Sperm motility index: a new parameter for human sperm evaluation

Transcription

1 r FERTILITY AND STERILITY Copyright 1991 The American Fertility Society Yol. 56, No.1, July 1991 Printed on acid-free paper in U.S.A. Sperm motility index: a ne parameter for human sperm evaluation Benjamin BartooY, Ph.D.* Jacob Ben-Barak, Ph.D.t Abraham Mayevsky, Ph.D.* Mordechai Sneider* Lea Yogev, Ph.D.:!: Abraham Lightman, M.D.t 11 Bar-Ilan University, Rambam Medical Center, and Faculty of Medicine-Technion, Israel Institute of Technology, Haifa, Israel Objective: The study as performed to evaluate the correlation beteen sperm motility index, a novel parameter of semen quality, and routine semen analysis parameters by microscopic evaluation. Design: Sperm motility index as measured by an electro-optical device, the Sperm Quality Analyzer (United Medical Systems Inc., Santa Ana, CA). Human semen samples covering the hole span of qualities ere analyzed prospectively and simultaneously by both methods. Setting: Samples ere collected from patients referred to university hospital infertility clinics. Patients, Participants: Nine hundred sixty-eight semen samples of 812 patients and healthy men ere analyzed. Main Outcome Measure(s): Sperm motility index is a measurement of optical density fluctuations caused by motile cells; therefore, a positive correlation as anticipated beteen its values and semen motility parameters. Results: Sperm motility index values demonstrated statistically significant correlation ith motile cell concentration, total cell concentration, and percent motile cells. They ere also shon to reliably represent semen quality assessment obtained by to experienced andrologists. Conclusions: The sperm motility index provides a reliable and objective reflection of semen motility parameters and quality. Fertil Steril 56:108, 1991 Correct diagnosis of a possible male factor in infertility ork-up is critically dependent on the accurate determination of the motility characteristics of spermatozoa. 1-3 The total count of motile cells and the velocity of their motility have been implicated as crucial parameters for fertility.4-6 The recognition of the significance of objective cell motility determination led to the development of several measuring devices. Hoever, most of these devices are expensive or require lengthy data processing. 7,8 Studies in domestic animals, primarily bulls and rams, indicated that motile cells in semen samples, hen examined by electro-optical means, cause analog ave-like disturbances in the optical density Received July 19, 1990; revised and accepted March 11, * Department of Biochemistry, Bar-Han University. t Faculty of Medicine-Technion, Israel Institute of Technology. :\: Maternity Hospital, Tel Aviv, Israel. Department of Obstetrics and Gynecology, Rambam Medical Center. II Reprint requests: Abraham Lightman, M.D., Department of Obstetrics and Gynecology, Rambam Medical Center, P.O. Box 9697, Haifa, Israel. (OD) of the examined specimen. 9,10 Motility of spermatozoa in these high cell density semen samples is characterized by collective movement of sperm cells. The frequency of the ave-like disturbances caused by sperm collective movement as found to be dependent on the concentration of motile cells and the intensity of their movement. This frequency provides, on analog to digital conversion, the sperm motility index of the examined semen. Sperm motility index values ere employed for the objective evaluation of cell concentration and motility in domestic animal semen.9,10 To extend the applicability of sperm motility index determinations to human semen evaluation, a device ith increased sensitivity as developed. This device, the Sperm Quality Analyzer (United Medical Systems Inc., Santa Ana, CA) is capable of measuring sperm motility index values in human semen samples hose cells are of lo density and are therefore independently motile, ith no collective movement. The goals of the present research ere to study the correlation beteen sperm motility index values and semen parameters and to assess the 108 Bartoov et al. Sperm motility index for semen evaluation

