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Effect of storage temperature on sperm cryopreservation
FERTILITY AND STERILITYt VOL. 70, NO. 6, DECEMBER 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Effect of storage temperature on sperm cryopreservation Harald Trummer, M.D.,* Kathleen Tucker, Ph.D.,† Christine Young, M.S.,† Norbert Kaula, M.S.,* and Randall B. Meacham, M.D.* University of Colorado Health Science Center, Denver, Colorado
Objective: To evaluate the influence of cryopreservation temperature on human sperm motility and morphology. Design: Controlled study, investigator was blinded to the type of cryopreservation. Setting: University-based andrology laboratory. Patient(s): Sixteen semen samples with normal motility and sperm count from men after a fertility work up. Intervention(s): Semen aliquots were either stored in a mechanical freezer at 270°C or in liquid nitrogen at 2196°C for 7 days or 3 months. Test yolk buffer was used as a cryoprotectant. With use of a programmable freezing unit, all samples were cooled at a controlled rate. Main Outcome Measure(s): Sperm motility and morphology. Result(s): After 7 days of cryopreservation, there was a greater decrease in sperm motility among specimens maintained at 270°C than among those maintained at 2196°C (47% versus 39% decrease). The difference in sperm motility was even greater after 3 months of cryopreservation (72% versus 39% decrease). No difference in postthaw sperm morphology was detected among sperm preserved at 270°C versus 2196°C. Conclusion(s): Sperm cryopreservation at 2196°C is superior to cryopreservation at 270°C. Sperm can be stored at 270°C for a short period of time with a relatively modest loss of motility. (Fertil Sterilt 1998;70: 1162– 4. ©1998 by American Society for Reproductive Medicine.) Key Words: Human sperm, cryopreservation, mechanical freezer, sperm motility, sperm morphology Received February 3, 1998; revised and accepted July 16, 1998. Supported by a travel scholarship from AESCA GmbH Traiskirchen, Austria, and Ferring Arzneimittel GesmbH Wien, Austria (H.T.). Presented at the 93rd Annual Meeting of the American Urological Association, San Diego, California, May 30 –June 4, 1998. Reprint requests and present address: Harald Trummer, M.D., Department of Urology, University of Graz, Auenbruggerplatz 7, A8036 Graz, Austria (FAX: 143-316-385-3550; E-mail: harald.trummer@kfunigraz .ac.at). * Division of Urology. † Department of Obstetrics and Gynecology. 0015-0282/98/$19.00 PII S0015-0282(98)00349-5
1162
Sperm cryopreservation may provide the opportunity for future fertility in a variety of situations. A relatively common cause of severe male factor infertility is the treatment of malignancy (1). Chemotherapy, radiation therapy, and retroperitoneal surgery may cause testicular failure or ejaculatory dysfunction. Cryopreservation of sperm collected before initiation of such therapy, however, may offer subsequent treatment options including insemination or IVF. These options have become more viable as advances in assisted reproductive technologies have allowed successful induction of pregnancy despite severely impaired semen quality. The standard method for preservation of human sperm is storage in liquid nitrogen at a temperature of 2196°C (2). A number of studies have documented that improvements in cooling technique and the use of improved cryopreservatives, as well as thawing at 37°C in a water bath improve postthaw sperm quality (3).
Unfortunately, liquid nitrogen cryopreservation units are not universally available. If, however, storage at 270°C proved to be an effective method for sperm storage, facilities that have access to a standard 270°C freezer could potentially offer sperm cryopreservation. The aim of the present study is to compare the effect of sperm cryopreservation at 270°C versus 2196°C.
MATERIALS AND METHODS Semen Collection and Assessment Semen specimens were obtained from 16 consecutive subjects, all of whom had normal sperm motility (.50%) and normal sperm count (.20 3 106/mL). All samples were collected by masturbation in a clean specimen container, allowed to liquefy, and immediately evaluated according to World Health Organization guidelines (4). Volume, viscosity, percent motility, concentration, and percent normal forms were determined for each sample.
Sperm concentration was determined with a hemocytometer (Brightline; Hausser Scientific, Horsham, PA). Slides were prepared for morphology evaluation by smearing a small volume of semen on the slide and allowing it to air dry. Dried slides were stained subsequently with a modified Giemsa stain (Diff-Quik; Dade Diagnostics, Aguada, PR). Sperm morphology was determined with use of Kruger’s strict criteria (5). Motility was determined as a percentage by evaluating 200 sperm per sample.
