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Testicular Morphology in Eunuchoidal and Infertile Men
Testicular Morphology in Eunuchoidal and Infertile Men Warren 0. Nelson, M.D.
BIOPSY of the human testis is rapidly attaining a role in the diagnosis of defects of male gonadal function which parallels that served by endometrial biopsy in the female. 2 • 11 • 12 • 20 • 21 • 25 Indeed, considerably more information can be obtained by careful study of the testicular biopsy than is possible from endometrial tissue, since the latter simply reflects the character of ovarian endocrine function without revealing defects of gametogenesis other than the failure of ovulation. With technics now available it is possible to determine, in the case of the testicular biopsy, not only the morphologic characteristics of endocrine and gametogenic defects but also the lipid, carbohydrate, and protein changes which are associated with testicular function. As investigations in these areas progress it is not too much to expect that at least some of the defects of reproduction in the male will be sufficiently understood to permit intelligent and efficacious treatment. In pursuing this thought it may be of interest to note the nature, distribution, and frequency of defects which are encountered in studies on biopsies of the human testis and to indicate the areas in which future studies may aid our understanding and treatment of these defects. With this in mind it is proposed to discuss briefly the morphologic findings in 623 men whose testicular biopsies have been studied in considerable detail. Included in this summary are patients whom we have studied as well as numerous cases in which biopsy material was referred to us for diagnostic opinion. A number of cases are not included because of insufficient data or because of unsatisfactory biopsy preparations. From the College of Medicine, State University of Iowa, Iowa City, Iowa. 477
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Patients who present themselves for biopsy fall into two major groups, men whose primary complaint is deficient production of androgenic hormone and men who are infertile. These two principal categories, eunuchoidism and infertility, will serve as a basis for discussing the most common types of testicular abnormalities which are encountered.
EUNUCHOIDISM Although most eunuchoidal patients present very similar pictures in so far as body configurations, lack of secondary sexual characters, etc., are concerned, the histologic picture of biopsy specimens may show several different conditions. The scrotal contents may lack testicular tissue entirely or show the scarcely recognizable atrophic elements found in men whose testes either failed to develop or atrophied very early in life. 10 More commonly testes can be identified, but show severe sclerosis of the tubules and poorly formed interstitial elements as in the eunuchoidal or moderately eunuchoidal types 23 of the Klinefelter-Albright-Reifenstein syndrome. The testicular condition which is most commonly encountered in eunuchoidal men is failure of tubular and Leydig cell maturation due to lack of adequate stimulation by hypophysial gonadotrophins. Table 1 shows the incidence of these three types of testicular conditions in 126 eunuchoidal men, and lists briefly the histologic characteristics which TABLE 1. Condition Testicular aplasia (early atrophy) Klinefelter (eunuchoidal and moderately eunuchoidal types) H ypogonadotrophic eunuchoidism
Testicular Conditions Associated with Eunuchoidism No. Cases
Tubules
15
None
28
Usually only remnants Infantile
83
Peritubular Conn. Tiss.
Marked increase Variable, tends to increase with age
Intertubular Cells
Ganadotrophins
None
High
Few or no Leydig cells Mesenchymal cells
High
Low
are seen in biopsy tissue. It will be noted that the levels of gonadotrophin (urinary) are high in the first two groups and low in the third group. In the case of testicular aplasia or atrophy or of the Klinefelter syndrome the defect is primary to the testis and the two conditions may be grouped together as primary or hypergonadotrophic hypogonadism. In the third group the defect is secondary to the testis and may be designated as secondary or hypo-
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gonadotrophic hypogonadism. Only in the latter group may treatment with gonadotrophic hormones be expected to induce improvement in the eunuchoidal condition.