2 capillary display b (Q) light \ / source Figure 1 The Sperm Quality Analyzer (a) and its schematic operational structure (b). usefulness of its determinations by the Sperm Quality Analyzer as an independent novel semen parameter for an objective and fast evaluation of human semen quality. Subjects MATERIALS AND METHODS Nine hundred sixty-eight semen samples of 812 men ere analyzed. The internal distribution of these samples as chosen to include semen samples covering the hole span of semen qualities. Seven hundred fifty-eight of the men ere referred to infertility clinics, 26 men attended in vitro fertilization programs because of female factors, 22 men ere subjects of proven fertility (examination as done during ife's pregnancy or ithin the 1st year after birth), and 6 men ere semen donors. Routine Semen Analysis Semen samples ere obtained by masturbation. Semen volume, cell density, concentration of motile and immotile cells, and progressive motility speed ere determined ithin 1 hour of semen collection using the Makler Chamber (Sefi Medical Instruments, Haifa, Israel) at X200 magnification. Cell velocity as measured by microscopic determination of the time (in seconds) required for a progressively motile cell to cross a distance of 0.1 mm. Cell velocity of the sample as defined as the mean velocity of 25 different cell measurements. Measurement of Sperm Motility Index Sperm motility index determinations ere performed simultaneously on the same semen samples using the Sperm Quality Analyzer. Semen as applied by capillary force into a thin glass capillary (internal depth 0.2 mm). A predetermined field of the specimen contained in the capillary as examined for 40 seconds by the photoelectric cell in the Sperm Quality Analyzer to detect variations in OD caused by the motility of spermatozoa. The frequency of the analog ave-like electrical signal as counted to provide, on analog to digital conversion, the sperm motility index value of the tested sample. All measurements ere done at room temperature. The Sperm Quality Analyzer and the principle of sperm motility index determinations by the device are depicted in Figure 1. To evaluate the correlation beteen routine semen analysis parameters and the sperm motility index values of the samples, linear regression analysis as applied using Pearson's analysis. I I I I I I I III II I III I II I III I TIMING PULSES a) Fertile Donor ~SMI=216 b) Infertile Donor~ SMI =35 Figure 2 Optical density fluctuations as detected by the Sperm Quality Analyzer caused by motile cells of fertile (upper channel) and subfertile donors (loer channel). SMI, sperm motility index. RESULTS Determination of Sperm Motility Index Values Sperm motility index is defined as the frequency of the OD disturbances caused by the motility of spermatozoa in the examined optical field. Figure 2 demonstrates representative analog electric signals obtained from semen sample of fertile and infertile Vol. 56, No.1, July 1991 Bartoov et al. Sperm motility index for semen evaluation 109

3 r donor. The semen of the fertile donor, hich contained 82 X 10 6 motile cells/ml ith mean progressive cell velocity of 31 J,lm/sec, produced analog signal of 216 deflections/sec (sperm motility index = 216 units). In contrast, the semen of the infertile donor, in hich as lo as 5 X 10 6 motile cells/ml ere present (velocity = 6 J,lm/sec) produced analog signal ith markedly reduced frequency (sperm motility index = 35 units). Sperm motility index values ere found to vary in the range of 0 sperm motility index units (in azoospermic or complete asthenozoospermic semen samples) to over 300 sperm motility index units in samples ith exceptionally high motile cell concentration and high cell velocity. Dependence of Sperm Motility Index Values On Sperm Cell Motility The mechanism of the electro-optical measurement dictates that only motile cells ill generate fluctuations in OD and generate sperm motility index readings. Dead or dormant cells ill not affect sperm motility index readings. Dead or dormant cells ill not affect sperm motility index values. This principle as tested by determining sperm motility index values in samples containing increasing concentrations of motile cells. A series of dilutions ere prepared from a single semen specimen obtained from a fertile male. The semen as divided into to equal volumes. One volume as maintained at room temperature and the other as subjected to 60 C for 5 minutes to abolish cell motility. The initial concentration of motile cells and the total loss of motility in the heat-treated semen ere evaluated microscopically. Semen aliquots ere mixed in eight tubes to generate a fixed level of total sperm concentration of 140 X 10 6 cells/ml ~ 180r ~~ J S9" =1~ ~ ~ 120.cr O mean!z 100..,0" ~ 80 0',., 8 60 "".0-0 ~ ~,~',- ~ 20,.