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Simultaneous Assessment of Human Sperm Motility and Morphology by Videomicrography
0022-5347 /81/1263-0357$02.00/0 THE JOURNAL OF UROLOGY
Vol. 126, September Printed in U.S. A.
Copyright© 1981 by The Williams & Wilkins Co.
SIMULTANEOUS ASSESSMENT OF HUMAN SPERM MOTILITY AND MORPHOLOGY BY VIDEOMICROGRAPHY JAMES W. OVERSTREET,* MICHAEL J. PRICE, WILLIAM F. BLAZAK, ERNEST L. LEWIS DAVID F. KATZ
AND
From the Departments of Human Anatomy, Obstetrics and Gynecology, and Urology, School of Medicine, University of California, Davis, California
ABSTRACT
Videotapes we.re analyzed to assess sperm motility and morphology in the semen of 5 fertile donors and 20 infertile patients. The slow motion playback control of the video recorder allowed simultaneous assessment of the motility and morphology of individual sperm cells. Similar values were obtained with this method for living sperm cells and for sperm on fixed, Papanicolaou-stained seminal smears. Spermatozoa with abnormal morphology more often were immotile or weakly motile than were the normal sperm in the same ejaculates. Normal oval sperm in the semen of infertile men were less likely to be motile and tended to swim more slowly than did morphologically normal sperm in the semen of fertile men. The association between semen quality and male infertility has been recognized for >30 years. 1 The major parameters of semen quality, sperm concentration, motility and morphology are correlated with fertility and with each other. 2- 5 However, the precise interrelationships among these parameters and their relative values in distinguishing between fertile and infertile men remain poorly understood. This is in large part owing to inaccuracy and imprecision in measurement, and lack of accepted standards for specific parameters of semen quality. The concentration of spermatozoa in the ejaculate is defined unambiguously, and may be determined accurately either by an automatic counting device or with a hemocytometer. However, only approximately 20 per cent of men referred for semen evaluation have sperm counts lower than the normal range. 6 Therefore, male fertility potential in the remaining 80 per cent must be assessed primarily by measurements of sperm motility and morphology. Current standards for these parameters a.re based almost entirely on subjective visual assessments. Significant errors result frequently from visual observation of sperm motility7 and morphology. 8 The recent, rapid development of video recorders and cameras for home entertainment has resulted in a variety of low cost, technically simple instruments and this technology recently has been applied to human semen evaluation. Sperm motility can be assessed from videotapes and accurate, objective measurements can be made of the percentage of motile cells and the mean swimming speed of the sperm population. 9 The morphology of living spermatozoa also can be assessed by videomicrography and the slow motion playback control of the video recorder provides the capability for simultaneous assessment of the morphology and motility ofliving spermatozoa. We herein describe experiments that demonstrate the potential value of this approach to human semen evaluation. MATERIALS AND METHODS
Materials. Semen samples from 25 men we.re studied in these experiments. Twenty were infertile patients who were referred to our laboratory for semen evaluation. All 20 men had normal sperm counts (>20 X 106 /ml.) but none had fathered a child. Five men were semen donors in our therapeutic artificial insemAccepted for publication November 7, 1980. Supported in part by National Institutes of Health Research Career Development Award HD 00224. * Requests for reprints: Department of Human Anatomy, School of Medicine, University of California, Davis, California 95616.
ination program. These 5 men had normal semen and the fertility of each had been proved within the previous 6 months. A single semen specimen was studied from each man and was examined within 1 hour of collection by masturbation. The semen of each infertile patient was evaluated clinically by l of us (J. W. 0.) with a sperm count (by hemocytometer), visual assessment of percentage motility (by scoring 100 consecutive cells for presence or absence of flagellar activity) and sperm morphology assessment from Papanicolaou-stained slides (100 cells with bright field, oil immersion optics). The same specimen was assessed simultaneously by videomicrography as described. Semen from the fertile donors was assessed by videomicrography alone. Based on the clinical evaluation the infertile patients were subdivided into those with normal and those with abnormal semen. The criteria for normal semen in this study were sperm count 20 to 250 X 106 /ml., motility >50 per cent and oval sperm >60 per cent. Assessment of sperm motility and morphology by videomicrography. All videomicrographic assessments of sperm motility and morphology were done by a single observer (M. J. P.). Two preparations were made from each semen specimen with a plain glass slide and a No. 1 ½ coverglass (22 X 22 mm.). The first slide was prepared with 7 µl. semen and the depth of this preparation was approximately 14 µm. Spermatozoa swimming in this preparation were videotaped for simultaneous assess·· ment of percentage motility, swimming speed and morphology. The second slide was prepared with l to 2 µl. semen. The shallow depth of this preparation immobilized the sperm and, thus, enabled more careful assessment of their morphology. All observations were made with phase contrast microscopy, using an Olympus Vanox Universal microscope equipped with a 40X plan-achromatic objective lens and a 2.5x ocular lens. An air curtain incubator was used to maintain the microscope stage at 37C. Ten to 15 independent microscopic fields on each slide were videotaped for 10 seconds each, using a Panasonic WV3300 color camera and NV8310 VHS recorder. Details of this equipment and methodology have been published previously. 