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Assessment of sperm for cryopreservation using the hypoosmotic viability test
FERTILITY AND STERILITY
Vol. 58, No.4, October 1992
Copyright <> 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Assessment of sperm for cryopreservation using the hypoosmotic viability test
Philip J. Chan, Ph.D.*t:j: Donald R. Tredway, M.D., Ph.D.*:j: Samuel C. Pang, M.D.*
Johannah Corselli, Ph.D.* Brian C. Su, M.D.*
Loma Linda University Sclwol of Medicine, Loma Linda, California
The cryopreservation methods for the freezing of sperm cells as backup specimens for assisted reproductive technologies, as client depositors, for husband or for donor inseminations have been well established (1). Cryopreservation is currently standard procedure to rule out acquired immune deficient syndrome virus from semen specimens. However, a reliable method to predict the outcome of the cryopreservation procedure or freezability of the sperm is lacking. Such a method is needed: [1] to provide better information on the eventual outcome of the cryopreservation procedure; [2] to determine the optimal time or specimen for cryopreservation after a course of medication or therapy in the male patient; and [3] to reduce time-consuming trial freezing. The objectives of the study were to ascertain if the hypoosmotic viability parameter (2) predicts the motility outcome of sperm cryopreservation and to compare the hypoosmotic viability parameter with the standard hypoosmotic sperm swelling test (3) in predicting the sperm cryopreservation outcome. The hypoosmotic viability parameter provides information concerning the integrity of the membranes at the sperm head (2), whereas the hypoosmotic swelling test is an indicator of sperm tail membrane integrity (3). The information obtained may provide a better understanding of the sperm membrane properties and the relationship to solute transport during freezing. The results of this study suggest a Received October 4, 1991; revised and accepted June 30, 1992.
* Department of Obstetrics and Gynecology.
t Reprint requests: Philip J. Chan, Ph.D., Department of Gynecology and Obstetrics, Lorna Linda University School of Medicine, Lorna Linda, California 92350. t Department of Physiology and Pharmacology. Vol. 58, No.4, October 1992
higher correlation between hypoosmotic viability and sperm freezability compared with the hypoosmotic swelling test and sperm freezability. MATERIALS AND METHODS
Semen Samples
Fresh semen specimens containing sperm cells were collected from 68 patients presenting for the sperm penetration assay (SPA) (n = 15 samples) and 53 patients for routine semen analyses. The specimens were liquefied at 37°C for 30 minutes and analyzed within the hour. The basic semen parameters are presented in Table 1. Supravital staining of sperm was performed as previously described using 0.5% eosin Y stain (4). Viability is defined here as the property of living sperm cells to exclude dyes that have low permeability in the sperm membrane lipid bilayer. The cryopreservation procedure was carried out as previously described (1). The steps involved gradual dilution of the raw semen specimen with an equal volume of freezing medium (TEST-yolk buffer with glycerol; Irvine Scientific, Irvine, CA). The freezing medium consisted of 48% (vol/vol) TEST, TRIS, 12% glycerol, 30% sodium citrate, 20% egg yolk, 2% fructose, 100 U jmL penicillin C, and 100 JlgjmL streptomycin sulfate. Each mixture was placed in marked 1.8-mL Nalgene cryovials (Nalge Co, Rochester, NY) and refrigerated at 4°C for 1 to 1.5 hours. The cryovials were then removed and stored in liquid nitrogen. The test-thaw procedure was carried out after 1 to 3 days of cryostorage. Each cryovial was placed in a 37°C water bath for 7 to 10 minutes. The contents of each cryovial were gently Chan et al.
