FACTORS AFFECTING SPERM MOTILITY. V. WASHING AND RESUSPENSION OF HUMAN SPERMATOZOA IN VARIOUS ARTIFICIAL MEDIA

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1 FERTILITY AND STERILITY Copyright c 1981 The American Fertility Society Vol. 35, No.4, April 1981 Printed in U.s.A. FACTORS AFFECTING SPERM MOTILITY. V. WASHING AND RESUSPENSION OF HUMAN SPERMATOZOA IN VARIOUS ARTIFICIAL MEDIA AMNON MAKLER, M.D.* PETER JAKOBI, M.D. t Infertility Institute, Department of Obstetrics and Gynecology, Rambam Medical Center, Technion Faculty. of Medicine, Haifa, Israel Fresh semen specimens from fertile donors were subjected to one-step and two-step washings in six various commonly used artificial media. It was found that washing procedures per se, in most cases, had an immediate and extended harmful effect on sperm motility which was much more prominent after the second washing. However, human albumin, when added, could usually protect spermatozoa {rom this deleterious effect. None of the tested media showed any stimulatory or "revitalization" effect, and the increase in 'sperm velocity after one washing in some of these media was attributed to a simple decrease in viscosity of the original seminal fluid. The possible mechanism of the deleterious effect of sperm washings as related to the property of the medium and some implications for practical clinical and research studies are discussed. Fertil Steril35:442, 1981 Separation of spermatozoa from the seminal fluid and their resuspension in foreign media is a very common procedure used by clinicians and investigators. 1-4 Exchange of seminal fluid with a foreign medium is performed after semen is centrifuged and sedimented spermatozoa are resuspended in the desired medium. However, in this one-step washing, only about 85% to 90% of the original fluid is eliminated, and this may not be adequate when completely washed spermatozoa are required. By repeating this procedure, i.e., two-step washing, approximately 97% of the original fluid can be eliminated and a suspension which is almost seminal fluid-free is thus obtained. These multi-stage procedures can hardly be considered innocuous to spermphysiology, motility, and viability, and deleterious effects have been reported by others. 5 The effect of the first stage, namely centrifugation per se at various speeds and durations on sperm motility, Received May 20, 1980; "revised August 19, 1980; revised and accepted November 26,1980. *Head of Infertility Institute. To whom reprint requests should be addressed. tsubmitted in partial fulfillment of the M.D. Thesis as required by the Sackler Medical School, Tel-Aviv University. was carefully studied by us and reported in a previous study.61t was found that sperm motility and viability was affected very little in specimens subjected to centrifugation at 320 x g for 10 or 20 minutes, by which an efficient separation and a minimum of sperm loss in the supernatant were provided. In the present study, the effect of both one-step and two-step washing with various artificial media on sperm motility and viability was investigated with the multiple-exposure photography (MEP) method for objective sperm motility determination. From these results an attempt was made to discern which of the common artificial media is the least harmful when used for clinical and research purposes. MATERIALS AND METHODS Semen Specimens. Fresh semen specimens were obtained by masturbation from healthy normospermic (sperm count above 20 million/ml) donorsafter 3 to 4 days of abstinence. According to standards established from results of previous objective measurements, a motility rate of 40% to 45% defines the borderline between the two groups.' 442

2 Vol. 35, No.4 FACTORS AFFECTING SPERM MOTILITY. V. 443 TABLE 1. Chemical Composition, ph, and Osmolar.ityofthe Six Media Used Medium Composition ph" Osmolarity" Glucose Nacl Na2HPO. NaH2PO. MgCl2 KCl CaCl2 Albumin M W, W2 M W, W 2 gm/liter mosmoleslkg water 5% Glucose Normal saline Buffered phos phate Baker's solu tion Tyrode's solu tion Tyrode's albumin Pooled semi nal fluid am, Medium alone; W 1> one-step washing; W 2, two-step washing. Each value represents the final value of the last two parameters. Washing Procedures. Within 1 hour after ejaculation,each specimen was divided into four equal portions. The first two portions served as experimental specimens, and both were subjected to a washing procedure as follows: after centrifugation at 320 x g (1500 rpm) for 10 minutes, approximately 85% to 90% of each supernatant was gently aspirated and equal volumes of each of the artificial media listed below were used for resuspension of the pellet. This one-step washing was followed by the same procedure, repeated on only one of the washed specimens, after which only 3% to 4% of the original seminal fluid remained in the two-step washed specimen. The other two portions were centrifuged only, at the same rate of 320 x g for 10 or 20 minutes, and served as control 1 and control 2 for the one-step and two-step washing procedures, respectively. Eight different specimens were included in each experiment and were washed with one of the six sterile media listed in Table 1. Table 1 contains data on the chemical compositions of these media, as well as ph and osmolarity before and after spermatozoal washings. Osmolarity was determined by the cryoscopy method, using the Fiske osmometer model 130. ph was determined with a glass electrode ph meter (Radiometer, model PHM 28, Copenhagen, Denmark). The rationale for selecting these media was to determine systematically how the supply or deprivation of some commonly used ingredients in various artificial physiologic media affects sperm survival. Sperm velocity and percentage of motility were analyzed in all specimens, kept at room temperature, before and at 60, 120, and 240 minutes after the experiments had been initiated. Assessment of Motility. Sperm motility was assessed with the MEP method described by us elsewhere. B 9 Each of two or three drops from a wellmixed specimen was placed in the improved 10- f.lm chamber 10 (manufactured by EL-OP, Israel Electrooptical Industry, Rehovoth, Israel), and four to six fields each containing 200 to 400 spermatozoa were photographed at predetermined locations. The film was exposed each time for 1 second during which time the sample was illuminated by six light pulses. Images of photographed spermatozoa were projected onto sheets of paper from which the percentage of motile spermatozoa and their velocity were calculated with the aid of a microcomputer described in detail elsewhere. ll In those cases where motility was significantly affected by the washing procedures, sperm viability was also analyzed by the combined supravital staining and the MEP method described by us elsewhere. 12 Each sample was mixed with 1% eosin Y for 2 minutes and photographed as described, using a green filter and color slides. From projected images the ratios of dead spermatozoa (red-stained), nonmotile living spermatozoa (unstained, appear green), and motile spermatozoa (chains) were determined simultaneously. Results from all experiments were evaluated statistically, and significance was determined using the paired t-test. RESULTS Figures 1 and 2 describe graphically the relative changes in sperm velocity and percentage of motility during 4 hours for washed spermatozoa as compared with curves for the control specimens. Washing procedures per se in most cases induced an immediate decrease in percentage motility which lasted for the subsequent 4 hours.