9 The videotapes of the sperm suspensions were analyzed on a Sony CVM1250 12-inch color monitor. Sperm morphology was assessed by direct comparison with photographs depicting the morphological types (fig. 1). Spermatozoa with a normal head (length 3 to 5 µm., width 2 to 3 µm.), mid piece and tail were classified as normal-oval. Spermatozoa with a head length >5 µm. and width <3 µm. were classified as tapering head. 5 Those sperm with a normal mid piece and tail but an irregularly
357
358
OVERSTREET AND ASSOCIATES
shaped head were scored as amorphous head, while those with a normal head and tail but an abnormal mid piece (excessive cytoplasm on the mid piece or abnormal head-mid piece angle) were classified as amorphous mid piece. The remaining morphological types, including abnormal tails, large and small heads, duplicate forms and so forth, were assigned to the "other abnormality" category. A total of 100 spermatozoa distributed equally among the microscopic fields (that is IO-second takes) was assessed for morphology in each shallow slide preparation. The spermatozoa swimming in the deeper preparations were evaluated simultaneously for motility and morphology. Each of those tapes was analyzed twice. During the first analysis sequence 100 sperm distributed equally among the takes were classified for presence or absence of motility9 and each was assigned to a morphological class. The tape then was replayed and 50 motile spermatozoa distributed equally among the microscopic fields again were classified for morphology and the swimming speeds were determined. Morphological classification of the motile sperm was facilitated by the variable slow motion control of the recorder. Swimming speeds were measured during a 0.4-second interval using a calibrated transparent overlay (fig. 2). 9 Spermatozoa with a swimming speed of ~25 µ,m. per second were classified as progressive. 10 To test the consistency of sperm morphology assessment by videomicrography and its relationship to conventional methods of morphology assessment the Papanicolaou-stained slides were coded, randomized and re-evaluated by videomicrography. A
FIG. 1. Phase contrast photomicrograph of human spermatozoa with 3 different head morphologies. A, normal, oval head. B, amorphous head. C, tapering head. Reduced from Xl,140.
total of 100 cells distributed in 10 independent microscopic fields was assessed by 1 of us (M. J. P.) at the same magnification and by the same criteria applied to the living cells. Statistical analysis of the data had several objectives. The equivalence of the different methods of morphology assessment was tested by chi-square analysis. Differences in the mean percentages of motile and progressive spermatozoa in different morphological categories, as well as between the different groups of men, were tested by Student's t test. Values were transformed to angles before such analyses. RESULTS
Sperm concentrations in the 3 groups of men. The average sperm concentration in the donor semen was 129 x 106 /ml., with a range of 75 to 232. The average sperm concentration in the group of infertile patients with clinically normal semen was 137 X 106 /ml., with a range of 66 to 250. The average sperm concentration for the patients with clinically abnormal semen also was 137 X 106 /ml. but the range was much broader (20 to 506). Two men with sperm concentrations of 381 X 106 and 506 x 106/ml. were considered to have abnormally high sperm counts. Sperm morphology assessment by videomicrography. The morphological classification of spermatozoa in the semen of the 20 patients was equivalent when living spermatozoa assessed by videomicrography were compared to fixed sperm evaluated on Papanicolaou-stained smears. Morphological assessment by
FIG. 2. Photograph of video screen illustrating use of analysis transparency for determination of swimming speed. Cross-hair is positioned at sperm head mid piece junction, tape is projected forward and final position of junction is noted. Swimming speed is this "straight line distance" divided by elapsed real time. Distance between concentric rings of transparency is determined by reference to stage micrometer on calibration tape. Sperm shown have normal morphology. Reduced from Xl,140.
TABLE 1. Percentages of spermatozoa in different morphological classes as assessed by uideomicrography (living sperm) or after fixation
and staining* Fertile Donors Living Sperm
Infertile Pts. With Normal Semen
Infertile Pts. With Abnormal Semen
Living Sperm
Normal Amorphous head Amorphous mid piece Tapering head Other abnormalities
Immobilized
Swimming Freely
71.6 7.6 11.9 4.6 4.3
79.4 3.8 11.6 1.8 3.4
Immobilized
Swimming Freely
65.3 6.4 19.4 2.2 6.7
68.0 5.1 21.0 2.1 3.8
Living Sperm Fixed Spermt 70.6 6.1 11.9 6.5 4.9
Immobilized
Swimming Freely
52.7 10.5 23.4 6.8 6.6
60.7 7.8 22.3 2.9 6.3
Fixed Spermt 57.1 12.1 14.4 12.0 4.4
* 100 cells evaluated by each method for each individual. A total of 500 sperm from the fertile donors, 800 from infertile patients with normal semen and 1,200 from the infertile patients with abnormal semen was classified. The different methods of assessing sperm morphology yielded similar results (p >0.50). as determined by chisquare analysis. t Papanicolaou stain was used.