Communications-in-brief
841
Table 1 Correlation Between Hypoosmotic Viability and Hypoosmotic Sperm Swelling Parameters and the Total Motility Outcome After Freeze-Thaw or Refrigeration of Human Sperm Cells Parameters correlated to thaw motility Freeze-thaw procedure Hypoosmotic viability (n = 15) Hypoosmotic swelling (n = 15) Supravital stain (n = 15) TEST -yolk refrigeration procedure Hypoosmotic viability (n = 53) Hypoosmotic swelling (n = 53) Supravital stain (n = 5)
Correlation coefficient
Coefficient of determination
0.7*
0.5
0.3
0.1
0.2
0.04
0.4
0.2
0.4
0.1
0.07
0.01
* P < 0.05. Linear equation: Thaw Motility = 4.80 + (0.76 X % Hypoosmotic Viable).
mixed and an aliquot pipetted on to a glass slide with cover slip. Total sperm motility was determined with the aid of a phase-contrast microscope set at 400X magnification. All sperm that exhibited movement were scored as motile. In each treatment group, at least five microscope fields totaling over 100 sperm were counted. The sperm count between specimens in the cryopreserved group (73.3 ± 18.2 X 106/mL) and in the refrigerated group (51.8 ± 6.2 X 106/mL) were similar. The TEST -yolk buffer refrigeration procedure was part of the SPA procedure. It involved the gradual mixing of 0.5 mL of refrigeration medium (Irvine Scientific) to 0.5 mL of raw semen specimen in a 1.8-mL cryovial. The refrigeration medium contained 80 mM TRIS, 176 mM TES, 9 mM dextrose, 1,000 U ImL penicillin C, 1 mg/mL streptomycin sulfate,20% (vol/vol) egg yolk. Each mixture in the cryovial was placed in a 100-mL bottle containing approximately 50 mL of distilled water at room temperature. The bottles were placed in the refrigerator for 48 hours. At the end of the refrigeration period, each cryovial was removed and added with prewarmed culture medium (Biggers, Whitten, and Whittingham [BWW] medium with 3.5% human serum albumin) and immediately placed in a 37°C incubator for 30 minutes. After the incubation period, the content of each cryovial was transferred to centrifuge tubes and centrifuged at 300 X g for 10 minutes. The pellets were resuspended with BWW medium and incubated for 3 hours with zona free 842
Chan et al.
Communications-in-brief
hamster oocytes at 37°C at 5% CO 2 in moist air mixture. After 3 hours, the sperm cells were again examined, and the total sperm motility was recorded. Hypoosmotic Viability Staining
The hypoosmotic solution (3) consisted of sodium citrate (7.35 giL; Sigma Chemical Co., St. Louis, MO) and fructose (13.51 giL; Sigma Chemical Co.). The hypoosmotic solution was modified for hypoosmotic viability testing by the addition of eosin Y (0.5% wt/vol) to the solution. The final osmolarity of the mixture adjusted to 150 mOsm/L using MilliQ/UF (Millipore Products Division, Bedford, MA) water. The pH was determined to be 7.9. The mixture was then filter sterilized through a Nalgene 0.2-#Lm filter (Nalge Co). Aliquots (0.1 mL) of the liquefied semen samples or processed sperm samples were individually mixed with 1.0 mL of the prewarmed hypoosmotic solution containing the stain in 2-mL eppendorf-capped tubes and incubated upright at 37°C for 30 minutes. After the incubation period, an aliquot (5 #LL) was pipetted from the bottom of the tube on to a glass slide and a cover slip placed on top of the droplet. We noted that the smaller the aliquot, the easier it was to make the microscopic observations because there was less streaming of the sperm. The percent hypoosmotic viability was calculated from the number of sperm with clear or white colored heads (unstained) divided by the total number of sperm (sperm with clear or white colored heads plus stained red colored heads) and multiplied by 100. A total of 100 sperm cells (irrespective of whether the cells were swollen or not) in several random fields was analyzed for each sample (light microscope at 400X magnification). The cutoff value for normal samples was previously reported (2) to be at 40% or more staining live during hypoosmotic treatment. The cumulative percent sperm swelling parameter, irrespective of the types of tail coiling, types B to G, was also determined (3). The normal criterion for the hypoosmotic sperm swelling test has previously been established at 60% or more sperm swelling (3). Data Analyses
The data were analyzed using linear regression correlation coefficient, and the significance of the slopes was tested using the t-test. Data were presented as means ± SEM. RESULTS
The results shown in Figure 1 indicated a significant correlation (P < 0.05) between hypoosmotic Fertility and Sterility
70
~1iO ~
J'"' I'"
i: 10
70
211
80
Figure 1 Predictive value of hypoosmotic viability parameter for the assessment of total sperm motility after cryopreservation and thawing.
