FACTORS AFFECTING SPERM MOTILITY. II. HUMAN SPERM VELOCITY AND PERCENTAGE OF MOTILITY AS INFLUENCED BY SEMEN DILUTION*

Transcription

1 FERTlJTY AND SrERLTY Copyright 1979 The American Fertility Society Vol. 32. No.4. October 1979 Printed in U.SA. FACTORS AFFECTNG SPERM MOTLTY.. HUMAN SPERM VELOCTY AND PERCENTAGE OF MOTLTY AS NFLUENCED BY SEMEN DLUTON* AMNON MAKLER, M.D.t ZEEV BLUMENFELD, M.D. JOSEPH M. BRANDES, M.D. ETAN PALD, M.D. nfertility nstitute, Department of Obstetrics and Gynecology, Rambam Medical Center, Technion Faculty of Medicine, Haifa, srael Spermatozoal velocity and percentage of motility were analyzed objectively with the multiple exposure photography method before and after specimens from fertile and infertile men were diluted in their own seminal plasma or normal saline. No significant change in percentage of motility was found in samples diluted up to 1:6 in both kinds of diluents. However, a significant relative increase (up to 25% of the original velocity) was found when a specimen was diluted with its own seminal plasma, and an even greater increase (up to 37% of the original velocity) was found when it was diluted with saline. Compared with undiluted specimens, there was no delayed effect on spermatozoal motility when semen was diluted with saline after up to 4 hours' incubation time. Contrary to the findings in animal and human semen described by others, there was no deleterious effect on sperm motility with this kind and rate of dilution and duration of time. The assumption that the increase in sperm velocity caused by dilution is not excitatory but is due only to a decrease of seminal fluid viscosity and a reduced number of spermatozoa which interfere with sperm free movement is discussed. We recommend evaluation of spermatozoal motility in diluted specimens in addition to evaluation of the original specimen in any routine semen analysis in order to determine true spermatozoal motility potential under optimal conditions. Fertil Steril 32:443, 1979 Spermatozoal motility evaluation is an essential part of a routine semen analysis. n most cases it is performed subjectively by estimating motility in a sample from an undiluted specimen. 1 n methods with which motility is determined objectively, semen must often be diluted in order to perform accurate quantitative assessment. 2-4 This dilution Received February 12, 1979; revised May 7,1979; accepted May 11, * Supported by Grant from the srael Commission for Basic Research. thead of nfertility nstitute. To whom reprint requests should be addressed. 443 induces several variables, some of which must be considered: (1) The viscosity of the medium is generally lowered by the diluent, and resistance to swimming spermatozoa is reduced. (2) The concentrations of metabolites and nutritive and other organic and inorganic substances are altered so that motility may be stimulated, unaffected, or depressed. (3) Distances between spermatozoa are increased, thus enabling the sperm to swim freely and progressively without colliding with or jostling each other. t is therefore necessary to examine the influence of all these factors carefully before the validity of motility determinations of diluted specimens can be accepted.