SPERM MOTILITY AND MORPHOLOGY ASSESSMENT BY VIDEOMICROGRAPHY
videomic:rography provided similar results whether the sperm were immobilized or swimming freely (table 1).
The percentage motility and progression of sperm with normal and abnormal morphology. Over-all, the mean percentage of motile sperm (all cells) in the donor semen was higher than in the abnormal patient semen (p <0.02) but was not significantly different from that of normal patient semen (table 2). The mean percentage motility of normal cells was than that of the abnormal cells in all 3 groups (p <0.01). The mean percentages of motile sperm swimming ~25 µ.m. per second were similar for the fertile donors and patients with normal semen but significantly fewer motile cells were progressive in the semen of the abnormal patients (p <0.05, table 3). The percentage progression of normal oval cells was gTeater than that of the abnormal cells in all 3 groups (p <0.01).
Semen evaluation by combined motility and morphology assessment. The data in table 4 were derived from simultaneous assessment of sperm morphology and motility in semen from the 3 groups of men. It can be seen that the mean percentage of spermatozoa from fertile donors with normal morphology and any evidence of motility was 66.2 per cent. The mean percentage of donor spermatozoa with normal morphology and . progressive motility was somewhat lower (50.6 per cent). Similar analysis of the sperm populations from the 2 groups of infertile patients resulted in lower values for these parameters, for example only 20.l per cent of the sperm in the abnormal semen specimens were oval and progressive (table 4). Statistical analysis of the data showed that the percentage motility and percentage progression of the normal cells were greater than 2. Percentage motility (mean ± standard error of the mean) of morphologically normal and abnormal spermatozoa as assessed by videomicrography *
TABLE
Normal Abnormal All cells
Fertile Donors (5 pts.)
Normal Se1nen (8 pts.)
Abnormal Semen (12 pts.)
82.9 ± 8.0 49.6 ± 8.3 76.0 ± 8.0
73.5 ± 7.2
68.0 ± 5.0
40.7 ± 9.0
28.l ± 4.8
63.1 ± 7.6
51.8 ± 4.7
"' Values are calculated with respect to cells of the same morphological type, that is a mean of 82.9 per cent of normal sperm from the fertile donors was motile. A total of 100 cells was examined per individual.
3. Relative percentages (mean ± standard error of the mean) of morphologically normal and abnormal spermatozoa displaying progressive motility as assessed by videomicrography *
TABLE
Fertile Donors (5 pts.) Normal Abnormal All cells
74.9 ± 3.8 31.5 ± 13.7 67.4 ± 5.1
Normal Semen (8 pts.)
Abnormal
69.2 ± 3.6 27.1 ± 4.5 63.2 ± 4.1
47.3 ± 4.9 26.6 ± 9.0 41.8 ± 5.4
Semen (12 pts.)
* Values rure calculated with respect to motile cells of the same morphological type. A progressive sperm is defined as one having a swimming speed ~25 µm. per second. 4. Absolute percentages (mean ± standard error of the mean) of motile and progressive morphologically normal and abnormal spermatozoa as assessed by videomicrography *
TABLE
Normal: Motile Progressive Abnormal: Motile Progressive All cells: Motile Progressive
Fertile Donors (5 pts.)
Normal Semen (8 pts.)
Abnormal Semen (12 pts.)
66.2 ± 7.4
50.0 ± 6.1
50.6 ± 7.7
33.6 ± 5.8
41.1 ± 3.4 20.l ± 5.2
9.8 ± 1.4 3.4 ± 2.1
13.l ± 3.6 3.9 ± 2.0
10.7 ± 1.9 2.7 ± 1.1
76.0 ± 8.0
63.1 ± 7.6 37.5 ± 5.5
51.8 ± 4.7 22.8 ± 5.0
54.0 ± 5.7
* Values are calculated with respect to all cells in the ejaculate, that is 66.2 per cent of all cells in ejaculates from fertile donors were motile, morphologically normal cells.