INFERTILITY The types of testicular defects which are found in infertile men are complex. It is advantageous to divide them in two groups on the basis of whether or not the seminal specimens contain sperm. In men whose semen samples lack spermatozoa one of four more or less typical pictures can be expected. These conditions which cause azoospermia, in the order in which the 119 cases reported here have been observed, are germinal cell aplasia ( 46 cases), germinal cell arrest ( 27 cases), generalized fibrosis ( 24 cases), and occlusion or absence of efferent ducts ( 22 cases). The testes of men whose azoospermia is due to germinal cell aplasia usually are approximately normal in size and the biopsy picture presents a remarkably uniform appearance. The tubules usually are uniform, although slightly reduced, in size and show very little peritubular fibrosis. The striking feature is the complete lack of germ cells, the tubules being populated by Sertoli cells only. In occasional cases a few tubules may show a sprinkling of germ cells and even spermatogenesis, but the number of sperm produced is so small that they escape detection in the ejaculate. The Leydig cells usually are normal in appearance and numbers, but occasionally show an apparent increase. The condition is presumed to be due either to failure of the primitive germ cells to migrate into the developing testes 3 or to very early death of the primitive germ cells. Germinal cell arrest is a curious condition in which the spermatogenic process fails to progress beyond one of the early stages of sperm formation. The testes usually are somewhat reduced in size and the tubules reflect this reduction. As a rule the spermatogonia are normal in numbers and apparent behavior, proceeding to the formation of primary spermatocytes. At this point, in most cases of this type, the process appears to break down. As a result the number of primary spermatocytes is increased, but few or no advanced stages are seen. Less frequently arrested development occurs at the secondary spermatocyte or spermatid stage. In any event the result is the same-very few or no sperm are produced. The arrested cells, whatever their stage of development, undergo fragmentation and slough into the tubular lumina. The cause of germinal cell arrest is unknown, but may
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represent one or more defects in the mechanisms of chromosomal synapse and division. Careful study of the abortive attempts of primary spermatocytes to carry out the complicated processes of the first maturation division is strongly suggestive of a serious disturbance in chromosomal behavior. Other possible causes of germinal cell arrest are failure of the Sertoli cells to provide the nutritive environment necessary for spermatogenesis or a defective action of the gonadotrophic hormones. The testes of men whose azoospermia is due to generalized peritubular fibrosis are smaller than normal and present a histologic picture of thickened peritubular connective tissues. A few tubules may be fairly large and contain the various stages of spermatogenesis, but the great majority are small and show only the earlier stages or are completely replaced by connective tissue. As a consequence either no sperm are produced or the number is so small as to escape detection in the ejaculate. Leydig cells show considerable variability, in some instances their numbers appear to be normal while in other cases they may be reduced or show an apparent increase. Peritubular fibrosis is a progressive process and appears to continue until all germ cells are eliminated and the tubules completely sclerosed. Its fundamental cause is unknown, but probably many factors may lead to initiation of the process. It is seen in men with histories of testicular trauma, orchitis, severe infections, and reduced vascular supply to the testes. The testes of men with the noneunuchoidal type of the Klinefelter syndrome show the picture of generalized fibrosis. In other cases no pertinent basis for the condition can be found. In about one of five men with azoospermia the principal reason for the condition is found to be bilateral absence or obstruction of the epididymis or vas deferens. In the majority of such cases the testicular condition as revealed by biopsy is such that fertility would be expected. 22 In some instances spermatogenesis is impaired, but the types of defects are similar to those seen in men with intact ducts and it seems probable that the lack of a complete and patent efferent duct system has no specific effect on spermatogenesis. In most instances the biopsy picture is quite normal even in men who have never possessed passages for their sperm. The condition is caused by congenital absence of some part of the duct system or by infection or trauma of the vas deferens or epididymis. Table 2 presents data on the incidence of these defects and summarizes the principal histologic features of each. It should be emphasized that some
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variation from the usual picture may be expected, but the large majority of cases of each category will correspond to the description. It is worth noting further that probably in none of these groups does the condition of azoospermia have an endocrine basis. The one possible exception is germinal cell arrest, but this defect probably will be found to have its basis in aberrations of chromosomal activity. Two of the conditions, generalized fibrosis and TABLE 2.
