Concentration of glycerol required for optimal survival and in vitro fertilizing capacity of frozen sperm is dependent on cryopreservation medium

Transcription

1 FERTILITY AND STERILITY Copyright e 1988 The American Fertility Society Printed in U.S.A. Concentration of glycerol required for optimal survival and in vitro fertilizing capacity of frozen sperm is dependent on cryopreservation medium Diane G. Hammitt, Ph.D.*t:l: David L. Walker, B.S.* Roger A. Williamson, M.D.* University of Iowa Hospitals and Clinic, Iowa City, Iowa Sperm survival and in vitro fertilizing capacity were examined following cryopreservation with three concentrations of glycerol (, 2, and %) and four cryopreservation media. Post-thaw motility and motility index increased with increasing concentrations of glycerol. Post-thaw velocity, linearity, percentage of zona-free hamster oocytes penetrated, and penetrations per oocyte were greater following freezing with 2 or % glycerol than with % glycerol. A significant interaction between media and glycerol was observed for motility, velocity, linearity, motility index, and percentage of oocytes penetrated. These results suggest that the concentration of glycerol required for optimal survival and in vitro fertilizing capacity of human sperm following cryopreservation is dependent on the type of medium used for freezing. Fertil Steril 49:68, 1988 Frozen-thawed sperm remain viable 1 and fertile 2 for shorter periods of time in vitro than nonfrozen sperm. The lower pregnancy rates following therapeutic insemination by donor (TID) achieved with frozen compared with fresh semen may be due to the shorter fertile life span of frozen sperm. Glycerol has been the primary cryoprotectant for freezing human semen since the first successful procedures were reported in The optimal concentration of glycerol for freezing human semen has been suggested to be between 5 and 1%. 4 This recommendation was based primarily on post-thaw motility prior to glycerol removal. Other more recent studies have called these find- Received August 17, 1987; revised and accepted December 16, *Department of Obstetrics and Gynecology. t Reprint requests: Diane G. Hammitt, Ph.D., Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, Iowa City, Iowa :j: Department of Urology. ings into question. 5-7 Sperm leave the glycerol in which they were frozen during their transit to the site of fertilization. Removal of human 5 and boar sperm 8 9 from glycerol appears to result in osmotic damage to sperm, which may, in turn, reduce the sperm's fertile life span. When boar sperm were frozen with greater than 6% glycerol, the extent of osmotic damage to sperm was so severe that a complete loss of fertilizing capacity occurred, while maximal fertility was retained with 1% to 2% glycerol. 8 9 Jeyendran et al. 5 recently compared human sperm viability and fertilizing capacity following cryopreservation with 1% glycerol and a medium buffered with zwitter ions devoid of glycerol (TEST-citrate [TESTCY]). Supplementation with glycerol resulted in higher motility immediately post-thaw, but lower motility following glycerol removal and in vitro incubation, reduced rates of penetration of zona-free hamster oocytes, and lower levels of acrosin/proacrosin. The results of this study suggested that lower concentrations of glycerol might increase the fertile life span of cryopreserved sperm by decreasing the 68 Hammitt et al. Cryopreservation of human sperm Fertility and Sterility

2 osmotic damage that sperm sustain upon glycerol removal. The current study was undertaken for the following reasons: (1) to compare human sperm viability and fertilizing capacity following cryopreservation with, 2, and % glycerol; (2) to examine whether cryoprotective media other than TESTCY could be used for freezing human semen in the absence of glycerol; (3) to examine whether the optimal concentration of glycerol for freezing semen was dependent on the chemical composition of the medium in which sperm were frozen; (4) to determine whether sperm motility, velocity, linearity, and/or motility index immediately following thawing were related to their