3 444 MAKLER AND JAKOBI April 1981,DISCUSSION ONE STEP WASHING (WI) GWC TWO STEP WASHINGS (W2) :==::::::::::==~""'CONT2 TYR-ALB o mini. TIME AFTER WASHINGS FIG. 1. Relative change in percentage motility with time after spermatozoa were washed once or twice as compared with standard curves constructed from all 48 controll (CONT 1) and control 2 (CONT 2) specimens, respectively. Each point on the experimental curves represents the mean of values for eight specimens washed in that medium and indicates the difference between the controls, being transformed and adjusted to the standard control curve. TYR -ALB, Tyrode's-albumin; NSaline, normal saline; B Phos, buffered phosphate; BAKER, Baker's solution; GLUC, glucose. This effect was much more marked in twicewashed spermatozoa. Sperm velocity was similarly affected but to a lesser extent~ Concerning both parameters, the effect varied among the media and was found to be least extensive in the medium containing all of the ingredients listed in Table 1. On the other hand, the medium containing only isotonic glucose induced the most dramatic effect on both sperm velocity and percentage of motility. The other media had intermediate negative effects that varied slightly and insignificantly. The histograms depicted in Figure 3 describe the ratio between motile, nonmotile live, and dead spermatozoa as analyzed with the combined supravital staining technique and the MEP method 1 hour after washing twice with Tyrode's-albumin or isotonic glucose solution. After washing with Tyrode's-albumin, motile spermatozoa became mainly nonmotile live, but the fraction of dead spermatozoa did not increase. However, after washing with glucose, most motile spermatozoa shifted either directly or indirectly to the fraction of dead spermatozoa. In both cases these effects were significant (P < 0.01). The harmful effect of washing on sperm motility and survival has been reported by others 5 but was noted mainly when two-step washing procedures were performed. The effect of one-step washing on sperm motility has been almost completely ignored by most investigators, probably because evaluations were made with the inaccurate subjective method. The harmful effect of washing is thought to be the same as that of extensive semen dilution: damage to the cell membrane of the spermatozoa and leakage of vital substances out of this delicate cell membrane. l3-16 The one exception was when human albumin was added to the washing medium, which prevented the harmful effects on sperm motility of one-step washing and most of those of two-step washing. The protective effect of albumin has been reported by Eliasson and Lindholmer 17 and was explained as due to coverage of the cellular membrane by molecules of albumin, thereby preventing any major damage to the washed spermatozoa. None of the other media was obviously superior to normal saline, even VELOCITY I~r ',-.:O:.::NE:S:T:.:E~P WASHING (WI) 100~===:::::::::::'- IZ 50 3 m- ALB CONTI BAKER ~~:::::::::::~::--... ;;;;;;;::--- NSalin. Bl'llOl GLUC ~ ~ o~ TWO STEPS WASHINGS (W2) ~ 120 ~~~~::: CONT TYR-ALB. ~======================::TYR NSalino =:'P':: ' GLUC min. TIME AFTER WASHINGS FIG. 2. Relative change in sperm velocity with time after spermatozoa were washed once or twice in each one of the tested media. Curves were constructed in the same way as in Figure 1. Abbreviations are defined in legend to Figure 1.

