Improvement of post-thaw sperm motility in poor quality human semen*

FERTILITY AND STERILITY Vol. 6, No.4, October 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U. S. A. Improvement of post-thaw sperm motility in poor quality human semen* Chiravudh Sawetawan, M,D, t Eric S. Bruns, M.S. Gail S, Prins, Ph.D.* Department of Obstetrics and Gynecology, University of Illinois College of Medicine and Michael Reese Hospital, Chicago, Illinois Objective: To find alternative cryopreservation methods to improve the post-thaw fertilizing capacity of poor quality human sperm. Design: Controlled clinical study. Setting: Fertility clinic of a teaching hospital. Patients: Men with poor quality semen samples, i.e., asthenozoospermia «4% motile sperm) and/or oligozoospermia «2 X 1 6 sperm/ml). Fertile sperm donors were used for comparison. Interventions: Semen samples were divided into four aliquots and slowly diluted 1:1 with: [1] n-tris (hydroxymethyl) methyl-2-amino ethane sulfonic acid (TES) and tris (hydroxymethyl) aminomethane (Tris)-citric acid-egg yolk buffer with 12% glycerol (TEST), [2] TEST + CryoSeeds (Cell Systems, Ltd., Cambridge, UK), [3] TEST + 1 mm dithiothreitol (DTT), or [4] TEST + CryoSeeds + 1 mm DTT. Cryovials were frozen using slow staged cooling and static vapor freeze and stored at -196 C. Main Outcome Measure: The frozen aliquots were randomly thawed and, after 15 minutes at 37 C, motion analysis was performed. Results: The percent motility after freeze-thaw in TEST was significantly decreased to 42 ± 5% of prefreeze motility (P <.1). Addition of CryoSeeds with holding at -5 C for 1 minutes resulted in 47 ± 6% of prefreeze motility, which was not different than TEST alone. Addition of DTT to TEST significantly improved post-thaw motility over TEST alone to 71 ± 7% of initial motility (P <.1). The combination of CryoSeeds and DTT further improved post-thaw motility to 8 ± 1% of initial motility, which was not different than the neat semen. Conclusion: The present results suggest that DTT, a reducing agent that prevents oxidation of sulfhydryl groups, protects poor quality spermatozoa from excessive cryodamage. Thus, DTT along with seeding may be a useful addition when long-term storage of poor quality semen is crucial for maintaining reproductive potential. Fertil Steril 1993;6:76-1 Key Words: Human sperm, cryopreservation, sperm motility Received March 4, 1993; revised and accepted June 24, 1993. * Presented at American Fertility Society Annual Meeting, New Orleans, Louisiana, November 1 to 3, 1992. t Present address: Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas, :j: Reprint requests: Gail S. Prins, Ph.D., Reproductive Biology Laboratory, Dreyfus Research Building, Room 812, Michael Reese Hospital, 2929 South Ellis Avenue, Chicago, Illinois 6616. Human sperm cryopreservation has been widely used and applied since the 196s. In the past decade, improved success of preserving sperm has been realized with better methodologies, equipment, and cryoprotective agents (1, 2). Under these optimized conditions, the mean motility rate after freezing and thawing is 6% to 7% of fresh specimens when normal semen from fertile donors is frozen. However, semen samples of initial poor quality, as obtained from men with testicular carcinoma, freeze with low success even when an optimized cryobuffer system is used. Accordingly, cryopreservation of subfertile specimens has not proved beneficial in its ability to achieve successful 76 Sawetawan et al. Post-thaw motility in poor quality sperm Fertility and Sterility

pregnancies (3, 4). Unfortunately, men who are diagnosed with testicular cancer, lymphoma, or Hodgkin's disease often have sperm that are either subfertile or considered infertile (5-7). Although the treatment modalities for these diseases may obtain long-term survival for the young patient, they are destructive to spermatogenesis and may render the patient permanently sterile. For these men, semen autopreservation before chemotherapy may be the only alternative to complete sterility. Since freezing conditions that are optimal for normospermic samples have not been adequate for the poor quality specimen, it is essential to find alternative cryopreservation methods that will improve the post-thaw fertilizing capacity of poor quality sperm. In the present study, we have investigated the effects of inducing ice nucleation (seeding) and/or the addition of dithiothreitol (DTT), a reducing agent, to an otherwise optimized cryobuffer system with regards to improving the postthaw motility of poor quality sperm, i.e., asthenozoospermia «4% motile) and/or oligozoospermia «2 X 1 6 /ml) in the neat semen. Both seeding and DTT have previously been shown to modestly improve post-thaw recovery of normal sperm (8, 9). We were therefore interested in determining iftheir protective effects would extend to