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Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa
Vol. 39, No.5, May 1983 Printed in U.SA.
FERTILITY AND STERILITY Copyright 0 1983 The American Fertility Society
Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa
Ronald L. Urry, Ph.D.*t Douglas T. Carrell:j: Dale B. Hull, B.S. * Richard G. Middleton, M.D. * Milo C. Wiltbank, M.S.:j: University of Utah School of Medicine, Salt Lake City, Utah, and Brigham Young University, Provo, Utah
Semen samples from 25 fertile donors and 34 infertility patients were analyzed and evaluated both fresh and frozen with the zona-free hamster egg penetration test (EPT) and the bovine cervical mucus penetration test (MPT). Cryopreserved sperm penetrated 47.2% (average) of the hamster eggs, and fresh sperm penetrated 55.9%. Frozen sperm retained 86% of the fresh EPT values (p, not significant), but only 38% of the fresh MPT values (P < 0.001). Cryopreserved sperm yielded the same fertility classification as fresh sperm in 94% of the cases with the EPT but only 43% with the MPT. The use of cryopreserved sperm simplifies the procedure and should enable more laboratories to utilize the EPT, but the poor results with the MPT preclude utilization of cryopreserued sperm for routine fertility evaluation with the MPT. Fertil Steril 39:690, 1983
Traditionally, semen analysis has been the main method utilized to evaluate men with fertility problems. A complete semen analysis includes determination of sperm count, number of motile sperm, number of normal forms, viability, volume measurement, white blood cells, agglutination, and viscosity. 1 The semen analysis, although useful i.n fertility workup, is imprecise in its measurement of fertility and may not directly simulate any of the events in the conception process. 2 Recently, new tests have been introduced that
Received September 13,1982; revised and accepted December 23, 1982. *Division of Urology, University of Utah School of Medicine. tReprint requests: Ronald L. Urry, Ph.D., Division ofUrology, University of Utah School of Medicine, Salt Lake City, Utah 84132. :j:Department of Zoology, Brigham Young University. 690
Urry et aI. Penetration by cryopreserved sperm
could provide a more accurate diagnosis of male fertility problems. The zona-free hamster egg penetration test (EPT), introduced by Yanagimachi et al.,3 appears to be effective in differentiating fertile and infertile men. 4 -7 This assay involves the incubation of zona-free hamster eggs with capacitated human sperm and subsequent evaluation of the eggs for penetration by the sperm. The EPT is usually performed with fresh, rather than frozen, human spermatozoa because of reduced motility and longevity of sperm that have been frozen in liquid nitrogen. 8 , 9 Previous studies have shown cryopreserved sperm to be less effective in penetrating zona-free hamster eggs, although there is nO agreement on the extent of this reduction. 9 - 11 Another test of sperm function uses bovine cervical mucus in flat capillary tubes for determination of the ability of human sperm to penetrate cervical mucus. 12 Like the EPT, the mucus peneFertility and Sterility
l tration test (MPT) is normally performed with fresh semen. Attempts to ~tilize frozen semen have demonstrated a large reduction in the ability to penetrate cervical mucus following freezing.la, 14 Although the above two tests may be useful in evaluating male infertility, one aspect that presents technical·difficulties is the rigid timetable that each assay requires. If semen samples could be frozen and saved for evaluation at a more optimal time, the procedure would be simplified for both the patient and the laboratory. The purpose of this study was twofold: (1) to evaluate the exact amount of change in the EPT and MPT results following freezing and (2) to assess the clinical usefulness of cryopreserved semen in the EPT and the MPT. Fresh and frozen sperm from each sample from fertile and suspected infertile individuals were compared. MATERIALS AND METHODS