2 444 U.'1,.. MAKLER ET AL. FG. 1. Collection of sperm-free seminal fluid into a test tube. The original specimen is filtered with the aid of a Millipore funnel containing a glass paper filter. Comparing spermatozoal motility from undiluted and diluted semen by subjective methods cannot be accepted as reliable, since estimation techniques are not sensitive enough to identify small variations. Recently a new multiple exposure photography (MEP) method for spermatozoa motility determination which is objective, sensitive, and accurate was developed by us. 5 n this study the effect of dilution on spermatozoal motility is investigated with the aid of the new MEP method. MATERALS AND METHODS n order to study any stimulatory or depressing effect of dilution on normal as well as subnormal semen, specimens were collected by masturbation from both fertile and infertile patients attending our infertility clinic. No specific etiology was encountered in these patients, and specimens from men with varicocele, genital tract infections, a history of mumps, and idiopathic asthenospermia were included. All specimens were analyzed after liquefaction and within 1 hour after ejaculation. The following experiments were performed: Experiment 1. To study how motility is affected by dilution without altering the medium, semen was diluted in its own seminal plasma. For this purpose a portion ofthe specimen was filtered with the aid of a small Millipore funnel (no. 13) containing a glass paper filter (Wartman FG/A). Drops of sperm-free seminal fluid were collected in a test tube (Fig. 1) to serve as the diluent for aliquots taken from the original stock. Spermatozoal motility was analyzed 5 minutes after making 1:1, 1:3, and 1:6 dilutions in test tubes. October 1979 Experiment 2. To study how spermatozoal motility is affected by dilution with an isotonic aqueous solution (normal saline), samples were analyzed 5 minutes after making 1:1, 1:3, and 1:6 dilutions with this solution in test tubes. Experiment 3. To study any delayed dilutional effect on spermatozoal motility, some of the samples from experiment 2 were further analyzed 1 and 4 hours after dilution. Motility Determination. All well-mixed samples were analyzed at room temperature (23 0 C) by the MEP method described in detail elsewhere. 5 Three different drops were analyzed from each test tube. Each drop was inserted into a 10-/illl counting chamber, and at least four randomly selected viewing areas at the corners of the grid were photographed. Each time, the shutter was opened for 1 full second, during which spermatozoa were illuminated by six stroboscopically induced light pulses. All 12 or more negative films containing images of approximately 200 to 400 spermatozoal specimen were projected and analyzed as described. The information included percentage of motile spermatozoa, average speed, and frequency distribution of spermatozoal velocity. Results from the three above-mentioned experiments were statistically evaluated. Significance of the data was determined by paired t-tests. RESULTS A total of 45 specimens with sperm concentrations that ranged from 12 to 210 x 10 6 /ml (average 73 x 106/ml) were analyzed. The percentage of motility ranged from 6 to 83 (average 43.8), and spermatozoal velocity from 12.1 to 41.3 pm/second (average 26.8 pm/second). From our findings in previous studies5,6 it was concluded that, when objectively determined, a value of 45% can be considered low borderline for normal percentage of motility. Accordingly, 18 of the 45 analyzed specimens (40%) were found to have subnormal motility, and this ratio (2:5) was taken approximately as a key when specimens were subgrouped for the various experiments. Table 1 lists data from eight different specimens. For each specimen, results of spermatozoal velocity and percentage of motility are given before and after dilution with its own seminal plasma and normal saline. Table 2 shows the means of velocity and percentages of motility from undiluted and diluted specimens obtained from 45 patients and divided into groups. There was no significant change in percentage of motility due to