viability and percent total motility of the sperm cells only in the cryopreserved-thawed group. The correlation coefficient was r = 0.7 (Table 1). The predictive linear equation generated for this group was: Percent Thawed Motility =
4.8
+ (0.76 X % Hypoosmotic Viable)
When the data was adjusted for initial prefreeze motility (prefreeze motility minus thawed motility), the correlation was also significant (P < 0.05). The linear correlation coefficient was r = -0.6 and the coefficient of determination was 0.31. The linear equation was: % Loss of Initial Motility
= 45.6 + (-0.49 X % Hypoosmotic Viable) In contrast, in a similar adjustment for hypoosmotic sperm swelling test data, there were no significant correlations observed. In addition, there was a lack of correlation between hypoosmotic viability and refrigerated-warmed sperm motility after 48 hours in TEST-yolk buffer refrigeration at 4°C. Similarly, there were no correlations between the hypoosmotic sperm swelling test and motility and prefreeze supravital staining for viability and motility.
ity parameter was not correlated to sperm motility after refrigeration. The disparity may be because of the differences between the freezing and refrigeration procedures. The freezing process places a greater demand on a more stable and intact sperm membrane than the refrigeration process. The different results observed between the hypoosmotic viability parameter and the hypoosmotic sperm swelling parameter suggested that the determinant factor in survival during cryopreservation might be the membranes at the sperm head rather than at the sperm tail. Damage to the sperm head may be assessed by release of glutamic-oxaloacetic transminase from the sperm (6). Indeed, it may be concluded that the hypoosmotic sperm swelling test, which tested only the tail membrane integrity, did not provide information on sperm survival after cryopreservation. The sperm head alters its configuration in response to freezing. Occasionally, cryoinjuries, such as swelling of the sperm membranes and acrosomal leakage, occur in the sperm. Information on the physical property of the membrane may be obtained from the calculated equation for hypoosmotic viability and the percent loss of initial (prefreeze ) sperm motility after thawing. The lower the percentage hypoosmotic viable, the greater the loss of the initial sperm motility. Conversely, the higher the percentage hypoosmotic viable, the better the percentage sperm cryosurvival. If the sperm cells showed no cryodamage and resumed all oftheir initial motility, the hypoosmotic viability parameter is calculated to be 93.1%, indicating that the ideal specimen for cryopreservation will show 93.1 % sperm survival in a hypoosmotic stress condition. The association between the ability of the sperm head to withstand hypoosmotic stressor condition and survive the cryopreservation process is evident from this study. Interestingly, the membranes at the sperm tail do not provide significant information regarding the ability of the sperm to regain their motility or survive after the cryopreservation process. SUMMARY
DISCUSSION
The value of the hypoosmotic viability parameter in predicting the thawed sperm motility after cryopreservation procedure was significantly better than the hypoosmotic sperm swelling test parameter. Sperm motility after thawing is an important parameter linked to favorable outcome after artificial insemination (5). However, the hypoosmotic viabilVol. 58, No.4, October 1992
In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated Chan et al.
Communications-in-brief
843
that the integrity of the sperm membranes at the head were more important than the tail membrane. Key Words: Sperm, cryopreservation, Test-yolk buffer, viability, hypoosmotic swelling, membrane integrity. Ackrwwledgments. The authors thank the staff at the Loma Linda University Center for Fertility and In Vitro Fertilization, and the Loma Linda University Gynecology and Obstetrics Medical Group, Inc. for their help and support in the preparation of this manuscript. REFERENCES 1. Prins GS, Weidel L. A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril 1986;46:147-9.
844
Chan et al.
Communications-in-brief
2. Chan PJ, Tredway DR, Su BC, Corselli J, Ren S. Identification of male factor semen samples prior to insemination and in vitro fertilization. J In Vitro Fert Embryo Transf 1991;8:37--40. 3. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984;70:219-28. 4. Eliasson R, Treichl L. Supravital staining of human spermatozoa. Fertil Steril1971;22:134-7. 5. Keel BA, Webster BW. Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates. Fertil Steril 1989;52:100-5. 6. Mortimer D, Bramley TA. Glutamic-oxaloacetic transaminase leakage from human spermatozoa as an indicator of cryodamage. Arch AndroI1981;6:337--41.