Semen samples were obtained from 25 known fertile donors and from 34 patients seen at a fertility evaluation clinic. All semen samples were divided following collection, with a portion (approximately 1 ml) saved for evaluation fresh and and equal portion frozen in liquid nitrogen for analysis approximately 1 week later (mean, 8.3 days). The portion to be frozen was first diluted with glycerol and Baker's buffer (1:1) to a final concentration of 7.5% glycerol, lowered into liquid nitrogen vapor for 20 to 30 minutes, and then stored in liquid nitrogen ( -196° C). At the time of the test, the frozen sample was thawed in water at 28° C for 10 minutes and then evaluated with the use of a procedure identical to that for the fresh sample. Both fresh and frozen portions were evaluated for the percentage of progressively (forward) motile' sperm before use in either test. 1 The EPT was performed as previously described by Yanagimachi et al. a with the exception of the modifications described below. The semen being tested in the EPT was diluted to six times its original volume with modified Biggers, Whitten and Whittingham (BWW) medium15 (10 mM HEPES buffer replacing 10 mM NaHCO a) to which was added 1.0% bovine serum albumin (Fraction V, Sigma Chemical Company, St. Louis, MO). This mixture was centrifuged at 500 x g for 5 minutes, and the supernatant was removed, leaving the sperm pellet. The pellet was washed twice and diluted to a progressively motile sperm Vol. 39, No.5, May 1983
concentration of 5 x 106 sperm/ml. A 0.2-ml aliquot of this sperm suspension was placed under mineral oil and preincubated for 2 hours at 37° C. Eggs for the EPT were obtained from mature female Golden hamsters. Hamsters were given injeetions of 40 IU of pregnant mare serum gonadotropin (Sigma) on the day of the postestrus vaginal discharge. Forty-eight to 56 hours later they were given injections of 50 IU of human chorionic gonadotropin (Sigma), and 16 hours later the oviducts were excised and the egg masses removed. The cumulus cells were dispersed by exposure to 0:1% bovine hyaluronidase. The zona pellucida was removed by treatment with 0.3% bovine pancreatic trypsin, ar:td the eggs were washed four times in BWW. Twenty-five to 50 eggs were then placed directly into each sperm suspension and incubated at 37° C for 5 hours. The eggs were then removed, pressed slightly under a gel-suspended coverslip, and examined under phase-contrast microscopy at x 400. The eggs were considered penetrated if a swollen sperm head or a male pronucleus was found in the egg. The percentage of the eggs that were penetrated and the total number of penetrating sperm per egg were recorded. Fifty semen. samples, 25 of which were from men of known fertility, were evaluated fresh and frozen in the EPT. Nine additional samples (one from a fertile donor and eight from suspected infertile patients) were eliminated from the results because of an insufficient motile sperm density follQwing freezing. The interassay variation was monitored by running a control sample from a previously tested fertile man with each group of samples. The MPT was performed by the method described by AlexanderY Flat capillary tubes, filled with bovine cervical mucus, were incubated with the semen for 90 minutes at room temperature. The penetration was reported as the distance (in millimeters) traveled by the vanguard spermatozoa. Means, standard deviations (SD), t-tests, and correlation coefficients were determined for the data, and correlation coefficients were converted to Z values, which were used to determine significance. RESULTS
The proportion of eggs penetrated in the EPT was 55.9% ± 3.2% (mean ± standard error) by Urry et al. Penetration by cryopreserved sperm
691
Table 1. Percentage of Samples Changing Various Increments When the Frozen Sperm Value Was Subtracted from the Fresh Sperm Value with the Hamster Egg Human Sperm EPT or with the Bovine Cervical MPT Percentage of total samples inEPT
Percentage of total samples inMPT
2
o o
20 38 24 14 2
2.5 10 40
o
45 2.5
Difference in EPT or MPT value (fresh EPT or MPT minus frozen EPT or MPT value)
< - 20% difference - 10% 0% 0% + 10% + 20% > + 40%
to - 20% difference to -10% difference to + 10% difference to + 20% difference to + 40% difference difference
