FERTILITY AND STERILITY
Vol. 58, No, 5, November 1992
Printed on acid-free paper in U.S.A.
Copyright © 1992 The American Fertility Society
Custom cryopreservation of human semen
Thomas F, Kolon, M,D. Kimberley A. Philips, B.S. Jeffrey P. Buch, M.D.* Surgical Research Center, Department of Surgery, Division of Urology, University of Connecticut Health Center, Farmington, Connecticut
Objective: To design a protocol to evaluate individual variability in human semen cryoprotection by native seminal plasma. Design: Post-thaw motility from the frozen semen of pregnancy-proven donors (n = 10) and patients referred for infertility screening (n = 10) was examined in three equal aliquots (per original ejaculate) that comprised varying ratios of native seminal plasma to TES and Tris (TEST)-yolk buffer (Irvine Scientific, Irvine, CA) in a dose-titration curve format. All aliquots from the same ejaculate contained final vol/vol 6% glycerol, had equal sperm density, and had undergone centrifugation for 5 minutes at 600 X g before buffer:semen ratio adjustment and standard precooling protocol for submersion in liquid nitrogen. Post-thaw measurement of percent original motility preserved (post-thaw percent motility/original percent motility X 100) was used for standardization of results. Results: In 14 of 20 specimens (70%), the maximal yield of original motility was obtained in 50% seminal plasma, with an average post-thaw motile yield of 50%. In 6 of 20 specimens (30%), the best preservation of original motility was noted at 100% seminal plasma, with an average postthaw motile yield of 58%. No specimen had a greatest percent motility preserved at 0% seminal plasma. Donor specimens have equal preference for either 50% or 100% seminal plasma, whereas patient specimens have a preference for 50% seminal plasma (P < 0.05). Conclusions: Native seminal plasma has variable cryoprotectant qualities for which custom cryopreservation can compensate. A simple two-point dose-titration test of cryopreservation buffer: seminal plasma ratio (Le., 50:50 versus 0:100) can determine the optimal mixture for cryopreservation of a given individual's semen. Fertil Steril 1992;58:1020-3 Key Words: Human semen, male infertility, sperm motility, cryopreservation (semen)
Cryopreservation of human semen plays an important role in the diagnosis and treatment of male infertility. In 1949, the accidental discovery and subsequent use of the polyhydric alcohol, glycerol, as a cryoprotective agent initiated the establishment of cryobanks for frozen mammalian spermatozoa (1, 2). A successful technique for cryopreservation of human spermatozoa was introduced in 1953 to 1954 by Sherman (1,3), who
Received January 24,1992; revised and accepted July 8, 1992.
* Reprint requests: Jeffrey P. Buch, M.D., Department of Surgery, Division of Urology, University of Connecticut Health Center' Farmington, Connecticut 06030-3955.
1020
Kolon et aI.
Custom cryopreservation of human semen
also demonstrated that spermatozoa were capable of fertilization and normal progeny after freezing and storing in dry ice. Conclusions from recent literature on spermatozoan survival after various methods of cryoprotection are conflicting at best. Controversy exists concerning the addition of cryoextenders, buffers, and cryoprotectants such as glycerol (4-7). We hypothesize that this failure of consensus is, in part, because of variability in cryoprotectant qualities of each individual's native seminal plasma. Herein, we report the results of our study designed to evaluate individual variability in cryoprotection afforded by native seminal plasma. Fertility and Sterility
MATERIALS AND METHODS
