A NEW MULTIPLE EXPOSURE PHOTOGRAPHY METHOD FOR OBJECTIVE HUMAN SPERMATOZOAL MOTILITY DETERMINATION

Transcription

1 FERTILITY AND STERILITY Copyright < 1978 The American Fertility Society Vol. 30, No.2, August 1978 Printed in U.S A. A NEW MULTIPLE EXPOSURE PHOTOGRAPHY METHOD FOR OBJECTIVE HUMAN SPERMATOZOAL MOTILITY DETERMINATION AMNON MAKLER, M.D.* Fertility Institute, Departments of Obstetrics and Gynecology "A" and "B," Rambam Medical Center, Technion, Faculty of Medicine, Haifa, Israel A new method for objective spermatozoal motility determination is described. In this method, spermatozoa from undiluted specimens are always examined under the same standard conditions by inserting them into a new counting chamber 10 pm thick. This constant thickness allows free, frictionless, horizontal movement of the spermatozoa to be seen clearly in one focal plane when microscopically observed. Using a phase-contrast microscope, these samples are photographed with a still camera and a multiple exposure technique, where six short light pulses are given during 1 second with the aid of a mechanical stroboscope. As a result, nonmotile spermatozoa appear 6 times brighter than do motile spermatozoa. The latter are seen as six-ringed chains; their length and shape describe the distance traveled by the spermatozoa during 1 second. From these pictures it is possible to obtain the exact percentage of motility, individual and average speed (expressed as microns per second), frequency of distribution of spermatozoal speed, and spermatozoal concentration. By statistical evaluation it was shown that a high degree of accuracy can be obtained from analyzing about four to six pictures, or 300 to 400 spermatozoa in each sample. It was also found that, when this objective motility determination was compared with subjective determination, human judgment tended to overestimate motility-about 20% more than did the objective method. This inexpensive, easy to perform, and highly informative technique can be of great value in the routine clinical work-up and in research studies. The average sperm speed may become a new index in any routine semen analysis. Fertil Steril30:192, 1978 In a previous study 1 we presented a new counting chamber for rapid semen analysis which is only 10 ~J-m deep instead of the 100!J-m of an ordinary hemocytometer. This shallow chamber enables analysis of undiluted samples and clear vision of all spermatozoa while they move freely in one focal plane and under the same conditions, common to all specimens examined. The importance of having the same conditions for all examined samples was stressed by us in another study 2 in which it was demonstrated how results of motility estimation were affected highly by the varying thickness of the drop, because friction and pressure impaired free movement of the spermatozoa. Standard conditions obtained with this chamber solved one of the main problems in Received January 10, 1978; accepted March 15, *Head of Fertility Institute. 192 sperm motility determination; however, the question of choosing the ideal method for measuring motility remained unanswered. The most popular method, that of simple subjective estimation, 3 is very inaccurate, cannot describe true sperm speed, has a very limited value in clinical studies, and is not suitable for research work. In this study we present a new objective method for sperm motility determination which combines the use of a 10-~J-m chamber developed by us with the use of a still camera and the multiple exposure photography (MEP) technique. In photography, this method of exposure is used to describe moving objects in a very clear way and is superior to that of time or double-exposure techniques. Until now, it has never been used in human or animal spermatozoal motility determination..-

2 Vol. 30, No. 2 MEP METHOD FOR SPERMATOZOAL MOTll..ITY DETERMINATION 193 ' FIG. 1. Preparation for semen analysis: a small drop from a well-mixed specimen is put in the center of the lower part of the 10-~J.m chamber with the aid of a wooden stick and is covered immediately. In this way the drop automatically has a thickness of 10 IJ.m. -. " MATERIALS AND METHODS A drop of semen from a well-mixed, undiluted sample is placed in the center of the 10-JLm chamber* and covered immediately (Fig. 1). The chamber is put on a stage of a phase-contrast microscope, and a x 10 objective and x 15 eyepiece are used. A still camera loaded with 100 ASA black and white film is attached with the aid of a bellows or extension rings to the microscope. The bellows or rings are adjusted so that the film will cover an area of approximately 0.5 x 0.35 sq mm. With the microscope fine adjustment knob, spermatozoa that are moving within the space of the chamber, rather than nonmotile spermatozoa lying on the bottom, are brought into focus; thus all spermatozoa can be seen clearly on the film. While exposed for 1 full second, six light pulses, each of 1 /200-second duration and at intervals of 1/ 6 second, are given. This multiple exposure technique is achieved by a stroboscope made of a six-slotted black disc placed between the light source and the condensor of the microscope and rotating at 60 rpm (1 rps) by an electric motor *Pending patent no ; manufactured by EL-OP, Rehovoth, Israel. (Fig. 2). As a result, during this 1 second, only about 1 / 30 of the light penetrates the camera, so that images of nonmotile spermatozoa will not c FIG. 2. Assembly of the system for multiple exposure photography: The specimen within the 10-~J.m chamber (B) is placed on the stage of a phase-contrast microscope (A). A still camera (D) is attached to the microscope. With the aid of a bellows (E) the proper enlargement is chosen, after which motile spermatozoa are brought into focus. A six-slotted disc attached to an electric motor of 60 (C) is placed between the microscope light source and the condenser. While the disc is rotating the shutter of the camera is opened for 1 full second.

