STERILITY-FERTILITY
SPERM HYPERACTIVATION AS QUALITY CONTROL FOR SPERM PENETRATION ASSAY PEIILIP J. CHAN, P1r.D. DONALD R. TREDWAY. BRIAN C. SU, M.D.
M.D.,
Prr.D.
JOHANNAH CORSELLI. Pir I). BERT J. DAVIDSON, M.D.. PII. D. MASAO SAKUGAWA. M.D.
From the AndrologyiMale Reproduction Laboratory. Loma Linda liniversity Gynecology and Obstetrics Medical Group, Inc., Ioma Linda, California; and the Department of Obstetrics and Gynecology, Ryukyu University School of h4edicine. Okinawa, Japan
ABSTRACTThe sperm penetration assay (SPA) is subject to considerable va riation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperactivation (HA) can Serve as a quality control check for the SPA. The objective was to determine if there was an association between the SP4 outcome and sperm HA measured at various times during the SPA procedurc. The data showed a significant correlation between percent sperm 11.4 and percent xona-free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm huperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after ten trifuge washing of sperm. The results sugge,st that it is possible to utilize sperm HA measured immediutely after the .sperm-oocyte interaction period as a quality control check of SP4 resu 1ts .
Human sperm undergo a distinctive form of motility termed hyperactivation (HA) which is characterized by star-spin or transitional trajectory motility with wide amplitude head and tail oscillatory movements. lm4 Recently, HA has been subdivided into four categories: helical, thrashing, circling, and star-spin.5 The hyperactive motility of sperm cells in the other animal species has been reviewed.e-lo The acquisition of hyperactive motility is postulated to be a marker of pre-capacitation events in a form of tail capacitation. The human sperm exhibit hyperactivation after sperm washing in defined culture medium and peaks between two to six hours after the start of incubation, the period associated with sperm capacitation.3 Sperm hyperactivation is influenced by inter-
donor variability, albumin concentration in the medium, calcium ions, temperature,” and hyperosmotic conditions. 5 Sperm hyperactivity has been observed during the sperm penetration assay (SPA) or zona-free hamster oocyte test. I2 The SPA results are subject to variation mainly because of the many variables such as length of abstinence and incubation temperature that can affect the SPA outcome.13 In the present study, it is proposed that sperm HA can serve as a supplemental quality control check for the SPA in addition to control sperm penetration rates. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during: the SPA procedure.
TABLE
I.
coefficients for sperm hyperactivation and sperm penetration assay (SPA)
Specimen Raw semen Immediately after sperm wash After sperm oocyte interaction Material
Type of Preincubation Short Long Short Long Short Long
and Methods
Semen samples Semen specimens were obtained from 20 patients for SPA processing. The abstinence period ranged from two days to eight days with a median of four days. Basic semen analysis was performed on all samples in accordance with the standards of the World Health 0rganization.r4 Sperm Penetration Assay The SPA procedure involved centrifugewashing of the sperm cells.15 The medium used was Biggers, Whitten, Whittingham (BWW) supplemented with 3.5% human mediumi serum albumin (HSA). The centrifuge-washing procedure consisted of mixing an equal volume of the liquefied semen with the BWW medium and centrifuging the mixture at 300 x g for ten minutes in 15 mL centrifuge tubes. The resultant pellets were resuspended in 2 mL of BWW medium and centrifuged again at 300 x g for ten minutes. The final pellets were each resuspended at a concentration of 10 million sperm/ mL and preincubated in a 5% CO2 in air mixture at 37” C for either three hours (short preincubation group) or twenty-one hours (long preincubation group). The hamster oocytes were obtained from commercially available Cryotech straws (Charles River, Wilmington, MA). The oocytes were treated with 0.1% trypsin to remove the zona pellucida. The zona-free oocytes were then incubated in 100 PL sperm preparations for three hours before being analyzed. Controls were carried out for each SPA run using cryopreserved fertile donor sperm. Data with SPA below 50 percent penetration in the controls were omitted. Sperm hyperactivation Aliquots of sperm were removed at the following stages: at the raw semen stage, immediately after centrifuge washing, and at the end
