IN THE STUDY of male fertility and infertility there are several criteria which

Cytologic Examination of Semen Irwin N. Frank, A.B., John A. Benjamin, M.D., and James E. Segerson, M.D. IN THE STUDY of male fertility and infertility there are several criteria which have been established to evaluate the adequacy and potency of an ejaculated semen specimen. Most investigators stress the importance of a large number of active spermatozoa per cc. 1 8 However, it is an established fact that patients with "low counts" occasionally have been proven fertile. Other standard criteria used to evaluate the semen specimen are type of sperm motility, and the ph, volume, odor, color, and viscosity of the ejaculate. Still another criterion is that of sperm morphology which has been especially stressed by Moench. In the examination of stained preparations of material obtained by prostatic massage, cells were noted which were believed to be sperm precursors.4 Further study of semen specimens stained in a similar manner showed varying numbers of these cells. A survey of the literature disclosed many publications discussing the morphology of mature spermatozoa, but relatively little investigation of the significance and relationship to fertility and infertility of the other cells seen in the ejaculate. The interesting observations made by Moench, Michael and Joel, and Maroulis, who have described and evaluated various cells seen in semen, furthered our interest in the subject. From the Department of Surgery, Division of Urology, The University of Rochester School of Medicine and Dentistry, Rochester, New York. This study was supported in part with funds from the Dr. Henry C. Buswell Memorial and Fluid Research Fund, and carried out in the Cytology Laboratory supported by the Monroe County Branch of the American Cancer Society and the Rochester Health Bureau. The authors wish to express their appreciation for the use of the unpublished material of Dr. Hannah Peters. 217

218 FRANK ET AL. Fertility & Sterility This cytologic study was undertaken in an effort to describe the more frequently encountered sperm precursors, and to determine if spermatidsperm relationships could prove of some value as another means of describing and evaluating the semen. MATERIAL AND METHOD From 33 patients being studied for infertility, with ages ranging from 22 to 52 years (average 33), a total of 70 semen specimens were obtained according to certain instructions. These included abstinence from intercourse for a period of 5 days, and then the use of coitus interruptus to obtain the semen physiologically in a clean, dry, wide-mouth bottle. Studies carried out 1 to 2 hours after ejaculations included determinations for volume, ph, color, odor, viscosity, morphology, and sperm counts, according to the methods described by Hotchkiss? The remaining portion of the specimen was then sent to our Cytology Laboratory. In the Cytology Laboratory, specimens were centrifuged for 10 minutes at approximately 20,000 r.p.m. A thin film of Mayer's albumin was placed on three slides, and smears were made of the centrifuged "button" with a wire loop. These smears were placed immediately into a 50-50 preparation of 95 per cent alcohol and ether. After a minimum 30-minute fixation, the slides were stained by the routine Papanicolaou staining procedure 6 employed in this laboratory. The stained specimens were then examined by one of us (LN.F.). The cytologic interpretation consisted of three parts: first, a low-power screening for smear distribution and presence of clumping and larger epithelial cells; second, a more detailed appraisal of cell types seen with the high dry objective was made; and last, a differential count on the spermatid types seen in relation to mature spermatozoa. This procedure is similar to the hematologic technic of reticulocyte counting. This latter step was carried out with the oil immersion lens, and enabled the examiner to get better cell detail necessary to differentiate the more obscure and transitional forms occasionally encountered. In doing the differential count, an effort was made to select areas wherein the cells were fairly well spaced, well preserved, and on the same plane. Fields were chosen whether they had spermatids or not. This is necessary if a "true count" is to be obtained. Several fields on each of the three slides were examined. A total of 500 spermatozoa were counted, and the spermatid

Vol. 5, No.3, 1954 CYTOLOGIC EXAMINATION OF SEMEN 219 types enumerated. From these data, the number of spermatids per cc. was determined based upon the number of mature spermatozoa per cc. A note was also made in each case as to the presence and relative number of epithelial and white blood cells. RESULTS Description of Cells With the low-power survey, the specimens obtained showed varying numbers of mature spermatozoa. In some cases they were very numerous and formed large clumped areas, while in specimens from patients with very low counts, they were absent or widely dispersed on the slide. In all of the satisfactory specimens (adequate number of mature spermatozoa for counting), spermatids were seen. In some cases, a large number of squamous cells were evident. Also seen on some of the specimens were cells believed to be earlier precursors (primary and scondary spermatocytes. spermatogonia, and those believed to be Sertoli cells). These cells were seen too infrequently to enable the examiner to attempt any conjecture as to the significance of their appearance in the particular cases in which they were seen. In most of the specimens examined, polymorphonuclear leukocytes were either absent or present in very small numbers. Occasionally, eosinophils were evident. However. in a few specimens, the number of polymorphonuclear leukocytes was greatly increased, and histiocytes were also present in conspicuous numbers. Theoretically, cells lining the passages through which the spermatozoa pass might also be expected to appear in the ejaculate. However, if these desquamated cells (columnar and cuboidal) appeared in the smear, it is believed that they would be very difficult to recognize because of degenerative changes. This is noted in many cases of material expressed by prostatic massage, wherein the columnar cells lose their cytoplasm and retain only a well-rounded pyknotic nucleus. The cells which were of greatest interest to us in this study were the spermatids in their many forms. Although relationships may exist between the presence of the other cells mentioned and biologic characteristics of the ejaculate, nothing will be said at this time regarding their qualitative or quantitative appearance in the semen specimens examined. For differential counting, the spermatids were classified as mononuclear; protruding, eccentric nucleus; binucleate; clover-leaf nuclei (3 nuclei);