359
that of the abnormal cells in every group (p <0.01). The percentage motility and the percentage progression of the normal cells were not significantly different for donors and patients with normal semen but the values for the donors were higher than those of patients with abnonnal semen (p <0.01 for percentage motility, p <0.001 for percentage progression). DISCUSSION
The diagnosis of male infertility currently is made by refer . ence to prescribed standards of semen quality. Generally, these are bulk measures such as sperm concentration, motile and percentage normal morphology. 5 A fertility u"'1''m"rn based on these standards is subjective, often wrong. The logic underlying this approach is sound, that is the fertility potential of a man should be related directly to the number of normal or fertile cells in the ejaculate. Unfortunatel.y, there are little experimental data and virtually no agreement of scientific opinion on what characteristics are unique to a fertile spermatozoon. Determination of any such parameter will require measurements on individual living sperm cells. Our approach in making simultaneous motility and morphology measurements on living sperm is an initial step in this direction . We know that the semen of fertile and infertile men contains significant numbers of senescent, poorly motile and abnormaI!y formed cells. A correlation between abnormal sperm morphology and male infertility has been documented but the data also indicate a close association between sperm morphology and the other parameters of semen quality. 2- 5 Studies of sperm stained with vital dyes have suggested that morphologically abnormal sperm are more likely to be nonviable, 11 and there is evidence of a significant inverse correlation between the incidence of abnormal sperm morphology and the average quality in an ejaculate. 12 However, the lack of direct observations on the motility of normal and abnormal sperm has made it sible to more than speculate on the natural history or ultimate fate of morphologically abnormal spermatozoa. The limited data obtained in our study suggest that abnormally spermatozoa are likely to be either immotile 01· weakly whether they are found in the semen of a fertile or an infertile man. These cells may be nonfunctional and, thus, a source "noise," which obscures the true concentration of functional. sperm in the ejaculate. Previous studies have suggested that there may be a significant overlap in the total number of normally shaped, motile spermatozoa in ejaculates from fertile and infertile men. Our current clinical standards for semen evaluation appear to be founded on the assumption that all motile sperm cells with normal morphology are physiologically equivalent. The limited data obtained in these experiments suggest that oval spermatozoa from infertile men, although normal in morphology, are less likely to be motile and may swim more slowly than .ouuua"' v shaped sperm from the semen of fertile men. This implies that normal sperm from fertile and infertile men may differ in their physiology. Further studies with larger, more well defined patient populations will be required to verify these preliminary findings and additional tests of sperm function will have to be applied. Videomicrography affords the unique capability for experimen-· tal studies of specific subgroups of spermatozoa within the ejaculate. This technology and its successors will be valuable tools for identifying the characteristics of fertile spermatozoa. REFERENCES
l. MacLeod, J. and Gold, R. Z.: The male factor in
infertility; semen quality and certain other factors in ease of conception. Fertil. Steril., 4: 10, 1953. 2. Freund, M.: Interrelationships among the characteristics of human semen and factors affecting semen-specimen quality. J. Reprod. Fertil., 4: 143, 1962.
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OVERSTREET AND ASSOCIATES
3. Hartman, C. G.: Correlations among criteria of semen quality. Fertil. Steril., 16: 632, 1965. 4. Santomauro, A. G., Sciarra, J. J. and Varma, A. 0.: A clinical investigation of the role of the semen analysis and postcoital test in the evaluation of male infertility. Fertil. Steril., 23: 245, 1972. 5. Eliasson, R.: Analysis of semen. In: Progress in Infertility. Edited by S. J. Behrman and R. W. Kistner. Boston: Little, Brown & Co., p. 691, 1975. 6. MacLeod, J. and Wang, Y.: Male fertility potential in terms of semen quality: a review of the past, a study of the present. Fertil. Steril., 31: 103, 1979. 7. Van Duijn, C., Jr., Van Voorst, C. and Freund, M.: Movement characteristics of human spermatozoa analysed from kinemicrographs. Eur. J. Obst. Gynec., 4: 121, 1971. 8. Freund, M.: Standards for the rating of human sperm morphology. A cooperative study. Int. J. Fertil., 11: 97, 1966. 9. Katz, D. F. and Overstreet, J. W.: Sperm motility assessment by videomicrography. Fertil. Steril., 35: 188, 1981. 10. Katz, D. F., Overstreet, J. W. and Hanson, F. W.: Variations within and amongst normal men of movement characteristics of seminal spermatozoa. J. Reprod. Fertil., 62: 221, 1981.
11. Fredricsson, B., Waxegiird, G., Brege, S. and Lundberg, I.: On the morphology of live spermatozoa of human semen. Fertil. Steril., 28: 168, 1977. 12. Bartak, V.: Sperm velocity and morphology in 1727 ejaculates with normal sperm count. Int. J. Fertil., 18: 116, 1973. EDITORIAL COMMENT This article provides data that will be necessary if we are to expand our knowledge of the motility and morphology of sperm, and the relationship between these factors. As the authors indicate conclusions based on bulk measurements, useful until now, can be measured more precisely by the method described. However, it does look like an expensive technique and it is doubtful whether it will ever be a part of a routine fertility evaluation. In the meantime, the information gained from its evaluation in sophisticated laboratories will contribute greatly to our understanding and the identification of fertile sperm. J. Walter Smyth Brady Urological Institute The Johns Hopkins Hospital Baltimore, Maryland
Vol. 126, September Printed in U.S. A.