3 Vol. 32, No.4 FACTORS AFFECTNG SPERM MOTLTY. 445 TABLE 1. Spermatozoal Velocity and Percentage of Motility in Eight Different Specimens Measured before and after Dilution in Various Proportions Average % motility Average velocity Patient Original sperm concentration Seminal plasma UD" 1:1 1:3 1:1 Normal saline Seminal plasma Normal saline 1:3 1:6 UD 1:1 1:3 1:1 1:3 1:6 x 10'; Mean ±SD ±20 ±17 ±19 ±22 P value NSb NS NS add, Undiluted. bns, Not significant. the effect 'Of dilutidn with any 'One 'Of the diluents (Tables 1 and 2). HDwever, the veldcity 'Of spermatdzda diluted with seminal plasma increased significantly, and an even greater increase in ve DCity was fdund when spermatdzda were diluted with saline (Figs. 2,3, and 4). The relative gain Dr DSS 'Of spermatdzoal mdtility caused by the dilutidn effect is demdnstrated in Figure 5. Only a relatively mindr change in percentage 'Of mdtility (ndt exceeding 5%) was fdund between undiluted and diluted specimens, whereas the relative increases in veldcity were apprdximately 25% and 37% fdr seminal plasma and saline dilutidns, respectively. Figure 6 describes hdw the frequency distributidn 'Of spermatdzdal veldcities is changed by dilutidn with seminal plasma Dr ndrmal saline at a 1:3 ratid. At any dilutidn the ndrmal distributidn was ±17 ±17 ±2.8 ±5.2 ±3.2 ±4.2 ±4.7 ±3.9 NS NS <0.02 <0.01 <0.01 <0.01 <0.01 preserved except fdr a gradual shift 'Of the mean td higher figures accdrding to the type 'Of the diluent. Figure 7 demdnstrates the spermatdzdal mdtility 'Of eight undiluted and diluted specimens fdl Dwed fdr up td 4 hdurs after dilutidn. BDth the percentage 'Of mdtility and the spermadzdal ve Dcity changed with time in a similar pattern fdr all cases. The difference between the higher initial veldcity 'Of diluted and Dwer initial veldcity 'Of undiluted spermatozda was preserved aldng the 4 hdurs 'Of the experiment. n all 'Of these experiments, there was nd significant difference amdng ndrmdspermic, DligDspermic, and asthendspermic specimens with regard td the effect 'Of dilutidn 'On percentage DfmDtility and spermatdzdal veldcity. n 'Only 'One case (patient 1 in Table 1) did veldcity ndt increase at any 'Of the dilutidns used. TABLE 2. Means of Spermatozoal Velocity and Percentage of Motility in Forty-Five Specimens, Divided into Six Groups, Measured before and after Dilution in Various Proportions Type of dilution Original aver- Average % motility Average velocity No. of age sperm cases concentration ± SD Undiluted Diluted Difference P value Undiluted Diluted Difference P value 1:1 Seminal plasma ± NSa <0.01 Normal saline 24 69± NS <0.01 1:3 Seminal plasma 8 68± NS <0.01 Normal saline 34 65± NS <0.01 1:6 Seminal plasma ± NS <0.01 Normal saline 17 94± NS <0.01 Mean 73 ± SEM ±3.3 ±2.3 ±0.8 ±1.6 ans, Not significant.

4 October 1979 MAKLER ET AL. 446 FG. 2. Photomicrograph of an original specimen made with the aid of the MEP method. Film was exposed for 1 full second while sperm were illuminated by six light pulses. Nonmotile spermatozoa are much more accentuated than are motile spermatozoa, which appear as six-ring chains whose length is proportional to their velocity. One millimeter = three micrometers. DSCUSSON The effect of various diluents on spermatozoal motility has been investigated extensively and FG. 4. Same specimen as in Figure 2 after being diluted with normal saline in ratio of 1:3. Note further extension in sperm tracks as compared with those in Figures 2 and 3. Same scale as in Figure 2. widely reviewed. 7 - O However, semen dilution per se without the addition of components to the original seminal fluid has been studied very little. nvestigations in which motility was evaluated mostly by estimation have been performed more often with animal spermatozoa than with human spermatozoa. n most of these studies, the conclusion was reached that dilution has a slightly or strongly deleterious effect on spermatozoal motility. This effect was attributed to the escape of vital intracellular substances into the media caused by loss oflipids from the surface, or to dilution of some protective proteins within the seminal fluid Others have described a transitory or prolonged Semnal pi. Saline FG. 3. Same specimen as in Figure 2 after being diluted with its own seminal plasma in ratio of 1:3. Note that distances between spermatozoa are increased and the lengths of motile sperm tracks are longer as compared with those in Figure 2. Same scale as in Figure 2. FG. 5. Histograms describing relative gain or loss of spermatozoal velocity and percentage of motility as a result of the dilution effect.

