TURBIDIMETRIC ANALYSIS OF HUMAN SPERM MOTILITY*

Transcription

1 FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society Vol. 28, No. 12, December 1977 Printed in V.SA. TURBIDIMETRIC ANALYSIS OF HUMAN SPERM MOTILITY* JOSEPH E. SOKOLOSKI, B.A. LUIS BLASCO, M.D. BAYARD T. STOREY, PH.D.t DON P. WOLF, PH.D.t Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania A turbidimetric method has been developed for determining rapidly the fraction of sperm in human ejaculates which show the most vigorous motility. The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette. This results in a time-dependent increase in turbidity in the medium which is recorded spectrophotometricaily as an increase in absorbance. The determination requires 10 minutes and yields both the fraction of rapidly moving sperm, F R.Il, and their average velocity, V R İl Examination of25 samples yielded FR.I! values of 10% or lower, whereas values for V R.1l averaged about 100 p.mlsecond. These vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity. It is suggested that this simple turbidimetric test be used in evaluation of human semen as a possible indicator offertilizing capacity. Motility is a characteristic of the sperm cell which is so readily observable that it continues to serve as a convenient indicator of the physiologic state of the cell, despite the fact that no firm correlation between motility and fertilizing capacity has been established. I - 4 There are two aspects to the study of sperm motility. One is the mathematical description of the flagellar wave which gives the sperm cell a characteristic swimming pattern and velocity.5 This has been accomplished by a combination of photomicrographic techniques 6 - lo and computer simulation. 11 The second aspect is the relation between the motility of a given sample of sperm cells.and the fertilizing capacity. This latter aspect carries obvious clinical relevance to the evaluation of human ejaculates and has given rise to several motility-rating methods. These include direct microscopic observation with various criteria for obtaining a numerical motility index,12-14 tracking Received June 13, 1977; revised August 8, 1977; accepted August 9, *Supported by United States Public Health Service Grants HD and HD and.contract N01-HD tto whom reprint requests should be addressed of sperm cells over a grid to determine the average velocity,15 and spectrophotometric methods which follow the return of sperm cells to random orientation after alignment by. flow l6, 17 or centrifugation.ls More recently, the laser has been utilized to quantitate motility by detection of angular light scattering to yield a distribution of swimming speeds. 19 In a fresh ejaculate, motile sperm show a wide range of velocity and of directional consistency, and one can define a "vigorous" population as that group of cells which shows the highest velocity and greatest capability for swimming in a given direction. It seems reasonable to suppose that sperm samples containing a high fraction of vigorous cells are the best candidates for high fertilizing capacity. But before this can be established, an objective method is needed to determine the fraction of rapidly moving cells in the sample and their average velocity, in order to provide quantitative data for eventual statistical correlation. In this paper, we report a rapid and convenient turbidimetric method for this determination, which yields a permanent instrumental record for ready reference.

2 1338 SOKOLOSKI ET AL. December 1977 MATERIALS AND METHODS Fresh ejaculates were obtained from healthy donors and allowed to liquify for 30 minutes at 37 C prior to use. Semen analysis was conducted by routine microscopic techniques: total cell number and sperm cell number by hemocytometer count, and over-all percentage motility on undiluted samples at x400. For the turbidimetric determination, ejaculates were either used directly after liquefaction or were diluted to 5 ml in Krebs-Ringer bicarbonate medium containing 0.2% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.; fraction V) at ph 7.6, washed by centrifugation at 100 x g for 10 minutes at room temperature, and then reconstituted to their original volume with medium. Turbidimetric measurement of the cell fraction with vigorous motility is based on two principles. First, a population of sperm moving into medium in the optical path will produce a time-dependent increase in absorbance. This increase is due to turbidity which is proportional to the average rate of movement of the cell population and to the concentration of cells. Second, sperm with the most vigorous motility and greatest directional consistency will be the first to swim upward into the optical path from the bottom of the cuvette and cause the initial increase in turbidity. The fraction and average velocity of these cells can, therefore, be determined at short recording times. Aging effects are avoided, and multiple samples can be processed in a short time. Control tests showed that the settling speed of nonmotile spermatozoa was negligibly slow as compared with the swimming speed of the very motile cells, so that gravitation plays a negligible role in this test. This method resembles that of Goodall and Roberts,20 which was designed to detect differences between X- and V-bearing spermatozoa, and is the analytic analog of the preparative method developed by Lopata et al.2 1 for obtaining motile sperm from human samples by vertical migration in columns of medium. Recordings of absorbance due to sperm cell turbidity were obtained with a Gilford model 240 spectrophotometer equipped with a tungsten light source and a jacketed cuvette holder maintained at 37 C. The wavelength chosen was 545 nm, which is also available on those spectrophotometers which use mercury arc lamps. A standard curve relating absorbance to cell concentration was determined, as described by Henle and Zittle.22 It was linear with a correlation coefficient of up to concentrations of 200 x 10 6 cells/ml. The Injection Port Icm 4mm -II-- Fill Line For I.Oml Sample ~>Iln1D1f!,Chamber...