Copyright© 1981 by The Williams & Wilkins Co.
SIMULTANEOUS ASSESSMENT OF HUMAN SPERM MOTILITY AND MORPHOLOGY BY VIDEOMICROGRAPHY JAMES W. OVERSTREET,* MICHAEL J. PRICE, WILLIAM F. BLAZAK, ERNEST L. LEWIS DAVID F. KATZ
AND
From the Departments of Human Anatomy, Obstetrics and Gynecology, and Urology, School of Medicine, University of California, Davis, California
ABSTRACT
Videotapes we.re analyzed to assess sperm motility and morphology in the semen of 5 fertile donors and 20 infertile patients. The slow motion playback control of the video recorder allowed simultaneous assessment of the motility and morphology of individual sperm cells. Similar values were obtained with this method for living sperm cells and for sperm on fixed, Papanicolaou-stained seminal smears. Spermatozoa with abnormal morphology more often were immotile or weakly motile than were the normal sperm in the same ejaculates. Normal oval sperm in the semen of infertile men were less likely to be motile and tended to swim more slowly than did morphologically normal sperm in the semen of fertile men. The association between semen quality and male infertility has been recognized for >30 years. 1 The major parameters of semen quality, sperm concentration, motility and morphology are correlated with fertility and with each other. 2- 5 However, the precise interrelationships among these parameters and their relative values in distinguishing between fertile and infertile men remain poorly understood. This is in large part owing to inaccuracy and imprecision in measurement, and lack of accepted standards for specific parameters of semen quality. The concentration of spermatozoa in the ejaculate is defined unambiguously, and may be determined accurately either by an automatic counting device or with a hemocytometer. However, only approximately 20 per cent of men referred for semen evaluation have sperm counts lower than the normal range. 6 Therefore, male fertility potential in the remaining 80 per cent must be assessed primarily by measurements of sperm motility and morphology. Current standards for these parameters a.re based almost entirely on subjective visual assessments. Significant errors result frequently from visual observation of sperm motility7 and morphology. 8 The recent, rapid development of video recorders and cameras for home entertainment has resulted in a variety of low cost, technically simple instruments and this technology recently has been applied to human semen evaluation. Sperm motility can be assessed from videotapes and accurate, objective measurements can be made of the percentage of motile cells and the mean swimming speed of the sperm population. 9 The morphology of living spermatozoa also can be assessed by videomicrography and the slow motion playback control of the video recorder provides the capability for simultaneous assessment of the morphology and motility ofliving spermatozoa. We herein describe experiments that demonstrate the potential value of this approach to human semen evaluation. MATERIALS AND METHODS
Materials. Semen samples from 25 men we.re studied in these experiments. Twenty were infertile patients who were referred to our laboratory for semen evaluation. All 20 men had normal sperm counts (>20 X 106 /ml.) but none had fathered a child. Five men were semen donors in our therapeutic artificial insemAccepted for publication November 7, 1980. Supported in part by National Institutes of Health Research Career Development Award HD 00224. * Requests for reprints: Department of Human Anatomy, School of Medicine, University of California, Davis, California 95616.
ination program. These 5 men had normal semen and the fertility of each had been proved within the previous 6 months. A single semen specimen was studied from each man and was examined within 1 hour of collection by masturbation. The semen of each infertile patient was evaluated clinically by l of us (J. W. 0.) with a sperm count (by hemocytometer), visual assessment of percentage motility (by scoring 100 consecutive cells for presence or absence of flagellar activity) and sperm morphology assessment from Papanicolaou-stained slides (100 cells with bright field, oil immersion optics). The same specimen was assessed simultaneously by videomicrography as described. Semen from the fertile donors was assessed by videomicrography alone. Based on the clinical evaluation the infertile patients were subdivided into those with normal and those with abnormal semen. The criteria for normal semen in this study were sperm count 20 to 250 X 106 /ml., motility >50 per cent and oval sperm >60 per cent. Assessment of sperm motility and morphology by videomicrography. All videomicrographic assessments of sperm motility and morphology were done by a single observer (M. J. P.). Two preparations were made from each semen specimen with a plain glass slide and a No. 1 ½ coverglass (22 X 22 mm.). The first slide was prepared with 7 µl. semen and the depth of this preparation was approximately 14 µm. Spermatozoa swimming in this preparation were videotaped for simultaneous assess·· ment of percentage motility, swimming speed and morphology. The second slide was prepared with l to 2 µl. semen. The shallow depth of this preparation immobilized the sperm and, thus, enabled more careful assessment of their morphology. All observations were made with phase contrast microscopy, using an Olympus Vanox Universal microscope equipped with a 40X plan-achromatic objective lens and a 2.5x ocular lens. An air curtain incubator was used to maintain the microscope stage at 37C. Ten to 15 independent microscopic fields on each slide were videotaped for 10 seconds each, using a Panasonic WV3300 color camera and NV8310 VHS recorder. Details of this equipment and methodology have been published previously. 9 The videotapes of the sperm suspensions were analyzed on a Sony CVM1250 12-inch color monitor. Sperm morphology was assessed by direct comparison with photographs depicting the morphological types (fig. 1). Spermatozoa with a normal head (length 3 to 5 µm., width 2 to 3 µm.), mid piece and tail were classified as normal-oval. Spermatozoa with a head length >5 µm. and width <3 µm. were classified as tapering head. 5 Those sperm with a normal mid piece and tail but an irregularly
