitute a small section of the preserved donor population. No difference was found in motility percentage before freezing (55% 1.1% and 57% 0.8% [mean SE]; median 55% and 57%, respectively; P¼.08) and after freezing–thawing and before storage (41% 2.2% and 35% 2.0%; median 41% and 35%, respectively; P¼.07), between the long- and short-term groups. Sperm motility was observed using the Makler chamber (Sefi Medical Instruments, Haifa, Israel). The percentage of DNA-damaged spermatozoa was determined by the
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1327
terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. The In Situ Cell Death Detection Kit (Roche Diagnostics, GmbH, Penzberg, Germany) was used for the TUNEL assay. This kit uses fluorescein-deoxyuridine triphosphate (red color) to label sites of DNA fragmentation according to the manufacturer’s instructions. Final suspensions of sperm were fixed with 4% paraformaldehyde and were permeabilized with 1% Triton X-100 in DDH2O. This was followed by incubation in the dark at 37 C for 1 hour in TUNEL reaction mixture containing enzyme solution (calf thymus terminal deoxynucleotidyl transferase) and label solution (nucleotides mixture). Negative (omitting the enzyme terminal transferase) and positive (using deoxyribonuclease I, 1 mg/mL for 10 minutes at room temperature) controls were performed in each experiment. A total of 200 cells per slide were randomly analyzed under an Olympus AX-70 microscope (Tokyo, Japan). Normal-shaped spermatozoa were identified initially, using phase contrast, and then analyzed by red fluorescent staining (detection range of 570–620 nm).
FIGURE 1 Box and whisker plot showing percentage of damaged DNA (upper panel) and motile spermatozoa (lower panel) thawed after long-term (9–13 years) and short-term (1–5 years) storage in liquid nitrogen. Specimens (washed and gradient isolated sperm) were observed immediately after thawing (0 time) and after 24 hours of incubation. The box encloses the middle half of the data (supplying first and third quartile) and is bisected by median value. The vertical lines indicate the range of ‘‘typical’’ data values. Density gradient ¼ after isolation on two-layer density gradient. aSignificant difference between long- and short-term storage of ‘‘washed’’ and ‘‘density gradient’’ samples at 0 time (P¼ .009 and P¼ .004, respectively) and after 24 hours of ‘‘washed’’ sample incubation (P¼ .023).
To compare frozen–thawed sperm DNA fragmentation after different sperm preparation methods (i.e., washed and treated with density gradient isolation), each thawed sperm sample was divided into two aliquots. One aliquot was washed twice with ‘‘medium’’ (human tubal fluid [Irvine Scientific] þ 1% human serum albumin [Kamapharm, Kibbutz Beit Kama, Israel]), and the pellet was suspended in 0.4 mL of medium. Washing out the freezing medium was essential for the TUNEL staining technique. The second aliquot was layered onto Isolate (Irvine Scientific) two-layer discontinuous gradient and centrifuged for 20 minutes at 400 g. The sperm pellet was resuspended in 1 mL of medium and washed twice. The effect of in vitro postthawed sperm specimen incubation on DNA fragmentation progress was monitored in all specimens after incubation of 24 hours at room temperature (8). Results are presented as mean SE. The statistical significance level was set to .05, and commercial software (SPSS 13.0 for Windows; SPSS, Chicago, IL) was used for the analysis. Comparisons between the two groups (short- and long-term cryostorage) were performed using the Mann-Whitney nonparametric test. Long-term cryopreservation of donor samples did not affect DNA integrity compared with the short-term group (37% 3.4% and 31% 3.2%, respectively; P¼.17; observed power: 0.248) (Fig. 1). After in vitro incubation a higher but nonsignificant percentage of sperm cells with damaged DNA was observed in the long-term group (54% 4.1%) compared with the short period of cryopreservation (45% 3.1%; P¼.08; observed power: 0.39). The minor difference between the two groups in terms of the percentage of sperm cells with damaged DNA was also not significant after isolation of a high-quality spermatozoa population from the samples (31% 2.9% and 26% 3.9% for long- and short-term storage, respectively). Conversely, 1328
Edelstein et al.
Correspondence
Edelstein. Long-Storage effect on Sperm Cells. Fertil Steril 2008.