ability to fertilize zona-free hamster oocytes; and (5) to determine whether 1-ml cryovials could be used rather than the.1-ml pellets used by Jeyendran et al. 5 for freezing semen without glycerol, since the latter method of freezing is not recommended for semen to be used for TID. 1 MATERIALS AND METHODS A two-factor crossed design was used with four types of media and three concentrations of glycerol. Three media, human sperm preservation medium (HSPM), 4 TESTCY, 5 11 and yolk-citrate-dextrose-glycine (YCDG), 12 were selected for use in this study because they had previously been used successfully for cryopreservation of semen for TID. One medium, TEST-dextrose (TESTDEX) was selected for use because it had previously been shown to result in an increased rate of penetration of zona-free hamster oocytes when used in conjunction with cooling to 2 C to 5 CP were prepared as described previously containing egg yolk were centrifuged for 3 minutes at 5 X g to remove large particles of yolk. Glycerol was added to each type of media to a final concentration of, 4, and 15% (vol/vol). The 12 mediaglycerol combinations were stored in 1-ml volumes at -2 C and used within 2 months of preparation. Semen was obtained from eight donors of normal fertility. Ejaculates were collected by masturbation after 2 to 5 days of sexual abstinence into sterile specimen containers (no. 461, American Pharmaseal Co., Valencia, CA). Minimal seminal quality for acceptance of ejaculates was ~1 ml volume, ~5 X 1 6 sperm/ml, ~5% motility, and ~6% normal-oval sperm. Ejaculates were obtained for semen cryopreservation from each of the eight donors on 5 separate days over a 4-week period. Three cryovials (no. D-5233, Sarstedt, Princeton, NJ) of semen were frozen on each of the 5 days for each media-glycerol combination. Following a 2- to 4-minute period of liquefaction, semen from the eight donors was pooled, thoroughly mixed, and diluted with an equal volume (.5 ml) of media in the cryovials. Semen was frozen at a controlled rate of -.5 C/min to -1 C, -5 C/min from -1 octo -4 C, and -1 C/min from -4 C to -1 C with a CryoMed freezer/controller (Mt. Clemens, MI). The interval between semen collection and freezing was always less than 2 hours. Semen was stored at -196 C in liquid nitrogen for 3 to 4 months prior to thawing. Semen was thawed in a 24 C water bath for 5 minutes, then transferred to a 37 C heat block. Twelve vials of semen, one for each media-glycerol combination, were thawed simultaneously. Seminal analyses were performed after the semen had incubated 5 to 25 minutes at 37 C (post-thaw) and, 4, 8, and 24 hours following sperm washing. A computerized semen analyzer (Cell Soft, Cryo Resources, New York, NY), calibrated and verified for accuracy on a daily basis, was used for determination of motility, velocity, linearity, and motility index. Sperm were washed with Biggers, Whitten, and Whittingham (BWW) medium supplemented with.4% bovine serum albumin (Cohn Fr V, Sigma Chemical Company, St. Louis, MO), as described previously. 15 Sperm penetration assays (SPAs) were performed according to Karp et au 5 after incubating the washed sperm for 4 hours at 37 C in 5% C 2 in air. The final insemination concentration was 5 X 1 6 sperm/mi. Three hours following insemination, oocytes were washed free of loosely bound sperm, mounted on slides, fixed with 25% acetic acid in ethanol for 18 to 24 hours, and stained with.25% lacmoid in 45% acetic acid. Thirty to 4 oocytes were evaluated for each media-glycerol combination for percentage of oocytes penetrated and penetrations per oocyte. Data for motility, velocity, linearity, and motility index were statistically analyzed by repeated measures analysis of variance 16 for between (glycerol, media, and interaction) and within (time and interaction) vial effects. Data for percentage of zona-free hamster oocytes penetrated and penetrations per oocyte were analyzed by two-way analysis ofvariance. 17 Tukey's "studentized range test" was used to evaluate differences among means for percent glycerol and type of media. 17 Hammitt et al. Cryopreservation of human sperm 681