Condition
No. Cases
Testicular Conditions in Men with Azoospermia (Leydig cells usually in normal range)
Spermatogonia
Germinal cell aplasia Germinal cell arrest
46
None
27
Numerous
Generalized fibrosis Obstructed or absent ducts
24
Reduced numbers Numerous
22
Spermatocytes
Spermatids Peritubular and Sperm Conn. Tiss.
Ganadotrophins
None
None
High
Increased primary; usually few secondary Reduced numbers Usually numerous
Few
Few Usually numerous
Usually normal Usually normal
Low normal to normal
Marked increase Variable
Usually high Normal
germinal cell aplasia, are associated with high urinary levels of gonadotrophins, but the latter is a secondary effect which is explained best by taking into account the failure of the germinal epithelium to utilize gonadotrophic hormone. Although Del Castillo, Trabucco, and de la Baize; and Howard, et al. 13 claimed that men with germinal cell aplasia have normal gonadotrophins it has been shown by Heller and Nelson 6 that such men have definitely elevated urinary levels of gonadotrophin. Apparently Howard, Sniffen, Simmons and Albright14 have more recently reassessed their patients in this category since they now report that men with germinal cell aplasia have elevated gonadotrophins. The earlier argument-which was advanced on the basis of the occurrence of normal levels of gonadotrophin as evidence in support of the theory that the Sertoli cells produce some pituitary-inhibiting substance (variously designated as inhibin, estrin or "X" hormone)would appear to require modification. Probably a great deal of the confusion which has existed in these cases stems from the variable application of the "too little" and "too much" method used in assaying gonadotrophins and the indecision regarding whether the method assays only FSH or a combination of gonadotrophins as other related procedures do. The misunderstandings which arise in evaluating various methods for assay of gonadotrophins are apparent in such publications as the recent one of Engle and Southam.
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The most complex and most poorly characterized pictures seen in testicular biopsies are those found in infertile men whose semen specimens contain spermatozoa in numbers which are usually below the range commonly regarded as characteristic of fertility. It should be emphasized that it is not possible at this time to set up clearly delineated standards and measurements which separate the infertile from the fertile male. The factors which must be considered are numerous, variable, and not always subject to control. However, in general it may be said that men whose sperm counts are regularly less than 50 to 60 million per cc. are likely to be infertile and may be expected to show recognizable defects in their testes. These may be present singly, but more frequently some combination of them is present. They are: regional peritubular fibrosis, disorganized spermatogenesis, sloughing of immature cells, incomplete germinal cell arrest, and germinal cell atrophy. An additional condition which may exist in the testes of men with oligospermia in combination with other defects is abnormalities of mitotic activity. This defect is seen also in men with higher sperm counts and in either case is always predictable on the basis of the percentage of abnormal sperm in the seminal specimen. Table 3 summarizes 314 cases in each of which the sperm count averaged less than 68 million per cc. and 64 cases in which the sperm count ranged TABLE 3. Testicular Conditions in Men with Oligospermia (Leydig cells and gonadotrophins usually in normal range) No.
Spermatogonia
Condition
Cases
Sloughing and disorganization
152
Numerous
Incomplete germinal cell arrest
74
Numerous
Regional fibrosis
58
Germinal cell atrophy Abnormal mitoses
30
Numerous except in regions of fibrosis Reduced numbers Numerous
64
Spermatocytes Numerous, much sloughing Increased primary; numerous or reduced secondary Numerous except in regions of fibrosis Reduced numbers Usually numerous, but many abnormal
Spermatids and Sperm
Peritubular Conn. Tiss.
