- Email: [email protected]
Sperm deoxyribonucleic acid fragmentation dynamics in fertile donors
MALE FACTOR Sperm deoxyribonucleic acid fragmentation dynamics in fertile donors Jaime Gos alvez, M.D.,a Elva Cort es-Gutierez, Ph.D.,b Carmen L opez-Fern andez, Ph.D.,a c d Jos e Luıs Fern andez, M.D., Pedro Caballero, M.D., and Rocio Nu~ nez, M.D.d a
Department of Biology, Genetics Unit, Universidad Autonoma de Madrid (UAM), Madrid, Spain; b Department of Genetics, Northwest Research Center of Biology, Mexican National Institute for Health, Monterrey, Mexico; c Seccion de Genetica y Unidad de Investigacion, Complejo Hospitalario Universitario Juan Canalejo, A Coru~na, Spain; and d Clinica Tambre, Madrid, Spain
Objective: To study the velocity of sperm DNA fragmentation in frozen-thawed sperm samples from male sperm donors of proved fertility. Design: Sperm DNA fragmentation assessment with use of sperm chromatin dispersion methodology after 0, 4, 8, and 24 hours of incubation in IVF medium. Setting: Academic biology and reproductive medicine centers. Patient(s): Twenty male fertility donors with proved fertility for a maximum of six births at the reproductive medicine center. Intervention(s): None. Main Outcome Measure(s): The velocity of sperm DNA fragmentation between two consecutive incubations was scored. Best adjustment of sperm DNA fragmentation index versus incubation time for linear, logarithmic, or exponential function was tested. Result(s): Increase of sperm DNA fragmentation through time accounted for a substantial percentage of the overall variation. The highest velocity of sperm DNA fragmentation was observed in the first 4 hours of incubation, decreasing by 50% during the second incubation period and being of the order of 1% in the final experimental period. The tendency to increase in sperm DNA fragmentation is not homogeneous among donors; they may adjust to a logarithmic, linear, or exponential function rendering high values for R2. Conclusion(s): Sperm DNA fragmentation occurs rapidly after thawing, and it is an important cause of the rapid decline of sperm quality. Thus, the use of sperm samples as quickly as possible after thawing is highly recommended in clinical practice. Different sperm DNA fragmentation dynamics among individuals were observed. (Fertil Steril 2009;92:170–3. 2009 by American Society for Reproductive Medicine.)
Sperm DNA fragmentation has been the subject of numerous studies because effects on fertility parameters appear to be correlated with a high frequency of sperm nuclei containing damaged DNA (1, 2). Sperm DNA fragmentation has always been referred to as a constant parameter of sperm quality, without any specification of the period of time that had passed between sperm quality assessment and use for ART. However, this could be untrue because the sperm DNA fragmentation index may change rapidly when samples for artificial insemination are handled. In fact, in mammalian species it has been reported that temperature excursion episodes dramatically increase the rate of sperm DNA degradation, and Received February 26, 2008; revised May 16, 2008; accepted May 19, 2008; published online August 11, 2008. J.G. has nothing to disclose. E.C.-G. has nothing to disclose. C.L.-F. has nothing to disclose. J.L.F. has nothing to disclose. P.C. has nothing to disclose. R.N. has nothing to disclose. n y Ciencia, Supported by BFU 2007-66340/BFI (Ministerio de Educacio Spain). lvez, M.D., Department of Biology, Genetics Reprint requests: Jaime Gosa noma de Madrid (UAM), 20849-Madrid, Spain Unit, Universidad Auto (FAX: þ34-91-497-83-44; E-mail: [email protected]).