--Cleor Window T E:::::~-CUliet --l 12mm I-- Mask Bottom BTS-:'~1. FIG. 1. Schematic of the turbidimetric cuvette as viewed along its optical path. The stippled area represents the window of clear Plexiglas; all other areas are of black Plexiglas to block the incident light. The sample chamber is 34 mm high and 4 mm wide, with an optical path of 8 mm. The injection port allows the concentrated sperm sample to be layered directly on the bottom of the cuvette by extrusion from a syringe through a 21-gauge needle inserted in the injection port with its tip touching the bottom of the cuvette. The bevel of the tip faces toward the sample chamber. Extrusion with a single, smooth motion of the syringe plunger spreads a layer of concentrated sperm on the bottom of the sample chamber, with minimal eddy formation. The I-mm mask shown blocks the incident light from the layering process, thus minimizing initial absorbance artifacts arising from eddies of concentrated sperm appearing transiently in the light path. The clear window is 1 cm high; above this height, the cuvette contents are blocked from the light beam by a black Plexiglas mask. The fill line for a 1.0-ml sample is indicated. concentrated sperm suspension was layered on the bottom of the spectrophotometer cuvette, and sperm were allowed to swim upward into the optical path through a distance of 1 cm. Although a standard spectrophotometric cuvette can be used in this method, the use of a self-masked cuvette with injection port, constructed for this study and shown in Figure 1, provided a precise, vertical, 1-cm path and avoided absorbance artifacts created by flow eddies when layering the sperm suspension underneath the culture medium. The volumes of suspension medium and semen or washed sperm were 1.0 ml and 40 ILl, respectively. The increase in absorbance observed upon. the addition of sperm was recorded with a Gilford model 6040 strip-chart recorder. The contents of the cuvette were then vigorously mixed with a stirring rod to give a uniform suspension for the final turbidity reading. The ph of the suspension was checked after the determination; it remained constant at 7.6 in every case.

3 Vol. 28, No. 12 TURBIDIMETRIC ANALYSIS OF HUMAN SPERM MOTILITY 1339 IA ~~m 13 T AA"' Change Sens.J -!Imin! t 1 ~ A=O- Mix T AA = TS-"W. FIG. 2. A, Recorder trace obtained with the spectrophotometer during turbidimetric determination of the fraction and average velocity of rapidly moving sperm in the sample. At the point indicated, the concentrated sperm sample is layered on the bottom of the cuvette shown in Figure 1. There is an initial increase in absorbance that is linear with time, the rate of which then decreases after about 2 minutes. At the point indicated, the contents of the cuvette are vigorously mixed, and the signal detector sensitivity is decreased by 2.5 times at the point marked Change Sens. B, Schematic of the trace in A showing the slope (daldt) of the first linear time-dependent absorbance increase, the final absorbance A 2, and the means for determining A,. The point ofintersection of the first two slopes is taken as the final absorbance A,. In those cases where the second slope is zero and a plateau is observed, the final constant absorbance is taken asa,. RESULTS The recording obtained in a typical motility determination is shown in Figure 2A. At the point indicated, a sperm suspension was injected into the bottom of the cuvette. There was a slight lag before any increase in optical density was recorded because of the 1-mm optical mask on the bottom of the cuvette. The rate of increase in absorbance was linear for the first few minutes of recording, then slowed to give a second linear rate. On mixing the contents of the cuvette, a much higher absorbance was obtained which corresponded to a uniform distribution of all cells in the suspension. The turbidity induced by the rapidly moving cells is given by the absorbance, A to which is estimated as shown in Figure '2B. The fraction, F RM, of the total sperm population which are rapidly moving isfrm = At/F.<A2' where Fs is the fraction of sperm cells in the whole sample and A 2 is the final absorbance obtained on mixing the contents of the cuvette (Fig. 2, A and B). The turbi~ity corresponding to absorbance A 2 is due to both sperm and non-sperm cells in the sample. In calculating F RM, it is assumed that all cells contribute equally to the turbidity. Comparison of A2 values obtained from samples with high or low fractions of sperm showed that this was a reasonable assumption. The average velocity of the rapidly moving cells, V RM, is calculated directly from the slope (daldt) of the first linear portion of the trace and At: V RM = (daldt)ia t. The second, shallower slope observed in recordings like that shown in Figure 2A showed no signs of leveling off in a number of the samples within the usual time of 10 minutes allotted to the experiments. In some samples, the curve showed a series of