fresh sperm and 47.2% ± 2.8% by frozen sperm (P < 0.05). The average decline in penetration by the frozen sperm was 8.8% + 1. 7% for the 50 samples. The frozen samples' EPT values subtracted from those of the fresh samples are given in Table 1. Fifty-eight percent of the frozen samples were found to be less than 10% different from the fresh values, and 84% were less than 20% different from the fresh values. The correlation between EPT results for the fresh and frozen portions was 0.85 (P < 0.0001). The polyspermia index was also calculated by dividing the total number of penetrated sperm by the number of eggs evaluated. The index averaged 0.88 ± 0.10 for the fresh sperm and 0.57 ± 0.05 for the frozen sperm (P < 0.01). Six percent of the sperm samples (3 of 50) declined from a penetration rate considered normal (above 20% to 25% of the eggs penetrated4 - 7 ) to a subfertile value following freezing. Using above 10% as a normal penetration rate,3, 5 none of the samples decre&sed from normal to subfertile following freezing. Nine of the 59 original samples (15%; 1 from a fertile donor and 8 from suspected infertile patients) were eliminated from the analysis because of an insufficient motile sperm concentration « 5 x 106 /ml) following freezing. Three of the nine samples were evaluated fresh and frozen with the EPT, and each experienced a significant drop in penetration rate following freezing (33% to 5%; 30% to 0%; 13% to 0%), which may have been due to the low motile sperm density. The control samples for the EPT were collected from one man over a period of 7 months (8 different semen samples, 23 different tests). These samples had an interassay variation (coefficient of variation) of 11.2% with an SD of7.3%. Two to five semen samples from each of four different 692
Urry et aI. Penetration by cryopreserved sperm
men were examined with the EPT, and the coefficient of variati'on between samples from the same man was 16.3% (7.6% SD). The mean MPT result for sperm from fresh semen was 32.9 ± 2.2 mm, and for frozen sperm it was 12.4 ± 1.2 mm. The migration distance for crY9preserved sperm, therefore, averaged 20.5 mm less than for fresh sperm (P < 0.0001). The difference between fresh MPT values and frozen values is given in Table 1. Only 12.5% of the frozen samples were less than 10 mm different from the fresh values. Fifty-eight percent of the values (23 of 40) declined from a penetration rate considered normal (above 15 mm penetration)12 to a subfertile value following freezing. The correlation between the MPT values for fresh sperm and frozen sperm was 0.56 (P< 0.0001). The percentage of recovery for sperm motility, MPT, and EPT results was determined by dividing the frozen value by the fresh value and multiplying by 100. The mean percentage of recovery and the SD for these three groups are compared in Table 2. The differences in the percentage of recovery of the three groups was significant (P < 0.0001), the EPT showing the highest recovery rate (mean, 86%) and the MPT the lowest (mean, 38%). Each group had a similar SD. The decline in sperm motility did not significantly correlate with the decline in the EPT results (r = 0.02; P > 0.90) but did with the decline in the MPT results (r = 0.48; P < 0.0002). No significant correlation was found between the EPT and the MPT (r = 0.27; P > 0.10). The EPT, MPT, and sperm motility results were evaluated for differences between known fertile men and patients with fertility problems (Table 3). The fertile and infertile groups were significantly different in all but postfreeze sperm motility and postfreeze MPT values. The range and SD were similar for the fresh and frozen EPT results but were different for the fresh and frozen MPT results. All fertile donors scored above our "normal" value in the EPT when either fresh or frozen samples were used. All fertile donors also Table 2. Comparison of the Percentage of Recovery (Frozen Value/Fresh Value x 100) for the EPT, MPT, and Motility Resultsa Test
% Recovery
EPT MPT
86.0 ± 21.0 38.0 ± 21.9 60.0 ± 18.2
Sperm motility aValues are mean ± SD.
Fertility and Sterility
Table 3. Comparison of the EPT, MPT, and Sperm Motility Values for Known Fertile and Suspected Infertile Individuals Known fertile
Fresh sperm Mean
SD Range Frozen sperm Mean
SD
Range
(n =
24)
Suspected infertile
(n =
26)
EPT
MPT
Motility % motile
EPT
MPT
Motility
% penetration
mm
% motile
% penetration
mm
66.9" 20.2 26-100
38.2b
58.1b
12.4 1~2
8.7 45-75
38.5 19.4 12-70
26.4 14.2 8-52
50.8 13.1 25-70·
57.2a
15.1c
35.9c
16.1 23-87
6.3 5-25
9.2 12-55
33.0 14.6 10-55
10.2 7.7 2-31
31.0 11.4 10-55
ap < 0.001 when compared with the "suspected infertile" value. bp < 0.01 when compared with the "suspected infertile" value. cp > 0.05 when compared with the "suspected infertile" value.