poor quality semen as well and thus aid in optimizing the postthaw recovery. MATERIALS AND METHODS Semen Specimens Semen samples collected from patients undergoing workup in a male infertility clinic were obtained by masturbation ~1 hour before semen analysis. Each specimen was allowed to liquify at 37 C and semen analysis was performed. Volume, ph, morphology, presence or absence of agglutination, and white blood cell count were measured by a trained technician. Samples were then analyzed for sperm concentration, total sperm count, and percent motility by videomicrography (CellSoft; CryoResources, Ltd., New York, NY). The CellSoft parameter settings used throughout this study were: 23 frames analyzed at an image-sampling frequency of 3 frames/second; 1 frame minimal sampling for motility; 1 frames minimal sampling for velocity and amplitude lateral head displacement (ALH); 8 Il/s threshold velocity for velocity and ALH measurements; 3 Il/s maximum velocity; and a cell size range of 4 to 25 pixels with a magnification calibration of.688 Il/pixel. Specimens with motility <4% and/or concentration <2 X 1 6 /ml (n = 2) were considered as poor quality semen and were included in the study. In addition, 1 normal semen samples from our donor program were collected and processed in a parallel experiment for comparison with the subfertile specimens. Cryoprotective Medium A zwitterionic buffer containing n-tris (hydroxymethyl) methyl-2-amino ethane sulfonic acid (TES) and tris (hydroxymethyl) amino methane (Tris) with citric acid, egg yolk, and 12% glycerol (TEST yolk buffer; Irvine Scientific, Santa Ana, CA) was used as a cryoprotective buffer for all samples undergoing freezing. This buffer system has been previously shown to be optimal for cryopreservation of normospermic human samples (1). Experimental Groups and Freezing Technique Before freezing, each semen sample was thoroughly mixed and divided into four aliquots. Each portion was slowly diluted 1:1 with either: [1] TEST egg yolk buffer with 12% glycerol (TEST), [2] TEST + CryoSeeds (Cell Systems Limited, Cambridge, UK), [3] TEST + 1 mm DTT, or [4] TEST + CryoSeeds + 1 mm DTT. Thus the final concentration of glycerol was 6% and DTT was 5 mm. Cryoseeds are micronized particles coated with xygon, which induce ice nucleation at -6 to -8 C. After addition of the buffer, the samples were placed in a water bath at room temperature (24 C) and slowly cooled at a rate of -5 C/min to 4 C. One milliliter of each sample was loaded into precooled (-5 C) plastic vials (Nunc, Naperville, IL), which were immersed in an ice bath (-5 C). After holding at -5 C for 1 minutes, the vials were suspended over static nitrogen vapor to allow samples to freeze at a rate of -2 C/min to -8 C. The samples were then plunged into liquid nitrogen for storage at -196 C. Thawing and Motility Analysis The four groups from each sample were stored and thawed together. After 1 week in storage, the samples were removed from the holding tanks, thawed in a 37 C water bath for 15 minutes, and analyzed within 3 minutes after thaw. The four aliquots were randomly selected and the observer Vol. 6, No.4, October 1993 Sawetawan et al. Post-thaw motility in poor quality sperm 77

Table 1 Prefreeze Semen Analysis Concentration (X1 6 /ml) Percent motile Velocity (I1m/s) Linearity ALH (mean) Beat-cross frequency * P <.1 versus donor. t P <.5 versus donor. Donor (n = 1) 113. ± 15 81. ± 3 42.4 ± 2.1 5.3 ±.2 2.4 ±.1 13 ±.4 Patient (n = 2) 21.6 ± 8* 33 ± 2.5* 37.7 ± 1.9 4.71 ±.2t 2.3 ±.1 11.8 ±.3t was blinded for analysis. A single observer performed motion analysis using CellSoft following a predetermined grid pattern on a MaIder specimen chamber (Irvine Scientific, Ltd., Santa Ana, CA). Statistical Analysis Individual motility scores for each sample were normalized to the percentage of motile spermatozoa in the fresh semen for that sample. The data were subjected to analysis of variance. Significant differences between the means were determined by the Scheffe Test. RESULTS Prefreeze semen analysis of the donor specimens and poor quality specimens are shown on Table 1. Sperm concentration, percent motility, linearity, and beat cross frequency were significantly lower in the poor quality patient samples than in the donor population. Sperm concentration and percent motility in the patient population were considered subfertile or of poor quality as defined by the W orid Health Organization (1) semen standards. Patient samples were subnormal in sperm concentration, initial percent motility, or both. Of the 2 patient samples, 4 had initial percent motility <2%, 12 possessed between 2% and 4% motile cells, and 4 contained between 41% and 54% motile sperm. With regards to sperm concentration, 11 samples were <2 X 1 6 sperm/ml, 3 were between 2 and 3 X 1 6 sperm/ml, and 6 had concentrations between 31 and 72 X 1 6 