3 RESULTS Prior to freezing, sperm motility, velocity, linearity, motility index, and concentration of the pooled semen for the 5 days of collection averaged 68.9 ± 9.2% (standard deviation), 45.1 ± 5.2 J.lml sec, 6.4 ±.5, 31.4 ± 6.8 J.lm/sec and X 1 6 ± 19.8 sperm/ml, respectively. Post-thaw motility, velocity, linearity, and motility index for all mediaglycerol combinations were 3, 93, 78, and 3% of their original prefreeze values, respectively. Motility, velocity, linearity, and motility index following freezing for all media-glycerol combinations over the entire 24-hour period of incubation averaged 13.1 ±.5% (standard error), 42. ± 1. J.lm/sec, 4.5 ±.1 and 6.2 ±.2 J.lm/sec, respectively. Results of analyses of post-thaw motility, velocity, linearity, motility index, and penetration of zona-free hamster oocytes following cryopreservation are presented in Tables 1 to 5 for each mediaglycerol combination, concentration of glycerol, and type of media. Data are presented for analyses performed immediately following thawing (postthaw) and after sperm washing. There was a significant effect of glycerol on motility (P <.1). For all time periods, motility Table 2 Sperm Velocity Post-thaw and, 4, 8, and 24 Hours Following Sperm Washing Hours post-wash - %glycerol Post-thaw HSPM- 41.1" HSPM HSPM TESTCY TESTCY TESTCY TESTDEX TESTDEX TESTDEX YCDG YCDG YCDG ' 47.8' 51.3' 48.6' 42.9' ' HSPM 47.o TESTCY TESTDEX 4.9' 45.o YCDG 37.9' 39.7' 39.1' X±SE 4 ± ± ± ± ± 1. b-e Means within columns, for a given main effect, with different superscripts are significantly different (P <.5). Table 1 Percent Sperm Motility Post-thaw and, 4, 8, and 24 Hours Following Sperm Washing Hours post-wash - %glycerol Post-thaw 4 8 HSPM- 9.5" HSPM HSPM TESTCY TESTCY TESTCY TESTDEX TESTDEX TESTDEX YCDG YCDG YCDG ' 1'.6'.6' HSPM 23.6' ' 11. TESTCY 2.1'.2' 13.5' 13.4 TESTDEX 18.7' 13.6' 13.st 13.5 YCDG 19.2' ' X±SE 2.4 ± ± ± ± ±.3 b-f Means within columns, for a given main effect, with different superscripts are significantly different (P <.5). increased with increasing concentrations of glycerol (% < 2% < %). A significant effect of media also was found (P <.3). The YCDG media performed the poorest when motilities were averaged over all concentrations of glycerol. The glycerol X media interaction was significant for motility (P <.1). The cause of this interaction appeared to be the higher motility for HSPM than for other media at % glycerol, and the larger increase in motility observed for YCDG than for other media with increasing concentrations of glycerol. There was a significant decrease in motility with time for all media-glycerol combinations (P <.1). A significant time X glycerol interaction was attributed to the larger decrease in motility observed with time with increasing concentrations of glycerol due to the higher post-thaw motilities for greater concentrations of glycerol. A time X media interaction also was observed (P <.1). This was due to the unequal decrease in motility over the 24-hour period of incubation for the four types of media. The decrease for HSPM, TESTCY, TESTDEX, and YCDG during the 24- hour period of incubation was 18, 12, 11, and 12%, 682 Hammitt et al. Cryopreservation of human sperm Fertility and Sterility

4 Table 3 Sperm Linearity Post-thaw and, 4, 8, and 24 Hours Following Sperm Washing % glycerol HSPM- HSPM-2 HSPM- TESTCY- TESTCY-2 TESTCY- TESTDEX- TESTDEX-2 TESTDEX- YCDG- YCDG-2 YCDG- 2 HSPM TESTCY TESTDEX YCDG Post-thaw b b o Hours post-wash b 5.4c b b d 4.9 d e 5.2d X±SE 5. ±.1 4.7±.1 4.8±.1 4.6± ±.1 b-f Means within columns, for a given main effect, with different superscripts are significantly different (P <.5). Table 4 Sperm Motility Index Post-thaw and, 4, 8, and 24 Hours Following Sperm Washing % glycerol HSPM- HSPM-2 HSPM- TESTCY- TESTCY-2 TESTCY- TESTDEX- TESTDEX-2 TESTDEX- YCDG- YCDG-2 YCDG- 2 HSPM TESTCY TESTDEX YCDG X±SE Post-thaw 4.3" b 1.3b 1.4c 7.3c 1d 1.d 11.6e ' ' ' ' 9.3 ± ±.1 Hours post-wash b-f Means within columns, for a given main effect, with different superscripts are significantly different (P <.5) ' 7.2' ± ± ±.2 Table 5 % glycerol HSPM- HSPM-2 HSPM- TESTCY- TESTCY-2 