FIGURE 1 Sperm motility as a function of duration and temperature of cryopreservation. Results are means 6 1 SD.
Procedure for Sperm Freezing and Thawing Each specimen was divided into four aliquots. The four aliquots then were cryopreserved as follows: 1 week at 270°C, 3 months at 270°C, 1 week at 2196°C, and 3 months at 2196°C. Shortly after liquefaction and after macroscopic and microscopic evaluation of the semen sample, test yolk buffer (Irvine Scientific, Irvine, CA) was added dropwise, in a 1:1 (vol:vol) ratio. The resulting solution (1-mL aliquots) was pipetted into cryovials (Nunclon, Roskilde, Denmark). Each sample underwent a period of equilibration (10 minutes) before the freezing procedure. All samples were frozen with use of a programmable Planar II freezer unit (TS Scientific, Perkasie, PA). In this study, two separate freezing procedures were used. In both procedures, samples were cooled to seeding temperature at a rate of 23.0°C per minute until seeding temperature was reached (26.5°C) as published by Hammit et al. (6). After induction of seeding, all specimens were cooled further to the desired end temperature at a rate of 10° per minute. In procedure 1, samples were cooled to 220°C and stored at 270°C. In procedure 2, samples are cooled in the same way as procedure 1; however, when the samples reached 236°C, they were plunged immediately into and stored in liquid nitrogen. Sample pairs (freezing procedure 1 and 2) were thawed after a storage period of 1 week or 3 months. Samples were thawed by placing the cryovials in a 37°C water bath for 5 minutes. Sperm motility and morphology were evaluated for all thawed samples. All evaluations were performed in a blinded fashion to avoid investigator bias. The semen used in this study would have been discarded under routine conditions. Specimen were analyzed anonymously and could not be traced back to the donors. Because it was not planned to use the semen for reproduction at any time and laboratory studies only were performed for scientific interest, there was no obvious need for approval by the Institutional Review Board.
Statistics Differences in motility and morphology among sperm preserved using the two storage procedures were assessed with use of a paired Student’s t-test. All analyses were performed with SAS Institute’s (Cary, NC) statistical software package. FERTILITY & STERILITYt
RESULTS The mean (6 SD) prestorage motility and morphology for all samples were 72.5% 6 14.8% and 12.5% 6 4.8%. After 7 days of storage at 270°C, mean (6 SD) sperm motility decreased to 38% 6 22.9%, which represents a decline of 47% from baseline. After the same period of storage at 2196°C, mean (6 SD) sperm motility decreased to 44% 6 22.7%, a decrease of 39% from baseline. This represented a significant decrease from baseline sperm motility values in both groups (P,.05). The loss of sperm motility between the two cryopreservation methods was also statistically significant (P,.05). After 3 months of storage, mean (6 SD) sperm motility decreased to 18% 6 13.1% among sperm stored at 270°C but remained at 44% 6 20% in the specimens that were cryopreserved at 2196°C (P,.05) (Fig. 1). The mean (6 SD) percentage of sperm with normal morphology after 7 days of cryopreservation decreased from a baseline of 12.5% to 9.4% 6 4.7% when preserved at 270°C. Specimens cryopreserved for 7 days at 2196°C showed a similar decrease in normal forms with a mean (6 SD) postthaw normal morphology rate of 9.8% 6 5.0%. After 3 months of cryopreservation, specimens stored at 270°C and 2196°C were found to have normal mean (6 SD) morphology values of 8.4% 6 3.5% and 8.6% 6 3.6%, respectively (Fig. 2). The decrease in normal forms was significant for both groups compared with prestorage values (P,.05). No statistically significant difference in morphology could be detected, however, between the two storage methods at 7 days or 3 months.