sperm/ml. In the poor quality patient specimens, significant differences were noted in the post-thaw motilities of sperm in the four treatment groups (Fig. 1). The percent motility after freeze-thaw in TEST buffer was significantly decreased to 42% ± 5% of the prefreeze motility (P <.1). Post-thaw motility was not improved when CryoSeeds alone were added to the TEST buffer to induce seeding at -6 C (47% ± 6%; mean ± SE). The addition ofdtt to TEST buffer significantly improved the post-thaw motility over TEST alone to 71 % ± 7% of initial motility (P <.1). The combination of DTT and Cryo Seeds further improved the post-thaw motility to 8% ± 1% of initial motility, which was not statistically different from the initial motility of the prefreeze samples. Post-thaw recoveries in the DTT and CryoSeeds group were further analyzed based on initial sperm concentration and motility to determine whether subpopulations existed in the poor quality patient samples. The percent of initial motility that was retained after freeze-thaw in the patients with sperm concentrations < 2 X 1 6 /ml, between 2 and 3 X 1 6 /ml, and between 31 and 54 X 1 6 /ml were 83%,69%, and 79%, respectively. The percent of initial motility that was retained after freezethaw in patients with initial motilities < 2%, between 2% and 4%, and between 41% and 54% were 84%, 78%, and 64%, respectively. None of these values were significantly different from each other. In the fertile donor samples, each of the treatment groups showed a significant decrease in postthaw motility when compared with the initial prefreeze motility (P <.1) with average results r-. c W N 12 1 :J 8 -< ::il :: o 3-6 ~ :J i= ::il ~ 4 2 o FRESH TEST SEED OTT SEED+DTT Figure 1 Post-thaw motility of poor quality semen samples frozen in various cryobuffer systems. Data is normalized to the percent motility of each initial sample before freeze (Fresh), which was considered 1%. **P <.1 versus Fresh, *P<.5 versus Fresh, +P <.1 versus Seed+DTT. 78 Sawetawan et al. Post-thaw motility in poor quality sperm Fertility and Sterility

1,..., c W N ::J..:( ::E 12 1 8 a::: z 6 '-" S i= ::E. ei\! 4 2 o FRESH TEST SEED OTT SEED+DTT Figure 2 Post-thaw motility of fertile donor semen samples frozen in various cryobuffer systems. Data is normalized to the percent motility of each initial sample before freeze (Fresh), which was considered 1%. *P <.1 versus Fresh. between 57% and 64% of the prefreeze value (Fig. 2). The addition of DTT and/or CryoSeeds to the TEST -yolk buffer did not improve the average post-thaw motilities in this population of sperm cells. DISCUSSION Normal semen samples appear to withstand freezing and thawing better than the poor quality specimen. Although decreased motility is seen post-thaw in normal samples, this apparent result of cryoinjury is exaggerated in samples with initial poor motility and sperm count. It may be likely that poor quality specimens have more spermatozoa with defective plasma membranes and internal organelles that render them a higher susceptibility for cryoinjury. In the present study, a marked improvement in post-thaw motility of poor quality semen samples was observed with the addition of DTT to the cryobuffer. No immediate improvement was noted in post-thaw motility when the samples were seeded before freezing. However, seeding did appear to have an additive beneficial effect when used in conjunction with DTT. The improvement was the same for oligozoospermic, asthenozoospermic, and oligoasthenozoospermic samples, suggesting that improvement in post-thaw motility relates to the overall suboptimal quality of the sperm produced. Interestingly, these beneficial effects were not observed in the normospermic samples. The result of freezing and thawing sperm depends on numerous factors, the most significant of which is the phase change of water from liquid to solid. Inducing extracellular ice formation (seeding) allows minimal supercooling of the medium, thereby shortening the time the cells are exposed to dehydration as well as intracellular ice formation, both of which contribute to cellular damage. Past investigators using other cell types have shown that seeding results in a higher post-thaw survival of these cells (9, 11-13). Critser et al. (8) studied the effects of holding human sperm at -5 C for 1 minutes with or without seeding. Improved motility immediately after thaw for seeded specimens was not observed; however, at 24 hours after thaw, seeded specimens had significantly higher motility than specimens that were not seeded. In the present study, poor quality frozen-thawed sperm samples did not survive for 24 hours after thaw, therefore, their motility could not be analyzed at that time. It is possible, however, that seeding may extend the life span of the poor quality spermatozoa after thawing. Another major factor in loss of motility during freezing