170
after some hours of sperm incubation at 37 C, the basal values for the sperm DNA fragmentation index may be doubled (3, 4). Thus, the level of sperm DNA fragmentation as analyzed when the semen is freshly obtained will increase over time, with variation among species and among different individuals within the same species (5). Reference to the time that has passed between semen collection and analysis seems to be imperative; otherwise, reported analysis results could be meaningless at the time of ART if this temporal factor is not considered seriously. In addition, comparison of results among different laboratories also could become highly biased. The aim of this investigation was to analyze the evolution of sperm DNA fragmentation values over time in donors with proved fertility in an attempt to stress the importance of considering time after ejaculation and/or after thawing of frozen samples as a factor influencing sperm DNA quality. MATERIALS AND METHODS Twenty donors from the Clinica Tambre (Madrid, Spain) were included in the analysis. Their semen samples had
Fertility and Sterility Vol. 92, No. 1, July 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.05.068
been used in different ART programs, achieving a maximum of six pregnancies with normal embryo development and birth. This is the maximum permitted by the Spanish law for one donor in an ART program. The cryopreservation of frozen sperm pellets was performed according to previously published methodology (6, 7). All samples were thawed by immersion in a 37 C water bath for 30 seconds and diluted to 10 to 15 106 spermatozoa/mL in IVF medium (MediCult, Jyllinge, Denmark). Two aliquots of the corresponding sperm samples were incubated in a humidified atmosphere of 5% CO2 in air at 37 C. The sperm DNA fragmentation index, the ratio of fragmented versus total spermatozoa in the analyzed sample, expressed as a percentage, was assessed after 0, 4, 8, and 24 hours of incubation (t0, t4, t8, and t24). This time period was selected to show the differences in sperm DNA damage within a reasonable time in which the sperm could be viable for ART. Replicates, processed and assessed by two different observers, were used as internal controls. The samples were assessed for sperm DNA fragmentation just after they were thawed (t0), and the corresponding sperm DNA fragmentation index was considered as basal for this particular donor. The sperm DNA fragmentation frequency was determined with use of the Halosperm kit (Halotech DNA SL, Madrid, Spain; Conception Technologies, San Diego, CA). Deoxyribonucleic acid haloes resulting after the sperm chromatin dispersion test were visualized by fluorescence microscopy with propidium iodide staining (2.5 mg/ mL in Vectashield; Vector Laboratories, Burlingame, CA). Descriptive statistics with use of box-and-whisker diagrams and regression analysis, including a comparison of individual regression equations, were conducted with use of Stat-Graphics Plus 5.1 (Academic Enterprise, StatPoint Inc., Herndon, VA). In addition, to test the significance of the increase in sperm DNA fragmentation index over the incubation at 37 C, the slopes and Y-axis intercepts of the regression equations for each individual were also compared. The velocity of sperm DNA fragmentation (VsDF) between two periods of incubation (t1 and t2) was determined according to the following equation: VsDF ¼ (Sperm DNA fragmentation index at t2) (Sperm DNA fragmentation index at t1)/Incubation time within the selected period, expressed in hours. These values correspond to the average of the slopes obtained by regression analysis.
RESULTS AND DISCUSSION The basal distribution of sperm DNA fragmentation index after thawing ranged from 8% to 28%. Deoxyribonucleic acid fragmentation started to increase after the samples had been diluted and incubated at 37 C (Fig. 1a). Regression analysis was conducted to investigate the relationship between sperm DNA fragmentation index and incubation time, considering time points t0 and t24. Variation in time accounted for a substantial percentage of the overall variation in sperm DNA fragmentation index (R2 ¼ 76.3%, P<.05; Fig. 1b). Y-axis intercepts showed significant differences between Fertility and Sterility
FIGURE 1 (a) Box-and-whisker plots for the sperm DNA fragmentation index observed at different incubation times (37 C) for spermatozoa and the rate for sperm DNA fragmentation observed at each interval. (b) Average sperm DNA fragmentation observed at different incubation times (blue line) and adjustments for logarithmic (red line), linear (gray line), or exponential (green line) functions. The corresponding equations are shown with the same color pattern. The pink line (donor 7) shows the maximum R2 value for a logarithmic function, and the light-green line (donor 15) shows the maximum R2 value for an exponential function. VsDF ¼ velocity of sperm DNA fragmentation.
Gos alvez. Dynamics of sperm DNA fragmentation. Fertil Steril 2009.