TESTCY- TESTDEX- TESTDEX-2 TESTDEX- YCDG- YCDG-2 YCDG- 2 HSPM TESTCY TESTDEX YCDG X±SE Results of Sperm Penetration Assay n Oocytes penetrated % 6.1" b.7c 12.5c ±.6 Penetrations per oocyte, b.17c.13c ±.1 b,c Means within columns, for a given main effect, with different superscripts are significantly different (P <.5). respectively. Before washing, motilities were highest for HSPM and TESTCY, but after washing, motilities were highest for TESTCY and TESTDEX. Sperm velocity was significantly greater for 2 and % glycerol than for % glycerol for all time periods (glycerol effect, P <.1). There was a significant effect of media on velocity (P <.1). Velocity was lower for YCDG than for HSPM, TESTCY, and TESTDEX. The significant interaction between media and glycerol (P <.1) for velocity appeared to be due to the higher velocity for HSPM than other media at % glycerol, and the requirement of YCDG for higher levels of glycerol than HSPM, TESTCY, or TESTDEX for maintenance of good velocity. There was a significant decrease in velocity with time for all media (P <.1). A significant time X glycerol interaction was found (P <.1). This was due to the greater decrease in velocity with time for % than for 2% or % glycerol (decrease of.6, 3.9, and 3.3 J.Lm/sec, respectively, over the 24-hour period of incubation). Linearity was significantly less for % glycerol than 2% or % glycerol for all time periods (glycerol effect, P <.1). The significant effect of Hammitt et al. Cryopreservation of human sperm 683

5 media on linearity (P <.1) was primarily due to linearity being lower for YCDG than other media. The significant interaction between glycerol and media appeared to be due to the lower linearity for YCDG than other media at % glycerol, and equivalent linearity with other media at 2 and % glycerol. Linearity decreased significantly with time for all media (P <.1). There was a greater decrease in linearity with time for % than 2% or % glycerol (, 1.2, and 1.1, respectively), resulting in a significant interaction between time and glycerol (P <.6). Motility index increased with increasing concentration of glycerol for all time periods (glycerol effect, P <.1). The overall effect of media on motility index was not significant (P <.9). There was a significant interaction between media and glycerol (P <.8). This was expected since this interaction was significant for motility and velocity, and motility index is the product of these values. Motility index decreased significantly over the 24-hour period of incubation for all media-glycerol combinations (P <.1). The significant interactions for time X glycerol (P <.1) and time X media (P <.1) were attributed to the same factors causing these interactions for motility. Significant effects were found for glycerol for percentage of zona-free hamster oocytes penetrated and penetrations per oocyte (P <.1). Higher penetration rates and penetrations per oocyte were found for 2 and % glycerol than % glycerol. The effect of media was not significant. As was the case for motility, the significant interaction between glycerol and media (P <.4) for percentage ofoocytes penetrated appeared to be due to the higher penetration rate for HSPM at % glycerol than other media, and the greater increase in penetration rate for YCDG than for other media with increasing concentration of glycerol. A similar trend (P <.1) was observed for penetrations per oocyte. The correlation between percentage of oocytes penetrated and penetrations per oocyte was.88 (P <.1). A "cumulative fertility score" was calculated for each media-glycerol combination by addition of means (over all times) for motility, velocity, linearity, motility index, percentage of oocytes penetrated, and penetrations per oocyte. These scores are presented in Table 6, along with the increase, which was observed in the score within medium as the concentration of glycerol was increased from % to 15%. A ranking of the media-glycerol combi- Table 6 Cumulative Fertility Scores and Ranking of -Glycerol Combinations Increase in cumulative - Cumulative score from % to %glycerol score" 15% glycerol Rank HSPM HSPM HSPM TESTCY