(j"".0' 0-0 ~ O~'~"~~ '~ -L---L---L---L---L---L--~ o SMI UNITS 100, , SD 80.0' ' 0-' mean >- 60 0'" ~ 50.t::f"'" -SD ;= 40 - ", 0-. o p".0-.0' == ~~ _ /'.t::f O.O' 10-/ "" O~~~'~_L L L L L L ~ o SMI UNITS Figure 4 Correlation beteen sperm motility index (SM!) values and total sperm cell concentration (top) or percent motile cells (bottom), (n = 968). Each filled circle represents the mean cell concentration (top) or the mean percent motile cells (bottom) of all semen samples grouped in 40 sperm motility index unit ranges (±SD, open circles). but ith variable concentrations of motile spermatozoa ranging from 0 to 120 X 10 6 cells/ml. Sperm motility index values in each of the solutions as determined in triplicates. The results of this experiment are shon in Figure 3. The response of sperm motility index value to increasing concentration of motile cells as found to be biphasic. The linear phase and, therefore, the highest sensitivity of the measurement is in the lo range of motile cell density range of 0 to 40 X 10 6 cells/ml. At higher motile cell concentrations the curve plateaus en 70 ~ 60 :::;, 50 :=;; 40 en CONCENTRATION OF MOTILE CELLS (x106/ml) Figure 3 Dependence of sperm motility index values on the concentration of motile cells in a given sample. Total cell concentration as kept constant by the addition of corresponding concentration of killed, immotile cells of the same specimen. Correlation of Sperm Motility Index Values With Total Cell Concentration and Percentage of Motile Cells Statistically significant correlation of total cell concentration and sperm motility index values as found (r = 0.765, P < 0.001, n = 968, Fig. 4, top). Each point in the curve represents the mean cell concentration (±SD) of semen samples hose sperm motility index values ere grouped in 40 unit ranges. The same group of human semen samples as used to analyze the correlation beteen sperm motility index values and percent motility (Fig. 4, bottom). The data are presented as the mean percent motility (±SD) of groups defined by ranges of Bartoov et al. Sperm motility index for semen evaluation

4 units. A significant correlation as found beteen sperm motility index values and percent motile cells (r = 0.685, P < 0.001, n = 968). Sensitivity and Specificity of Sperm Motility Index for the Detection of Subnormal Cell Concentration In light of the observation that sperm motility index values are highly correlated ith total cell concentration, the capability of the Sperm Quality Analyzer to serve as a diagnostic tool for the diagnosis of subnormal cell concentration as examined. The study group as divided into subnormal «20 X 10 6 cells/ml) and normal (>20 X 10 6 cells/ml) populations (World Health Organization definition ll ), and the diagnostic poer of sperm motility index values as analyzed. The sensitivity, specificity, and negative predictive values of diagnosing semen ith subnormal cell concentration ere calculated. The curves ere generated by calculating, for every given sperm motility index value: (1) the percentage of semen samples ith subnormal cell concentration that produced sperm motility index readings loer than that given value (sensitivity); (2) the percentage of semen samples ith normal cell concentration that produced sperm motility index readings higher than that given value (specificity); and (3) the percentage of semen samples that produced sperm motility index readings higher than that given value that indeed had normal cell concentration (negative predictive value). Taking the value of 80 units as a cutoff, it as found that 322 of 346 semen samples ith cell concentration < 20 X 10 6 cells/ml indeed produce readings in the range of 0 to 80 sperm motility index units (sensitivity of 93%). Five hundred eight of 622 samples ith cell concentration> 20 X 10 6 cells/ml produce sperm motility index readings > 80 units (specificity of 82%). Five hundred eight of 532 samples ith >80 sperm motility index units indeed possess normal cell concentration (negative predictive value of 85.5%). Correlation of Sperm Motility Index Values With Overall Semen Assessment Semen quality and, indeed, sperm motility index values are determined by the combination of several parameters. A deficient quality of one parameter (e.g., the percent of motile cells) can be compensated by an improved quality of another (e.g., the intensity of their motility). To test if sperm motility index values ill correlate ith an overall microscopic semen assessment, 64 semen samples obtained from both patients and healthy donors ere evaluated by to experienced andrologists. They ere asked to rank in a comprehensive manner (in the range of 0 to 10) the quality of each ofthe 64 samples according to the folloing six parameters obtained from routine semen analysis: (1) sperm concentration; (2) percent motile sperm; (3) type of motility (1 to 4); (4) vitality (live/dead); (5) volume; and (6) ph. A regression analysis as applied to study the correlation beteen the subjective scores given by the andrologists and the sperm motility index readings obtained from the same specimens independently (Fig. 5). It is evident that sperm motility index values are highly correlated ith the andrologists' assessments. DISCUSSION Data presented in this study obtained from 968 semen samples covering the hole span of semen qualities demonstrate the significance of sperm motility index as a novel diagnostic parameter for