64
(HA)
Correlation
No. of Assays
Correlation Coefficient
14 17 14 17 14 17
0.143 0.106 0.379 0.430 0.688 0.179
Significance P P P P P P
> > = = = >
0.25 0.25 0.07 0.06 0.001 0.25
of the three-hour sperm-zona-free oocyte interaction period for both short- and long-preincubated sperm. Large droplets of sperm (about 100 pL) were placed between glass slides and cover slips where there was ample room for HA motility. The sperm were visually evaluated by light microscopy for HA (star-spin and transitional motility with high amplitude of lateral head displacement and reduced forward progression) . For consistency, the same technician was used throughout the study. The percent HA was based on the number of hyperactive sperm divided by the total number of motile sperm. Data analyses Linear regression statistic with regression correlation coefficient was utilized in the analyses of the data. Significance was determined using the t-test analysis of the regression slope. Outliers were tested according to the residuals method of Grubbs and Beck.i7 As with all correlation studies, indications of correlation does not necessarily imply direct cause and effect. Results The data presented in Table I indicate a significant correlation (r = 0.688, P = 0.001) between sperm HA and SPA results for the short preincubation group. The regression equation between sperm HA (denoted by x) and SPA results (denoted by y) is: y = 1.42 + 0.89x where 0.89 is the slope. The equation was valid for SPA results ranging from 0 to 50 percent. Above 50 percent penetrations, the percent HA plateau off. There was a low correlation (r = 0.179) between sperm HA and SPA results for the long preincubated sperm treatment group. Low correlation was also observed between sperm HA and SPA results when the HA was determined at the raw semen stage (r = 0.106 to 0.143) and immediately after centrifuge sperm washing for both short and long preincubation sperm treatment groups (r = 0.379 to 0.430). The mean
UROLOGY
i
JANUARY
1992 i
VOLUME
XXXIX, NUMBER
1
TABLE II.
Sperm hyperactivation (HA) observed at different times during sperm, penetration (SPA) procedure Raw
Parameter % HA % Motility
Semen Stage 5.2 51.4
f f
1.0 2.4
Immediately After Sperm Washing
After 6 Hrs of Incubation
After 24 Hrs of Incubation
16.3
29.2 53.6
12.3 45.5
+ 1.8 nla
+ 4.0 + 4.9
f 4.9 zk 5.5
Values ;are expressed as mean + S. E.M.
percent HA and percent total motility of the sperm samples in the study are presented in Table II. Comment There is a need for more quality control monitoring of the SPA procedure to provide accurate reporting of SPA results. At the present time, the only quality control for SPA is the percent SPA penetration by control sperm. The significant correlation between percent sperm HA and percent penetration for short preincubation SPA in the range of O-50 percent penetrations observed in the present study suggests that it is possible to utilize sperm HA as a quality control check for SPA results. The advantage of using sperm HA measurements is that hyperactivity can be easily determined from the incubated sperm specimen. One drawback of using sperm HA for SPA quality control is that it cannot be used for thawed sperm after cryopreservation in Test Yolk Buffer-Glycerol Medium possibly due to alterations in post-thaw sperm membrane which changes motility characteristics. The linear regression equation derived for the association between sperm HA and SPA results for short preincubated sperm provides an approximate 1:l association for a rule-ofthumb purpose which means that a 15 percent penetration in the SPA will be associated with about 15 percent sperm hyperactivity in the sample at the end of the three-hour spermzona-free oocyte interaction period. For patients undergoing the IVF procedure, the SPA results should show 20 percent or greater zonafree oocyte penetration.18 Applying the equation, the quality control check using sperm HA should also show 20 percent hyperactivity. Interestingly this value coincides with the mean percent HA of fertile patients (20.2 % ) reported by. Burkman.‘!’ A patient’s semen sample processed for SPA subsequent to this study yielded a 12.5 percent penetration. The quality control check based on the visual determination of sperm HA showed 15 percent hyperactivity, a