DISCUSSION Cryopreservation of human spermatozoa has many potential applications in the management of male infertility. Long1163
FIGURE 2 Sperm morphology as a function of duration and temperature of cryopreservation. Results are means 6 1 SD.
transcription after 24 hours of cryopreservation. It is significant that they noted good preservation of DNA transcription among sperm that had been preserved at both 270°C and 2196°C. This led the investigators to conclude that optimal cryopreservation of buffalo sperm only requires that the storage temperature is less than 220°C. Sperm cryopreservation in liquid nitrogen at 2196°C has become standard protocol in most andrology facilities. Because this is now accepted technique, little consideration has been given to the potential effectiveness of short- or longterm storage of sperm at higher temperatures. Unfortunately, not all facilities have consistent access to liquid nitrogen cryopreservation facilities. As described above, the debate about the relative effectiveness of sperm storage at 2196°C versus 270°C has not been resolved definitively. Therefore, if sperm storage at a temperature higher than that of liquid nitrogen is to be considered, it is important to establish whether this is consistent with acceptable levels of sperm survival.
term storage in liquid nitrogen at 2196°C consistently causes a reduction in postthaw motility (7). It appears, however, that this loss of sperm motility does not increase with prolonged periods of cryopreservation, making long-term storage feasible. Because liquid nitrogen cryopreservation facilities are not uniformly available, the option of cryopreserving sperm in 270°C freezers may be attractive to some facilities. In 1945, Parkes and associates (8) indicated that sperm cryopreservation at 2196°C offered no advantage over storage at 279°C. In 1954, Bunge and coworkers (9) reported data that supported the conclusions of Parkes and, in fact, concluded that cryopreservation of sperm at 278°C resulted in better postthaw motility than did cryopreservation at 2196°C. In the same year, Sherman (10) published a comparison of sperm storage at 2196°C versus 270°C and similarly reported cryopreservation at 270°C to be superior in preservation of postthaw sperm motility. In 1963, however, Sherman (11) again reported on the effect of cryopreservation temperature on sperm motility but in this report concluded that storage at 2196°C appeared to offer an advantage. In 1969, Fjallbrant and Ackerman (12) compared the results of the cervical mucus penetration assay using sperm that had been stored at 2196°C versus sperm stored at 290°C. Although sperm cryopreserved at 2196°C appeared to perform better on the mucus penetration assay, sperm motility was not assessed in this study. In 1995, Pironcheva and coworkers (13) evaluated the impact of cryopreservation of buffalo sperm when stored at 4°C, 220°C, 270°C, or 2196°C. To evaluate the effect of storage temperature on DNA integrity, they measured DNA
1164 Trummer et al.
Temperature and sperm cryopreservation
The results of the present study indicate that sperm storage at 270°C is linked with a greater loss of sperm motility than is storage at 2196°C. The reduction in motility noted at 1 week, however, was fairly modest. Unfortunately, the loss of sperm motility increased dramatically after 3 months of storage. These results indicate that short-term storage of sperm at 270°C may be a viable option if liquid nitrogen storage facilities are not available. Long-term storage at this temperature, however, appears to be linked with an unacceptable reduction in sperm motility. References 1. Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer-criteria change. Fertil Steril 1992;58:1024 –7. 2. Sherman JK. Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 1973;24:397– 412. 3. Leffler KS, Walters CA. A comparison of time, temperature, and refreezing variables on frozen sperm motility recovery. Fertil Steril 1996;65:272– 4. 4. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus Interaction. 3rd ed. New York: Cambridge University Press, 1992:3–23. 5. Kruger TF, Menkveld R, Standerl FS, Lombard CJ, Van der Merwe JP, Van Zyll JA, et al. Sperm morphologic features as a prognostic factor in vitro fertilization. Fertil Steril 1986;46:1118 –23. 6. Hammitt DG, Hade DK, Williamson RA. Survival of human sperm following controlled- and noncontrolled-rate cryopreservation. Andrologia 1989;21:311–7. 7. Keel BA, Black JB. Reduced motility longevity in thawed human spermatozoa. Arch Androl 1980;4:213–5. 8. Parkes AS. Preservation of human spermatozoa at low temperatures. Br Med J 1945;2:212–3. 9. Bunge RG, Keettele WC, Sherman JK. Clinical use of frozen semen. Fertil Steril 1954;5:520 –9. 10. Sherman JK. Freezing and freeze-drying of human spermatozoa. Fertil Steril 1954;5:357–71. 11. Sherman JK. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril 1963;14:49 – 64. 12. Fjallbrant B, Ackerman DR. Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimen. J Reprod Fertil 1969;20:515–7. 13. Pironcheva GL, Miteva K, Vaisberg C, Russev G. Influence of freezing at different temperatures on the transcription activity of buffalo sperm chromatin. Cytobios 1995;83:207–10.