Semen was collected after 2 to 3 days of abstinence from 10 men referred for infertility screening and from 10 pregnancy-proven donors. After liquefaction at 37°C, a standard semen analysis was performed. Two aliquots (1.0 mL and 0.5 mL) of semen were then centrifuged for 10 minutes at 600 X g. The seminal plasma was immediately removed from the 1.0-mL aliquot for subsequent dilutions (0% and 50% seminal plasma) of the pellet, which was resuspended in TES and Tris (TEST)-yolk buffer (Irvine, Scientific, Irvine, CA). The pellet from the 0.5-mL aliquot was simply resuspended without removing seminal plasma (100% seminal plasma). This procedure allowed for equal sperm density in all dilutions to be tested. Six percent glycerol vol! vol was added to each aliquot before cryopreservation, with final aliquot volumes of 0.5 mL. On the first several specimens, a seven-point dosetitration assay was performed, beginning with 0% seminal plasma and followed by 5%, 10%, 25%, 50%, 75%, and 100% seminal plasma. After analyzing the initial data, we found no trend benefit to include the 5%, 10%, 25%, and 75% seminal plasma dose-titration points. Peak post-thaw motility was achieved either at 50% or 100% seminal plasma dilution points, depending on the individual specimen. A three-point dose-titration assay at 0%, 50%, and 100% seminal plasma was performed on the remaining specimens. Each of the aliquots was drawn into 0.5-mL cryopreservation straws (TS Scientific, Perkasie, PA) and sealed. The straws were placed in 15-mL conical tubes in a 37°C incubator for 30 minutes to equilibrate. The tubes were then placed in a beaker of 37°C water and cooled at 4°C for 90 minutes. Straws were then further cooled in the vapor phase of liquid nitrogen for 10 to 15 minutes before submersion in the liquid phase. After at least 48 hours in liquid nitrogen, the straws were removed and thawed at room temperature for 30 minutes before equilibration at 37°C for 15 minutes. Percent sperm motility and forward progression (scale 0 to 4) were then assessed and recorded by a single observer. Statistical analysis was performed using a one-way ANOVA for comparison of multiple groups and Student's t-test for further individual comparison of groups. RESULTS
Specimens were collected, and semen analyses were performed on semen from fertile donors Vol. 58, No.5, November 1992
Table 1
Donor Percent Motility Preserved * Seminal plasma
Specimen 1 2 3 4 5 6 7 8 9 10 Mean ± SEM
0%
50%
100%
28.0 25.0 15.0 7.0 27.0 59.0 23.0 44.0 28.0 3.0
78.0 50.0 46.0 27.0 47.0 76.0 31.0 50.0 56.0 40.0
94.0 88.0 77.0 40.0 7.0 6.0 15.0 13.0 44.0 47.0
28.9 ± 4.6
50.1 ± 5.3
43.1 ± 10.6
* Percent post-thaw motility/percent original motility X 100.
(n = 10) and patients referred for fertility screening (n = 10). The original percent motility (mean ± SEM) was 78.0% ± 2.8% for the donors and 51.7% ± 5.9% for the patients. Both donor and patient semen specimens were then cryopreserved in varying ratios of native seminal plasma:TEST -yolk buffer in a dose-titration curve format as outlined. The specimens were thawed and semen analyses repeated on each. Donor post-thaw percent motilities (mean ± SEM) for 0%,50%, and 100% seminal plasma were 23.0% ± 4.0%, 40.0% ± 5.4%, and 34.0% ± 8.8%, respectively (Table 1). Similarly, patient post-thaw percent motilities were 14.5% ± 4.4%, 27.1 % ± 5.0%, and 13.6% ± 7.2% for 0%,50%, and 100% seminal plasma, respectively (Table 2). Results were analyzed by Student's t-test at a 95%
Table 2
Patient Percent Motility Preserved * Seminal plasma
Specimen 1 2 3 4 5 6 7 8 9 10 Mean± SEM
0%
50%
100%
40.0 10.0 9.0 2.0 42.0 14.0 17.0 0.1 42.0 70.0
57.0 24.0 17.0 70.0 58.0 29.0 67.0 20.0 63.0 90.0
100.0 0.0 17.0 0.0 8.0 4.0 25.0 0.0 20.0 40.0
24.6 ± 7.2
49.5 ± 7.9t
21.4 ± 9.7
* Percent post-thaw motility/percent original motility X 100. t p < 0.05. Kolon et al.