is cellular damage, especially damage to the plasma membrane. This has been described by several investigators and may be related to generation of superoxide radicals and peroxides in membrane lipids during freezing (14, 15). Rao and David (9) have shown that the addition of DTT, a reducing agent that prevents oxidation of sulfhydryl groups, significantly improved post-thaw motility in healthy donor sperm. Dithiothreitol has been used in other fields of study relating to cell injury caused by radiation or other substances toxic to the cell. Dithiothreitol may exert its protective effect by scavenging free radicals and preventing lipid peroxidation and destruction of disulfide bonds of membrane proteins and enzymes (9). Improvements in post-thaw motility due to CryoSeeds and DTT addition to TEST-yolk buffer were not observed when the present experiments were extended to semen samples from normal fertile donors. Apparently, maximum cryoprotection was achieved with the TEST -yolk buffer system. Because the normospermic samples were not examined 24 hours after thaw, the possibility remains that Cryoseeds and DTT may improve the longevity of these thawed sperm cells. Perhaps the factors that render poor quality semen more susceptible to cryoinjury, factors that may not be present in Vol. 6, No.4, October 1993 Sawetawan et al. Post-thaw motility in poor quality sperm 79

normospermic samples, are the site of action of the protective effects of DTT. Differences are indeed apparent in the nuclear maturity of sperm cells. Sub fertile semen samples have been shown to have a higher proportion of immature sperm as well as those with nuclear chromatin defects (16). During the maturation process of spermatozoa, one aspect that has been demonstrated is the stabilization and condensation of the nuclear material by formation of disulfide bonds between the nuclear protamines (17). Although the current methods of cryopreservation appear adequate for most normal semen specimens, there is a pressing need for optimization of cryoconditions for poor quality spermatozoa. The present results suggest that, for the poor quality semen samples from patients who require sperm cryopreservation, the added effort of seeding and addition of DTT to the cryobuffer may prove to be of great benefit in improving the conception potential. Acknowledgments. We thank Ms. Mary Coppolillo and Ms. Lynn Birch at Michael Reese Hospital for assistance during preparation of the manuscript. REFERENCES 1. Weidel L, Prins GS. Cryosurvival of human spermatozoa frozen in eight different buffer systems. J AndroI1987;8:41-7. 2. Pilikian S, Czyba JC, Guerin JF. Effect of various concentration of glycerol on post-thaw motility and velocity of human spermatozoa. Cryobiology 1982;19:147-53. 3. Reed E, Sanger WG, Armitage JO. Results of semen cryopreservation in young men with testicular carcinoma and lymphoma. J Clin Oncol 1986;4:537-9. 4. Redman JR, Bajorunas DR, Goldstein MC, Evenson DP, Gralla RJ, Lacher MJ. Semen cryopreservation and artificial insemination for Hodgkin's disease. J Clin Oncol 1987;5:233-8. 5. Hendry WF, Stedronska J, Jones CR, Blackmore CA, Barrett A, Peckham MJ. Semen analysis in testicular cancer and Hodgkin's disease: pre and post treatment findings and implication for cryopreservation. Br J Urol 1983;55:769-73. 6. Scammel GE, White N, Stedronska J, Hendry WF, Edmonds DK, Jeffcoate SL. Cryopreservation of semen in men with testicular tumour or Hodgkin's disease results of artificial insemination of their patients. Lancet 1985; 2:31-2. 7. Berhelsen JG. Testicular cancer and fertility. Int J Androl 1987;1:371-8. 8. Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction. Fertil Steril 1987;47:656-63. 9. Rao B, David G. Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol. Cryobiology 1984;21:536-4l. 1. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 3rd ed. Cambridge, England: Cambridge University Press, 1992. 11. Mazur P. Theoretical and experimental effects of cooling and warming velocity on the survival of frozen and thawed cells. Cryobiology 1966;2:181-92. 12. Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 1977;14:251-72. 13. Fiser PS, Hansen C, Underhill KL, Shrestha NB. The effect of induced ice nucleation (seeding) on the post-thaw motility and acrosome integrity of boar spermatozoa. Anim Reprod Sci 1991;24:293-34. 14. Jones R, Mann T. Damage to ram spermatozoa byperoxidation on endogenous phospholipids. J Reprod FertiI1977;5: 261-8. 15. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987;81:459-69. 16. Liu DY, Baker HWG. Tests of human sperm function and fertilization in vitro. Fertil Steril 1992;58:465-83. 17. Bedford LM, Calvin It, Cooper GW. The maturation of spermatozoa in the human epididymis. J Reprod Fertil 1973;18:199-213. 71 Sawetawan et al. Post-thaw motility in poor quality sperm Fertility and Sterility