TESTCY TESTCY TESTDEX TESTDEX TESTDEX YCDG YCDG YCDG a Obtained by addition of means, over all times, for motility, velocity, linearity, motility index, percentage of oocytes penetrated, and penetrations per oocyte. nations by score also is shown. The media X glycerol interaction, reported previously for all the individual parameters, is readily apparent in this table. The glycerol X media interaction appeared to be primarily due to (1) the better performance of sperm frozen with HSPM than other media at % glycerol; (2) the large increase in sperm performance for YCDG compared with other media as glycerol was increased from % to %, and (3) the better performance of TESTCY at 2% glycerol than % or % glycerol, in contrast to all other media. DISCUSSION Motilities and penetration rates of zona-free hamster oocytes were lower in the present study than for some studies reported in the past for cryopreserved sperm The freezing procedures used were partially responsible for these lower values, as this study was performed prior to recent publications describing optimal rates of freezing human semen in cryovials. 19 In addition, motility was evaluated objectively in this study, which has been found by our laboratory and others 19 to result in an approximate 1% reduction in motility readings compared with subjective evaluation of motility. The procedure used for washing sperm in this study probably resulted in greater osmotic damage to sperm than what would occur in vivo. The penetration rate of zona-free hamster oocytes by cryopreserved sperm can be increased over that ob- 684 Hammitt et al. Cryopreservation of human sperm Fertility and Sterility

6 served in the present study by adding wash medium in.5-ml aliquots at 5-minute intervals (personal observation). The penetration rate also can be increased by decreasing the preinsemination incubation time, since a high percentage of frozen-thawed sperm have undergone the acrosome reaction immediately following washing. 2 Maximal penetration of zona-free hamster oocytes by frozen sperm occurs at hours post-wash. 2 Lastly, a low insemination concentration (5 X 1 6 sperm/ml) was used in this study so that fertility differences among the various media-glycerol combinations would be more apparent. For all of these reasons, the 1 to % minimal rate of penetration of zona-free hamster oocytes, set as the lower limit of the fertile range for nonfrozen semen, 15 2 cannot be used in the present study to distinguish between fertile and nonfertile media-glycerol combinations. The increase in motility observed in this study, with increasing concentrations of glycerol without a corresponding increase in sperm fertilizing capacity, has been observed previously for human 5 and boar sperm. 8 9 The data from this study indirectly support conclusions from these studies that (1) sperm are osmotically damaged when they are removed from glycerol and (2) measurement of sperm motility in the presence of glycerol may not be indicative of fertilizing capacity. Sperm velocity and linearity were better predictors of fertilizing capacity in this study than sperm motility. Postthaw motility of human sperm has been demonstrated by others to be unrelated to the sperm's ability to penetrate zona-free hamster oocytes Jeyendran et al reported that motility following washing and incubation for 2 hours more closely paralleled the oocyte penetrating capacity of sperm than motility immediately following thawing. In our study, motility was a poor predictor of fertilizing capacity before and after sperm washing. Although no relationship was found in this study or others between post-thaw motility and in vitro fertilizing capacity, significant correlations have been demonstrated between post-thaw motility and pregnancy rates following TID of cryopreserved semen In these studies, correlations between pregnancy outcome and various parameters of seminal quality were examined. Post-thaw motility was found to be more highly correlated with pregnancy than prefreeze seminal quality (seminal volume, sperm per ejaculate, motility, morphology), post-thaw motile sperm per inseminate, or decrease