Semen was collected by masturbation from 33 infertile patients undergoing diagnostic evaluation MATERIALS AND METHODS

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1 FERTILITY AND STERILITY Copyright Clt989 The American Fertility Society Printed in U.S.A. Variation of movement characteristics with washing and capacitation of spermatozoa. I. Univariate statistical analysis and detection of sperm hyperactivation * Kenneth A. Ginsburg, M.D.t Anthony G. Sacco, Ph.D. Kamran S. Moghissi, M.D. Suzanne Sorovetz, B.S. The Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University and Hutzel Hospital, Detroit, Michigan Movement alterations as spermatozoa are washed and capacitated in preparation for the zonafree sperm penetration assay were investigated using a videomicrographic computerassisted system for tracking and analysis of sperm trajectories. Thirtythree semen samples from infertile men and 17 fertile controls were studied. Despite significant differences on the sperm penetration assay, univariate comparisons of movement characteristics between infertile and control samples failed to detect differences. For all samples combined, significant alterations in movement characteristics were detected after washing. After an 18hour sperm capacitation, mean velocity, head yawing amplitude, and head yawing frequency still were significantly increased over seminal plasma values. Videomicrographic motion analysis thus allows detection of changes that are consistent with hyperactivated motility, as has been described for capacitating spermatozoa. Fertil Steril51:869, 1989 Alterations in movementi 4 have been described after removal of spermatozoa from seminal plasma, including the detection of hyperactivated motility in capacitating human 5 and animal 6 9 spermatozoa. Hyperactivated motility is found in approximately 20% of fertile sperm during capacitation, 5 preceding the acrosome reaction that begins after spermzona interaction. 10 Differences in the proportion of hyperactivated sperm between fertile and infertile samples 5 suggest that determination of hyperacti Received September 2, 1988; revised and accepted January 6, * Presented in part at the FortyThird Annual Meeting of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada. t Reprint requests: Kenneth A. Ginsburg, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University and Hutzel Hospital, St. Antoine, Detroit, Michigan vated motility may be a useful indicator of fertility status. In the past, however, direct methods that analyze individual spermatozoa were required to detect hyperactivated motility because only a minority of cells exhibit this motility pattern. This objective, quantitative description of sperm trajectories has been difficult and thus was limited to research laboratories. Computerassisted videomicrographic techniques have simplified the acquisition of statistically sound measurements of sperm motion, and the validity of this methodology now has been established Using such techniques, in this study we report the quantitative changes in sperm motility seen during a standard washing and capacitation procedure. MATERIALS AND METHODS Semen was collected by masturbation from 33 infertile patients undergoing diagnostic evaluation Ginsburg et al. Motion characteristics of spermatozoa 869

2 by sperm penetration assay (SPA)/ 4 and from 17 control males (who had previously been shown to perform well on the SPA) analyzed concurrently with the patient samples. Semen was allowed to liquify at room temperature, then diluted with an equal volume of Biggers, Whitten, and Whittingham media containing 0.3% human serum albumin (Sigma Chemical Company, St. Louis, MO) and centrifuged at 600 X g for 5 minutes. The sperm pellet was resuspended in 1.0 ml of medium and centrifuged again. The supernatent was discarded, and the resulting pellet gently loosened. One milliliter fresh media was layered over it and the sperm was allowed to "swim up" into the media over 1 hour at 37 C. The upper 0.4 to 0.6 ml was removed, an aliquot counted, and the concentration of spermatozoa adjusted to 1 X 10 6 sperm/mi. To capacitate sperm, this preparation was incubated for 18 hours at 37oC, after which 0.2 ml of the suspension was used for insemination of zonafree hamster oocytes in the SPA as described by Y anagimachi et al. 14 Videomicrographic computerassisted semen analysis (CASA) was performed at three steps in the above procedure: (1) on the sperm in seminal plasma (RAW), (2) on the sample after washing, swimup, and adjustment of concentration (WASH), and (3) after 18hour in vitro capacitation immediately before oocyte insemination (CAP). A 10 1 aliquot of each sample was placed on a prewarmed Makler Chamber (Sefi Medical Industries, Haifa, Israel). The sample was viewed through an Olympus BHS microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with phasecontrast optics, lox SPlan objective lens, 6.7X photo lens, and a Panasonic Model WV1410 videocamera (Matsushita Electric Industries, Secaucus, NJ) for movement analysis at a framing rate of 30 Hz. Details of the hardware and operational settings used during CASA with the CellSoft Automated Semen Analyzer (CRYOResources, New York, NY) are provided in a previous report. 