5 Vol. 32, No.4 FACTORS AFFECTNG SPERM MOTLTY. 447 % Undiluted 0~------L-~No~r-~~~sa~li~ne~1:~3~ O~~~~~~~~~.- ~~ Vmic/sec FG. 6. Frequency distribution of sperm velocities before and after dilution of semen with seminal plasma or normal saline in ratio of 1:3. excitatory dilutional effect, at least for the initial part of the experiment.14 n our study the effect of dilution per se, i.e., dilution of a specimen with its own seminal plasma or with normal saline, was investigated. No foreign component, with the exception of NaC to retain normal osmolarity, was added. Neither a depressing nor an excitatory effect on percentage of motility due to semen dilution was found to be significant in any case. On the contrary, spermatozoal velocity was positively affected by dilution, and two stages of increment were found: (1) an increase in velocity with a decrease of sperm concentration; (2) a further increase in velocity after the addition of aqueous solutions, which always leads to a decrease in viscosity of seminal fluid. That spermatozoal velocity is reduced in highly concentrated specimens because the spermatozoa jostle each other has already been mentioned by Tampion and Gibbons. 11 They believed that, with counts above 20 x 106/ml, spermatozoal velocity is somewhat lower because of this phenomenon. However, an increase in velocity after dilution with various aqueous media was not explained by the same mechanism. Several authors attribute this phenomenon to the "exciting effect" of some ingredients included in the diluent,14 and Joel15 even cited this effect to distinguish between nonmotile and dead spermatozoa and called it a "revitalization test."15 n our opinion, there is no reason to believe that isotonic saline is excitatory to spermatozoa, since it is the most inert physiologic medium known; we believe that the increase in velocity is due simply to the lowered viscosity of the diluted specimen. The velocity of moving bodies within a fluid is affected by the viscosity of the fluid. This is a purely physical phenomenon, and spermatozoa are not excepted. Therefore, any assumption that an increase in velocity is due to a stimulatory effect of the diluent can be confirmed only after careful investigation including a comparison of various media and normal saline. Such a study is in progress in our institute. n experiment 3, motility varied with time in a similar manner in both undiluted and diluted samples. This fact excluded any immediate or delayed deleterious or stimulatory effect of dilution per se on moving and nonmoving spermatozoa. There are some important implications of our study: 1. Semen can be diluted for clinical and investigative purposes whenever required with no significant change in percentage of motility, provid- lor r- u 30 l- u E 20 r-.> lor- Hours undiluted..... line :6- FG. 7. Means of sperm velocity and percentage of motili ty in eight undiluted and diluted specimens (with saline only) followed for up to 4 hours. J

6 448 MAKLER ET AL. ing that the ratio of dilution does not exceed our figures. We did not extend our dilution ratios beyond 1:6 because further dilution of human semen reduces sperm concentrations to levels where motility cannot be determined accurately. Also, for all practical purposes there seems to be no need to use higher dilutions. However, it should be kept in mind that spermatozoal velocity in diluted specimens differs significantly from that in undiluted specimens. 2. Motility is unaffected by dilution throughout the first 4 hours. This is important information for the investigator who performs long-duration experiments with diluents. These surprising data imply that one-sixth the original concentration of nutrients is still adequate for normal spermatozoal activity or that spermatozoa may not need external energy sources at all and only intracellular sources are used for this purpose. n order to prove this assumption, completely washed spermatozoa must be resuspended in normal saline and analyzed, but not before the effect of centrifugation on spermatozoal motility is studied carefully. We did not extend our study beyond 4 hours because any effect after that time would be secondary to bacterial contamination, change in ph, etc., as was discussed by us in a previous report. l6 3. Seminal fluid is not an ideal environment for moving spermatozoa. Viscosity, antibodies, bacterial toxins, debris, or jostling of too-concentrated spermatozoa may interfere with their free movement. Spermatozoa spend only a minor part of their