individuals (P¼.000), indicating variation in the basal level of sperm DNA fragmentation index. To test the homogeneity of the rate of sperm DNA fragmentation between different periods of incubation, the velocity of sperm DNA fragmentation between two consecutive incubations was compared. Results showed that there are differences among different consecutive incubation times (P<.05; Fig. 1a). The highest rate of sperm DNA fragmentation increase was observed in the first 4 hours of incubation (velocity of sperm DNA fragmentation ¼ 8.3% per hour). This rate decreased by 50% during the second incubation period (velocity of sperm DNA fragmentation ¼ 4.1% per hour) and was of the order of 1% in the final experimental period (Fig. 1a). After performing regression analysis using a linear adjustment for each sperm sample, we found different intercepts for 171
different slopes (data not shown). This indicates that some individuals may present a more intense rate of sperm DNA damage than others may, and, conversely, some sperm samples are more resistant to DNA fragmentation than others are. This also explains the large deviation in the lowest and highest values for sperm DNA fragmentation index values and the differences in the lower and upper quartiles observed, especially after 4 and 8 hours of incubation (see box-andwhisker diagrams in Fig. 1a). This deviation was lower at t0 because, at this time, sperm DNA fragmentation index is related directly to basal interindividual variations in sperm DNA fragmentation and does not depend on the incubation time, which explains a large part of the variation observed in this experiment. The global increasing tendency is for sperm DNA fragmentation values (blue line in Fig. 1b) to be fitted to a logarithmic function (red line in Fig. 1b; R2 ¼ 0.992; y ¼ 47.03 ln[x] þ 17.3). The values of R2 for linear (gray line in Fig. 1b) or exponential functions (dark green in Fig. 1b) were lower (Fig. 1b). When each sample was analyzed separately, R2 values >0.9 were achieved in 60% of the samples when a logarithmic function was used, 30% in the case of a linear function, and 20% for an exponential function. In Figure 1b, one sperm sample (D7) with the maximum R2 value for a logarithmic function (pink line in Fig. 1b; y ¼ 49.363 ln[x] þ 15.03; R2 ¼ 0.9852) and another (D15) with the maximum R2 value (y ¼ 7.995e0.6183x; R2 ¼ 0.9791) with use of an exponential function (light green line in Fig. 1b) are shown. For an exponential trend, sperm longevity would be higher than with a logarithmic tendency, and this could be critical for DNA stability during the first hours of sperm incubation. The results obtained in this experiment indicate that DNA fragmentation is an important effector of the rapid decline of sperm quality, because sperm DNA tends to degrade very quickly after thawing. In practice, the experimental conditions used in this experiment emulate those used for ART, and sperm DNA degradation could be detected at the onset of temperature recovery to 37 C. This degradation therefore could exert a negative influence on fertility. In fact, given the high rate for sperm DNA fragmentation observed during the first 4 hours of incubation, it seems highly probable that any sperm sample would contain 50% of sperm cells with damaged DNA. Loss of sperm quality in samples, with use of other sperm parameters, has been reported in most animals analyzed, especially when sperm samples are exposed to increased temperatures. For example, the fertility of bull sperm is well preserved for 3 to 5 days when semen is stored at ambient temperatures. After this time, fertility declines at a rate of 3% to 6% per day, irrespective of whether the sperm are stored at 5 C or at 15 C. However, this effect is greater when storage temperatures exceed 25 C (8). Therefore, it seems clear that the temperature, or, more precisely, episodes of temperature change, may produce deleterious effects on sperm viability. In humans, it has been demonstrated that col172
Gosalvez et al.
Dynamics of sperm DNA fragmentation
lection of ejaculated semen at distant sites away from the laboratory where the artificial insemination will be performed, with overnight mail delivery, might lead to sperm DNA damage (9). The effects of the relative short longevity of sperm DNA quality detected in certain clinical situations could also be related to and even amplified by the effects described here. It has been reported that DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4- and 24-hour incubations (10). In fact, under these circumstances, the immediate use of testicular sperm in intracytoplasmic sperm injection is highly recommended. Special care must be paid to the fact that sperm characteristics considered as sufficient for fertilization at the onset of sperm quality assessment may change rapidly between insemination and fertilization. The results of this investigation have shown that different sperm samples exhibit different sperm DNA fragmentation dynamics. This tendency has been also reported in other mammalian species such as horses and donkeys, which also exhibit fast loss of sperm DNA quality (3, 4). In fact, the hypothesis to test is that those individuals presenting an exponential increase in sperm DNA damage might present higher reproductive fitness than those that exhibit a logarithmic increase in sperm DNA damage. If this is true, this experimental design could be used to select sperm samples or donors that exhibit the best long-term DNA stability for artificial insemination. This may present a new experimental strategy to assess the longevity of any sperm sample for sperm DNA quality. In any case, it seems obvious and imperative for any clinic to use sperm samples as quickly as possible after thawing. Finally, the time of sperm DNA fragmentation analysis should be specified in reported sperm DNA fragmentation data, because this could be a limitation when comparisons between techniques to assess sperm DNA fragmentation or results among different laboratories are performed. Moreover, this applies not only to humans but also to other species. Without any explicit reference to the time between sperm collection and sperm quality assessment, as well as the conditions of storage and handling, comparisons of sperm DNA fragmentation results among different laboratories, or correlations with fertilization, embryo quality, and development of pregnancies, could be meaningless. REFERENCES 1. Evenson DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 2006;65:979–91. 2. Velez de la Calle JF, Muller A, Walschaerts M, Clavere JL, Jimenez C, Wittemer C, et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil Steril. Published online December 29, 2007. 3. Lopez-Fernandez C, Crespo F, Arroyo F, Fernandez JL, Arana P, Johnston SD, et al. Dynamics of sperm DNA fragmentation in domestic animals II. The stallion. Theriogenology 2007;68:1240–50. 4. Cortes-Gutierrez EI, Crespo F, Gosalvez A, Davila-Rodrıguez MI, Lopez-Fernandez C, Gosalvez J. DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leones, a breed at risk of extinction. Theriogenology 2008;69:1022–32.