220 FRANK ET At. Fertility & Sterility and multinucleated giant-cell forms (more than 3 nuclei). These cells result from the amitotic division of the secondary spermatocytes.5 The average size of the spermatid is approximately 2-3 sperm heads in diameter, with a range of from 1 to 4 sperm heads. The cells with ;more nuclei are not always larger, and very often are smaller in size than the mononuclear types. Spermatids with one nucleus are more frequent, and eccentric location of the nucleus, with or without protrusion, is a commonly encountered type. Binucleated and clover-leaf types are rarer, and multinucleated spermatids are seldom present, seen only in a few specimens. The centrally located nucleus of the mononuclear spermatid (Fig. 1) occupies about three fourths of the cell, stains darkly basophilic (blueblack), and is homogeneous in character. The relatively small rim of cytoplasm that surrounds the nucleus is well outlined, giving the cell a uniformly round appearance. In general, the cytoplasm is also homogeneous in appearance, and stains deeply eosinophilic. Occasionally, one may note a very fine foamy appearance to the cytoplasm if careful examination is made with oil-immersion magnification. The cell with an eccentric and occasionally protruding nucleus (Fig. 2) is characterized by a smaller nucleus of similar appearance to those of the mononuclear spermatids, but very often having a somewhat more elliptical outline. Bulging of the cell outline by the nucleus in the "protruding forms" may be in any stage of development, and frequently "protoplasmic droplets" are seen which probably represent the final stage of complete nuclear extrusion with round, pink-staining, cytoplasmic bodies remaining (Fig. 3). The binucleated spermatid (Fig. 4) is either round or slightly oval, and in the latter case have nuclei which frequently are at opposite poles. In general, the nuclei are somewhat smaller in size, equal, but are still similar in appearance to those seen in the mononuclear cells. The cytoplasm is also the same as described in the mononuclear-type cell. The clover-leaf arrangement of the equal nuclei in the trinucleated spermatid (Fig. 5) seems to be a function of the small cell space available. Minimal cytoplasm is present between the peripheral nuclear borders and at the center of the cell. In occasional cases, where this cell type is seen with a larger diameter, the clover-leaf appearance is not present, and any nuclear arrangement may replace it. Again, the nuclei and cytoplasm maintain their homogeneous character as in the other forms. The spermatids containing more than three nuclei are classified as multi-

2 4 6 Fig. 1. Mononuclear spermatid with central nucleus. (x 1500) Fig. 2. Mononuclear spermatid with eccentric nucleus. (X 1500) Fig. 3. "Protoplasmic droplet" remaining after complete nuclear extrusion. (X 1500) Fig. 4. Binucleated spermatid with nuclei at opposite poles. (x 1500) Fig. 5. Trinucleated spermatid with clover-leaf nuclear arrangement. (x 1500) Fig. 6. Multinucleated spermatid with six nuclei. (x 1500)

222 FRANK ET AL. Fertility & Sterility nucleated forms (Fig. 6), and are very rare, usually present in specimens containing an unusually large number of sperm precursors. No particular nuclear arrangement is characteristic, and very often a cell which appears to contain three nuclei may have a fourth which is demonstrated on another plane by focusing up and down. Some of the spermatids which appear to have many small nuclei occasionally actually contain what appears to be Fig. 7. Large numbers of sperm precursors of many types in a semen specimen. (X 100) irregular nuclear fragments. In general, true nuclei are smaller in size than those described, and the multinucleated cell itself shows no particular tendency for greater diameter. The cells described may be confused with white blood cells, especially the clover-leaf type spermatid which may resemble a polymorphonuclear leukocyte, and the mononuclear form which is similar to the lymphocyte. However, the eosinophilic cytoplasm, and homogeneous, well-delineated

Vol. 5, No.3, 1954 CYTOLOGIC EXAMINATION OF SEMEN 223 basophilic nucleus make interpretation possible when one is familiar with the staining qualities of these particular cells. Occasionally a specimen is obtained which has an overwhelming number of sperm precursors. An example of such a situation is illustrated in Fig. 7 which represents a low-power field characteristic of the preparation. Such a picture may readily be misinterpreted as an example of a prostatitis if an unstained specimen were examined in evaluating semen in the clinic. A Fig. 8. Composite drawing illustrating several types of spermatids. (1) Mononuclear, (2) eccentric nucleus, (3) binucleated, (4) trinucleated, (5) multinucleated, and (6) protoplasmic droplet. composite drawing of the several spermatid types described is illustrated in Fig. 8 for purposes of comparison. Spermatid-Sperm Relationships The values obtained are represented in Fig. 9. In all cases illustrated, only the mononuclear form was considered because of its predominance in the specimens examined, or, in other words, the relationship is really one of mononuclear spermatids to mature sperm. This figure shows that pregnancy occurred only in the cases wherein the spermatid count was 1 million per cc. or less, as indicated by a circle. Such was the case in 7 specimens from 3 patients. It is of further interest to note that although sperm counts in these 3 patients were 17, 48, and 125 million per cc. respectively at the approximate time of conception, in all 3 cases the spermatid counts remained in the range noted above. Several cases showed what was considered to be