Vol. 70, No. 6, December 1998
Effect of storage temperature on sperm cryopreservation Harald Trummer, M.D.,* Kathleen Tucker, Ph.D.,† Christine Young, M.S.,† Norbert Kaula, M.S.,* and Randall B. Meacham, M.D.* University of Colorado Health Science Center, Denver, Colorado
Objective: To evaluate the influence of cryopreservation temperature on human sperm motility and morphology. Design: Controlled study, investigator was blinded to the type of cryopreservation. Setting: University-based andrology laboratory. Patient(s): Sixteen semen samples with normal motility and sperm count from men after a fertility work up. Intervention(s): Semen aliquots were either stored in a mechanical freezer at 270°C or in liquid nitrogen at 2196°C for 7 days or 3 months. Test yolk buffer was used as a cryoprotectant. With use of a programmable freezing unit, all samples were cooled at a controlled rate. Main Outcome Measure(s): Sperm motility and morphology. Result(s): After 7 days of cryopreservation, there was a greater decrease in sperm motility among specimens maintained at 270°C than among those maintained at 2196°C (47% versus 39% decrease). The difference in sperm motility was even greater after 3 months of cryopreservation (72% versus 39% decrease). No difference in postthaw sperm morphology was detected among sperm preserved at 270°C versus 2196°C. Conclusion(s): Sperm cryopreservation at 2196°C is superior to cryopreservation at 270°C. Sperm can be stored at 270°C for a short period of time with a relatively modest loss of motility. (Fertil Sterilt 1998;70: 1162– 4. ©1998 by American Society for Reproductive Medicine.) Key Words: Human sperm, cryopreservation, mechanical freezer, sperm motility, sperm morphology Received February 3, 1998; revised and accepted July 16, 1998. Supported by a travel scholarship from AESCA GmbH Traiskirchen, Austria, and Ferring Arzneimittel GesmbH Wien, Austria (H.T.). Presented at the 93rd Annual Meeting of the American Urological Association, San Diego, California, May 30 –June 4, 1998. Reprint requests and present address: Harald Trummer, M.D., Department of Urology, University of Graz, Auenbruggerplatz 7, A8036 Graz, Austria (FAX: 143-316-385-3550; E-mail: harald.trummer@kfunigraz .ac.at). * Division of Urology. † Department of Obstetrics and Gynecology. 0015-0282/98/$19.00 PII S0015-0282(98)00349-5
1162
Sperm cryopreservation may provide the opportunity for future fertility in a variety of situations. A relatively common cause of severe male factor infertility is the treatment of malignancy (1). Chemotherapy, radiation therapy, and retroperitoneal surgery may cause testicular failure or ejaculatory dysfunction. Cryopreservation of sperm collected before initiation of such therapy, however, may offer subsequent treatment options including insemination or IVF. These options have become more viable as advances in assisted reproductive technologies have allowed successful induction of pregnancy despite severely impaired semen quality. The standard method for preservation of human sperm is storage in liquid nitrogen at a temperature of 2196°C (2). A number of studies have documented that improvements in cooling technique and the use of improved cryopreservatives, as well as thawing at 37°C in a water bath improve postthaw sperm quality (3).
Unfortunately, liquid nitrogen cryopreservation units are not universally available. If, however, storage at 270°C proved to be an effective method for sperm storage, facilities that have access to a standard 270°C freezer could potentially offer sperm cryopreservation. The aim of the present study is to compare the effect of sperm cryopreservation at 270°C versus 2196°C.
MATERIALS AND METHODS Semen Collection and Assessment Semen specimens were obtained from 16 consecutive subjects, all of whom had normal sperm motility (.50%) and normal sperm count (.20 3 106/mL). All samples were collected by masturbation in a clean specimen container, allowed to liquefy, and immediately evaluated according to World Health Organization guidelines (4). Volume, viscosity, percent motility, concentration, and percent normal forms were determined for each sample.
Sperm concentration was determined with a hemocytometer (Brightline; Hausser Scientific, Horsham, PA). Slides were prepared for morphology evaluation by smearing a small volume of semen on the slide and allowing it to air dry. Dried slides were stained subsequently with a modified Giemsa stain (Diff-Quik; Dade Diagnostics, Aguada, PR). Sperm morphology was determined with use of Kruger’s strict criteria (5). Motility was determined as a percentage by evaluating 200 sperm per sample.