Vol. 92, No. 1, July 2009
5. Gosalvez J, Fernandez JL, Gosalbez A, Arrollo F, Agarwal A, LopezFernandez C. Dynamics of sperm DNA fragmentation in mammalian species as assessed by the SCD methodology. Fertil Steril 2007;88:S365. 6. Nagase H, Niwa T. Deep freezing of bull semen in concentrated pellet form. Proceedings of the 5th International Congress on Animal Reproduction and Artificial Insemination 1964;4:410–5. 7. Caballero P, N u~ nez R, Vazquez I. Coordinacion de un Banco de Semen. In: Dexeus JM, Barri PN, eds. Fertilidad, analogos Gn RH–periconceptologıa–endoscopia. Barcelona: Masson-Salvat Medicina, 1993: 83–92.
Fertility and Sterility
8. Vishwanath R, Shannon P. Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperatures. Reprod Fertil Dev 1997;9:321–31. 9. Young KE, Robbins WA, Xun L, Elashoff D, Rothmann, Perreault SD. Evaluation of chromosome breakage and DNA integrity in sperm: an investigation of remote semen collection conditions. J Androl 2003;24: 853–61. 10. Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SEM. Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril 2004;82:1443–5.
173
Department of Biology, Genetics Unit, Universidad Autonoma de Madrid (UAM), Madrid, Spain; b Department of Genetics, Northwest Research Center of Biology, Mexican National Institute for Health, Monterrey, Mexico; c Seccion de Genetica y Unidad de Investigacion, Complejo Hospitalario Universitario Juan Canalejo, A Coru~na, Spain; and d Clinica Tambre, Madrid, Spain
Objective: To study the velocity of sperm DNA fragmentation in frozen-thawed sperm samples from male sperm donors of proved fertility. Design: Sperm DNA fragmentation assessment with use of sperm chromatin dispersion methodology after 0, 4, 8, and 24 hours of incubation in IVF medium. Setting: Academic biology and reproductive medicine centers. Patient(s): Twenty male fertility donors with proved fertility for a maximum of six births at the reproductive medicine center. Intervention(s): None. Main Outcome Measure(s): The velocity of sperm DNA fragmentation between two consecutive incubations was scored. Best adjustment of sperm DNA fragmentation index versus incubation time for linear, logarithmic, or exponential function was tested. Result(s): Increase of sperm DNA fragmentation through time accounted for a substantial percentage of the overall variation. The highest velocity of sperm DNA fragmentation was observed in the first 4 hours of incubation, decreasing by 50% during the second incubation period and being of the order of 1% in the final experimental period. The tendency to increase in sperm DNA fragmentation is not homogeneous among donors; they may adjust to a logarithmic, linear, or exponential function rendering high values for R2. Conclusion(s): Sperm DNA fragmentation occurs rapidly after thawing, and it is an important cause of the rapid decline of sperm quality. Thus, the use of sperm samples as quickly as possible after thawing is highly recommended in clinical practice. Different sperm DNA fragmentation dynamics among individuals were observed. (Fertil Steril 2009;92:170–3. 2009 by American Society for Reproductive Medicine.)