value within the percent penetration + 6 percent interval range (in this case, 6.5-18.5%) based on 1 standard deviation, thus supporting the SPA result. Generally, SPA results in the range of O-50 percent become suspect if they differ from the observed percent HA by 10 percent or more. In the present study, sperm Hi4 was weakly correlated to SPA results for the long preincubation treatment group. One possible reason may be due to the decline in HA” which coincides with an overall decline in sperm motility after prolonged incubation. There were two extreme data points in the group which when checked, l7 did not appear to be regression outliers but rather biological variations. A larger sample size will be utilized in a future study to further examine the relationship of HA to SPA in sperm in the long preincubation treatment group. An interesting observation in the study was the differences in correlation between SPA results and sperm HA determined at different times during the SPA procedure. The time for observing sperm HA has not been standardized. While it is not useful to measure HA in the liquefied raw semen sample, the percent HA determined immediately after sperm centrifuge washing does indicate a low correlation to SPA results for both the short and long preincubation groups. More studies will be needed to determine the usefulness of sperm HA measured after centrifuge washing as a quality control step. It is postulated that sperm hyperactive motility is a marker of the ability of the sperm to fertilize oocytes. Indirect evidence supporting this hypothesis comes from recent studies showing a positive correlation between sperm HA and fertile males. I9 Wolf” described a failed in vitro fertilization (IVF) case which was associated with low sperm HA. Robertson, Wolf, and Tash4 proposed that the mechanism for sperm HA may be a removal of absorbed seminal plasma components from the sperm surface which triggers changes in intracellular
regulatory components such as cyclic adenosine 3 ‘, 5 ‘-monophosphate or Ca2+ ions that influence motility patterns. The acquisition of hyperactive motility is postulated to be an initial step toward capacitation and acrosome reaction, the latter steps being necessary for sperm penetration of oocytes. The present study is consistent with this postulate by showing a positive correlation between HA and sperm penetration of oocytes. In summary, the data showed a strong linear correlation between SPA results in the range of O-50 percent and sperm HA measured immediately after sperm-zona-free oocyte interaction for short preincubation SPA. The results suggest the possibility of using percent sperm HA as a quality control check of the SPA results. Loma Linda, California 92354 (DR. CHAN) ACKNOWLEDGMENT. To nurses Carole Green, Barbara Shipp, Jean Fortuna, and technicians Kim Werges and Monica Spentz for their technical help and support.
References 1. Perrault S, and Rogers BJ: Capacitation pattern of human spermatozoa, Fertil Steril 38: 258 (1982). 2. Mortimer D, Courtot AM, Giovangrandi Y, and Jeulin C: Do capacitated human spermatozoa show an “activated” pattern of motilitv?. in Martinus AT (Ed): The Suerm Cell. Hineham. D , MA, Nijhbff Publishers, 1983,‘pp’349-352: 3. Burkman LJ: Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations, Arch Androl 13: 153 (1984). 4. Robertson L, Wolf DP, and Tash JS: Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm, Biol Reprod 39: 797 (1988). 5. Burkman LJ: Movement characteristics of four classes of hu-
66
man sperm hyperactivation: criteria for computerized idegtification, Proc 45th Annu Mtg Am Fertil Sot 1989, p 66. 6. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation, J Reprod Fertil 23: 193 (1970). 7. Fraser LR: Motility patterns in mouse spermatozoa before and after capacitation, J Exp Zoo1 202: 439 (1977). 8. lohnson LL. Katz DF. and Overstreet IW: The movement characteristics of rabbit spermatozoa before and after activation, Gamete Res 4: 275 (1981). 9. Cummins JM: Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes, Gamete Res 6: 53 (1982). 10. Suarez SS, Vincenti L, and Ceglia MW: Hyperactivated motility induced in mouse sperm by calcium A23187 is reversible, J Exp Zoo1 244: 331 (1987). 