in motility during cryopreservation Velocity and linearity, which appeared to be more closely related to fertilizing potential in the present study, were not measured in these studies We are presently evaluating the relationship between pregnancy outcome following TID and post-thaw motility, velocity, linearity, and motility index to determine whether, similar to the situation in vitro, velocity and linearity are more highly correlated with in vivo fertility than with motility. Serafini et au 8 suggested that post-thaw motility could be used as a clinical marker of freezing quality following observance of a significant correlation (r =.98) between post-thaw motility and sperm with intact heads. In their study, however, glycerol was not removed from sperm before determination of motility. Because motility and in vitro fertilizing capacity of sperm frozen in glycerol remain high as long as sperm are kept in glycerol, 21 it is uncertain whether motility and sperm with intact heads would correlate following removal of glycerol, as occurs in vivo during sperm transit to the site of fertilization. A study by Jeyendran et al., 21 demonstrating a lower penetration of zona-free hamster oocytes by frozen compared with nonfrozen sperm at equal concentrations of motile sperm, suggests that damage to the sperm head and tail occur independently during cryopreservation. Hence, it seems reasonable to propose that damage to the sperm head and tail must be evaluated separately after glycerol removal. Damage to the sperm head can be evaluated with the SPA, 5 21 transmission electron microscopy/ 8 and/or loss of acrosin/proacrosin from sperm. 23 Based on the results ofthe present study, none of the components of YCDG (yolk, citrate, dextrose, glycine) have appreciable cryoprotective properties on human sperm. Lipoproteins contained in the yolk of YCDG reportedly protect sperm against cold shock, 4 13 but may provide little protection during the periods of extracellular and intracellular ice formation. Cryoprotectants are required during these periods to protect sperm from the damaging effects of increasing salt concentrations and ice crystal formation. Sucrose and albumin 4 in HSPM and zwitter ion buffers 7 in TESTCY and TEST DEX have been thought to protect cells during the periods of extracellular and intracellular ice formation. The ability of TESTCY to better support sperm fertilizing capacity with 2% than % or % glycerol in this study was in agreement with results of a recent report by Lucena and Obando. 6 They tested Hammitt et al. Cryopreservation of human sperm 685

7 glycerol concentrations ranging from % to 5%, and found 3.5% superior for freezing human semen in TESTCY. The interaction observed in this study between glycerol and media suggests that the optimal concentration of glycerol for maintenance of maximal post-thaw fertility differs among the various cryoprotective media currently used for freezing human semen. Thus, the optimal concentration of glycerol for freezing human semen should be determined separately for each type of cryopreservation medium. Some of the differences reported in the past among cryopreservation media in their ability to support sperm survival/fertilizing capacity 4 7 may have been due to comparison of media without prior optimization of concentration of glycerol for each medium. The greatly reduced sperm survival/fertilizing capacity observed in this study for sperm frozen with % glycerol compared with 2% or % glycerol contrasts with results of an earlier study by J eyendran et al. 5 They obtained higher rates of penetration of zona-free hamster oocytes and higher motility 2 hours after sperm washing when sperm were frozen in TESTCY devoid of glycerol than for sperm frozen in 1% glycerol. Similar to the present study, immediate post-thaw motility was lower for TESTCY than 1% glycerol. However, in their study, post-thaw motility for TESTCY was 69% and 84% of that for 1% glycerol (for two different experiments), while in the present study post-thaw motility for % glycerol was only 26, 12, 11, and 5% of that for % glycerol for HSPM, TESTCY, TESTDEX, and YCDG, respectively. The clearly superior results achieved by Jeyendran et al. 5 with TESTCY devoid of glycerol, compared with all media in the present study devoid of glycerol, was most likely due to their use of.1-ml pellets for freezing semen, rather than the 1-ml cryovials used in the present study. Pelletfreezing was not used in this study because we were attempting to develop optimal procedures for freezing human semen for TID. Pellet-freezing is not recommended for TID use due to the problems of handling, labeling, storage, and contamination of semen. 