Fertility and Sterility
Vol. 58, No.4, October 1992
Copyright <> 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Assessment of sperm for cryopreservation using the hypoosmotic viability test
Philip J. Chan, Ph.D.*t:j: Donald R. Tredway, M.D., Ph.D.*:j: Samuel C. Pang, M.D.*
Johannah Corselli, Ph.D.* Brian C. Su, M.D.*
Loma Linda University Sclwol of Medicine, Loma Linda, California
The cryopreservation methods for the freezing of sperm cells as backup specimens for assisted reproductive technologies, as client depositors, for husband or for donor inseminations have been well established (1). Cryopreservation is currently standard procedure to rule out acquired immune deficient syndrome virus from semen specimens. However, a reliable method to predict the outcome of the cryopreservation procedure or freezability of the sperm is lacking. Such a method is needed: [1] to provide better information on the eventual outcome of the cryopreservation procedure; [2] to determine the optimal time or specimen for cryopreservation after a course of medication or therapy in the male patient; and [3] to reduce time-consuming trial freezing. The objectives of the study were to ascertain if the hypoosmotic viability parameter (2) predicts the motility outcome of sperm cryopreservation and to compare the hypoosmotic viability parameter with the standard hypoosmotic sperm swelling test (3) in predicting the sperm cryopreservation outcome. The hypoosmotic viability parameter provides information concerning the integrity of the membranes at the sperm head (2), whereas the hypoosmotic swelling test is an indicator of sperm tail membrane integrity (3). The information obtained may provide a better understanding of the sperm membrane properties and the relationship to solute transport during freezing. The results of this study suggest a Received October 4, 1991; revised and accepted June 30, 1992.
* Department of Obstetrics and Gynecology.
t Reprint requests: Philip J. Chan, Ph.D., Department of Gynecology and Obstetrics, Lorna Linda University School of Medicine, Lorna Linda, California 92350. t Department of Physiology and Pharmacology. Vol. 58, No.4, October 1992
higher correlation between hypoosmotic viability and sperm freezability compared with the hypoosmotic swelling test and sperm freezability. MATERIALS AND METHODS
Semen Samples
Fresh semen specimens containing sperm cells were collected from 68 patients presenting for the sperm penetration assay (SPA) (n = 15 samples) and 53 patients for routine semen analyses. The specimens were liquefied at 37°C for 30 minutes and analyzed within the hour. The basic semen parameters are presented in Table 1. Supravital staining of sperm was performed as previously described using 0.5% eosin Y stain (4). Viability is defined here as the property of living sperm cells to exclude dyes that have low permeability in the sperm membrane lipid bilayer. The cryopreservation procedure was carried out as previously described (1). The steps involved gradual dilution of the raw semen specimen with an equal volume of freezing medium (TEST-yolk buffer with glycerol; Irvine Scientific, Irvine, CA). The freezing medium consisted of 48% (vol/vol) TEST, TRIS, 12% glycerol, 30% sodium citrate, 20% egg yolk, 2% fructose, 100 U jmL penicillin C, and 100 JlgjmL streptomycin sulfate. Each mixture was placed in marked 1.8-mL Nalgene cryovials (Nalge Co, Rochester, NY) and refrigerated at 4°C for 1 to 1.5 hours. The cryovials were then removed and stored in liquid nitrogen. The test-thaw procedure was carried out after 1 to 3 days of cryostorage. Each cryovial was placed in a 37°C water bath for 7 to 10 minutes. The contents of each cryovial were gently Chan et al.
Communications-in-brief
841
Table 1 Correlation Between Hypoosmotic Viability and Hypoosmotic Sperm Swelling Parameters and the Total Motility Outcome After Freeze-Thaw or Refrigeration of Human Sperm Cells Parameters correlated to thaw motility Freeze-thaw procedure Hypoosmotic viability (n = 15) Hypoosmotic swelling (n = 15) Supravital stain (n = 15) TEST -yolk refrigeration procedure Hypoosmotic viability (n = 53) Hypoosmotic swelling (n = 53) Supravital stain (n = 5)
Correlation coefficient
Coefficient of determination
0.7*
0.5
0.3
0.1
0.2
0.04
0.4
0.2
0.4
0.1
0.07
0.01
* P < 0.05. Linear equation: Thaw Motility = 4.80 + (0.76 X % Hypoosmotic Viable).