scored above our "normal" value in the MPT when fresh sperm were used; however, 11 of the 19 (58%) dropped below this value when frozen sperm were used. DISCUSSION
The results of this study indicate that 94% of the patients retained their fertile or subfertile classification (below 20% penetration) in the EPT when frozen sperm were used instead of fresh. In other reports utilizing the EPT it has been suggested that patient samples with a minimum of 11%,5 15%,4 or 20%6 egg penetration are considered fertile. After evaluating our data from known fertile donors and all EPT values analyzed to date in our laboratory, we currently consider an egg penetration above 20% to 25% to be normal. In this study, frozen samples from all fertile donors gave a penetration value above 20%. Our technique for the EPT utilized a short preincubation period for the sperm (2 hours), as contrasted with the 16- to 24-hour "overnight" preincubation period often used. 3-7 Freeze-thawed sperm do not often survive the longer incubation period. B,9 Even with frozen sperm and the shorter incubation period, our fertile donor EPT results are similar to those obtained by others3-7 using a longer preincubation period and fresh sperm. Our results demonstrated a low interassay variability. This is consistent with the findings of others6, 7 and indicates that the assay is reproducible. We did include a control sample (usually frozen) with each assay and feel that it is important to have at least a positive control to assure the reliability of each test. Many of our assays also had a negative control, with inactivated Vol. 39, No.5, May 1983
spermatozoa, although the necessity of this is questionable. The difference between the fresh and frozen EPT results was also evaluated by determining what percentage of the penetration rate with fresh sperm was retained following freezing. The recovery rate in our study was over 85%, which agrees with the results of Hall (85%)11 and Binor et al. (80%).9 Cohen et al. lO showed a lower postfreeze recovery of 37%, but this was probably due to reduced sperm motility after the 5-hour preincubation period. It appears that the preincubation period for frozen sperm in this assay should not exceed 2 hours.9 The results of the MPT were dramatically different following freezing. This is consistent with the results of Zavos and Cohen,14 who used bovine cervical mucus, and Fjallbrant and Ackerman,13 who used human cervical mucus. All these results support the conclusion that cryopreserved sperm are less efficacious than fresh sperm in penetrating cervical mucus. Our results demonstrated that all fresh samples from fertile donors yielded "normal" MPT values (> 15 mm of penetration l2 ), but less than half of the frozen samples from fertile donors gave "normal" values. It appears that cervical mucus penetration should not be evaluated with cryopreserved semen in routine fertility assessment because of this large . drop in penetrability. It has been postulated that if the reduction in the ability of frozen sperm to penetrate cervical mucus in the MPT was also noted in situ, there could be a decreased number of spermatozoa available to fertilize the eggP It has been suggested that this might explain the lower pregnancy rates obtained when cryopreserved sperm, Urry et aI. Penetration by cryopreseroed sperm
693
rather than fresh sperm, are used for artificial insemination. 13 In our study, frozen samples from fertile donors penetrated from a high of 25 mm to a low of 5 mm with the MPT. It is not known whether the-frozen samples that scored higher in the MPT would yield better conception rates with artificial insemination than samples that exhibit a major drop. Additional research in this area seems warranted. The differences in the recovery of sperm motility, cervical mucus penetration, and zona-free hamster egg penetration demonstrate that different properties of sperm are affected separately by freezing. The drop in motility following freezing was correlated with the decrease in MPT results, although, as noted by others,14 there was a greater decrease in mucus penetration than in sperm motility. The decrease in sperm motility did not correlate with the decrease in the EPT results. This is probably due to the standardization of all semen samples used in the EPT to a specific motile sperm concentration. There were a few samples (15%) that did not show sufficient recovery of motile sperm following freezing. Our results show that these samples do not yield accurate EPT results and should not be evaluated after freezing with the use of this technique. The lack of correlation between the EPT and MPT results may indicate that these two tests measure different sperm properties. Both tests add useful information in fertility evaluation. REFERENCES 1. Cockett ATK, Netto ICV, Dougherty KA, Urry RL: Se-
men analysis: a review of samples from 225 men seen at an infertility clinic. J Urol 114:560, 1975