Sperm DNA fragmentation has been the subject of numerous studies because effects on fertility parameters appear to be correlated with a high frequency of sperm nuclei containing damaged DNA (1, 2). Sperm DNA fragmentation has always been referred to as a constant parameter of sperm quality, without any specification of the period of time that had passed between sperm quality assessment and use for ART. However, this could be untrue because the sperm DNA fragmentation index may change rapidly when samples for artificial insemination are handled. In fact, in mammalian species it has been reported that temperature excursion episodes dramatically increase the rate of sperm DNA degradation, and Received February 26, 2008; revised May 16, 2008; accepted May 19, 2008; published online August 11, 2008. J.G. has nothing to disclose. E.C.-G. has nothing to disclose. C.L.-F. has nothing to disclose. J.L.F. has nothing to disclose. P.C. has nothing to disclose. R.N. has nothing to disclose. n y Ciencia, Supported by BFU 2007-66340/BFI (Ministerio de Educacio Spain). lvez, M.D., Department of Biology, Genetics Reprint requests: Jaime Gosa noma de Madrid (UAM), 20849-Madrid, Spain Unit, Universidad Auto (FAX: þ34-91-497-83-44; E-mail: [email protected]).
170
after some hours of sperm incubation at 37 C, the basal values for the sperm DNA fragmentation index may be doubled (3, 4). Thus, the level of sperm DNA fragmentation as analyzed when the semen is freshly obtained will increase over time, with variation among species and among different individuals within the same species (5). Reference to the time that has passed between semen collection and analysis seems to be imperative; otherwise, reported analysis results could be meaningless at the time of ART if this temporal factor is not considered seriously. In addition, comparison of results among different laboratories also could become highly biased. The aim of this investigation was to analyze the evolution of sperm DNA fragmentation values over time in donors with proved fertility in an attempt to stress the importance of considering time after ejaculation and/or after thawing of frozen samples as a factor influencing sperm DNA quality. MATERIALS AND METHODS Twenty donors from the Clinica Tambre (Madrid, Spain) were included in the analysis. Their semen samples had
Fertility and Sterility Vol. 92, No. 1, July 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.05.068
been used in different ART programs, achieving a maximum of six pregnancies with normal embryo development and birth. This is the maximum permitted by the Spanish law for one donor in an ART program. The cryopreservation of frozen sperm pellets was performed according to previously published methodology (6, 7). All samples were thawed by immersion in a 37 C water bath for 30 seconds and diluted to 10 to 15 106 spermatozoa/mL in IVF medium (MediCult, Jyllinge, Denmark). Two aliquots of the corresponding sperm samples were incubated in a humidified atmosphere of 5% CO2 in air at 37 C. The sperm DNA fragmentation index, the ratio of fragmented versus total spermatozoa in the analyzed sample, expressed as a percentage, was assessed after 0, 4, 8, and 24 hours of incubation (t0, t4, t8, and t24). This time period was selected to show the differences in sperm DNA damage within a reasonable time in which the sperm could be viable for ART. Replicates, processed and assessed by two different observers, were used as internal controls. The samples were assessed for sperm DNA fragmentation just after they were thawed (t0), and the corresponding sperm DNA fragmentation index was considered as basal for this particular donor. The sperm DNA fragmentation frequency was determined with use of the Halosperm kit (Halotech DNA SL, Madrid, Spain; Conception Technologies, San Diego, CA). Deoxyribonucleic acid haloes resulting after the sperm chromatin dispersion test were visualized by fluorescence microscopy with propidium iodide staining (2.5 mg/ mL in Vectashield; Vector Laboratories, Burlingame, CA). Descriptive statistics with use of box-and-whisker diagrams and regression analysis, including a comparison of individual regression equations, were conducted with use of Stat-Graphics Plus 5.1 (Academic Enterprise, StatPoint Inc., Herndon, VA). In addition, to test the significance of the increase in sperm DNA fragmentation index over the incubation at 37 C, the slopes and Y-axis intercepts of the regression equations for each individual were also compared. The velocity of sperm DNA fragmentation (VsDF) between two periods of incubation (t1 and t2) was determined according to the following equation: VsDF ¼ (Sperm DNA fragmentation index at t2) (Sperm DNA fragmentation index at t1)/Incubation time within the selected period, expressed in hours. These values correspond to the average of the slopes obtained by regression analysis.