Custom cryopreservation of human semen
1021
confidence interval (CI) with 9 df. A comparison of donor original percent motility versus post-thaw percent motility at 0%, 50%, and 100% seminal plasma showed each of the differences to be statistically significant (P < 0.05). A similar comparison of the patient samples again resulted in statistically significant differences (P < 0.05). The percent original motility preserved was determined by dividing the post-thaw percent motility by the original percent motility and multiplying the result by 100. In 6 of 20 specimens, the greatest percent motility preserved was noted at 100% seminal plasma, whereas 13 of 20 specimens had their greatest percent motility preserved at 50% seminal plasma. In one specimen, the greatest percent motility preserved was the same for the 50% and the 100% seminal plasma groups. None ofthe 20 specimens showed a greatest percent motility preserved at 0% seminal plasma. An ANOVA was performed on the three assay groups for the donor and patient populations. For the donor specimens, the percent motility preserved (mean ± SEM) of the 0%, 50%, and 100% seminal plasma groups was 28.9% ± 14.5%,50.1 % ± 16.7%, and 43.1 % ± 33.6%, respectively (P = 0.136). Likewise, the percent motility preserved for the patients in the same three assay groups was 24.6% ± 22.7%, 49.5% ± 25.1%, and 21.4% ± 30.6%. This is a statistically significant difference at the 99% CI (P < 0.01). Comparison of percent motility preserved for donor versus patient at 0%, 50%, and 100% seminal plasma revealed no statistically significant differences. However, at the 100% seminal plasma ratio, a trend was indicated (P = 0.15, 85% CI). The post-thaw motile sperm density was calculated by multiplying sperm density (no. X 106 sperm/mL of semen) by percent motile sperm postthaw. The mean (±SEM) donor original sperm density was 132.8 ± 24.6, whereas the mean original density for the patient population was 102.8 ± 32.4. There was no significant difference between the two groups in this regard (P = 0.5). The patient group's motile sperm densities (mean ± SEM) were 15.4 ± 5.4, 27.3 ± 9.2, and 11.1 ± 4.2 for 0%, 50%, and 100% seminal plasma, respectively. These same respective measurements for the donor specimens (mean ± SEM) were 32.2 ± 7.9, 54.9 ± 12.0, and 52.6 ± 19.6. Student's t-test analysis of this data showed a statistically significant greater sperm density when compared with patient specimens at 100% seminal plasma (P = 0.05). The 0% and 50% seminal plasma groups showed a similar (but not significant) 1022
Kolon et al.
Custom cryopreservation of human semen
trend between donor and patient specimens for the post-thaw motile sperm density (P < 0.10). DISCUSSION
Cryopreservation of human semen is invaluable to the field of male infertility. However, opinion is divided concerning the best cryopreservation protocol for human semen (4-9). Each individual's native seminal plasma may have variable cryoprotectant qualities that make it difficult to select one best cryopreservation technique for all specimens. Therefore, we designed a protocol to use a patient's native seminal plasma for cryopreservation and evaluate its individual variability. To compare the efficacy of all future cryopreservation protocols, we propose a standardized method for reporting results. The standard that should be used is percent original motility preserved as outlined earlier. A 75% seminalplasma:25% TEST-yolkbufferratio was not included in the final assay because the first six specimens studied revealed that an initial rise in post-thaw percent motility from 50% to 75% seminal plasma continued as an upward trend for the 100% seminal plasma measurement. Likewise, a decrease in the percent motility from 50% to 75% seminal plasma continued as a downward trend for the 100% seminal plasma measurement. Therefore, we simplified the protocol to include only 0%, 50%, and 100% seminal plasma dilutions. Our results demonstrate that native seminal plasma has variable