3 194 MAKLER August 1978 FIG. 3. Photomicrograph of normal human specimen with sperm count of 1~5 x.106~ml, take~ with the MEP method: nonmo~ile spermatozoa are much brighter (A) than are motile sperm (B), which appear hke s1x-rmged chams; the shape and length descr1be their courses during 5 / 6 second. appear overexposed, motile spermatozoa not underexposed, and both are clearly represented on the film. Practically, images of the nonmotile spermatozoa appear 6-fold brighter than do images of motile spermatozoa; the latter are seen as small six-ringed chains, and their length describes the true distance they traveled during this 1 second (Fig. 3). For clinical and research purposes, as many films as desired by the observer are exposed at different areas of the same or of various drops. From enlarged prints and after marking the tracks of moving spermatozoa with ink (Fig. 4) the following data can be determined: (1) number and percentage of motile and nonmotile spermatozoa, (2) length and shape of the course of each spermatozoon, and (3) average speed of sperm expressed as microns per second. This is calculated by measuring the total length of sperm tracks and dividing by the number of moving spermatozoa, according to the formula V = TIN xs/(p - 1) where V = average speed in microns per second; T = total length of tracks of moving spermatozoa, described in micrometers as calculated from the magnification factor; N = number of marked motile spermatozoa; S = number of slots in the disc of the stroboscope, or frequency of light pulses per second; and P = number of heads seen in each "chain" of the fastest moving spermatozoa (this figure is constant for each picture). As an example: the total length of tracks is 852 mm; from the magnification factor it was found that each millimeter equals 2 ~-tm; then T = If the number of motile sperm= 63, the number of slots in the disc = 6, and the number of heads in each chain = 6, then V = 1704/63 x 6/5 = 32.4 JLm/second

4 Vol. 30, No. 2 MEP METHOD FOR SPERMATOZOAL MOTILITY DETERMINATION FIG. 4. Same photomicrograph as in Figure 3; moving spermatozoa are traced and marke.d with ink so that measuring the length of individual and total tracks as well as counting nonmotile and motile spermatozoa is possible. When the concentration is more than 100 x 10 6 /ml, spermatozoa may appear too crowded on the film. The courses of very active spermatozoa may intermingle and induce difficulties in tracing them. In this case any of the following steps is advised: (1) The disc of the stroboscope can be replaced by one with 12 slots and exposure time can be reduced to 0.5 second. This will shorten the tracks and their crossover effect (Fig. 5). (2) Seminal fluid can be diluted with its own plasma. This can be done easily by filtering part of the specimen with the aid of a 13-mm Millipore funnel and glass filter paper (Whatman, GF/A). This procedure will not alter the physical properties and viscosity of the seminal fluid, as was proved by us. A small drop of the filtrate is put in the center of the lower glass of the chamber and mixed with another drop of the original specimen. There is no need to make a quantitative dilution, since motility evaluation is not based on knowing the sperm concentration (Fig. 6). (3) A magnifying glass will help the naked eye in identifying the tracks of moving spermatozoa as they appear on the picture. Other data obtained from the pictures include ( 4) grouping spermatozoa according to frequency of distribution oftheir speed. In this way, grading of motility of histograms can be obtained. (5) Sperm concentration, expressed in millions per milliliter, can easily be calculated in the.undiluted specimen by counting all of the spermatozoa within the area of the picture. This is because each picture represents a constant volume of semen known from the area covered by it and the 10-JLm thickness of the chamber. All of these informative data are described in Table 1 and Figure 7. Time and expense can be saved by getting this information directly from the negative film, using a 90 turned slide projector; details are marked on a sheet of paper, and measurements are then taken (Fig. 8). RESULTS In Table 2, results obtained from two different samples--one of 72 x 10 6 and the other of 68 x are demonstrated. Three drops from each sample were analyzed. Each drop was photo-