11. Wolf DP: Sperm hyperactivation as a component of capacitation: diagnostic significance, in: Advances in Sperm-Egg Interaction: Diagnostic and Treatment Approaches in Andrology and Embryo Culture, Course V. Twenty-First Annual Postgraduate Course, October 8 to 9, 1988; Atlanta, Georgia. Sponsored by The American Fertility Society, 1988, Norfolk, VA, pp 45-70. 12. Jinno M, Burkman LJ, and Coddington CC: Correlation between hyperactivation-like (HAL) motility of human spermatozoa and penetration of hamster ova, Proc 5th World Congress on In Vitro Fertil and Embryo Transfer 1987, p 137. 13. Rogers BJ: The sierm penetration -assay: its usefulness reevaluated. Fertil Steril 43: 821 (1985). 14. World Health Organization‘: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, Cambridge University Press, Cambridge, 1987. 15. Margolioth EJ, et al: The long-term predictive value of the zona-free hamster ova sperm penetration assay, Fertil Steril 52: 490 (1989). 16. Biggers JD, Whitten WK, and Whittingham DG: The culture of mouse embryos in vitro, in Daniel JD (Ed): Methods of Mammalian Embryology, San Francisco, Freeman and Sons, 1971, pp 86-116. 17. Grubbs FE, and Beck G: Extension of sample sizes and percentage points for significance tests of outlying observations, Technometrics 14: 847 (1972). 18. Yang YS, Rojas FJ, and Stone SC: Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test, Fertil Steril 50: 954 (1988). 19. Burkman LJ: Experimental approaches to evaluation and enhancement of sperm function, in Jones HW Jr, Jones GS, Hodgen GD, and Rosenwaks Z (Eds): In Vitro Fertilization: Norfolk, Williams and Wilkins Publishers, Baltimore, MD, 1986, pp 201-214.
UROLOGY
i JANUARY
1992
i
VOLUME
XXXIX. NUMBER
1
SPERM HYPERACTIVATION AS QUALITY CONTROL FOR SPERM PENETRATION ASSAY PEIILIP J. CHAN, P1r.D. DONALD R. TREDWAY. BRIAN C. SU, M.D.
M.D.,
Prr.D.
JOHANNAH CORSELLI. Pir I). BERT J. DAVIDSON, M.D.. PII. D. MASAO SAKUGAWA. M.D.
From the AndrologyiMale Reproduction Laboratory. Loma Linda liniversity Gynecology and Obstetrics Medical Group, Inc., Ioma Linda, California; and the Department of Obstetrics and Gynecology, Ryukyu University School of h4edicine. Okinawa, Japan
ABSTRACTThe sperm penetration assay (SPA) is subject to considerable va riation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperactivation (HA) can Serve as a quality control check for the SPA. The objective was to determine if there was an association between the SP4 outcome and sperm HA measured at various times during the SPA procedurc. The data showed a significant correlation between percent sperm 11.4 and percent xona-free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm huperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after ten trifuge washing of sperm. The results sugge,st that it is possible to utilize sperm HA measured immediutely after the .sperm-oocyte interaction period as a quality control check of SP4 resu 1ts .
Human sperm undergo a distinctive form of motility termed hyperactivation (HA) which is characterized by star-spin or transitional trajectory motility with wide amplitude head and tail oscillatory movements. lm4 Recently, HA has been subdivided into four categories: helical, thrashing, circling, and star-spin.5 The hyperactive motility of sperm cells in the other animal species has been reviewed.e-lo The acquisition of hyperactive motility is postulated to be a marker of pre-capacitation events in a form of tail capacitation. The human sperm exhibit hyperactivation after sperm washing in defined culture medium and peaks between two to six hours after the start of incubation, the period associated with sperm capacitation.3 Sperm hyperactivation is influenced by inter-
donor variability, albumin concentration in the medium, calcium ions, temperature,” and hyperosmotic conditions. 5 Sperm hyperactivity has been observed during the sperm penetration assay (SPA) or zona-free hamster oocyte test. I2 The SPA results are subject to variation mainly because of the many variables such as length of abstinence and incubation temperature that can affect the SPA outcome.13 In the present study, it is proposed that sperm HA can serve as a supplemental quality control check for the SPA in addition to control sperm penetration rates. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during: the SPA procedure.