cryoprotective qualities. Although 30% of all specimens had maximal yield at 100% seminal plasma, 70% of the specimens had maximal yield at 50% seminal plasma. Most importantly, no specimens had a greatest percent motility preserved at 0% seminal plasma (100% TEST-yolk buffer), a previous buffer of choice (7). A breakdown of the donor and patient groups showed an equal preference of donor sperm for either 50% or 100% seminal plasma ratios. However, patient sperm clearly had a preference for 50% seminal plasma (P < 0.01 by ANOVA and P < 0.05 by ttest). Analysis of motile sperm density data confirmed these conclusions. Patient specimens at 50% seminal plasma averaged 27.3 ± 9.2 (mean ± SEM) X 106 motile sperm/mL of semen (P < 0.05 by ANOVA). Similarly, the greatest post-thaw motile sperm density for donor sperm was seen at 50% and 100% seminal plasma. Although percent motility preserved showed no statistically significant difference comparing donor and patient, motile sperm density of the donor was Fertility and Sterility
significantly better than that ofthe patient at 100% seminal plasma (P = 0.05). This trend was also seen in the 0% and 50% seminal plasma groups (P < 0.10). The post-thaw motile sperm density may be a better measure of sperm vitality in the comparison of fertility outcomes from cryopreserved specimens. Exactly what factor(s) enables some individuals to cryopreserve better in native seminal plasma remains to be determined. No single identifiable component of the semen analysis (i.e., sperm density, percent original motility, forward progression) explains why the percent motility preserved of two donor specimens decreased so greatly in the 100% seminal plasma group. Previous studies have shown that post-thaw percent motility increased along with the sperm count up to 120 X 106 , after which no further effect was seen (10-12). This is contradicted by our data, which showed no relationship between sperm count and percent motility preserved in either the donor or patient populations. Likewise, Keel and Karow (10), among others (13), suggested a relationship between sperm count and prefreeze motility, but our data did not confirm this. Further research must identify the specific factors within semen that confer the cryoprotection qualities. Lee et al. (14) identified two protein groups by two-dimensional polyacrylamide gel electrophoresis in the seminal plasma of fertile donors that were absent in vasectomized men. Perhaps this same technique could be employed to identify different protein patterns associated with cryoprotective qualities in variable specimens. Additional information is needed to further optimize cryopreservation protocols. Similar to the cryoprotectant literature, various studies have differed on the issue of controlled-rate/staged freezing versus vapor freezing (8, 9, 15, 16). Our next line of investigation will compare freezing modalities as well as buffer:seminal plasma ratios. It is clear from our data that native seminal plasma has cryoprotectant qualities that either exceed or enhance the properties of TEST -yolk buffer. Individual variability exists as to whether 50% or 100% seminal plasma is best. Custom cryopreservation protocols can compensate for the variable cryoprotectant qualities of native seminal plasma. Because none of the specimens preserved well in the absence of seminal plasma, a simple two-point dosetitration test of cryopreservation buffer:seminal plasma ratio (i.e., 50:50 and 0:100) can determine
Vol. 58, No.5, November 1992
the optimal cryoprotectant mixture for a given individual's semen. Furthermore, standardization of protocols and reporting of results are critical in this field of research. Standardized reporting of percent original motility preserved will aid our understanding of the effects of freezing and thawing on the physical characteristics and fertility potential of human spermatozoa. REFERENCES 1. Sherman JK. Synopsis of the use of frozen human semen
2.
3.
4.
5.
6.
7.
8.
9. 10.
11.
12. 13. 14.
15.
16.