decreasing slopes. In other samples, the initial linear absorbance increase was followed by a plateau. This diversity of behavior after the initial linear absorbance change reflects the fact that different sperm samples have populations of widely varying motility. Some populations apparently have a simple distribution of one rapidly moving and one slowly moving group, each with a characteristic average velocity. Others have a greater range of average velocities. But all motile samples showed an initial linear change from which FRM and V RM could be calculated, even if F RM was small. Control experiments with sperm rendered nonmotile by hypotonic or heat treatment showed no change in absorbance until after the contents of the cuvette had been mixed. When examined under the microscope, samples of untreated sperm taken from the cuvette after the determination showed motility equivalent to that of a control sample retained without dilution. Human sperm apparently were not damaged by dilution in these determinations, as were bull sperm under similar conditions during the diffusion experiments reported by Nevo and Mohan.23 The results from application of this method to 25 sperm samples are shown in Table 1. The fractionfrm is expressed as a percentage, and the samples are listed in order of increasing percentage of rapidly moving sperm cells. Velocities V RM are listed, along with the other parameters obtained in the routine analysis of the samples by direct microscopic observation. There is considerable variation between samples from

4 1340 SOKOLOSKI ET AL. December 1977 TABLE 1. Percentage and Velocity of Rapidly Moving Sperm Cells in Samples Rated for Sperm Count, Over-all Percentage Motility," and Percentage Sperm Cells" in Sample" Spectrophotometric measurement Microscopic measurement Sample no. % Rapidly Velocity Over-all Sperm count, % Sperm moving cells; V H1f, % motility 10" cells/ml in sample FHII x 100,urn/sec " e ge llf f " " h " " h " 24h "Over-all percentage motility includes only those cells which show some degree of progessive motility. bpercentage sperm cell values have been shown by replicate counts on the same sample to be within ± 10%. PEach sample was collected from a different donor, except where indicated below. "Samples from donor 26. esamples from donor 14. fsamples from donor 19. "Samples from donor 25. hsamples from donor 22. different donors and even between samples from the same donor obtained at different times. The swimming velocity determined for the rapidly moving samples was on the order of 100 JLrn! second, with a range of 42 to 280 JLm/second. These values are 2 to 3 times greater than those obtained for human sperm by microscopic measurements,12, 15 reflecting the fact that the turbidimetric method selects for rapidly moving sperm. There is no correlation between V RM and F RM: those sperm which comprise the fraction F RM all swim with about the same speed. Except for the first two samples in Table 1, whose low values of F RM were reflected in low over-all motilities, there was no obvious correlation between overall motility and FR!W in this group of ejaculates. The variation in over-all motility is limited, however, ranging from 40% to 80%, with most samples in the 60% to 80% range. In this respect, F RM does provide a parameter with a wider range of variation than over-all motility, which could make it a more discriminating indicator. The sperm count and percentage of sperm in the sample correlate neither with over-all sperm motility nor with F RM DISCUSSION The turbidimetric method described in this paper is rapid and requires only a simple spectrophotometer and cell. It provides a quantitative measure of the fraction of rapidly moving spermatozoa in the sample and a characteristic average velocity. This measure is obtained from the whole sample, unlike the microscopic techniques which focus on a small number of cells in the sample. A more complete description of the motility of a sperm sample can be obtained by combination of this method with stopped-flow reorientation measurements, also obtained spectrophotometrically.16,17 The method described by Atherton17 for bull sperm and recently extended to human sperm24 requires only the addition of a special flow cell to the spectrophotometric apparatus. The stopped-flow reorientation method yields a relaxation time characteristic of the average velocity of all the motile cells and so complements the turbidimetric method. Both methods provide an objective, quantitative parameter to characterize sperm motility, so that measurements obtained in different laboratories and clinics can be directly compared. This should facilitate the accumulation of a statistically significant number of samples to test correlations between motility parameters and fertilizing capacity. It would also allow a test of the following suggested hypothesis. The fraction F RM and velocity V RM, obtained by the turbidimetric method described here, characterize the "elite" sperm in the sample, with high swimming speed and directional consistency. Since only one spermatozoon is required for fertilization, the fraction and velocity of rapidly moving sperm in the sample should be the best indicator offertilizing capacity, as compared with average velocity or over-all percentage motility. The simplicity of the turbidimetric method recommends its inclusion in the clinical evaluation of human semen samples, and it is hoped that this hypothesis can eventually be tested with a statistically significant number of samples.