active life-span within seminal fluid, while most of it is spent in the female reproductive tract; therefore, it is unjustified to determine spermatozoal motility only in the seminal fluid. The real potential of motility under optimal conditions should be evaluated as well. For the time being, and unless proved otherwise, these conditions include low viscosity and a sperm count not exceeding 20 x 10 6 /ml, achieved by simple dilution. We recommend that the quality or grade of motility in a routine semen analysis should be determined twice: first in the native environment and second after dilution with an isotonic aqueous solution. n this way the above-mentioned factors are eliminated and spermatozoa in all specimens are analyzed under the same standard conditions. The better of the two measurements is to be considered the representative velocity under optimal conditions. A prospective study can tell how these figures are related to fertility rate. Obviously, testing spermatozoal motility in diluted specimens cannot substitute for in vivo or in October 1979 vitro tests of sperm penetration into cervical mucus. However, the latter tests cannot always yield enough information about spermatozoal motility, since cervical mucus does not have uniform properties, tends to vary cyclically, and is affected by many intrinsic and extrinsic factors. Therefore, although it is of utmost importance, the postcoital test is performed to analyze the quality and characteristics of the mucus-sperm interrelationship rather than sperm motility per se. Finally, from our findings there is reason to believe that dilution of semen may have some important implications for homologous artificial insemination. f diminished motility is a factor in a couple's infertility, then clearly, improvement in the average velocity of spermatozoa might well increase the likelihood of pregnancy. This is the rationale behind the technique of washing spermatozoa and resuspending them in buffer, used by some physicians in treating these couples. n conclusion, there is no immediate or delayed dilutional effect on the percentage of human sper" matozoal motility when semen is diluted to ratio of 1:6, other than an increase in velocity between 25% and 37% due to a decrease in both viscosity and sperm concentration which enables sperm to reach their maximal motile capacity. These facts can be taken into consideration whenever semen is diluted for clinical or investigative purposes, as well as for homologous artificial insemination. REFERENCES 1. Eliasson R: Semen analysis. n Progress in nfertility, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1975, p Baker FN, Cragle RG, Salisbury GW, VanDemark NL: Spermatozoan velocities in vitro. Fertil Steril 8:149, Harvey CJ: The speed of human spermatozoa and the effect on it of various diluents. J Reprod Fertil 1:84, JanickJ, MacLeodJ: Measurement of human spermatozoa motility. Fertil Steril 21:140, Makler A: A new multiple exposure photography method for objective human spermatozoa motility determination. Fertil Steril 30:192, Makler A, tskovitz J, Brandes JM, Paldi E: Sperm velocity and percentage of motility in 100 normospermic specimens analyzed by the multiple exposure photography (MEP) method. Fertil Steril 31:155, Mann T: The Biochemistry of Semen and the Male Reproductive Tract. London, Methuen and Co Ltd, 1964, P MarcusM,BishopMWH, WaltonH: Metabolism and motility of mammalian spermatozoa. n Marshall's Physiology of Reproduction, Vol 1, Part 2, Edited by AS Parker. London, Longmans Green and Co, 1960, p Nelson L: Sperm motility. n fertilization, Vol 1, Edited by CB Metz, A Monroy. New York, Academic Press, 1967, P 61

7 Vol. 32, No.4 FACTORS AFFECTNG SPERM MOTUTY White G, MacLeod J: Composition and physiology of semen. n Mechanisms Concerned with Conception, Edited by CG Hartman. Oxford, Pergamon Press, 1963, p Tampion D, Gibbons RA: Swimming rate of bull spermatozoa in various media and the effect of dilution. J Reprod Fertil 5:259, Wales RG, White G: Viability of diluted dog spermatozoa in vitro. J Reprod Fertil 5:67, Lindholmer CH: The importance of seminal plasma for human sperm motility. Bio Reprod 10:533, Freund M, Wiederman J: Factors affecting the dilution, freezing, and storage of human semen. J Reprod Fertil 11:1, Joel CA: Fertility Disturbances in Men and Women. Basel; S Karger, 1971, p Makler A, Zeidise, Paldi E, Brandes JM: Factors affecting sperm motility.. n vitro change in motility with time after ejaculation. Fertil Steril 31:147, 1979