since 1964: state of the art of human semen banking. Fertil Steril 1973;24:397-412. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164:666. Sherman JK. Cryopreservation in human semen. In: Hafez E, editor. Techniques of human andrology. Amsterdam: North Holland, 1977:399-420. Phillip M, Hermoni D, Potashnik G. Comparison of postthaw sperm motility after freezing in liquid nitrogen with protective media of either glycerol or glycerol-egg yolk-citrate. Int J FertiI1983;28:156-60. Mahadevan M, Trounson AO. Effect of cryoprotective media and dilution methods on the preservation of human spermatozoa. Andrologia 1984;16:52-60. Jeyendran RS, Van der Van HH, Kennedy W, Perez-Pelaez M, Zanevald LJ. Comparison of glycerol and a zwitter-ion buffer system as cryoprotectant media for human spermatozoa. J AndroI1984;5:1-7. Prins GS, Weidel L. A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril 1986;46:147-9. Cohen J, Felten P, Zielmaker GH. In vitro fertilizing capacity of fresh and cryopreserved human spermatozoa: a comparative study of freezing and thawing procedures. Fertil Steril 1981;36:356-62. Thachil JV, Jewett MAS. Preservation techniques of human semen. Fertil Steril 1981;35:546-8. Keel BA, Karow AM. Motility characteristics of human sperm, nonfrozen and cryopreserved. Arch Androl 1980;4: 205-12. Beck WW Jr, Silverstein I. Variable motility recovery of spermatozoa following freeze preservation. Fertil Steril 1975;26:863-7. Steinberger E, Perloff WHo Preliminary experience with a human sperm bank. Am J Obstet Gynecol 1965;92:577-9. Smith JD, Steinberger E. Survival of spermatozoa in a human sperm bank. JAMA 1973;223:774-7. Lee LM, Salama M, Lamb DJ, Guevara J Jr, Lipshultz LI, Coburn M. Analysis of human seminal fluid proteins using two-dimensional gel electrophoresis [Abstract]. J Urol 1990;143:284A. Taylor PJ, Wilson J, Laycock R, Weber J. A comparison of freezing and thawing methods for the cryopreservation of human semen. Fertil Steril 1982;37:100-3. Serafini P, Marrs RP. Computerized staged-freezing technique improves sperm survival and preserves penetration of zona-free hamster ova. Fertil SteriI1986;45:854-8.
Kolon et al.
Custom cryopreservation of human semen
1023
Vol. 58, No, 5, November 1992
Printed on acid-free paper in U.S.A.
Copyright © 1992 The American Fertility Society
Custom cryopreservation of human semen
Thomas F, Kolon, M,D. Kimberley A. Philips, B.S. Jeffrey P. Buch, M.D.* Surgical Research Center, Department of Surgery, Division of Urology, University of Connecticut Health Center, Farmington, Connecticut
Objective: To design a protocol to evaluate individual variability in human semen cryoprotection by native seminal plasma. Design: Post-thaw motility from the frozen semen of pregnancy-proven donors (n = 10) and patients referred for infertility screening (n = 10) was examined in three equal aliquots (per original ejaculate) that comprised varying ratios of native seminal plasma to TES and Tris (TEST)-yolk buffer (Irvine Scientific, Irvine, CA) in a dose-titration curve format. All aliquots from the same ejaculate contained final vol/vol 6% glycerol, had equal sperm density, and had undergone centrifugation for 5 minutes at 600 X g before buffer:semen ratio adjustment and standard precooling protocol for submersion in liquid nitrogen. Post-thaw measurement of percent original motility preserved (post-thaw percent motility/original percent motility X 100) was used for standardization of results. Results: In 14 of 20 specimens (70%), the maximal yield of original motility was obtained in 50% seminal plasma, with an average post-thaw motile yield of 50%. In 6 of 20 specimens (30%), the best preservation of original motility was noted at 100% seminal plasma, with an average postthaw motile yield of 58%. No specimen had a greatest percent motility preserved at 0% seminal plasma. Donor specimens have equal preference for either 50% or 100% seminal plasma, whereas patient specimens have a preference for 50% seminal plasma (P < 0.05). Conclusions: Native seminal plasma has variable cryoprotectant qualities for which custom cryopreservation can compensate. A simple two-point dose-titration test of cryopreservation buffer: seminal plasma ratio (Le., 50:50 versus 0:100) can determine the optimal mixture for cryopreservation of a given individual's semen. Fertil Steril 1992;58:1020-3 Key Words: Human semen, male infertility, sperm motility, cryopreservation (semen)
Cryopreservation of human semen plays an important role in the diagnosis and treatment of male infertility. In 1949, the accidental discovery and subsequent use of the polyhydric alcohol, glycerol, as a cryoprotective agent initiated the establishment of cryobanks for frozen mammalian spermatozoa (1, 2). A successful technique for cryopreservation of human spermatozoa was introduced in 1953 to 1954 by Sherman (1,3), who
Received January 24,1992; revised and accepted July 8, 1992.
* Reprint requests: Jeffrey P. Buch, M.D., Department of Surgery, Division of Urology, University of Connecticut Health Center' Farmington, Connecticut 06030-3955.
1020
Kolon et aI.