224 FRANK ET At. Fertility & Sterility consistently increased spermatid to sperm ratios of a marked and possibly significant type. It was also in the latter group of cases that the trinucleated and multinucleated spermatid types were most frequently encountered. DISCUSSION Many criteria are used in evaluating the reproductive capacity of a semen specimen. However, the use of these established standards often proves inadequate. Therefore, it is desirable to search out other factors 250 235 220 195 U 180 u... 165 z 0 150 :::i...j :!i 135 0 120 l- Z => 10 0 u 90 Fig. 9. :!i a:: w 75 CL I/) 0 60 45 0 30 0 15 Jeo 2 3 4 5 6 7 8 9 10 SPERMATID MILLION ICC. Spermatid to sperm relationship. Note that pregnancies (indicated by cirdli) occurred in cases with 1 million or less spermatid per cc. which may possibly aid in arriving at a more reliable conclusion reganling fertility or infertility. Perhaps a further knowledge of the cell constituents of semen and their variants may provide some of the information, now lacking in the accepted procedures. Moench and others have reported the presence of cells other than mature spermatozoa in the ejaculated specimen. Many of these cells are believed to be sperm precursors, which appear to be increased in number in infertjle

Vol. 5, No.3, 1954 CYTOLOGIC EXAMINATION OF SEMEN 225 cases. Also the presence of abnormal spermatid types are considered to be an indication of impaired spermatogenesis. The examination of semen specimens stained by the Papanicolaou method verifies the presence of these precursors. Furthermore, it is of interest that there exists certain specific spermatid types which may be readily identified. Differential counts and sperm-spermatid relationships were studied as other possible means of semen evaluation. The information derived from the differential counts would indicate that spermatids are present in all specimens studied which contained mature spermatozoa, and that the more bizarre forms are present in those specimens characterized by increased numbers of sperm precursors. It is also suggested that the latter cases represent the group wherein infertility may be frequently encountered despite high sperm counts. This appears to be further verified by the spermspermatid relationships. Although only a few cases were considered, it is of interest to note that the pregnancies which occurred in our study represented patients with low spermatid counts regardless of sperm count. It is believed that differential spermatid counts and sperm-spermatid relationships do not in themselves offer the solution to the problem of semen evaluation. However, they may provide added information in certain cases of infertility, and further our knowledge of the mechanisms of spermatogenesis. Furthermore, the awareness of the possible existence of large numbers of precursor cells in the unstained semen specimen may prevent the occasional faulty diagnosis of prostatitis. One might speculate that large numbers of sperm precursors represent stimulation of spermatogenesis with the presence of large numbers of prema~ure forms in the ejaculate. On the other hand, the appearance of these cells might represent the arrest of spermatogenesis or an indication of the presence of some pathologic process. It is believed that increased numbers. of bizarre and multinucleated forms support the latter conditions. Therefore, if there is an increased spermatid count, or the presence of abnormal forms are present in repeated specimens from a patient with adequate rest periods between specimens, who is receiving no hormonal therapy, and whose wife is considered fertile, one may infer that faulty spermatogenesis exists, which may be temporary or permanent. SUMMARY Many criteria are available for the evaluation of a semen specimen. A,c)' ~ulogic method using the Papanicolaou staining technic is discussed, and

226 FRANK ET AL. Fertility & Sterility the sperm precursors present in the ejaculate are described. Further data derived from differential counts and spermatid-sperm relationships are evaluated and their possible clinical significance is discussed. REFERENCES 1. FARRIS, E. J. Human Fertility and Problems of the Male. White Plains, New York, Authors Press, Inc., 1950. 2. HOTCHKISS, R. S. Fertility in Men. Philadelphia, Lippincott, 1944. 3. MAROULIS, B. C. Round multinucleated spermatogenic cells. Fertil. & Steril. 4:412, 1953. 4. MICHAEL, M., and JOEL, K. Zelformen in normelen und pathologischen Ejakulaten und ihre klinische Bedeutung. Schweiz. med. Wchnschr. 62:757, 1937. 5. MOENCH, C. L. The relation of certain seminal findings to fertility with special reference to sperm concentration and the significance of testicular epithelial cells in s.emen. Am. J. Surg. 47:586, 1940. 6. PAPANICOLAOU, G. N., and T~UT, H. F. Diagnosis of Uterine Cancer by the Vaginal Smear. New York, Commonwealth Fund, 1943. 7. PETERS, H., and FRANK, I. N. The cytologic interpretation of the prostatic smear. Surg., Cynec. & Obst. 94:69, 1952. 8. WEISMAN, A. I. Spermatozoa and Sterility: A Clinical Manual. New York, Hoeber, 1941.