1 Pellet-freezing may result in better survival of sperm frozen with % glycerol because sperm are frozen and thawed at a faster, more uniform rate, compared with cryovials or straws. 24 In addition, with pellet-freezing, there is little or no supercooling of semen during freezing. 8 Although the degree of supercooling can be reduced when sperm are frozen in cryovials or straws by injecting large amounts of liquid nitrogen into the freezing system at the specimen's freezing point (with a controlled rate freezer), it cannot be eliminated completely. Supercooling is one ofthe major causes of damage to sperm during freezing. 8 It may be that glycerol provides protection to human sperm during supercooling. Recent studies indicate that human semen also cannot be successfully frozen in straws with % glycerol. 6 7 The inability to successfully freeze human semen with % glycerol in cryovials or straws, in contrast to successful cryopreservation with pellets, indicates there may be an interaction between optimal concentration of glycerol for freezing human semen and rates of freezing/thawing and/or degree of supercooling during freezing. Although it appears that human semen cannot be successfully frozen in media devoid of glycerol using cryovials or straws, the results of the present study and other recent studies suggest that the optimal concentration of glycerol for freezing human semen may be lower than the 5 to 1% concentration currently used by most semen banks. Cryoprotective media, with concentrations of glycerol ranging from % to % have recently been compared for their ability to support sperm survival following freezing. 6 7 The optimal concentration of glycerol in both studies was 3%. Lucena and Obando 6 found 2% glycerol to be inferior to 3., 3.5, and 4.%, when testing concentrations ranging from % to 5%. A 2% concentration of glycerol was used in the present study because it resulted in maximal fertility of frozen boar sperm and human and boar sperm appear to respond similarly to cryopreservation with glycerol The 2% concentration of glycerol was the optimal concentration in the present study for the TESTCY medium only. Based on the findings of Lucena and Obando, 6 other media in this study also may have performed better with an intermediate concentration of glycerol (than with % or % glycerol) if the intermediate concentration had been slightly higher. Recent recommendations by the American Association of Tissue Banks and Centers for Disease Control that frozen semen be used for TID to decrease the likelihood of disease transmission 1 have resulted in renewed interest in developing better procedures for freezing human semen. Scant attention appears to have been given to the presence of interactions among various cryopreservation 686 Hammitt et al. Cryopreservation of human sperm Fertility and Sterility

8 methodologies in developing optimal procedures for freezing human semen. In contrast, the importance of these interactions in developing effective methods for freezing bovine semen is well known. Interactions have been reported for freezing container X cooling rate, dilution rate, thaw rate; freezing rate X storage temperature, glycerol concentration, thaw rate; thaw rate X glycerol concentration, equilibration time; and media X glycerolization temperature. 