Custom cryopreservation of human semen
also demonstrated that spermatozoa were capable of fertilization and normal progeny after freezing and storing in dry ice. Conclusions from recent literature on spermatozoan survival after various methods of cryoprotection are conflicting at best. Controversy exists concerning the addition of cryoextenders, buffers, and cryoprotectants such as glycerol (4-7). We hypothesize that this failure of consensus is, in part, because of variability in cryoprotectant qualities of each individual's native seminal plasma. Herein, we report the results of our study designed to evaluate individual variability in cryoprotection afforded by native seminal plasma. Fertility and Sterility
MATERIALS AND METHODS
Semen was collected after 2 to 3 days of abstinence from 10 men referred for infertility screening and from 10 pregnancy-proven donors. After liquefaction at 37°C, a standard semen analysis was performed. Two aliquots (1.0 mL and 0.5 mL) of semen were then centrifuged for 10 minutes at 600 X g. The seminal plasma was immediately removed from the 1.0-mL aliquot for subsequent dilutions (0% and 50% seminal plasma) of the pellet, which was resuspended in TES and Tris (TEST)-yolk buffer (Irvine, Scientific, Irvine, CA). The pellet from the 0.5-mL aliquot was simply resuspended without removing seminal plasma (100% seminal plasma). This procedure allowed for equal sperm density in all dilutions to be tested. Six percent glycerol vol! vol was added to each aliquot before cryopreservation, with final aliquot volumes of 0.5 mL. On the first several specimens, a seven-point dosetitration assay was performed, beginning with 0% seminal plasma and followed by 5%, 10%, 25%, 50%, 75%, and 100% seminal plasma. After analyzing the initial data, we found no trend benefit to include the 5%, 10%, 25%, and 75% seminal plasma dose-titration points. Peak post-thaw motility was achieved either at 50% or 100% seminal plasma dilution points, depending on the individual specimen. A three-point dose-titration assay at 0%, 50%, and 100% seminal plasma was performed on the remaining specimens. Each of the aliquots was drawn into 0.5-mL cryopreservation straws (TS Scientific, Perkasie, PA) and sealed. The straws were placed in 15-mL conical tubes in a 37°C incubator for 30 minutes to equilibrate. The tubes were then placed in a beaker of 37°C water and cooled at 4°C for 90 minutes. Straws were then further cooled in the vapor phase of liquid nitrogen for 10 to 15 minutes before submersion in the liquid phase. After at least 48 hours in liquid nitrogen, the straws were removed and thawed at room temperature for 30 minutes before equilibration at 37°C for 15 minutes. Percent sperm motility and forward progression (scale 0 to 4) were then assessed and recorded by a single observer. Statistical analysis was performed using a one-way ANOVA for comparison of multiple groups and Student's t-test for further individual comparison of groups. RESULTS
Specimens were collected, and semen analyses were performed on semen from fertile donors Vol. 58, No.5, November 1992
Table 1
Donor Percent Motility Preserved * Seminal plasma
Specimen 1 2 3 4 5 6 7 8 9 10 Mean ± SEM
0%
50%
100%
28.0 25.0 15.0 7.0 27.0 59.0 23.0 44.0 28.0 3.0
78.0 50.0 46.0 27.0 47.0 76.0 31.0 50.0 56.0 40.0
94.0 88.0 77.0 40.0 7.0 6.0 15.0 13.0 44.0 47.0
28.9 ± 4.6
50.1 ± 5.3
43.1 ± 10.6
* Percent post-thaw motility/percent original motility X 100.