RESULTS AND DISCUSSION The basal distribution of sperm DNA fragmentation index after thawing ranged from 8% to 28%. Deoxyribonucleic acid fragmentation started to increase after the samples had been diluted and incubated at 37 C (Fig. 1a). Regression analysis was conducted to investigate the relationship between sperm DNA fragmentation index and incubation time, considering time points t0 and t24. Variation in time accounted for a substantial percentage of the overall variation in sperm DNA fragmentation index (R2 ¼ 76.3%, P<.05; Fig. 1b). Y-axis intercepts showed significant differences between Fertility and Sterility
FIGURE 1 (a) Box-and-whisker plots for the sperm DNA fragmentation index observed at different incubation times (37 C) for spermatozoa and the rate for sperm DNA fragmentation observed at each interval. (b) Average sperm DNA fragmentation observed at different incubation times (blue line) and adjustments for logarithmic (red line), linear (gray line), or exponential (green line) functions. The corresponding equations are shown with the same color pattern. The pink line (donor 7) shows the maximum R2 value for a logarithmic function, and the light-green line (donor 15) shows the maximum R2 value for an exponential function. VsDF ¼ velocity of sperm DNA fragmentation.
Gos alvez. Dynamics of sperm DNA fragmentation. Fertil Steril 2009.
individuals (P¼.000), indicating variation in the basal level of sperm DNA fragmentation index. To test the homogeneity of the rate of sperm DNA fragmentation between different periods of incubation, the velocity of sperm DNA fragmentation between two consecutive incubations was compared. Results showed that there are differences among different consecutive incubation times (P<.05; Fig. 1a). The highest rate of sperm DNA fragmentation increase was observed in the first 4 hours of incubation (velocity of sperm DNA fragmentation ¼ 8.3% per hour). This rate decreased by 50% during the second incubation period (velocity of sperm DNA fragmentation ¼ 4.1% per hour) and was of the order of 1% in the final experimental period (Fig. 1a). After performing regression analysis using a linear adjustment for each sperm sample, we found different intercepts for 171
different slopes (data not shown). This indicates that some individuals may present a more intense rate of sperm DNA damage than others may, and, conversely, some sperm samples are more resistant to DNA fragmentation than others are. This also explains the large deviation in the lowest and highest values for sperm DNA fragmentation index values and the differences in the lower and upper quartiles observed, especially after 4 and 8 hours of incubation (see box-andwhisker diagrams in Fig. 1a). This deviation was lower at t0 because, at this time, sperm DNA fragmentation index is related directly to basal interindividual variations in sperm DNA fragmentation and does not depend on the incubation time, which explains a large part of the variation observed in this experiment. The global increasing tendency is for sperm DNA fragmentation values (blue line in Fig. 1b) to be fitted to a logarithmic function (red line in Fig. 1b; R2 ¼ 0.992; y ¼ 47.03 ln[x] þ 17.3). The values of R2 for linear (gray line in Fig. 1b) or exponential functions (dark green in Fig. 1b) were lower (Fig. 1b). When each sample was analyzed separately, R2 values >0.9 were achieved in 60% of the samples when a logarithmic function was used, 30% in the case of a linear function, and 20% for an exponential function. In Figure 1b, one sperm sample (D7) with the maximum R2 value for a logarithmic function (pink line in Fig. 1b; y ¼ 49.363 ln[x] þ 15.03; R2 ¼ 0.9852) and another (D15) with the maximum R2 value (y ¼ 7.995e0.6183x; R2 ¼ 0.9791) with use of an exponential function (light green line in Fig. 1b) are shown. For an exponential trend, sperm longevity would be higher than with a logarithmic tendency, and this could be critical for DNA stability during the first hours of sperm incubation. The results obtained in this experiment indicate that DNA fragmentation is an important effector of the rapid decline of sperm quality, because sperm DNA tends to degrade very quickly after thawing. In practice, the experimental conditions used in this experiment emulate those used for ART, and sperm DNA degradation could be detected at the onset of temperature recovery to 37 C. This degradation therefore could exert a negative influence on fertility. In fact, given the high rate for sperm DNA fragmentation observed during the first 4 hours of incubation, it seems highly probable that any sperm sample would contain 50% of sperm cells with damaged DNA. Loss of sperm quality in samples, with use of other sperm parameters, has been reported in most animals analyzed, especially when sperm samples are exposed to increased temperatures. For example, the fertility of bull sperm is well preserved for 3 to 5 days when semen is stored at ambient temperatures. After this time, fertility declines at a rate of 3% to 6% per day, irrespective of whether the sperm are stored at 5 C or at 15 C. However, this effect is greater when storage temperatures exceed 25 C (8). Therefore, it seems clear that the temperature, or, more precisely, episodes of temperature change, may produce deleterious effects on sperm viability. In humans, it has been demonstrated that col172
Gosalvez et al.