11 Data collected for each sample at each of the three times during preparation (RAW, WASH, CAP) included sperm concentration (X10 6 /cc); percent motile spermatozoa (MOT); curvilinear velocity (microns/sec); linearity index (the straight/curvilinear velocity ratio for all cells, giving a measure of average progressivity or path straightness); maximum amplitude of lateral head displacement (ALH; microns); mean ALH (microns); and head beat frequency (cycles/sec). Kinematic description of these various motion determi 870 Ginsburg et al. Motion characteristics of spermatozoa nants has been reported elsewhere Data for curvilinear velocity (VEL), linearity index (LIN), maximum ALH (XALH), mean ALH (MALH), and head beat frequency (HBF) represent values averaged over the entire population of spermatozoa examined. The latter measurements (XALH, MALH, HBF) reflect the amplitude and frequency of flagellar beating by tracking and measuring the corresponding opposite movements of the sperm head. In all cases, at least 225 cells were examined. Average hamster egg penetration rates were compared between control and patient samples using Student's unpaired ttest. Movement measurements for all 50 samples were compared at various times in sperm processing using univariate analysis of variance with an orthogonal contrast algorithm to further compare patient and control groups at each of the three sampling intervals. Allowance was made for unequal sample sizes in the statistical analysis of group contrasts. Associations between combined (patient and control) motion measurements and SPA outcome were investigated using univariate correlation analysis. Washed and capacitated concentration data were excluded from statistical analysis because these values had been adjusted to optimize the hamster egg penetration test. RESULTS The seminal plasma, washed, or capacitated movement measurements did not differ significantly between the patient and control groups (Table 1). However, mean ± standard error of the mean SPA rates for the patient and control groups were significantly different (8.9 ± 2.5 versus 35.8 ± 5.0, respectively, P < 0.001). Data for MOT, VEL, LIN, HBF, XALH, and MALH are presented in Figure 1. In each case, the data compare the combined (patient and control) values at each sampling interval, since the groups were not different (see above). As shown in Figure 1, the mean motility increased by nearly 13% (P < 0.001) as sperm were washed and allowed to separate by a swimup procedure. After an 18hour incubation to allow capacitation, percent motility had declined 16% (P < 0.001) back to prewashing values, so that capacitated and seminal plasma measurements were not different (P = 0.26). For the velocity data, the same trend holds with a substantial increase in sperm velocity of 13 m/ sec (P < 0.001) after washing. After capacitation, velocity fell toward prewash values, although a sig Fertility and Sterility

3 Table 1 Comparison Between Patient (n = 33) and Control (n = 17) Movement Measurements for Seminal, Washed, and acitated Sperm Samples Seminal spermatozoa (RAW)" Washed spermatozoa (WASH)" acitated spermatozoa (CAP)" Patients Controls Patients Controls Patients Controls Mean±SEM' Mean±SEM Concentration ± ± 7.70 Motility ± ± 5.03 Velocity ± ± 2.70 Linearity 5.43 ± ± 0.25 MaximumALH 2.48 ± ± 0.14 MeanALH 2.03 ± ± 0.12 Head beat frequency ± ± 0.47 " Comparisons between patient and control samples were not significant for any measurement (P > 0.05) using analysis of variance with contrasts. b SEM, standard error of the mean. Mean±SEM Mean±SEM Mean±SEM Mean±SEM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.56 c Concentration measurements for washed and capacitated samples are not reported since these values had been adjusted after washing. nificant (P < 0.001) change between capacitated and seminal plasma velocity still was maintained. Large error terms minimized the observed changes in linearity, or path trajectory straightness (Figure 1); in fact, differences between seminal plasma and washed samples were just significant (P = 0.044) )50 50 l '; I I J 50 II" Jl :: J. :: Jzz li 20 Figure 1 li 20,...L 60,_.z 8 f 6 f 4.J 2 0 I 16 l J 12 I II 10,., Mean± standard error of the mean motility, velocity, linearity, maximum and mean amplitude of lateral head displacement, and head beat cross frequency data for three processing steps. RAW, seminal sperm; WASH, washed and swimup sperm; CAP, 18hour capacitated sperm. Brackets indicate significant comparisons using analysis of variance with contrasts, *** = P < 0.001, ** = P < 0.01, * = P < Comparisons not shown were not significant (P > 0.05). Ginsburg et al. Motion characteristics of spermatozoa 871