mixed and an aliquot pipetted on to a glass slide with cover slip. Total sperm motility was determined with the aid of a phase-contrast microscope set at 400X magnification. All sperm that exhibited movement were scored as motile. In each treatment group, at least five microscope fields totaling over 100 sperm were counted. The sperm count between specimens in the cryopreserved group (73.3 ± 18.2 X 106/mL) and in the refrigerated group (51.8 ± 6.2 X 106/mL) were similar. The TEST -yolk buffer refrigeration procedure was part of the SPA procedure. It involved the gradual mixing of 0.5 mL of refrigeration medium (Irvine Scientific) to 0.5 mL of raw semen specimen in a 1.8-mL cryovial. The refrigeration medium contained 80 mM TRIS, 176 mM TES, 9 mM dextrose, 1,000 U ImL penicillin C, 1 mg/mL streptomycin sulfate,20% (vol/vol) egg yolk. Each mixture in the cryovial was placed in a 100-mL bottle containing approximately 50 mL of distilled water at room temperature. The bottles were placed in the refrigerator for 48 hours. At the end of the refrigeration period, each cryovial was removed and added with prewarmed culture medium (Biggers, Whitten, and Whittingham [BWW] medium with 3.5% human serum albumin) and immediately placed in a 37°C incubator for 30 minutes. After the incubation period, the content of each cryovial was transferred to centrifuge tubes and centrifuged at 300 X g for 10 minutes. The pellets were resuspended with BWW medium and incubated for 3 hours with zona free 842
Chan et al.
Communications-in-brief
hamster oocytes at 37°C at 5% CO 2 in moist air mixture. After 3 hours, the sperm cells were again examined, and the total sperm motility was recorded. Hypoosmotic Viability Staining
The hypoosmotic solution (3) consisted of sodium citrate (7.35 giL; Sigma Chemical Co., St. Louis, MO) and fructose (13.51 giL; Sigma Chemical Co.). The hypoosmotic solution was modified for hypoosmotic viability testing by the addition of eosin Y (0.5% wt/vol) to the solution. The final osmolarity of the mixture adjusted to 150 mOsm/L using MilliQ/UF (Millipore Products Division, Bedford, MA) water. The pH was determined to be 7.9. The mixture was then filter sterilized through a Nalgene 0.2-#Lm filter (Nalge Co). Aliquots (0.1 mL) of the liquefied semen samples or processed sperm samples were individually mixed with 1.0 mL of the prewarmed hypoosmotic solution containing the stain in 2-mL eppendorf-capped tubes and incubated upright at 37°C for 30 minutes. After the incubation period, an aliquot (5 #LL) was pipetted from the bottom of the tube on to a glass slide and a cover slip placed on top of the droplet. We noted that the smaller the aliquot, the easier it was to make the microscopic observations because there was less streaming of the sperm. The percent hypoosmotic viability was calculated from the number of sperm with clear or white colored heads (unstained) divided by the total number of sperm (sperm with clear or white colored heads plus stained red colored heads) and multiplied by 100. A total of 100 sperm cells (irrespective of whether the cells were swollen or not) in several random fields was analyzed for each sample (light microscope at 400X magnification). The cutoff value for normal samples was previously reported (2) to be at 40% or more staining live during hypoosmotic treatment. The cumulative percent sperm swelling parameter, irrespective of the types of tail coiling, types B to G, was also determined (3). The normal criterion for the hypoosmotic sperm swelling test has previously been established at 60% or more sperm swelling (3). Data Analyses
The data were analyzed using linear regression correlation coefficient, and the significance of the slopes was tested using the t-test. Data were presented as means ± SEM. RESULTS
The results shown in Figure 1 indicated a significant correlation (P < 0.05) between hypoosmotic Fertility and Sterility
70
~1iO ~
J'"' I'"
i: 10
70
211
80
Figure 1 Predictive value of hypoosmotic viability parameter for the assessment of total sperm motility after cryopreservation and thawing.