4 Vol. 35, No.4 FACTORS AFFECTING SPERM MOTILITY. V. 445 PERCENT MOTILE 0 NML~ DEAD. CONTROL TVR"'ALB GLUCOSE FIG. 3. Histograms describing ratio between motile, nonmotile live (NML), and dead spermatozoa 1 hour after being subjected to two-step washing in Tyrode's-albumin (TYR ALB) or in 5% glucose as compared with the controls. Values represent mean values from four different specimens determined with the combined supravital staining and the MEP method. though important ingredients such as potassium, phosphate, calcium, and magnesium were supplied. Also, the addition of glucose to the buffered medium (Baker's solution) was of no value to the washed spermatozoa. A solution containing only glucose induced a very harmful effect, presumably due to deprivation of vital inorganic components, despite the fact that isotonicity and normal ph were preserved. This caused a direct toxic effect to the washed spermatozoa, as was demonstrated in results determined by the supravital staining technique (Fig. 3). Our finding that in some media sperm velocity increased after one-step washing does not necessarily reflect an excitatory or stimulating effect. It seems to be the result of a decrease in seminal fluid viscosity as was found in a previous study18 in which the effects of simple semen dilutions were investigated. There is no doubt that natural seminal fluid is not the ideal medium for spermatozoa: it is more viscous than other aqueous media and therefore impairs sperm progression; it may contain components harmful to normal sperm physiology such as antibodies, microorganisms, leukocytes, and other deleterious secretions from the seminal vesicles19 or have a suboptimal ph. Therefore investigators have been experimenting with various artificial media to replace seminal fluid in order to improve sperm motility and viability.2o,21 Despite our careful objective and quantitative investigation we could not confirm any "revitalization" or improvement of sperm motility and viability by any of the most commonly used media, in contradiction to reports by others.22, 23 This discrepancy may be explained by one of two possibilities: (1) The increased sperm velocity may have been misinterpreted because the natural seminal fluid was replaced by a less viscous aqueous medium as was mentioned above. (2) The procedure of washing includes several steps, and each one may be essentially unharmful; however, the cumulative effect of these subliminal stages may exceed a level that finally might induce a remarkable decrease in sperm motility. It may be concluded that if washings are performed just to improve sperm motility and viability, other media should be sought. A study in which some natural media are being investigated for this purpose is now in progress at our Institute. REFERENCES 1. Shulman S, Harlin B, Davis P, Reyniak JV: Immune infertility and new approach to treatment. Fertil Steril 29:309, Rozin S, Rozin R: The influence of body fluids on motility of spermatozoa. Int J Fertil1:327, Homonay TZ, Paz G, Sofer A, Kreices PF: Effect of caffeine on motility, velocity, oxygen consumption and glycolysis of ejaculated human normokynetic spermatozoa. Int J Fertil 21:163, Lindholmer CH: The importance of seminal plasma for human sperm motility. BioI Reprod 10:533, Mann T: The Biochemistry of Semen and the Male Reproductive Tract. London, Methuen and Co, 1964, p Makler A, J akobi P: Effects of shaking and centrifugation on human sperm motility. Arch Androl 4:85, Makler A, Itzkovitz J, Brandes JM, Paldi E: Sperm velocity and percentage of motility in 100 normospermic specimens analyzed by the multiple exposure photography method. Fertil Steril 31:155, Makler A: A new multiple exposure photography IMEP) method for objective sperm motility determination. Fertil Steril 30:192, Makler A: Use of the elaborated multiple exposure photography method in routine sperm motility analysis and for research purposes. Fertil Steril 33:160, Makler A: The improved 10-f.lm chamber for rapid sperm analysis. Fertil Steril 33:337, Makler A: Use of a microcomputer in combination with the MEP method for sperm motility determination. J Urol 124:372, 1980

5 446 MAKLER AND JAKOB! April Makler A: Simultaneous differentiation between motile, nonmotile live and dead spermatozoa by combining the supravital staining and the MEP method. Int J Androl 2:32, Singer R, Bonnet M, AllaloufP, Chowers T: Sensitivity of human spermatozoa to various isolation procedures: differences in relation to sperm count. IntJ Fertil24:33, Jones RC, Holt WV: The effect of washing on the ultrastructure and cytochemistry of ram spermatozoa. J Reprod Fertil 41:159, Wales RG, White IG: Viability of diluted dog spermatozoa in vitro. J Reprod Fertil 5:67, Tampion D, Gibbons RA: Swimming rate of bull spermatozoa in various media and the effect of dilution. J Reprod Fertil 5:259, Eliasson R, Lindholmer CH: Human seminal plasma and sperm survival and transport. In The Biology of Spermatozoa, Edited by ESE Hafez. Basel, Karger, 1975, p Makler A, Blumenfeld Z, Brandes JM, Paldi E: Factors affecting sperm motility. II. Sperm velocity and percentage of motility as affected by semen dilution. Fertil Steril 32:443, Eliasson R, Lindholmer CH: Distribution and properties of spermatozoa in different fractions of split ejaculates. Fertil Steril 23:252, Hafez ESE: Human Semen and Fertility Regulation in Man. St Louis, CV Mosby Co, 1976, p Schirren C: Practical Andrology. Berlin, Verlag Bruder Hartman, 1972, p Freund M, Wiedeman J: Factors affecting the dilution, freezing and storage of human semen. J Reprod Fertilll: 1, Joel CA: Fertility Disturbances in Men and Women. Basel, Karger, 1971, p 80