357
358
OVERSTREET AND ASSOCIATES
shaped head were scored as amorphous head, while those with a normal head and tail but an abnormal mid piece (excessive cytoplasm on the mid piece or abnormal head-mid piece angle) were classified as amorphous mid piece. The remaining morphological types, including abnormal tails, large and small heads, duplicate forms and so forth, were assigned to the "other abnormality" category. A total of 100 spermatozoa distributed equally among the microscopic fields (that is IO-second takes) was assessed for morphology in each shallow slide preparation. The spermatozoa swimming in the deeper preparations were evaluated simultaneously for motility and morphology. Each of those tapes was analyzed twice. During the first analysis sequence 100 sperm distributed equally among the takes were classified for presence or absence of motility9 and each was assigned to a morphological class. The tape then was replayed and 50 motile spermatozoa distributed equally among the microscopic fields again were classified for morphology and the swimming speeds were determined. Morphological classification of the motile sperm was facilitated by the variable slow motion control of the recorder. Swimming speeds were measured during a 0.4-second interval using a calibrated transparent overlay (fig. 2). 9 Spermatozoa with a swimming speed of ~25 µ,m. per second were classified as progressive. 10 To test the consistency of sperm morphology assessment by videomicrography and its relationship to conventional methods of morphology assessment the Papanicolaou-stained slides were coded, randomized and re-evaluated by videomicrography. A
FIG. 1. Phase contrast photomicrograph of human spermatozoa with 3 different head morphologies. A, normal, oval head. B, amorphous head. C, tapering head. Reduced from Xl,140.
total of 100 cells distributed in 10 independent microscopic fields was assessed by 1 of us (M. J. P.) at the same magnification and by the same criteria applied to the living cells. Statistical analysis of the data had several objectives. The equivalence of the different methods of morphology assessment was tested by chi-square analysis. Differences in the mean percentages of motile and progressive spermatozoa in different morphological categories, as well as between the different groups of men, were tested by Student's t test. Values were transformed to angles before such analyses. RESULTS
Sperm concentrations in the 3 groups of men. The average sperm concentration in the donor semen was 129 x 106 /ml., with a range of 75 to 232. The average sperm concentration in the group of infertile patients with clinically normal semen was 137 X 106 /ml., with a range of 66 to 250. The average sperm concentration for the patients with clinically abnormal semen also was 137 X 106 /ml. but the range was much broader (20 to 506). Two men with sperm concentrations of 381 X 106 and 506 x 106/ml. were considered to have abnormally high sperm counts. Sperm morphology assessment by videomicrography. The morphological classification of spermatozoa in the semen of the 20 patients was equivalent when living spermatozoa assessed by videomicrography were compared to fixed sperm evaluated on Papanicolaou-stained smears. Morphological assessment by
FIG. 2. Photograph of video screen illustrating use of analysis transparency for determination of swimming speed. Cross-hair is positioned at sperm head mid piece junction, tape is projected forward and final position of junction is noted. Swimming speed is this "straight line distance" divided by elapsed real time. Distance between concentric rings of transparency is determined by reference to stage micrometer on calibration tape. Sperm shown have normal morphology. Reduced from Xl,140.
TABLE 1. Percentages of spermatozoa in different morphological classes as assessed by uideomicrography (living sperm) or after fixation
and staining* Fertile Donors Living Sperm
Infertile Pts. With Normal Semen
Infertile Pts. With Abnormal Semen
Living Sperm
Normal Amorphous head Amorphous mid piece Tapering head Other abnormalities
Immobilized
Swimming Freely
71.6 7.6 11.9 4.6 4.3
79.4 3.8 11.6 1.8 3.4
Immobilized
Swimming Freely
65.3 6.4 19.4 2.2 6.7
68.0 5.1 21.0 2.1 3.8
Living Sperm Fixed Spermt 70.6 6.1 11.9 6.5 4.9
Immobilized
Swimming Freely
52.7 10.5 23.4 6.8 6.6
60.7 7.8 22.3 2.9 6.3
Fixed Spermt 57.1 12.1 14.4 12.0 4.4
* 100 cells evaluated by each method for each individual. A total of 500 sperm from the fertile donors, 800 from infertile patients with normal semen and 1,200 from the infertile patients with abnormal semen was classified. The different methods of assessing sperm morphology yielded similar results (p >0.50). as determined by chisquare analysis. t Papanicolaou stain was used.