FIGURE 1 Sperm motility as a function of duration and temperature of cryopreservation. Results are means 6 1 SD.
Procedure for Sperm Freezing and Thawing Each specimen was divided into four aliquots. The four aliquots then were cryopreserved as follows: 1 week at 270°C, 3 months at 270°C, 1 week at 2196°C, and 3 months at 2196°C. Shortly after liquefaction and after macroscopic and microscopic evaluation of the semen sample, test yolk buffer (Irvine Scientific, Irvine, CA) was added dropwise, in a 1:1 (vol:vol) ratio. The resulting solution (1-mL aliquots) was pipetted into cryovials (Nunclon, Roskilde, Denmark). Each sample underwent a period of equilibration (10 minutes) before the freezing procedure. All samples were frozen with use of a programmable Planar II freezer unit (TS Scientific, Perkasie, PA). In this study, two separate freezing procedures were used. In both procedures, samples were cooled to seeding temperature at a rate of 23.0°C per minute until seeding temperature was reached (26.5°C) as published by Hammit et al. (6). After induction of seeding, all specimens were cooled further to the desired end temperature at a rate of 10° per minute. In procedure 1, samples were cooled to 220°C and stored at 270°C. In procedure 2, samples are cooled in the same way as procedure 1; however, when the samples reached 236°C, they were plunged immediately into and stored in liquid nitrogen. Sample pairs (freezing procedure 1 and 2) were thawed after a storage period of 1 week or 3 months. Samples were thawed by placing the cryovials in a 37°C water bath for 5 minutes. Sperm motility and morphology were evaluated for all thawed samples. All evaluations were performed in a blinded fashion to avoid investigator bias. The semen used in this study would have been discarded under routine conditions. Specimen were analyzed anonymously and could not be traced back to the donors. Because it was not planned to use the semen for reproduction at any time and laboratory studies only were performed for scientific interest, there was no obvious need for approval by the Institutional Review Board.
Statistics Differences in motility and morphology among sperm preserved using the two storage procedures were assessed with use of a paired Student’s t-test. All analyses were performed with SAS Institute’s (Cary, NC) statistical software package. FERTILITY & STERILITYt
RESULTS The mean (6 SD) prestorage motility and morphology for all samples were 72.5% 6 14.8% and 12.5% 6 4.8%. After 7 days of storage at 270°C, mean (6 SD) sperm motility decreased to 38% 6 22.9%, which represents a decline of 47% from baseline. After the same period of storage at 2196°C, mean (6 SD) sperm motility decreased to 44% 6 22.7%, a decrease of 39% from baseline. This represented a significant decrease from baseline sperm motility values in both groups (P,.05). The loss of sperm motility between the two cryopreservation methods was also statistically significant (P,.05). After 3 months of storage, mean (6 SD) sperm motility decreased to 18% 6 13.1% among sperm stored at 270°C but remained at 44% 6 20% in the specimens that were cryopreserved at 2196°C (P,.05) (Fig. 1). The mean (6 SD) percentage of sperm with normal morphology after 7 days of cryopreservation decreased from a baseline of 12.5% to 9.4% 6 4.7% when preserved at 270°C. Specimens cryopreserved for 7 days at 2196°C showed a similar decrease in normal forms with a mean (6 SD) postthaw normal morphology rate of 9.8% 6 5.0%. After 3 months of cryopreservation, specimens stored at 270°C and 2196°C were found to have normal mean (6 SD) morphology values of 8.4% 6 3.5% and 8.6% 6 3.6%, respectively (Fig. 2). The decrease in normal forms was significant for both groups compared with prestorage values (P,.05). No statistically significant difference in morphology could be detected, however, between the two storage methods at 7 days or 3 months.