694
Urry et aI. Penetration by cryopreseroed sperm
2. Smith KD, Rodriguez-Rigau LJ, Steinberger E: Relation between indices of semen analysis and pregnancy rate in infertile couples. Fertil Steril 28:1314, 1977 3. Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. BioI Reprod 15:471, 1976 4. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620,1981 5. Stenchever MA, Spadoni LR, Smith WD, Karp LE, Shy KK, Moore DE, Berger R: Benefits of the sperm (hamster ova) penetration assay in the evaluation of the infertile couple. Am J Obstet Gynecol 143:91, 1982 6. Hall JL: Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 35:457, 1981 7. Cohen J, Weber RF A, van der Vijver JCM, Zeilmaker GH: In vitro fertilizing capacity of human spermatozoa with the use of zona-free hamster ova: interassay variation and prognostic value. Fertil Steril 37:565, 1982 8. Keel BA, Karow AM: Reduced motility longevity in thawed human spermatozoa. Arch Androl 4:213, 1980 9. Binor Z, Sokoloski JE, Wolf DP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, 1980 10. Cohen J, Felten P, Zeilmaker GH: In vitro fertilizing capacity of fresh and cryopreserved human spermatozoa: a comparative study of freezing and thawing procedures. Fertil Steril 36:356, 1981 11. Hall JL: Unpublished data 12. Alexander NJ: Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril36:201, 1981 13. Fjallbrant B, Ackerman DR: Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J Reprod Fertil 20:515, 1969 14. Zavos PM, Cohen MR: Bovine mucus penetration test: an assay for fresh and cryopreserved human spermatozoa. Fertil Steril 34:175, 1980 15. Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, W. H. Freeman, 1971, p 101
Fertility and Sterility
FERTILITY AND STERILITY Copyright 0 1983 The American Fertility Society
Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa
Ronald L. Urry, Ph.D.*t Douglas T. Carrell:j: Dale B. Hull, B.S. * Richard G. Middleton, M.D. * Milo C. Wiltbank, M.S.:j: University of Utah School of Medicine, Salt Lake City, Utah, and Brigham Young University, Provo, Utah
Semen samples from 25 fertile donors and 34 infertility patients were analyzed and evaluated both fresh and frozen with the zona-free hamster egg penetration test (EPT) and the bovine cervical mucus penetration test (MPT). Cryopreserved sperm penetrated 47.2% (average) of the hamster eggs, and fresh sperm penetrated 55.9%. Frozen sperm retained 86% of the fresh EPT values (p, not significant), but only 38% of the fresh MPT values (P < 0.001). Cryopreserved sperm yielded the same fertility classification as fresh sperm in 94% of the cases with the EPT but only 43% with the MPT. The use of cryopreserved sperm simplifies the procedure and should enable more laboratories to utilize the EPT, but the poor results with the MPT preclude utilization of cryopreserued sperm for routine fertility evaluation with the MPT. Fertil Steril 39:690, 1983
Traditionally, semen analysis has been the main method utilized to evaluate men with fertility problems. A complete semen analysis includes determination of sperm count, number of motile sperm, number of normal forms, viability, volume measurement, white blood cells, agglutination, and viscosity. 1 The semen analysis, although useful i.n fertility workup, is imprecise in its measurement of fertility and may not directly simulate any of the events in the conception process. 2 Recently, new tests have been introduced that
Received September 13,1982; revised and accepted December 23, 1982. *Division of Urology, University of Utah School of Medicine. tReprint requests: Ronald L. Urry, Ph.D., Division ofUrology, University of Utah School of Medicine, Salt Lake City, Utah 84132. :j:Department of Zoology, Brigham Young University. 690
Urry et aI. Penetration by cryopreserved sperm
could provide a more accurate diagnosis of male fertility problems. The zona-free hamster egg penetration test (EPT), introduced by Yanagimachi et al.,3 appears to be effective in differentiating fertile and infertile men. 4 -7 This assay involves the incubation of zona-free hamster eggs with capacitated human sperm and subsequent evaluation of the eggs for penetration by the sperm. The EPT is usually performed with fresh, rather than frozen, human spermatozoa because of reduced motility and longevity of sperm that have been frozen in liquid nitrogen. 8 , 9 Previous studies have shown cryopreserved sperm to be less effective in penetrating zona-free hamster eggs, although there is nO agreement on the extent of this reduction. 9 - 11 Another test of sperm function uses bovine cervical mucus in flat capillary tubes for determination of the ability of human sperm to penetrate cervical mucus. 12 Like the EPT, the mucus peneFertility and Sterility