25 In developing improved methods for cryopreservation of human semen, it must be kept in mind that (1) alteration in one step of cryopreservation processing can affect the optimal conditions for other steps in processing and (2) development of innovative technologies for freezing human semen may only be applicable for the set of procedures they were developed in conjunction with and may not be transferrable to another set of procedures using different methodologies. REFERENCES 1. Keel BA, Black JB: Reduced motility longevity in thawed human spermatozoa. Arch Androl 4:213, Critser JK, Arneson BW, Aaker DV, Huse-Benda AR, Ball GD: Cryopreservation of human spermatozoa. II. Postthaw chronology of motility and of zona-free hamster ova penetration. Fertil Steril 47:98, Bunge RG, Sherman JK: Fertilizing capacity of frozen human spermatozoa. Nature 172:767, Mahadevan M, Trounson AO: Effect of cryoprotective media and dilution methods on the preservation of human spermatozoa. Andrologia 15:355, Jeyendran RS, Vander Ven HH, Kennedy W, Perez-Pelaez M, Zaneveld LJD: Comparison of glycerol and a zwitter ion buffer system as cryoprotective media for human spermatozoa. J Androl 5:1, Lucena E, Obando H: Comparative analysis of different glycerol levels when used as cryoprotective agents on human spermatozoa. In Andrology: Male Fertility and Sterility, Edited by JD Paulson, A Negro, E Lucena, L Martini. New York, Academic Press, 1986, p Prins GS, Weidel L: A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril 46:7, Graham EF, Crabo BG, Pace MM: Current status of semen preservation in the ram, boar and stallion. XII Biennial Symposium on Animal Reproduction. J Anim Sci, Suppl. II, p. 8, Wilmut I, Polge C: The low temperature preservation of boar spermatozoa. I. The motility and morphology of boar spermatozoa frozen and thawed in the presence of permeating protective agents. Cryobiology :471, Sherman JK: Current status of clinical cryobanking of human semen. In Andrology: Male Fertility and Sterility, Edited by JD Paulson, A Negro, E Lucena, L Martini. New York, Academic Press, 1986, p McCoshen JA, Wodzicki A, Tyson JE: Effectiveness of human semen frozen in TEST-Yolk buffered medium on AID outcome (Abstr). Fertil Steril 42:164, Mayaux MJ, Schwartz D, Czyglik F, David G: Conception rate according to semen characteristics in a series of 15,364 insemination cycles: results of a multivariate analysis. Andrologia 17:9, Bolanos JR, Overstreet JW, Katz DF: Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 C to 5 C. Fertil Steril 39:536, Biggers JD, Whitten WK, Whittingham DF: The culture of mouse embryos in vitro. In Methods of Mammalian Embryology, Edited by JC Daniel. New York, Raven Press, 1971, p Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: The sperm penetration assay: a useful test in the evaluation of male infertility. Obstet Gynecol 57:62, Gill JL, Hafs HD: Analysis of repeated measures of animals. J Anim Sci 33:331, Snedecor GW, Cochran WG: Statistical Methods (7th edition). Ames, la, The Iowa State University Press, Serafini PC, Hauser D, Moyer D, Marrs RP: Cryopreservation of human spermatozoa: correlations of ultrastructural sperm head configuration with sperm motility and ability to penetrate zona-free hamster ova. Fertil Steril46:691, Serafini P, Marrs RP: Computerized staged-freezing technique improves sperm survival and preserves penetration of zona-free hamster ova. Fertil Steril 45:854, Rogers BJ, Van Campen H, Ueno M, LambertH, Bronson R, Hale R: Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil Steril 32:664, Jeyendran RS, VanDer Ven HH, Perez-Pelaez M, Zaneveld LJD: Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa. Andrologia 17:241, David G, Czyglik F, Mayaux MJ, Schwartz D: The success of AID and semen characteristics: study on 89 cycles and 192 ejaculates. Int J Androl 3:613, Goodpasture JC, Zavos PM, Cohen MR, Zaneveld LJD: Effects of various conditions of semen storage on the acrosin system of human spermatozoa. J Rep rod Fertil 63:397, Graham EF: Fundamentals of the preservation of spermatozoa. Proc Natl Acad Sci USA 47:4, Saacke RG: Factors affecting spermatozoan viability from collection to use. Proceedings of the Seventh Technical Conference on Artificial Insemination and Reproduction, April -15, 1978, The National Association of Animal Breeders, Inc. Columbia, MO, 1978, p 3 Hammitt et al. Cryopreservation of human sperm 687