Dynamics of sperm DNA fragmentation
lection of ejaculated semen at distant sites away from the laboratory where the artificial insemination will be performed, with overnight mail delivery, might lead to sperm DNA damage (9). The effects of the relative short longevity of sperm DNA quality detected in certain clinical situations could also be related to and even amplified by the effects described here. It has been reported that DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4- and 24-hour incubations (10). In fact, under these circumstances, the immediate use of testicular sperm in intracytoplasmic sperm injection is highly recommended. Special care must be paid to the fact that sperm characteristics considered as sufficient for fertilization at the onset of sperm quality assessment may change rapidly between insemination and fertilization. The results of this investigation have shown that different sperm samples exhibit different sperm DNA fragmentation dynamics. This tendency has been also reported in other mammalian species such as horses and donkeys, which also exhibit fast loss of sperm DNA quality (3, 4). In fact, the hypothesis to test is that those individuals presenting an exponential increase in sperm DNA damage might present higher reproductive fitness than those that exhibit a logarithmic increase in sperm DNA damage. If this is true, this experimental design could be used to select sperm samples or donors that exhibit the best long-term DNA stability for artificial insemination. This may present a new experimental strategy to assess the longevity of any sperm sample for sperm DNA quality. In any case, it seems obvious and imperative for any clinic to use sperm samples as quickly as possible after thawing. Finally, the time of sperm DNA fragmentation analysis should be specified in reported sperm DNA fragmentation data, because this could be a limitation when comparisons between techniques to assess sperm DNA fragmentation or results among different laboratories are performed. Moreover, this applies not only to humans but also to other species. Without any explicit reference to the time between sperm collection and sperm quality assessment, as well as the conditions of storage and handling, comparisons of sperm DNA fragmentation results among different laboratories, or correlations with fertilization, embryo quality, and development of pregnancies, could be meaningless. REFERENCES 1. Evenson DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 2006;65:979–91. 2. Velez de la Calle JF, Muller A, Walschaerts M, Clavere JL, Jimenez C, Wittemer C, et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil Steril. Published online December 29, 2007. 3. Lopez-Fernandez C, Crespo F, Arroyo F, Fernandez JL, Arana P, Johnston SD, et al. Dynamics of sperm DNA fragmentation in domestic animals II. The stallion. Theriogenology 2007;68:1240–50. 4. Cortes-Gutierrez EI, Crespo F, Gosalvez A, Davila-Rodrıguez MI, Lopez-Fernandez C, Gosalvez J. DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leones, a breed at risk of extinction. Theriogenology 2008;69:1022–32.
Vol. 92, No. 1, July 2009
5. Gosalvez J, Fernandez JL, Gosalbez A, Arrollo F, Agarwal A, LopezFernandez C. Dynamics of sperm DNA fragmentation in mammalian species as assessed by the SCD methodology. Fertil Steril 2007;88:S365. 6. Nagase H, Niwa T. Deep freezing of bull semen in concentrated pellet form. Proceedings of the 5th International Congress on Animal Reproduction and Artificial Insemination 1964;4:410–5. 7. Caballero P, N u~ nez R, Vazquez I. Coordinacion de un Banco de Semen. In: Dexeus JM, Barri PN, eds. Fertilidad, analogos Gn RH–periconceptologıa–endoscopia. Barcelona: Masson-Salvat Medicina, 1993: 83–92.
Fertility and Sterility
8. Vishwanath R, Shannon P. Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperatures. Reprod Fertil Dev 1997;9:321–31. 9. Young KE, Robbins WA, Xun L, Elashoff D, Rothmann, Perreault SD. Evaluation of chromosome breakage and DNA integrity in sperm: an investigation of remote semen collection conditions. J Androl 2003;24: 853–61. 10. Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SEM. Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril 2004;82:1443–5.
173