l tration test (MPT) is normally performed with fresh semen. Attempts to ~tilize frozen semen have demonstrated a large reduction in the ability to penetrate cervical mucus following freezing.la, 14 Although the above two tests may be useful in evaluating male infertility, one aspect that presents technical·difficulties is the rigid timetable that each assay requires. If semen samples could be frozen and saved for evaluation at a more optimal time, the procedure would be simplified for both the patient and the laboratory. The purpose of this study was twofold: (1) to evaluate the exact amount of change in the EPT and MPT results following freezing and (2) to assess the clinical usefulness of cryopreserved semen in the EPT and the MPT. Fresh and frozen sperm from each sample from fertile and suspected infertile individuals were compared. MATERIALS AND METHODS
Semen samples were obtained from 25 known fertile donors and from 34 patients seen at a fertility evaluation clinic. All semen samples were divided following collection, with a portion (approximately 1 ml) saved for evaluation fresh and and equal portion frozen in liquid nitrogen for analysis approximately 1 week later (mean, 8.3 days). The portion to be frozen was first diluted with glycerol and Baker's buffer (1:1) to a final concentration of 7.5% glycerol, lowered into liquid nitrogen vapor for 20 to 30 minutes, and then stored in liquid nitrogen ( -196° C). At the time of the test, the frozen sample was thawed in water at 28° C for 10 minutes and then evaluated with the use of a procedure identical to that for the fresh sample. Both fresh and frozen portions were evaluated for the percentage of progressively (forward) motile' sperm before use in either test. 1 The EPT was performed as previously described by Yanagimachi et al. a with the exception of the modifications described below. The semen being tested in the EPT was diluted to six times its original volume with modified Biggers, Whitten and Whittingham (BWW) medium15 (10 mM HEPES buffer replacing 10 mM NaHCO a) to which was added 1.0% bovine serum albumin (Fraction V, Sigma Chemical Company, St. Louis, MO). This mixture was centrifuged at 500 x g for 5 minutes, and the supernatant was removed, leaving the sperm pellet. The pellet was washed twice and diluted to a progressively motile sperm Vol. 39, No.5, May 1983
concentration of 5 x 106 sperm/ml. A 0.2-ml aliquot of this sperm suspension was placed under mineral oil and preincubated for 2 hours at 37° C. Eggs for the EPT were obtained from mature female Golden hamsters. Hamsters were given injeetions of 40 IU of pregnant mare serum gonadotropin (Sigma) on the day of the postestrus vaginal discharge. Forty-eight to 56 hours later they were given injections of 50 IU of human chorionic gonadotropin (Sigma), and 16 hours later the oviducts were excised and the egg masses removed. The cumulus cells were dispersed by exposure to 0:1% bovine hyaluronidase. The zona pellucida was removed by treatment with 0.3% bovine pancreatic trypsin, ar:td the eggs were washed four times in BWW. Twenty-five to 50 eggs were then placed directly into each sperm suspension and incubated at 37° C for 5 hours. The eggs were then removed, pressed slightly under a gel-suspended coverslip, and examined under phase-contrast microscopy at x 400. The eggs were considered penetrated if a swollen sperm head or a male pronucleus was found in the egg. The percentage of the eggs that were penetrated and the total number of penetrating sperm per egg were recorded. Fifty semen. samples, 25 of which were from men of known fertility, were evaluated fresh and frozen in the EPT. Nine additional samples (one from a fertile donor and eight from suspected infertile patients) were eliminated from the results because of an insufficient motile sperm density follQwing freezing. The interassay variation was monitored by running a control sample from a previously tested fertile man with each group of samples. The MPT was performed by the method described by AlexanderY Flat capillary tubes, filled with bovine cervical mucus, were incubated with the semen for 90 minutes at room temperature. The penetration was reported as the distance (in millimeters) traveled by the vanguard spermatozoa. Means, standard deviations (SD), t-tests, and correlation coefficients were determined for the data, and correlation coefficients were converted to Z values, which were used to determine significance. RESULTS
The proportion of eggs penetrated in the EPT was 55.9% ± 3.2% (mean ± standard error) by Urry et al. Penetration by cryopreserved sperm
691
Table 1. Percentage of Samples Changing Various Increments When the Frozen Sperm Value Was Subtracted from the Fresh Sperm Value with the Hamster Egg Human Sperm EPT or with the Bovine Cervical MPT Percentage of total samples inEPT
Percentage of total samples inMPT
2
o o
20 38 24 14 2
2.5 10 40
o
45 2.5
Difference in EPT or MPT value (fresh EPT or MPT minus frozen EPT or MPT value)
< - 20% difference - 10% 0% 0% + 10% + 20% > + 40%