5 Vol. 28, No. 12 Acknowledgments. The authors are grateful to Miss Sallyann Schweitzer for expert technical assistance and to Mr. Joe Pili of the Department of Physiology Machine Shop for construction of the self-masked turbidimetric cuvette. REFERENCES 1. Chang MC: Fertilizing capacity of spermatozoa. In Endocrinology of Reproduction, Edited by CW Lloyd. New York, Academic Press, 1959, P Howe GR: Efforts to relate spermatozoan motility to fertilizing capacity. Int J Fertil 18:188, Linford E, Glover EA, Bishop C, Stewart DL: The relationship between semen evaluation methods and fertility in the bull. J Reprod Fertil 47:283, Nelson L: Spermatozoan motility. In Handbook ofphysiology, Edited by DW Hamilton, RO Green, Sect 7: Endocrinology, Vol 5: Male Reproductive System. Washington DC, American Physiological Society, 1975, P Gray J: Introduction: flagellar propulsion. In Spermatozoan Motility, Edited by DW Bishop. Washington DC, AAAS, 1962, P 1 6. Rothschild L: The movements of spermatozoa. In Mammalian Germ Cells, Edited by GEW Wolstenholme. Boston, Little, Brown and Co, 1953, P Rikmenspoel R: Microscopic chamber for simultaneous measurement of motility and respiration of spermatozoa. Rev Sci Instrum 35:44, Rikmenspoel R: Electronic analyser for measuring velocities and the concentration of spermatozoa. Rev Sci Instrum 35:52, Branham JM: Movements of free-swimming rabbit spermatozoa. J Reprod Fertil 18:97, Katz DR, Dott HM: Methods of measuring swimming speed of spermatozoa. J Reprod Fertil 45:263, Brokaw CJ: Computer simulation of flagellar movement. IV. Properties of an oscillatory two-state cross-bridge model. Biophys J 16:1029, 1976 TURBIDIMETRIC ANALYSIS OF HUMAN SPERM MOTILITY Bishop MHW, Walton H: Metabolism and motility of spermatozoa. In Marshall's Physiology of Reproduction Edited by AS Parkes. London, Longmans Green, 1960: p Dougherty KA, Emilson LBV, Cockett ATK, Urry RL: A comparison of subjective measurements of human sperm motility and viability with two live-dead staining techniques. Fertil Steril 26:700, Dougherty KA, Cockett ATK, Urry RL: Caffeine, theophylline, and human sperm motility. Fertil Steril 27:541, Harvey C: The speed of human spermatozoa and the effect on it of various diluents, with some preliminary observations on clinical material. J Reprod Fertil 1:84, Timourian H, Watchmaker G: Determination of spermatozoan motility. Dev BioI 21:62, Atherton RW: An objective method for evaluating Angus and Hereford sperm motility. Int J Fertil 20:119, Nelson L: Neurochemical control of Arbacia motility. Exp Cell Res 74:269, Shimizu H, Matsumoto G: Light scattering study on motile spermatozoa. IEEE Trans Biomed Eng 24:153, Goodall H, Roberts AM: Differences in motility of X- and Y-bearing spermatozoa. J Reprod Fertil 48:433, Lopata A, Patullo MJ, Chang A, James B: A method for collecting motile spermatozoa from human semen. Fertil Steril 27:677, Henle G, Zittle CA: Studies of the metabolism of bovine epididymal spermatozoa. Am J Physiol 136:70, Nevo AC, Mohan R: Migration of motile spermatozoa into sperm-free medium and the dilution effect. J Reprod Fertil 18:379, Atherton RW, Radany EW, Polakoski KL: Quantitation of human sperm motility (abstr). Fertil Steril 28:298, 1977