TABLE
I.
coefficients for sperm hyperactivation and sperm penetration assay (SPA)
Specimen Raw semen Immediately after sperm wash After sperm oocyte interaction Material
Type of Preincubation Short Long Short Long Short Long
and Methods
Semen samples Semen specimens were obtained from 20 patients for SPA processing. The abstinence period ranged from two days to eight days with a median of four days. Basic semen analysis was performed on all samples in accordance with the standards of the World Health 0rganization.r4 Sperm Penetration Assay The SPA procedure involved centrifugewashing of the sperm cells.15 The medium used was Biggers, Whitten, Whittingham (BWW) supplemented with 3.5% human mediumi serum albumin (HSA). The centrifuge-washing procedure consisted of mixing an equal volume of the liquefied semen with the BWW medium and centrifuging the mixture at 300 x g for ten minutes in 15 mL centrifuge tubes. The resultant pellets were resuspended in 2 mL of BWW medium and centrifuged again at 300 x g for ten minutes. The final pellets were each resuspended at a concentration of 10 million sperm/ mL and preincubated in a 5% CO2 in air mixture at 37” C for either three hours (short preincubation group) or twenty-one hours (long preincubation group). The hamster oocytes were obtained from commercially available Cryotech straws (Charles River, Wilmington, MA). The oocytes were treated with 0.1% trypsin to remove the zona pellucida. The zona-free oocytes were then incubated in 100 PL sperm preparations for three hours before being analyzed. Controls were carried out for each SPA run using cryopreserved fertile donor sperm. Data with SPA below 50 percent penetration in the controls were omitted. Sperm hyperactivation Aliquots of sperm were removed at the following stages: at the raw semen stage, immediately after centrifuge washing, and at the end
64
(HA)
Correlation
No. of Assays
Correlation Coefficient
14 17 14 17 14 17
0.143 0.106 0.379 0.430 0.688 0.179
Significance P P P P P P
> > = = = >
0.25 0.25 0.07 0.06 0.001 0.25
of the three-hour sperm-zona-free oocyte interaction period for both short- and long-preincubated sperm. Large droplets of sperm (about 100 pL) were placed between glass slides and cover slips where there was ample room for HA motility. The sperm were visually evaluated by light microscopy for HA (star-spin and transitional motility with high amplitude of lateral head displacement and reduced forward progression) . For consistency, the same technician was used throughout the study. The percent HA was based on the number of hyperactive sperm divided by the total number of motile sperm. Data analyses Linear regression statistic with regression correlation coefficient was utilized in the analyses of the data. Significance was determined using the t-test analysis of the regression slope. Outliers were tested according to the residuals method of Grubbs and Beck.i7 As with all correlation studies, indications of correlation does not necessarily imply direct cause and effect. Results The data presented in Table I indicate a significant correlation (r = 0.688, P = 0.001) between sperm HA and SPA results for the short preincubation group. The regression equation between sperm HA (denoted by x) and SPA results (denoted by y) is: y = 1.42 + 0.89x where 0.89 is the slope. The equation was valid for SPA results ranging from 0 to 50 percent. Above 50 percent penetrations, the percent HA plateau off. There was a low correlation (r = 0.179) between sperm HA and SPA results for the long preincubated sperm treatment group. Low correlation was also observed between sperm HA and SPA results when the HA was determined at the raw semen stage (r = 0.106 to 0.143) and immediately after centrifuge sperm washing for both short and long preincubation sperm treatment groups (r = 0.379 to 0.430). The mean
UROLOGY
i
JANUARY
1992 i
VOLUME
XXXIX, NUMBER
1
TABLE II.