Reduced numbers
Variable
Reduced numbers
Usually normal
Numerous except in regions of fibrosis Reduced numbers Reduced normal cells; increased abnormal forms
Marked increase in some regions Variable Variable
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up to 110 million per cc., but in which at least 23 per cent of the sperm was abnormal. In classifying these cases for purposes of tabulation a given case was placed in one or another of the categories on the basis of the principal defect. Frequently more than·one histologic disturbance could be identified, for example, increased peritubular connective tissue commonly coexisted with disorganization and sloughing and abnormal patterns of mitosis were frequently seen in cases of incomplete germinal cell arrest. Two of the categories, incomplete germinal cell arrest and regional fibrosis, present similar pictures to the more severe variety of these conditions described in men with azoospermia. The pathologic changes involve lesser amounts of the sperm producing elements and consequently the effects are less complete. In many cases, at least, the process of regional fibrosis is a progressive one and eventually may be expected to involve larger numbers of tubules, as has been shown by serial biopsies. 23 Although peritubular fibrosis has commonly been regarded as irreversible 5 it has been shown recently by Heller, et aP that severe fibrosis which occurred in testes of men receiving testosterone propionate disappeared after cessation of treatment. Although further study may reveal the existence of different types of peritubular fibrosis it is clear that this serious defect is not necessarily irreversible. The most common histologic pattern in the testes of oligospermic men is one of disorganized spermatogenesis with sloughing of immature cells. In these cases the orderly pattern of spermatogenesis is severely disturbed and immature forms (particularly spermatocytes) slough into the tubular lumina. These defects give the seminiferous epithelium a jumbled appearance and many tubules appear to be plugged with sloughed cells. Some degree of peritubular fibrosis is encountered in many of these cases. Germinal cell atrophy is a condition seen occasionally in oligospermic men. It is characterized by a general reduction in the number of germ cells of all stages. The impression is strong that the spermatogonia are unable to propagate at a normal rate. As a result the numbers of more mature cells are greatly reduced and the germinal epithelium presents a very loose, poorly populated appearance. In the testes of men whose seminal specimens show unusually large numbers of abnormal cells it is possible regularly to observe striking aberrations of cell division and sperm maturation. These disturbances include aberrant chromosomal behavior, large numbers of multinucleated cells, and
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alterations in size of spermatozoa and are seen in the testes of men with normal as well as low sperm counts. DISCUSSION By way of commenting upon the various histologic patterns which are encountered in the testes of eunuchoidal and infertile men it may be of interest to indicate the direction in which further studies may solve some of the problems which now confront us. In eunuchoidal men the testicular patterns are well defined and once established indicate clearly both the nature of the defect and the course of treatment. In men with testicular aplasia and in cases of the Klinefelter syndrome the testes are beyond salvage and only such benefits can be expected as may accrue to the eunuchoidal state from treatment with androgen. On the other hand, the infantile testes of hypogonadotrophic eunuchoidism may be expected to undergo maturation when the missing factors, the gonadotrophic hormones, are supplied. It has been shown by Heller and Nelson7 • 8 that chorionic gonadotrophin will stimulate the development and function of Leydig cells and that follicle-stimulating hormone will initiate spermatogenesis in such testes. No other course of treatment has been found to produce these results in proved cases of hypogonadotrophic eunuchoidism. The scattered cases in which the use of testosterone has been said to result in the initiation of spermatogenesis15 • 17 • 26 all are too incompletely documented to permit proper evaluation. Unless such cases are proved to belong in the category of hypogonadotrophic eunuchoidism by testicular biopsy prior to treatment and unless treatment with testosterone is uncomplicated by other procedures it is impossible to determine the actual effect of testosterone on the prepuberal type of testis. In proved cases of this type which we have observed before and following treatment with testosterone or chorionic gonadotrophin it has been noted that these procedures do cause an increase in testicular size, an effect which is reflected in an enlargement of the seminiferous tubules and an apparent increase in the differentiation of Sertoli cells. However, the spermatogenic process has not appeared to have been initiated unless FSH also was given. Admittedly this point needs more study, but it is important to establish definitely the pretreatment condition of the testes if the effect of any measure is to be properly evaluated. It is worth noting briefly that in men with hypogonadotrophic eunuchoid-