5 196 MAKLER August 1978 FIG. 5. Same specimen as in Figure 3, taken by the MEP method, except that the 6-slotted disc was replaced by a 12-slotted disc and the shutter of the camera opened for only 0.5 second. These tracks are shorter by about one-half of those in Figure 3, and fewer intersections of tracks are seen. graphed four times in various fields, and 1800 and 1600 spermatozoa, respectively, were analyzed. There was no difference in mean and standard deviation when data were obtained from all 12 pictures or only from 3 pictures, that is, from 300 to 400 spermatozoa analyzed. Data gained from one picture in which only 90 to 140 sperm were analyzed were not reliable. From this and other analytic work done by us, we came to the conclusion that accuracy does not depend on the number ofpictures taken but on the total number of sperm analyzed. At least 300 to 400 spermatozoa should appear on the pictures independently of the number of pictures taken, and it is obvious that oligospermic samples require more pictures. In Table 3 the results obtained from pictures are compared with those estimated subjectively by using the 10-p,m chamber, in 20 different samples. The subjective estimation yielded a motility rate higher by about 20% than those determined objectively, which means that human judgment tends to overestimate motility because the eyes are focused more intently on motile spermatozoa than on nonmotile spermatozoa; film is not influenced by this effect. DISCUSSION Many methods for objective sperm motility evaluation have been described in past decades. TABLE 1. Results of Analysis of a Photomicrograph with the MEP Method Sperm count % Motile spermatozoa Velocity (J.Lrnlsec) Average Distribution Total sperm counted 72 million

6 Vol. 30, No. 2 MEP METHOD FOR SPERMATOZOAL MOTILITY DETERMINATION 197 FIG. 6. Same specimen as in Figure 3 after it was diluted with its own seminal plasma. Tracing of moving spermatozoa is much easier, whereas the ratio of nonmotile to motile spermatozoa is the same as in the original sample... Some of them need very sophisticated and expensive equipment Nevertheless, the most popular methods are those using direct visual techniques in which the microscope is the main part of the system. A few of them, like stopwatch techniques and migratory techniques, were criticized because the dilution of semen with foreign diluents was necessary and spermatozoa were deprived of their natural environment. Other direct visual techniques include various % motility FIG. 7. Results shown in Table 1 are exhibited in the form of a histogram. Each column represents the percentage of spermatozoa of various speeds betwen 0 and 80 ~m/second. photographic methods, either cinematographic or still camera techniquesy- 24 When cinematographic, time exposure, and double-exposure techniques are compared with our technique, the \ / ) ---- \ I. \ I / ~ / I --. I -- / /I \ ~ -- \ SOJA FIG. 8. Tracks of motile spermatozoa were marked on a sheet of paper after the negative film was projected with the aid of a slide projector. Nonmotile spermatozoa are represented by dots. All necessary information can be obtained from this drawing.