(n = 10) and patients referred for fertility screening (n = 10). The original percent motility (mean ± SEM) was 78.0% ± 2.8% for the donors and 51.7% ± 5.9% for the patients. Both donor and patient semen specimens were then cryopreserved in varying ratios of native seminal plasma:TEST -yolk buffer in a dose-titration curve format as outlined. The specimens were thawed and semen analyses repeated on each. Donor post-thaw percent motilities (mean ± SEM) for 0%,50%, and 100% seminal plasma were 23.0% ± 4.0%, 40.0% ± 5.4%, and 34.0% ± 8.8%, respectively (Table 1). Similarly, patient post-thaw percent motilities were 14.5% ± 4.4%, 27.1 % ± 5.0%, and 13.6% ± 7.2% for 0%,50%, and 100% seminal plasma, respectively (Table 2). Results were analyzed by Student's t-test at a 95%
Table 2
Patient Percent Motility Preserved * Seminal plasma
Specimen 1 2 3 4 5 6 7 8 9 10 Mean± SEM
0%
50%
100%
40.0 10.0 9.0 2.0 42.0 14.0 17.0 0.1 42.0 70.0
57.0 24.0 17.0 70.0 58.0 29.0 67.0 20.0 63.0 90.0
100.0 0.0 17.0 0.0 8.0 4.0 25.0 0.0 20.0 40.0
24.6 ± 7.2
49.5 ± 7.9t
21.4 ± 9.7
* Percent post-thaw motility/percent original motility X 100. t p < 0.05. Kolon et al.
Custom cryopreservation of human semen
1021
confidence interval (CI) with 9 df. A comparison of donor original percent motility versus post-thaw percent motility at 0%, 50%, and 100% seminal plasma showed each of the differences to be statistically significant (P < 0.05). A similar comparison of the patient samples again resulted in statistically significant differences (P < 0.05). The percent original motility preserved was determined by dividing the post-thaw percent motility by the original percent motility and multiplying the result by 100. In 6 of 20 specimens, the greatest percent motility preserved was noted at 100% seminal plasma, whereas 13 of 20 specimens had their greatest percent motility preserved at 50% seminal plasma. In one specimen, the greatest percent motility preserved was the same for the 50% and the 100% seminal plasma groups. None ofthe 20 specimens showed a greatest percent motility preserved at 0% seminal plasma. An ANOVA was performed on the three assay groups for the donor and patient populations. For the donor specimens, the percent motility preserved (mean ± SEM) of the 0%, 50%, and 100% seminal plasma groups was 28.9% ± 14.5%,50.1 % ± 16.7%, and 43.1 % ± 33.6%, respectively (P = 0.136). Likewise, the percent motility preserved for the patients in the same three assay groups was 24.6% ± 22.7%, 49.5% ± 25.1%, and 21.4% ± 30.6%. This is a statistically significant difference at the 99% CI (P < 0.01). Comparison of percent motility preserved for donor versus patient at 0%, 50%, and 100% seminal plasma revealed no statistically significant differences. However, at the 100% seminal plasma ratio, a trend was indicated (P = 0.15, 85% CI). The post-thaw motile sperm density was calculated by multiplying sperm density (no. X 106 sperm/mL of semen) by percent motile sperm postthaw. The mean (±SEM) donor original sperm density was 132.8 ± 24.6, whereas the mean original density for the patient population was 102.8 ± 32.4. There was no significant difference between the two groups in this regard (P = 0.5). The patient group's motile sperm densities (mean ± SEM) were 15.4 ± 5.4, 27.3 ± 9.2, and 11.1 ± 4.2 for 0%, 50%, and 100% seminal plasma, respectively. These same respective measurements for the donor specimens (mean ± SEM) were 32.2 ± 7.9, 54.9 ± 12.0, and 52.6 ± 19.6. Student's t-test analysis of this data showed a statistically significant greater sperm density when compared with patient specimens at 100% seminal plasma (P = 0.05). The 0% and 50% seminal plasma groups showed a similar (but not significant) 1022
Kolon et al.