SPERM MOTILITY AND MORPHOLOGY ASSESSMENT BY VIDEOMICROGRAPHY
videomic:rography provided similar results whether the sperm were immobilized or swimming freely (table 1).
The percentage motility and progression of sperm with normal and abnormal morphology. Over-all, the mean percentage of motile sperm (all cells) in the donor semen was higher than in the abnormal patient semen (p <0.02) but was not significantly different from that of normal patient semen (table 2). The mean percentage motility of normal cells was than that of the abnormal cells in all 3 groups (p <0.01). The mean percentages of motile sperm swimming ~25 µ.m. per second were similar for the fertile donors and patients with normal semen but significantly fewer motile cells were progressive in the semen of the abnormal patients (p <0.05, table 3). The percentage progression of normal oval cells was gTeater than that of the abnormal cells in all 3 groups (p <0.01).
Semen evaluation by combined motility and morphology assessment. The data in table 4 were derived from simultaneous assessment of sperm morphology and motility in semen from the 3 groups of men. It can be seen that the mean percentage of spermatozoa from fertile donors with normal morphology and any evidence of motility was 66.2 per cent. The mean percentage of donor spermatozoa with normal morphology and . progressive motility was somewhat lower (50.6 per cent). Similar analysis of the sperm populations from the 2 groups of infertile patients resulted in lower values for these parameters, for example only 20.l per cent of the sperm in the abnormal semen specimens were oval and progressive (table 4). Statistical analysis of the data showed that the percentage motility and percentage progression of the normal cells were greater than 2. Percentage motility (mean ± standard error of the mean) of morphologically normal and abnormal spermatozoa as assessed by videomicrography *
TABLE
Normal Abnormal All cells
Fertile Donors (5 pts.)
Normal Se1nen (8 pts.)
Abnormal Semen (12 pts.)
82.9 ± 8.0 49.6 ± 8.3 76.0 ± 8.0
73.5 ± 7.2
68.0 ± 5.0
40.7 ± 9.0
28.l ± 4.8
63.1 ± 7.6
51.8 ± 4.7
"' Values are calculated with respect to cells of the same morphological type, that is a mean of 82.9 per cent of normal sperm from the fertile donors was motile. A total of 100 cells was examined per individual.
3. Relative percentages (mean ± standard error of the mean) of morphologically normal and abnormal spermatozoa displaying progressive motility as assessed by videomicrography *
TABLE
Fertile Donors (5 pts.) Normal Abnormal All cells
74.9 ± 3.8 31.5 ± 13.7 67.4 ± 5.1
Normal Semen (8 pts.)
Abnormal
69.2 ± 3.6 27.1 ± 4.5 63.2 ± 4.1
47.3 ± 4.9 26.6 ± 9.0 41.8 ± 5.4
Semen (12 pts.)
* Values rure calculated with respect to motile cells of the same morphological type. A progressive sperm is defined as one having a swimming speed ~25 µm. per second. 4. Absolute percentages (mean ± standard error of the mean) of motile and progressive morphologically normal and abnormal spermatozoa as assessed by videomicrography *
TABLE
Normal: Motile Progressive Abnormal: Motile Progressive All cells: Motile Progressive
Fertile Donors (5 pts.)
Normal Semen (8 pts.)
Abnormal Semen (12 pts.)
66.2 ± 7.4
50.0 ± 6.1
50.6 ± 7.7
33.6 ± 5.8
41.1 ± 3.4 20.l ± 5.2
9.8 ± 1.4 3.4 ± 2.1
13.l ± 3.6 3.9 ± 2.0
10.7 ± 1.9 2.7 ± 1.1
76.0 ± 8.0
63.1 ± 7.6 37.5 ± 5.5
51.8 ± 4.7 22.8 ± 5.0
54.0 ± 5.7
* Values are calculated with respect to all cells in the ejaculate, that is 66.2 per cent of all cells in ejaculates from fertile donors were motile, morphologically normal cells.