4 Table 2 Correlation of Movement Measurements a with Hamster Egg Penetration Assay (SPA) Rates Movement measurement Concentration Motility Velocity Linearity MaximumALH MeanALH Head beat frequency Correlation coefficient of motion measurements with SPA RAW WASH CAP c c a Data for patient and control samples combined, n = 50. b Not reported because concentrations had been adjusted to 1 million sperm/cc for all samples. c Significant correlation, P < Over the capacitation period, linearity then fell, and there was a significant difference (P < 0.001) after the incubation when comparing capacitated and washed linearity, but no difference between capacitated and seminal plasma linearity (P = 0.19). Head beat frequency measurements (Fig. 1) paralleled closely the changes in sperm velocity and overall motility. Head (and thus flagellar) beat frequency increased after washing, but the increase was reversed after 18 hours of incubation. The head beat frequency measurements after in vitro capacitation still remained significantly different from seminal plasma values (P = 0.038). The patterns of alteration for maximum (XALH) and mean (MALH) lateral head displacement measurements were different than that for the other measurements. Maximum ALH and MALH (Fig. 1) increased as sperm were washed (P < for both measurements), and a small but insignificant further increase, as opposed to decrease, occurred after 18hour capacitation (P = 0.24 for XALH, P = 0.36 for MALH). For both measurements, capacitated data were significantly different from seminal plasma data (P < 0.001). The correlations between combined patient and control movement measurements and SPA rates are shown in Table 2 for seminal plasma, washed, and capacitated spermatozoa. All correlations are weak, and only those for washed VEL (r = 0.289) and washed XALH (r = 0.341) reached statistical significance (P < 0.05). DISCUSSION We have shown that, as spermatozoa are washed, the proportion of motile cells and their average ve locity substantially increases, coincident with an increase in flagellar beat amplitude and frequency. Linearity also was increased corresponding to a straighter path trajectory immediately after washing. Similar findings have been reported by other investigators, using manual path trajectory analysis methods. After prolonged capacitation, maximum and mean amplitude of lateral head displacement measurements increased further, while average values for the other four measurements (motility, velocity, linearity, head beat frequency) returned back toward seminal plasma values. acitated motility and path straightness were indistinguishable from seminal plasma values, while curvilinear velocity and head beat frequency, along with maximum and mean amplitude oflateral head displacement, remained significantly elevated over seminal values. Although velocity, head beat amplitude and frequency, and linearity differences were detected after sperm capacitation in this study, these changes are not described on a persperm basis. It must be emphasized that the data presented here represent values averaged over the entire population of sperm in each sample. Episodes of hyperactivated motility may alternate with periods of normal or even nonprogressive motility for a particular spermatozoa,8 and average data may obscure both withinsperm and betweensperm changes. Incisive study of individual sperm paths, possible with the CellSoft Research Module, might allow identification and measurement of individual hyperactivated sperm trajectories similar to those described by other investigators using manual movement analysis. These criticisms aside, removal of sperm from seminal plasma appears to cause an increase in velocity, increased head yawing (and flagellar beating) amplitude and frequency, and decreased path straightness, changes that are consistent with hyperactivating spermatozoa. The fact that the proportion of motile sperm decreases substantially after prolonged capacitation suggests that such conditions may not be optimal for maintenance of a hyperactivated state. It is known that sperm populations from different men optimally capacitate under different conditions, 18 and neither the time course of hyperactivation onset nor its duration in capacitating human sperm populations is known. Our results appear to be a reflection of this, and suggest that differences in capacitation and hyperactivation kinetics between men, and even between subpopulations of sperm in a particular ejaculate, may be important. Such 872 Ginsburg et al. Motion characteristics of spermatozoa Fertility and Sterility