viability and percent total motility of the sperm cells only in the cryopreserved-thawed group. The correlation coefficient was r = 0.7 (Table 1). The predictive linear equation generated for this group was: Percent Thawed Motility =
4.8
+ (0.76 X % Hypoosmotic Viable)
When the data was adjusted for initial prefreeze motility (prefreeze motility minus thawed motility), the correlation was also significant (P < 0.05). The linear correlation coefficient was r = -0.6 and the coefficient of determination was 0.31. The linear equation was: % Loss of Initial Motility
= 45.6 + (-0.49 X % Hypoosmotic Viable) In contrast, in a similar adjustment for hypoosmotic sperm swelling test data, there were no significant correlations observed. In addition, there was a lack of correlation between hypoosmotic viability and refrigerated-warmed sperm motility after 48 hours in TEST-yolk buffer refrigeration at 4°C. Similarly, there were no correlations between the hypoosmotic sperm swelling test and motility and prefreeze supravital staining for viability and motility.
ity parameter was not correlated to sperm motility after refrigeration. The disparity may be because of the differences between the freezing and refrigeration procedures. The freezing process places a greater demand on a more stable and intact sperm membrane than the refrigeration process. The different results observed between the hypoosmotic viability parameter and the hypoosmotic sperm swelling parameter suggested that the determinant factor in survival during cryopreservation might be the membranes at the sperm head rather than at the sperm tail. Damage to the sperm head may be assessed by release of glutamic-oxaloacetic transminase from the sperm (6). Indeed, it may be concluded that the hypoosmotic sperm swelling test, which tested only the tail membrane integrity, did not provide information on sperm survival after cryopreservation. The sperm head alters its configuration in response to freezing. Occasionally, cryoinjuries, such as swelling of the sperm membranes and acrosomal leakage, occur in the sperm. Information on the physical property of the membrane may be obtained from the calculated equation for hypoosmotic viability and the percent loss of initial (prefreeze ) sperm motility after thawing. The lower the percentage hypoosmotic viable, the greater the loss of the initial sperm motility. Conversely, the higher the percentage hypoosmotic viable, the better the percentage sperm cryosurvival. If the sperm cells showed no cryodamage and resumed all oftheir initial motility, the hypoosmotic viability parameter is calculated to be 93.1%, indicating that the ideal specimen for cryopreservation will show 93.1 % sperm survival in a hypoosmotic stress condition. The association between the ability of the sperm head to withstand hypoosmotic stressor condition and survive the cryopreservation process is evident from this study. Interestingly, the membranes at the sperm tail do not provide significant information regarding the ability of the sperm to regain their motility or survive after the cryopreservation process. SUMMARY
DISCUSSION
The value of the hypoosmotic viability parameter in predicting the thawed sperm motility after cryopreservation procedure was significantly better than the hypoosmotic sperm swelling test parameter. Sperm motility after thawing is an important parameter linked to favorable outcome after artificial insemination (5). However, the hypoosmotic viabilVol. 58, No.4, October 1992
In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated Chan et al.
Communications-in-brief
843
that the integrity of the sperm membranes at the head were more important than the tail membrane. Key Words: Sperm, cryopreservation, Test-yolk buffer, viability, hypoosmotic swelling, membrane integrity. Ackrwwledgments. The authors thank the staff at the Loma Linda University Center for Fertility and In Vitro Fertilization, and the Loma Linda University Gynecology and Obstetrics Medical Group, Inc. for their help and support in the preparation of this manuscript. REFERENCES 1. Prins GS, Weidel L. A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril 1986;46:147-9.
844
Chan et al.
Communications-in-brief
2. Chan PJ, Tredway DR, Su BC, Corselli J, Ren S. Identification of male factor semen samples prior to insemination and in vitro fertilization. J In Vitro Fert Embryo Transf 1991;8:37--40. 3. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984;70:219-28. 4. Eliasson R, Treichl L. Supravital staining of human spermatozoa. Fertil Steril1971;22:134-7. 5. Keel BA, Webster BW. Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates. Fertil Steril 1989;52:100-5. 6. Mortimer D, Bramley TA. Glutamic-oxaloacetic transaminase leakage from human spermatozoa as an indicator of cryodamage. Arch AndroI1981;6:337--41.
Fertility and Sterility