7 198 MAK:LER August 1978 No. of pictures analyzed TABLE 2. Results of Analysis of Two Different Specimens with the MEP Methoda Specimen I (sperm concentration 78 million/ml) Specimen II (sperm concentration 62 million/ml) No. of spermatozoa No. of spermatozoa Average velocityb %Motile/' Average velocityb %Motile/' Total Per pictureb Total Per picture! p.mlsec 4x ± ± ± ± ± ± 4.6 2x ± ± ± ± ± ± X ± ± ± ± ± ± ± ± "Each specimen was photographed 12 times (3 pictures of each of 4 different drops). bvalues are means± standard deviation. p.m!sec "' following differences are to be pointed out: (1) The cinematographic method needs frame-by-frame evaluation which is very inconvenient and timeconsuming. (2) In the time exposure technique, there is an imbalance between images of motile and nonmotile spermatozoa because of the relatively long duration of exposure: the amount of light necessary to reveal motile spermatozoa clearly will overexpose nonmotile spermatozoa and they appear like blurred spots even when dark-field illumination is used. 20 On the other hand, if nonmotile spermatozoa are to be seen clearly, the intensity of the light has to be so low that motile spermatozoa appear too faint to be defined, if at all. Therefore, this method was used to measure the motility of moving spermatozoa only, and it was very difficult to determine the ratio between motile and nonmotile spermatozoa. (3) A double exposure of 1-second interval can give clear images of spermatozoa, but in popu- TABLE 3. Spermatozoal Motility in Specimens from Twenty Patients as Evaluated by Subjective Estimation and Objective Determination with MEP Method Sample no. Sperm count By subjective estimation %Motile By objective measurement million!ml Average lated specimens it is impossible to match couples of the same spermatozoon, or to differentiate between motile and nonmotile spermatozoa. (4) In pictures obtained by our MEP method, there is no need for dark-field illumination. Both motile and nonmotile spermatozoa are seen very clearly and distinctly, all necessary details can be defined easily, and all measurements can be taken with high degree of accuracy. Combining the MEP technique with the 10-J.tm chamber, which gives constant thickness to the examined undiluted semen, increases further the accuracy and the reliability of the method, as is demonstrated in Table 2. According to Van Demark,24 "an ideal method of evaluating sperm motility should include: (1) total numbers of sperm per unit volume, (2) number of motile sperm and non-motile sperm, (3) mean speed of motility displayed by the moving sperm, ( 4) the distribution of the total sperm with regard to kind and speed of motility." It seems that the above-mentioned technique fulfills all of these requirements very simply. Two more points of advantage may be added, namely: (5) storage of information, re-examination ofdata, and comparison of results are possible; (6) the method uses inexpensive basic equipment, is easy to perform, and the cost per analysis is low. All these can make the MEP a useful method for routine semen analysis at any moderate class institute. It is hoped that a new parameter added to the ordinary semen analysis-mean velocity of sperm expressed in microns per second-will lead to a new index of great value in describing the male potential of fertility. REFERENCES 1. Makler A: A new chamber for rapid sperm count and motility evaluation. Fertil Steril. In press 2. Makler A: The thickness of microscopically examined sample and its relationship to motility estimation. Int J Androl. In press..

8 Vol. 30, No. 2 MEP METHOD FOR SPERMATOZOAL MOTILITY DETERMINATION Eliasson R: Semen analysis. In Progress in Infertility, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co., 1975, p 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 20:109, Rothschild NM: A new method of measuring sperm speeds. J Exp Bioi 30:312, Bosselaar CA, Spronk N: A physical method for determination of the motility and concentration of spermatozoa. Nature 169:18, Dubois M, Jouannet P, Berge P, Volochine B, Serres C, _ David G: Methode et appareillage de mesure objective de la motilite des spermatozoides humains. Ann Physiol Bioi Med 9:19, Jouannet P, Volochine B, Deguent P: Light scattering determination of various characteristic parameters of spermatozoa motility in a series of human sperm. Andrologia 9:36, Bartak V: Sperm velocity test in.clinical practice. Int J Fertil 16:107, Branham JMA: Method of rating sea urchin sperm motility. Bioi Bull 127:363, Nevo AC, Mohan R: Migration of motile spermatozoa into sperm free medium and the dilution effect. J Reprod Fertil 18:379, Baker FN, Cragle RG, Salisbury GW, Van Demark ML: Spermatozoa velocities in vitro-a simple method of measurement. Fertil Steril 8:149, Harvey CJ: The speed of human spermatozoa and the effect on it of various diluents. J Reprod Fertil 1:84, Kremer RJ: The in vitro spermatozoal penetration test in fertility investigation. Doctoral dissertation, University ofgroeningen, The Netherlands, 1968, Van Dender Ulstein M: Sperm penetration of cervical mucus as a criterion of male fertility. Acta Obstet Gynecol Scand 51:335, Rothschild NM: The movement of spermatozoa in ma:mmalian germ cells. Ciba Foundation Symposium. London, J and A Churchill, 1953, p Rikmenspoel R, Harren GV: A microscopic film of the movement of bull sperm cells in dark field illumination. Experienta 12:160, Branham JM: Movement of free swimming rabbit spermatozoa. J Reprod Fertil 18:97, Janick J, MaCleod J: Measurement of human spermatozoa motility. Fertil Steril 21:140, Van Duijn C, VanVoorst C, Freund M: Movement characteristic of human spermatozoa from kinemicrograph. Eur J Obstet Gynecol 4:121, Bwjos MH, Tovar ES: Sperm motility in the rat epididymis. Fertil Steril25:985, Castenholz A: Photokymographische registrier Methode zur Derstettung und Analyse der Spermatozoen Bewegungen, Andrologia 6:155, Van Demark NL, Salisbury GW, Moeller AN: Exploration of electronic methods for evaluating sperm motility. Science 127:286, 1953