Sperm hyperactivation (HA) observed at different times during sperm, penetration (SPA) procedure Raw
Parameter % HA % Motility
Semen Stage 5.2 51.4
f f
1.0 2.4
Immediately After Sperm Washing
After 6 Hrs of Incubation
After 24 Hrs of Incubation
16.3
29.2 53.6
12.3 45.5
+ 1.8 nla
+ 4.0 + 4.9
f 4.9 zk 5.5
Values ;are expressed as mean + S. E.M.
percent HA and percent total motility of the sperm samples in the study are presented in Table II. Comment There is a need for more quality control monitoring of the SPA procedure to provide accurate reporting of SPA results. At the present time, the only quality control for SPA is the percent SPA penetration by control sperm. The significant correlation between percent sperm HA and percent penetration for short preincubation SPA in the range of O-50 percent penetrations observed in the present study suggests that it is possible to utilize sperm HA as a quality control check for SPA results. The advantage of using sperm HA measurements is that hyperactivity can be easily determined from the incubated sperm specimen. One drawback of using sperm HA for SPA quality control is that it cannot be used for thawed sperm after cryopreservation in Test Yolk Buffer-Glycerol Medium possibly due to alterations in post-thaw sperm membrane which changes motility characteristics. The linear regression equation derived for the association between sperm HA and SPA results for short preincubated sperm provides an approximate 1:l association for a rule-ofthumb purpose which means that a 15 percent penetration in the SPA will be associated with about 15 percent sperm hyperactivity in the sample at the end of the three-hour spermzona-free oocyte interaction period. For patients undergoing the IVF procedure, the SPA results should show 20 percent or greater zonafree oocyte penetration.18 Applying the equation, the quality control check using sperm HA should also show 20 percent hyperactivity. Interestingly this value coincides with the mean percent HA of fertile patients (20.2 % ) reported by. Burkman.‘!’ A patient’s semen sample processed for SPA subsequent to this study yielded a 12.5 percent penetration. The quality control check based on the visual determination of sperm HA showed 15 percent hyperactivity, a
value within the percent penetration + 6 percent interval range (in this case, 6.5-18.5%) based on 1 standard deviation, thus supporting the SPA result. Generally, SPA results in the range of O-50 percent become suspect if they differ from the observed percent HA by 10 percent or more. In the present study, sperm Hi4 was weakly correlated to SPA results for the long preincubation treatment group. One possible reason may be due to the decline in HA” which coincides with an overall decline in sperm motility after prolonged incubation. There were two extreme data points in the group which when checked, l7 did not appear to be regression outliers but rather biological variations. A larger sample size will be utilized in a future study to further examine the relationship of HA to SPA in sperm in the long preincubation treatment group. An interesting observation in the study was the differences in correlation between SPA results and sperm HA determined at different times during the SPA procedure. The time for observing sperm HA has not been standardized. While it is not useful to measure HA in the liquefied raw semen sample, the percent HA determined immediately after sperm centrifuge washing does indicate a low correlation to SPA results for both the short and long preincubation groups. More studies will be needed to determine the usefulness of sperm HA measured after centrifuge washing as a quality control step. It is postulated that sperm hyperactive motility is a marker of the ability of the sperm to fertilize oocytes. Indirect evidence supporting this hypothesis comes from recent studies showing a positive correlation between sperm HA and fertile males. I9 Wolf” described a failed in vitro fertilization (IVF) case which was associated with low sperm HA. Robertson, Wolf, and Tash4 proposed that the mechanism for sperm HA may be a removal of absorbed seminal plasma components from the sperm surface which triggers changes in intracellular
regulatory components such as cyclic adenosine 3 ‘, 5 ‘-monophosphate or Ca2+ ions that influence motility patterns. The acquisition of hyperactive motility is postulated to be an initial step toward capacitation and acrosome reaction, the latter steps being necessary for sperm penetration of oocytes. The present study is consistent with this postulate by showing a positive correlation between HA and sperm penetration of oocytes. In summary, the data showed a strong linear correlation between SPA results in the range of O-50 percent and sperm HA measured immediately after sperm-zona-free oocyte interaction for short preincubation SPA. The results suggest the possibility of using percent sperm HA as a quality control check of the SPA results. Loma Linda, California 92354 (DR. CHAN) ACKNOWLEDGMENT. To nurses Carole Green, Barbara Shipp, Jean Fortuna, and technicians Kim Werges and Monica Spentz for their technical help and support.