Custom cryopreservation of human semen
trend between donor and patient specimens for the post-thaw motile sperm density (P < 0.10). DISCUSSION
Cryopreservation of human semen is invaluable to the field of male infertility. However, opinion is divided concerning the best cryopreservation protocol for human semen (4-9). Each individual's native seminal plasma may have variable cryoprotectant qualities that make it difficult to select one best cryopreservation technique for all specimens. Therefore, we designed a protocol to use a patient's native seminal plasma for cryopreservation and evaluate its individual variability. To compare the efficacy of all future cryopreservation protocols, we propose a standardized method for reporting results. The standard that should be used is percent original motility preserved as outlined earlier. A 75% seminalplasma:25% TEST-yolkbufferratio was not included in the final assay because the first six specimens studied revealed that an initial rise in post-thaw percent motility from 50% to 75% seminal plasma continued as an upward trend for the 100% seminal plasma measurement. Likewise, a decrease in the percent motility from 50% to 75% seminal plasma continued as a downward trend for the 100% seminal plasma measurement. Therefore, we simplified the protocol to include only 0%, 50%, and 100% seminal plasma dilutions. Our results demonstrate that native seminal plasma has variable cryoprotective qualities. Although 30% of all specimens had maximal yield at 100% seminal plasma, 70% of the specimens had maximal yield at 50% seminal plasma. Most importantly, no specimens had a greatest percent motility preserved at 0% seminal plasma (100% TEST-yolk buffer), a previous buffer of choice (7). A breakdown of the donor and patient groups showed an equal preference of donor sperm for either 50% or 100% seminal plasma ratios. However, patient sperm clearly had a preference for 50% seminal plasma (P < 0.01 by ANOVA and P < 0.05 by ttest). Analysis of motile sperm density data confirmed these conclusions. Patient specimens at 50% seminal plasma averaged 27.3 ± 9.2 (mean ± SEM) X 106 motile sperm/mL of semen (P < 0.05 by ANOVA). Similarly, the greatest post-thaw motile sperm density for donor sperm was seen at 50% and 100% seminal plasma. Although percent motility preserved showed no statistically significant difference comparing donor and patient, motile sperm density of the donor was Fertility and Sterility
significantly better than that ofthe patient at 100% seminal plasma (P = 0.05). This trend was also seen in the 0% and 50% seminal plasma groups (P < 0.10). The post-thaw motile sperm density may be a better measure of sperm vitality in the comparison of fertility outcomes from cryopreserved specimens. Exactly what factor(s) enables some individuals to cryopreserve better in native seminal plasma remains to be determined. No single identifiable component of the semen analysis (i.e., sperm density, percent original motility, forward progression) explains why the percent motility preserved of two donor specimens decreased so greatly in the 100% seminal plasma group. Previous studies have shown that post-thaw percent motility increased along with the sperm count up to 120 X 106 , after which no further effect was seen (10-12). This is contradicted by our data, which showed no relationship between sperm count and percent motility preserved in either the donor or patient populations. Likewise, Keel and Karow (10), among others (13), suggested a relationship between sperm count and prefreeze motility, but our data did not confirm this. Further research must identify the specific factors within semen that confer the cryoprotection qualities. Lee et al. (14) identified two protein groups by two-dimensional polyacrylamide gel electrophoresis in the seminal plasma of fertile donors that were absent in vasectomized men. Perhaps this same technique could be employed to identify different protein patterns associated with cryoprotective qualities in variable specimens. Additional information is needed to further optimize cryopreservation protocols. Similar to the cryoprotectant literature, various studies have differed on the issue of controlled-rate/staged freezing versus vapor freezing (8, 9, 15, 16). Our next line of investigation will compare freezing modalities as well as buffer:seminal plasma ratios. It is clear from our data that native seminal plasma has cryoprotectant qualities that either exceed or enhance the properties of TEST -yolk buffer. Individual variability exists as to whether 50% or 100% seminal plasma is best. Custom cryopreservation protocols can compensate for the variable cryoprotectant qualities of native seminal plasma. Because none of the specimens preserved well in the absence of seminal plasma, a simple two-point dosetitration test of cryopreservation buffer:seminal plasma ratio (i.e., 50:50 and 0:100) can determine
Vol. 58, No.5, November 1992
the optimal cryoprotectant mixture for a given individual's semen. Furthermore, standardization of protocols and reporting of results are critical in this field of research. Standardized reporting of percent original motility preserved will aid our understanding of the effects of freezing and thawing on the physical characteristics and fertility potential of human spermatozoa. REFERENCES 1. Sherman JK. Synopsis of the use of frozen human semen
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