5 differences would have practical significance both in terms of performance of the SPA and fertilization of human oocytes in an in vitro fertilization program. Given the importance of sperm movement, it was surprising that none of the six motion characteristics examined from RAW, WASH, or CAP sperm differed between patient and control samples. By themselves, these measurements did not suggest any defect in sperm function. We also did not observe any strong correlations between motion data and SPA outcome. The similarity of measurements between these two groups, which differed significantly in functional assessment using the SPA, implies that no single movement measurement can predict the fertilizing ability of a sperm population. Because movement is a complex process involving vigor (velocity, motility) and pattern (path trajectory) determinants in threedimensional space, it would be anticipated that it cannot be adequately quantitated using single twodimensional measurements. Further, it is possible that sperm movement patterns may be more important in migration to the site of fertilization, than in the actual fertilization process itself. In summary, computerautomated videomicrographic systems for tracking and analysis of sperm trajectories appear capable of detecting alterations in movement as sperm capacitate. The pattern of alteration, with increased average velocity, increased amplitude and frequency of flagellar beating, and decreased progression, is consistent with hyperactivation. Further testing and refinement of this methodology could add an important tool to the assessment of sperm function in male infertility. REFERENCES 1. Jeulin C, Feneux D, Serres C, Jouannet P, GuilletRosso F, BelaischAllart J, Frydman R, Testart J: Sperm factors related to failure of human invitro fertilization. J Reprod Fertil 76:735, Aitken RJ, Sutton M, Warner P, Richardson DW: Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zonafree hamster oocytes. J Reprod Fertil 73:441, Mack SO, Wolf DP, Tash JS: Quantitation of specific pa rameters of motility in large numbers of human sperm by digital image processing. Bioi Reprod 38:270, Burke RK, Kapinos LJ: The effect of in vitro sperm capacitation on sperm velocity and motility as measured by an inoffice, integrated, microcomputerized system for semen analysis. Int J Fertil30:10, Burkman LJ: Characterization of hyperactivated motility by human spermatozoa during capacitation: a comparison of fertile and oligozoospermic sperm populations. Arch Androl13:153, Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil23:193, Cooper GW, Overstreet JW, Katz DJ: The motility of rabbit spermatozoa recovered from the female reproductive tract. Gamete Res 2:35, Cummins JM: Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes. Gamete Res 6:53, Katz DF, Yanagimachi R: Movement characteristics of hamster spermatozoa within the oviduct. Bioi Reprod 22: 759, Byrd W, Wolf DP: Acrosomal status in fresh and capacitated human ejaculated sperm. Bioi Reprod 34:859, Ginsburg KA, Moghissi KS, Abel EL: Computerassisted human semen analysis sampling errors and reproducibility. J Androl 9:82, Knuth UA, Yeung CH, Nieschlag E: Computerized semen analysis: objective measurement of semen characteristics is biased by subjective parameter setting. Fertil Steril48:118, Vantman D, Koukoulis G, Dennison L: Computerassisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil Steril49:510, Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zonafree animal ova as a test system for assessment of the fertilizing capacity of human spermatozoa. Bioi Reprod 15: 471, Katz DF, Davis RO: Automatic analysis of human sperm motion. J Androl8:170, Katz DF, Davis RO, Delandmeter BA, Overstreet JW: Realtime analysis of sperm motion using automatic video image digitization. Comput Methods Programs Biomed 21: 173, Mortimer D, Curtis EF, Ralston A: Semiautomated analysis of manually reconstructed tracks of progressively motile human spermatozoa. Hum Reprod 3:303, Perreault SD, Rogers BJ: The capacitation pattern of human spermatozoa. Fertil Steril 38:258, 1982 Ginsburg et al. Motion characteristics of spermatozoa 873