motility percentage was significantly lower in the long-term storage group than in the short-term storage group immediately after washing (28% 2.0% and 36% 2.2%, respectively; P¼.009), gradient isolation (49% 1.9% and 58% Vol. 90, No. 4, October 2008
2.2%, respectively; P¼.004), and after incubation of washed specimens (15% 2.1% and 22% 1.8%, respectively; P¼.023). This difference proved to be not significant after incubation of the gradient isolated fraction (37% 2.3% and 42% 3.0%, respectively) (Fig. 1). In the present study sperm DNA was not damaged after a very long-term storage period, compared with shortterm storage. The stability of sperm DNA during extended storage may encourage a long period of cryopreservation for fertility rescue when needed. Nonetheless, the above findings were demonstrated in a relatively small group. Pregnancy rates after IUI, IVF, and intracytoplasmic sperm injection were inversely related to sperm DNA damage (9, 10). Nonetheless, there is still some controversy concerning the importance of DNA integrity for pregnancy rate (11, 12). However, it may be concluded that as long as enough total progressive–motile sperm cells survive, the use of IUI is adequate. In cases of decreased motility, pooling of samples is recommended to achieve the amount of progressive–motile cells that is needed for IUI (7). It has been shown that cryopreservation causes deterioration of sperm chromatin quality (1, 13). The high rate of damaged sperm DNA may contribute to the difference between the number of IUI cycles needed for pregnancy achievement when using frozen–thawed instead of fresh donor sperm (3, 14, 15). However, a different observation was reported concerning the potential damage of the freezing– thawing process: whereas freezing and thawing of normal specimens triggered the apoptotic machinery, no significant changes were observed in DNA fragmentation (16). These diverse findings may be a result of the disparate methods used, as well as different inclusion criteria for patients recruited to the study groups. Cryopreservation of human semen induces damage at different cellular levels. Moreover, it was recently found that some cryoinjuries occurred shortly after the freezing process, whereas others may occur later (17). In this recent research, samples cryostored for more than 15 weeks showed a significant increase in a-tubulin immunodetection compared with samples thawed after a shorter period (17). The TUNEL assay labels DNA strand breaks from any insult, in both apoptotic and nonapoptotic cells. It has been reported that sperm DNA fragmentation activity continued after ejaculation during the 24-hour in vitro incubation of the fresh samples (6). The involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation was suggested. Reactive oxygen species may explain the deterioration of the frozen–thawed semen during incubation as well, given that freezing and thawing procedures are known to release oxygen species (18). Concerning fertilization, to date only one report has indicated a small decrease in bull conception after a storage Fertility and Sterility
period of >5 years (19), whereas no impact was shown by others (5, 20). The relatively minor effects on fertility of long-term semen storage in liquid nitrogen may escape its detection. Density gradient centrifugation techniques may enrich the normal sperm population by separating those with nicked DNA from poorly condensed chromatin cells. Having enough progressive motile sperm cells in the cryopreserved donor specimens may ensure successful IUI treatments.To the best of our knowledge, the present study demonstrates for the first time that human sperm DNA integrity can be successfully preserved during very long-term cryostorage in liquid nitrogen, whereas in sperm motility of the same samples a limited deterioration was observed. The capability of the density gradient isolation technique to amend this damage is emphasized.
Acknowledgments: The authors thank Mrs. Bella Gore of The Institute for the Study of Fertility for her skillful and excellent technical assistance; Mr. Doron Comaheshter of The Statistics Service, Tel Aviv Medical Center, for statistical assistance; and Mrs. Sigalit Siso of The Graphic Unit, Tel Aviv Medical Center, for the skilled graphic illustration.
Avital Edelstein, M.Sc. Haim Yavetz, M.D. Sandra E. Kleiman, Ph.D. Ron Hauser, M.D. Amnon Botchan, M.D. Gedalia Paz, Ph.D. Leah Yogev, Ph.D. The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel REFERENCES 1. Gandini L, Lombardo F, Lenzi A, Spano M, Dondero F. Cryopreservation and sperm DNA integrity. Cell Tissue Bank 2006;7:91–8. 2. Donnelly ET, Steele EK, McClure N, Lewis EM. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001;16:1191–9. 3. Duru NK, Morshedi MS, Schuffner A, Oehninger S. Cryopreservationthawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation. J Androl 2001;22:646–51. 4. Clarke GN, Liu DY, Baker HWG. Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen. Fertil Steril 2006;86: 721–2. 5. Murtori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl 2003;24:253–62. 6. Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, et al. The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod 2000;15:1112–6. 7. Yogev L, Kleiman S, Shabtai E, Botchan A, Gamzu R, Paz G, et al. Seasonal variations in pre- and post-thaw donor sperm quality. Hum Reprod 2004;19:880–5.
1329
8. Petrella C, Hsieh J, Thrift K, Jarow JP, Zacur H, Zhao Y. Optimizing incubation conditions for the preservation of sperm motility in processed semen samples. Fertil Steril 2005;84:513–5. 9. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001;10: 2160–5. 10. O’brien J, Zini A. Sperm DNA integrity and male infertility. Urology 2005;65:16–22. 11. Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 2004;19:1409–17. 12. Payne JF, Raburn DJ, Couchman GM, Price TM, Jamison MG, Walmer DK. Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques. Fertil Steril 2005;84:356–64. 13. De Paula TS, Bertolla RP, Spaine DM, Cunha MA, Schor N, Cedenho AP. Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia. Fertil Steril 2006;86:597–600.
1330
Edelstein et al.
Correspondence
14. Botchan A, Hauser R, Gamzu R, Yogev L, Paz G, Yavetz H. Results of 6139 artificial insemination cycles with donor spermatozoa. Hum Reprod 2001;16:2298–304. 15. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006;21:3876–81. 16. Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJ, et al. Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod 2004;71:1828–37. 17. Desrosier P, Legare C, Leclerc P, Sullivan R. Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertil Steril 2006;85:1744–52. 18. Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev 2001;59:451–8. 19. Haugan T, Gro¨hn YT, Kommisurd E, Ropstad E, Reksen O. Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception. Anim Reprod Sci 2007;97:1–11. 20. Rofeim O, Gilbert BR. Long-term effect of cryopreservation on human spermatozoa. Fertil Steril 2005;84:536–7.
Vol. 90, No. 4, October 2008