to - 20% difference to -10% difference to + 10% difference to + 20% difference to + 40% difference difference
fresh sperm and 47.2% ± 2.8% by frozen sperm (P < 0.05). The average decline in penetration by the frozen sperm was 8.8% + 1. 7% for the 50 samples. The frozen samples' EPT values subtracted from those of the fresh samples are given in Table 1. Fifty-eight percent of the frozen samples were found to be less than 10% different from the fresh values, and 84% were less than 20% different from the fresh values. The correlation between EPT results for the fresh and frozen portions was 0.85 (P < 0.0001). The polyspermia index was also calculated by dividing the total number of penetrated sperm by the number of eggs evaluated. The index averaged 0.88 ± 0.10 for the fresh sperm and 0.57 ± 0.05 for the frozen sperm (P < 0.01). Six percent of the sperm samples (3 of 50) declined from a penetration rate considered normal (above 20% to 25% of the eggs penetrated4 - 7 ) to a subfertile value following freezing. Using above 10% as a normal penetration rate,3, 5 none of the samples decre&sed from normal to subfertile following freezing. Nine of the 59 original samples (15%; 1 from a fertile donor and 8 from suspected infertile patients) were eliminated from the analysis because of an insufficient motile sperm concentration « 5 x 106 /ml) following freezing. Three of the nine samples were evaluated fresh and frozen with the EPT, and each experienced a significant drop in penetration rate following freezing (33% to 5%; 30% to 0%; 13% to 0%), which may have been due to the low motile sperm density. The control samples for the EPT were collected from one man over a period of 7 months (8 different semen samples, 23 different tests). These samples had an interassay variation (coefficient of variation) of 11.2% with an SD of7.3%. Two to five semen samples from each of four different 692
Urry et aI. Penetration by cryopreserved sperm
men were examined with the EPT, and the coefficient of variati'on between samples from the same man was 16.3% (7.6% SD). The mean MPT result for sperm from fresh semen was 32.9 ± 2.2 mm, and for frozen sperm it was 12.4 ± 1.2 mm. The migration distance for crY9preserved sperm, therefore, averaged 20.5 mm less than for fresh sperm (P < 0.0001). The difference between fresh MPT values and frozen values is given in Table 1. Only 12.5% of the frozen samples were less than 10 mm different from the fresh values. Fifty-eight percent of the values (23 of 40) declined from a penetration rate considered normal (above 15 mm penetration)12 to a subfertile value following freezing. The correlation between the MPT values for fresh sperm and frozen sperm was 0.56 (P< 0.0001). The percentage of recovery for sperm motility, MPT, and EPT results was determined by dividing the frozen value by the fresh value and multiplying by 100. The mean percentage of recovery and the SD for these three groups are compared in Table 2. The differences in the percentage of recovery of the three groups was significant (P < 0.0001), the EPT showing the highest recovery rate (mean, 86%) and the MPT the lowest (mean, 38%). Each group had a similar SD. The decline in sperm motility did not significantly correlate with the decline in the EPT results (r = 0.02; P > 0.90) but did with the decline in the MPT results (r = 0.48; P < 0.0002). No significant correlation was found between the EPT and the MPT (r = 0.27; P > 0.10). The EPT, MPT, and sperm motility results were evaluated for differences between known fertile men and patients with fertility problems (Table 3). The fertile and infertile groups were significantly different in all but postfreeze sperm motility and postfreeze MPT values. The range and SD were similar for the fresh and frozen EPT results but were different for the fresh and frozen MPT results. All fertile donors scored above our "normal" value in the EPT when either fresh or frozen samples were used. All fertile donors also Table 2. Comparison of the Percentage of Recovery (Frozen Value/Fresh Value x 100) for the EPT, MPT, and Motility Resultsa Test
% Recovery
EPT MPT
86.0 ± 21.0 38.0 ± 21.9 60.0 ± 18.2
Sperm motility aValues are mean ± SD.
Fertility and Sterility
Table 3. Comparison of the EPT, MPT, and Sperm Motility Values for Known Fertile and Suspected Infertile Individuals Known fertile
Fresh sperm Mean
SD Range Frozen sperm Mean
SD
Range
(n =
24)
Suspected infertile
(n =
26)
EPT
MPT
Motility % motile
EPT
MPT
Motility
% penetration
mm
% motile
% penetration
mm
66.9" 20.2 26-100
38.2b
58.1b
12.4 1~2
8.7 45-75
38.5 19.4 12-70
26.4 14.2 8-52
50.8 13.1 25-70·
57.2a
15.1c
35.9c
16.1 23-87
6.3 5-25
9.2 12-55
33.0 14.6 10-55
10.2 7.7 2-31
31.0 11.4 10-55
ap < 0.001 when compared with the "suspected infertile" value. bp < 0.01 when compared with the "suspected infertile" value. cp > 0.05 when compared with the "suspected infertile" value.