DISCUSSION Cryopreservation of human spermatozoa has many potential applications in the management of male infertility. Long1163
FIGURE 2 Sperm morphology as a function of duration and temperature of cryopreservation. Results are means 6 1 SD.
transcription after 24 hours of cryopreservation. It is significant that they noted good preservation of DNA transcription among sperm that had been preserved at both 270°C and 2196°C. This led the investigators to conclude that optimal cryopreservation of buffalo sperm only requires that the storage temperature is less than 220°C. Sperm cryopreservation in liquid nitrogen at 2196°C has become standard protocol in most andrology facilities. Because this is now accepted technique, little consideration has been given to the potential effectiveness of short- or longterm storage of sperm at higher temperatures. Unfortunately, not all facilities have consistent access to liquid nitrogen cryopreservation facilities. As described above, the debate about the relative effectiveness of sperm storage at 2196°C versus 270°C has not been resolved definitively. Therefore, if sperm storage at a temperature higher than that of liquid nitrogen is to be considered, it is important to establish whether this is consistent with acceptable levels of sperm survival.
term storage in liquid nitrogen at 2196°C consistently causes a reduction in postthaw motility (7). It appears, however, that this loss of sperm motility does not increase with prolonged periods of cryopreservation, making long-term storage feasible. Because liquid nitrogen cryopreservation facilities are not uniformly available, the option of cryopreserving sperm in 270°C freezers may be attractive to some facilities. In 1945, Parkes and associates (8) indicated that sperm cryopreservation at 2196°C offered no advantage over storage at 279°C. In 1954, Bunge and coworkers (9) reported data that supported the conclusions of Parkes and, in fact, concluded that cryopreservation of sperm at 278°C resulted in better postthaw motility than did cryopreservation at 2196°C. In the same year, Sherman (10) published a comparison of sperm storage at 2196°C versus 270°C and similarly reported cryopreservation at 270°C to be superior in preservation of postthaw sperm motility. In 1963, however, Sherman (11) again reported on the effect of cryopreservation temperature on sperm motility but in this report concluded that storage at 2196°C appeared to offer an advantage. In 1969, Fjallbrant and Ackerman (12) compared the results of the cervical mucus penetration assay using sperm that had been stored at 2196°C versus sperm stored at 290°C. Although sperm cryopreserved at 2196°C appeared to perform better on the mucus penetration assay, sperm motility was not assessed in this study. In 1995, Pironcheva and coworkers (13) evaluated the impact of cryopreservation of buffalo sperm when stored at 4°C, 220°C, 270°C, or 2196°C. To evaluate the effect of storage temperature on DNA integrity, they measured DNA
1164 Trummer et al.
Temperature and sperm cryopreservation
The results of the present study indicate that sperm storage at 270°C is linked with a greater loss of sperm motility than is storage at 2196°C. The reduction in motility noted at 1 week, however, was fairly modest. Unfortunately, the loss of sperm motility increased dramatically after 3 months of storage. These results indicate that short-term storage of sperm at 270°C may be a viable option if liquid nitrogen storage facilities are not available. Long-term storage at this temperature, however, appears to be linked with an unacceptable reduction in sperm motility. References 1. Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer-criteria change. Fertil Steril 1992;58:1024 –7. 2. Sherman JK. Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 1973;24:397– 412. 3. Leffler KS, Walters CA. A comparison of time, temperature, and refreezing variables on frozen sperm motility recovery. Fertil Steril 1996;65:272– 4. 4. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus Interaction. 3rd ed. New York: Cambridge University Press, 1992:3–23. 5. Kruger TF, Menkveld R, Standerl FS, Lombard CJ, Van der Merwe JP, Van Zyll JA, et al. Sperm morphologic features as a prognostic factor in vitro fertilization. Fertil Steril 1986;46:1118 –23. 6. Hammitt DG, Hade DK, Williamson RA. Survival of human sperm following controlled- and noncontrolled-rate cryopreservation. Andrologia 1989;21:311–7. 7. Keel BA, Black JB. Reduced motility longevity in thawed human spermatozoa. Arch Androl 1980;4:213–5. 8. Parkes AS. Preservation of human spermatozoa at low temperatures. Br Med J 1945;2:212–3. 9. Bunge RG, Keettele WC, Sherman JK. Clinical use of frozen semen. Fertil Steril 1954;5:520 –9. 10. Sherman JK. Freezing and freeze-drying of human spermatozoa. Fertil Steril 1954;5:357–71. 11. Sherman JK. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril 1963;14:49 – 64. 12. Fjallbrant B, Ackerman DR. Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimen. J Reprod Fertil 1969;20:515–7. 13. Pironcheva GL, Miteva K, Vaisberg C, Russev G. Influence of freezing at different temperatures on the transcription activity of buffalo sperm chromatin. Cytobios 1995;83:207–10.
Vol. 70, No. 6, December 1998