the quick and objective evaluation of sperm cell motility and semen quality. Inherent in the principle of sperm motility index measurement is the fact that its values are generated by motile cells only. Immotile cells, regardless oftheir density, ill not cause fluctuations in the OD of the examined sample and ill not, therefore, affect sperm motility index readout. Indeed, it as observed (Fig. 3) that semen containing as high as 140 X 10 6 immotile cells/ml pro- Z 9 o >= ~ 7 ;i. 6 ~ 5 Z 4 W ::;; 3 ~ 2 10r ~~.. ~ 10 Z 9 0 B ~ ::J -' ;; Z ::;; Cf) 2 R = n = 64 p<o.ool R = n = 64 p<o.ool Andrologist 1 Andrologist ao lao SMI Figure 5 Correlation beteen semen assessment performed by to andrologists and sperm motility index (SM/) values of the same semen samples measured independently (64 samples). Semen quality assessment as based on routine semen analysis data. Semen quality ranking by the andrologists is in the range of 0 to 10. Vol. 56, NO.1, July 1991 Bartoov et ai. Sperm motility index for semen evaluation 111

5 duced a sperm motility index value of zero. Sperm motility index values ere shon to increase ith increasing the concentration of motile cells. A biphasic response as observed ith a linear phase at o to 40 X 10 6 motile cells/ml. It is likely that the nonlinear response observed at motile cell concentrations higher than 40 X 10 6 motile cells/ml results from the significant number of cell collisions occurring ith increasing cell density. Vantman et a1.,8 using automated computer-assisted semen analyzer, also observed that representative velocity estimation can be obtained only at cell concentration < 40 X 10 6 cells/ml. The near zero electronic background and the linearity of sperm motility index response at the lo range of cell concentration (0 to 40 X 10 6 cell/ml) give the Sperm Quality Analyzer high resolution poer for the diagnosis of complete or partial asthenozoospermic semen samples. Although immotile cells do not contribute to sperm quality index readouts, a high correlation as found beteen sperm quality index and total cell concentration and percent motility. This observation most likely stems from the fact that lo motile cell concentration and lo cell velocity, hich produce lo sperm motility index values, are usually accompanied by lo total sperm count and lo percentage of motile cells. Indeed, approximately 90% of semen samples examined ere found to be in the zone defined by the mean value ± 1 SD of cell concentration and percent motility. The Sperm Quality Analyzer can thus serve for the estimation of the isolated semen parameters of cell concentration and percent motility. Moreover, sperm motility index values can be employed to diagnose ith high sensitivity, specificity, and negative predictive value semen samples ith subnormal cell concentration. Sperm motility index is affected by both the concentration of motile cells and their type of motility. It is theoretically possible, and indeed frequently encountered, that an examined semen sample ill yield markedly higher sperm motility index value than another sample ith identical concentration of motile cells because of the better quality of motility. Similarly, to semen samples may yield identical sperm motility index readouts, despite the difference in motile cell concentration because of the difference in motility quality. It appears, therefore, that the significance of sperm motility index as an independent semen parameter does not stem solely from its ability to estimate parameters measured by routine semen analysis but, rather, from its being a reflection of the inherent comprehensive motility status of the examined semen. It is significant to point out in this context the high correlations found beteen sperm motility index values and semen quality evaluation done by to experienced andrologists. When evaluating the quality of the examined semen, the various parameters obtained in routine semen analysis are taken into consideration. The high correlation obtained beteen sperm motility index measurement and semen quality evaluation (r = 0.89, Fig. 5) is likely to reflect the high significance the evaluators place on the motility parameters of the examined semen,l--u the same parameters measured by the Sperm Quality Analyzer. In conclusion, data presented in this study demonstrate that sperm motility index as determined by the Sperm Quality Analyzer is a novel parameter for the evaluation of semen motility and quality. Acknoledgment. The technical assistance of Dina Aminpur, M.Sc., Haim Lilos, and Naomi Bar-Yosef and the secretarial assistance of Liat Dobrin from the Medical Faculty ofthe Technion, Haifa, Israel, are acknoledged. REFERENCES 1. Blasco L: Clinical tests of sperm fertilizing ability. 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