scored above our "normal" value in the MPT when fresh sperm were used; however, 11 of the 19 (58%) dropped below this value when frozen sperm were used. DISCUSSION
The results of this study indicate that 94% of the patients retained their fertile or subfertile classification (below 20% penetration) in the EPT when frozen sperm were used instead of fresh. In other reports utilizing the EPT it has been suggested that patient samples with a minimum of 11%,5 15%,4 or 20%6 egg penetration are considered fertile. After evaluating our data from known fertile donors and all EPT values analyzed to date in our laboratory, we currently consider an egg penetration above 20% to 25% to be normal. In this study, frozen samples from all fertile donors gave a penetration value above 20%. Our technique for the EPT utilized a short preincubation period for the sperm (2 hours), as contrasted with the 16- to 24-hour "overnight" preincubation period often used. 3-7 Freeze-thawed sperm do not often survive the longer incubation period. B,9 Even with frozen sperm and the shorter incubation period, our fertile donor EPT results are similar to those obtained by others3-7 using a longer preincubation period and fresh sperm. Our results demonstrated a low interassay variability. This is consistent with the findings of others6, 7 and indicates that the assay is reproducible. We did include a control sample (usually frozen) with each assay and feel that it is important to have at least a positive control to assure the reliability of each test. Many of our assays also had a negative control, with inactivated Vol. 39, No.5, May 1983
spermatozoa, although the necessity of this is questionable. The difference between the fresh and frozen EPT results was also evaluated by determining what percentage of the penetration rate with fresh sperm was retained following freezing. The recovery rate in our study was over 85%, which agrees with the results of Hall (85%)11 and Binor et al. (80%).9 Cohen et al. lO showed a lower postfreeze recovery of 37%, but this was probably due to reduced sperm motility after the 5-hour preincubation period. It appears that the preincubation period for frozen sperm in this assay should not exceed 2 hours.9 The results of the MPT were dramatically different following freezing. This is consistent with the results of Zavos and Cohen,14 who used bovine cervical mucus, and Fjallbrant and Ackerman,13 who used human cervical mucus. All these results support the conclusion that cryopreserved sperm are less efficacious than fresh sperm in penetrating cervical mucus. Our results demonstrated that all fresh samples from fertile donors yielded "normal" MPT values (> 15 mm of penetration l2 ), but less than half of the frozen samples from fertile donors gave "normal" values. It appears that cervical mucus penetration should not be evaluated with cryopreserved semen in routine fertility assessment because of this large . drop in penetrability. It has been postulated that if the reduction in the ability of frozen sperm to penetrate cervical mucus in the MPT was also noted in situ, there could be a decreased number of spermatozoa available to fertilize the eggP It has been suggested that this might explain the lower pregnancy rates obtained when cryopreserved sperm, Urry et aI. Penetration by cryopreseroed sperm
693
rather than fresh sperm, are used for artificial insemination. 13 In our study, frozen samples from fertile donors penetrated from a high of 25 mm to a low of 5 mm with the MPT. It is not known whether the-frozen samples that scored higher in the MPT would yield better conception rates with artificial insemination than samples that exhibit a major drop. Additional research in this area seems warranted. The differences in the recovery of sperm motility, cervical mucus penetration, and zona-free hamster egg penetration demonstrate that different properties of sperm are affected separately by freezing. The drop in motility following freezing was correlated with the decrease in MPT results, although, as noted by others,14 there was a greater decrease in mucus penetration than in sperm motility. The decrease in sperm motility did not correlate with the decrease in the EPT results. This is probably due to the standardization of all semen samples used in the EPT to a specific motile sperm concentration. There were a few samples (15%) that did not show sufficient recovery of motile sperm following freezing. Our results show that these samples do not yield accurate EPT results and should not be evaluated after freezing with the use of this technique. The lack of correlation between the EPT and MPT results may indicate that these two tests measure different sperm properties. Both tests add useful information in fertility evaluation. REFERENCES 1. Cockett ATK, Netto ICV, Dougherty KA, Urry RL: Se-
men analysis: a review of samples from 225 men seen at an infertility clinic. J Urol 114:560, 1975
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Urry et aI. Penetration by cryopreseroed sperm
2. Smith KD, Rodriguez-Rigau LJ, Steinberger E: Relation between indices of semen analysis and pregnancy rate in infertile couples. Fertil Steril 28:1314, 1977 3. Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. BioI Reprod 15:471, 1976 4. Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620,1981 5. Stenchever MA, Spadoni LR, Smith WD, Karp LE, Shy KK, Moore DE, Berger R: Benefits of the sperm (hamster ova) penetration assay in the evaluation of the infertile couple. Am J Obstet Gynecol 143:91, 1982 6. Hall JL: Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 35:457, 1981 7. Cohen J, Weber RF A, van der Vijver JCM, Zeilmaker GH: In vitro fertilizing capacity of human spermatozoa with the use of zona-free hamster ova: interassay variation and prognostic value. Fertil Steril 37:565, 1982 8. Keel BA, Karow AM: Reduced motility longevity in thawed human spermatozoa. Arch Androl 4:213, 1980 9. Binor Z, Sokoloski JE, Wolf DP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, 1980 10. Cohen J, Felten P, Zeilmaker GH: In vitro fertilizing capacity of fresh and cryopreserved human spermatozoa: a comparative study of freezing and thawing procedures. Fertil Steril 36:356, 1981 11. Hall JL: Unpublished data 12. Alexander NJ: Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril36:201, 1981 13. Fjallbrant B, Ackerman DR: Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J Reprod Fertil 20:515, 1969 14. Zavos PM, Cohen MR: Bovine mucus penetration test: an assay for fresh and cryopreserved human spermatozoa. Fertil Steril 34:175, 1980 15. Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, W. H. Freeman, 1971, p 101
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