References 1. Perrault S, and Rogers BJ: Capacitation pattern of human spermatozoa, Fertil Steril 38: 258 (1982). 2. Mortimer D, Courtot AM, Giovangrandi Y, and Jeulin C: Do capacitated human spermatozoa show an “activated” pattern of motilitv?. in Martinus AT (Ed): The Suerm Cell. Hineham. D , MA, Nijhbff Publishers, 1983,‘pp’349-352: 3. Burkman LJ: Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations, Arch Androl 13: 153 (1984). 4. Robertson L, Wolf DP, and Tash JS: Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm, Biol Reprod 39: 797 (1988). 5. Burkman LJ: Movement characteristics of four classes of hu-
66
man sperm hyperactivation: criteria for computerized idegtification, Proc 45th Annu Mtg Am Fertil Sot 1989, p 66. 6. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation, J Reprod Fertil 23: 193 (1970). 7. Fraser LR: Motility patterns in mouse spermatozoa before and after capacitation, J Exp Zoo1 202: 439 (1977). 8. lohnson LL. Katz DF. and Overstreet IW: The movement characteristics of rabbit spermatozoa before and after activation, Gamete Res 4: 275 (1981). 9. Cummins JM: Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes, Gamete Res 6: 53 (1982). 10. Suarez SS, Vincenti L, and Ceglia MW: Hyperactivated motility induced in mouse sperm by calcium A23187 is reversible, J Exp Zoo1 244: 331 (1987). 11. Wolf DP: Sperm hyperactivation as a component of capacitation: diagnostic significance, in: Advances in Sperm-Egg Interaction: Diagnostic and Treatment Approaches in Andrology and Embryo Culture, Course V. Twenty-First Annual Postgraduate Course, October 8 to 9, 1988; Atlanta, Georgia. Sponsored by The American Fertility Society, 1988, Norfolk, VA, pp 45-70. 12. Jinno M, Burkman LJ, and Coddington CC: Correlation between hyperactivation-like (HAL) motility of human spermatozoa and penetration of hamster ova, Proc 5th World Congress on In Vitro Fertil and Embryo Transfer 1987, p 137. 13. Rogers BJ: The sierm penetration -assay: its usefulness reevaluated. Fertil Steril 43: 821 (1985). 14. World Health Organization‘: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, Cambridge University Press, Cambridge, 1987. 15. Margolioth EJ, et al: The long-term predictive value of the zona-free hamster ova sperm penetration assay, Fertil Steril 52: 490 (1989). 16. Biggers JD, Whitten WK, and Whittingham DG: The culture of mouse embryos in vitro, in Daniel JD (Ed): Methods of Mammalian Embryology, San Francisco, Freeman and Sons, 1971, pp 86-116. 17. Grubbs FE, and Beck G: Extension of sample sizes and percentage points for significance tests of outlying observations, Technometrics 14: 847 (1972). 18. Yang YS, Rojas FJ, and Stone SC: Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test, Fertil Steril 50: 954 (1988). 19. Burkman LJ: Experimental approaches to evaluation and enhancement of sperm function, in Jones HW Jr, Jones GS, Hodgen GD, and Rosenwaks Z (Eds): In Vitro Fertilization: Norfolk, Williams and Wilkins Publishers, Baltimore, MD, 1986, pp 201-214.
UROLOGY
i JANUARY
1992
i
VOLUME
XXXIX. NUMBER
1