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that of the abnormal cells in every group (p <0.01). The percentage motility and the percentage progression of the normal cells were not significantly different for donors and patients with normal semen but the values for the donors were higher than those of patients with abnonnal semen (p <0.01 for percentage motility, p <0.001 for percentage progression). DISCUSSION
The diagnosis of male infertility currently is made by refer . ence to prescribed standards of semen quality. Generally, these are bulk measures such as sperm concentration, motile and percentage normal morphology. 5 A fertility u"'1''m"rn based on these standards is subjective, often wrong. The logic underlying this approach is sound, that is the fertility potential of a man should be related directly to the number of normal or fertile cells in the ejaculate. Unfortunatel.y, there are little experimental data and virtually no agreement of scientific opinion on what characteristics are unique to a fertile spermatozoon. Determination of any such parameter will require measurements on individual living sperm cells. Our approach in making simultaneous motility and morphology measurements on living sperm is an initial step in this direction . We know that the semen of fertile and infertile men contains significant numbers of senescent, poorly motile and abnormaI!y formed cells. A correlation between abnormal sperm morphology and male infertility has been documented but the data also indicate a close association between sperm morphology and the other parameters of semen quality. 2- 5 Studies of sperm stained with vital dyes have suggested that morphologically abnormal sperm are more likely to be nonviable, 11 and there is evidence of a significant inverse correlation between the incidence of abnormal sperm morphology and the average quality in an ejaculate. 12 However, the lack of direct observations on the motility of normal and abnormal sperm has made it sible to more than speculate on the natural history or ultimate fate of morphologically abnormal spermatozoa. The limited data obtained in our study suggest that abnormally spermatozoa are likely to be either immotile 01· weakly whether they are found in the semen of a fertile or an infertile man. These cells may be nonfunctional and, thus, a source "noise," which obscures the true concentration of functional. sperm in the ejaculate. Previous studies have suggested that there may be a significant overlap in the total number of normally shaped, motile spermatozoa in ejaculates from fertile and infertile men. Our current clinical standards for semen evaluation appear to be founded on the assumption that all motile sperm cells with normal morphology are physiologically equivalent. The limited data obtained in these experiments suggest that oval spermatozoa from infertile men, although normal in morphology, are less likely to be motile and may swim more slowly than .ouuua"' v shaped sperm from the semen of fertile men. This implies that normal sperm from fertile and infertile men may differ in their physiology. Further studies with larger, more well defined patient populations will be required to verify these preliminary findings and additional tests of sperm function will have to be applied. Videomicrography affords the unique capability for experimen-· tal studies of specific subgroups of spermatozoa within the ejaculate. This technology and its successors will be valuable tools for identifying the characteristics of fertile spermatozoa. REFERENCES
l. MacLeod, J. and Gold, R. Z.: The male factor in
infertility; semen quality and certain other factors in ease of conception. Fertil. Steril., 4: 10, 1953. 2. Freund, M.: Interrelationships among the characteristics of human semen and factors affecting semen-specimen quality. J. Reprod. Fertil., 4: 143, 1962.
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OVERSTREET AND ASSOCIATES
3. Hartman, C. G.: Correlations among criteria of semen quality. Fertil. Steril., 16: 632, 1965. 4. Santomauro, A. G., Sciarra, J. J. and Varma, A. 0.: A clinical investigation of the role of the semen analysis and postcoital test in the evaluation of male infertility. Fertil. Steril., 23: 245, 1972. 5. Eliasson, R.: Analysis of semen. In: Progress in Infertility. Edited by S. J. Behrman and R. W. Kistner. Boston: Little, Brown & Co., p. 691, 1975. 6. MacLeod, J. and Wang, Y.: Male fertility potential in terms of semen quality: a review of the past, a study of the present. Fertil. Steril., 31: 103, 1979. 7. Van Duijn, C., Jr., Van Voorst, C. and Freund, M.: Movement characteristics of human spermatozoa analysed from kinemicrographs. Eur. J. Obst. Gynec., 4: 121, 1971. 8. Freund, M.: Standards for the rating of human sperm morphology. A cooperative study. Int. J. Fertil., 11: 97, 1966. 9. Katz, D. F. and Overstreet, J. W.: Sperm motility assessment by videomicrography. Fertil. Steril., 35: 188, 1981. 10. Katz, D. F., Overstreet, J. W. and Hanson, F. W.: Variations within and amongst normal men of movement characteristics of seminal spermatozoa. J. Reprod. Fertil., 62: 221, 1981.
11. Fredricsson, B., Waxegiird, G., Brege, S. and Lundberg, I.: On the morphology of live spermatozoa of human semen. Fertil. Steril., 28: 168, 1977. 12. Bartak, V.: Sperm velocity and morphology in 1727 ejaculates with normal sperm count. Int. J. Fertil., 18: 116, 1973. EDITORIAL COMMENT This article provides data that will be necessary if we are to expand our knowledge of the motility and morphology of sperm, and the relationship between these factors. As the authors indicate conclusions based on bulk measurements, useful until now, can be measured more precisely by the method described. However, it does look like an expensive technique and it is doubtful whether it will ever be a part of a routine fertility evaluation. In the meantime, the information gained from its evaluation in sophisticated laboratories will contribute greatly to our understanding and the identification of fertile sperm. J. Walter Smyth Brady Urological Institute The Johns Hopkins Hospital Baltimore, Maryland