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ism initiation of testicular activity, androgen production in particular, by gonadotrophins is sometimes followed by spontaneous and continued testicular activity when treatment is withdrawn. Thus it is now our practice to recommend that a period of treatment (four to five months) be followed by a period of rest during which observations for continued improvement are made. Of interest, too, are rare instances of this syndrome in which treatment with large amounts of gonadotrophin does not produce evidences of testicular stiii?-ulation. It is presumed that these represent examples of failure of end-organs to respond to trophic stimuli. We have in one man observed the presence of one testis in which spermatogenesis was in progress while the opposite testis contained Leydig cells, but in other respects was similar to the testis of a hypogonadotrophic eunuchoid. This would appear to be a case of unilateral unresponsiveness of the tubules, but not of the Leydig cells. The testicular defects in which new approaches and new methods are most necessary are those conditions which are associated with infertility. It is clear that nothing can be done to salvage spermatogenesis in testes in which germ cells are absent due to congenital failure or to complete fibrosis. In men with defects of the efferent duct system fertility must depend primarily upon the possible establishment of patent ducts. However, men whose azoospermia is due to germinal cell arrest, or who are oligospermic, have testes which may be improved when the nature and cause of the defects are understood. At this juncture it is important to note that there is very little reason to believe that any deficiency of the known endocrine factors or disturbance of endocrine relationships plays a role in the vast majority of cases of men who are studied because of infertility. This statement, of course, excludes men whose testes do show marked disturbance which can be related to such endocrine diseases as tumors of the hypophysis. 1 The conditions of germinal cell atrophy and germinal cell arrest may have some endocrine basis, but this remains to be demonstrated. The histologic appearance of the biopsies suggests inadequate stimulation by gonadotrophic hormone, but the levels of gonadotrophin associated with these conditions are usually normal and the administration of gonadotrophins has thus far produced disappointing , responses. It is important, however, to remember that the available preparations of FSH are likely to produce antigonadotrophic hormones and thus sharply limit the gametogenic response. 16 It is clear that better preparations
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of FSH are highly desirable for use in conditions which may be due to deficient gonadotrophins. In all probability the recognized endocrine deficiencies are of less significance than disturbances which influence the behavior of the peritubular connective tissues and the function of the Sertoli cells. The tendency of the tissues making up the basement membrane and tunica propria of the human testicular tubule to undergo increased thickness and hyalinization presents a serious problem. Perhaps more refined cytochemical technics will reveal the nature of these changes and indicate the reason for their proliferation. As noted in an earlier section the recent demonstration that severe fibrosis need not be permanent lends encouragement to efforts directed toward halting the progression of the disturbance. It is probable that many instances of defective spermatogenesis may be due to disturbed function of the Sertoli cells. Although there has been a recent trend to ascribe the production of hormones ( inhibin, estrin, "X" hormone, etc.) to these cells 1 • 14 • 15 the point is far from proved that these cells do produce such hormones under normal conditions. Indeed, the array of evidence which can be marshaled against that probability at least equals the arguments in its favor. On the other hand there is ample and substantial evidence which indicates that the Sertoli cells are sustentacular and nutritive elements whose function it is to provide support and nourishment for the germinal elements as they proceed away from the basement membrane in their development. 4 • 24 Thus the form and structure of the Sertoli cell and the distribution of its lipids correspond closely to the cyclic waves of spermatozoon maturation. It seems likely that the further application of cytologic and cytochemical methods will reveal that some disturbances of spermatogenesis such as disorganization, premature sloughing, germinal cell arrest, and germinal cell atrophy may be due in part at least to defective Sertoli cell function. We may also expect revelations which will have an important bearing on our understanding of spermatogenesis as studies are made which employ methods for determining the distribution and utilization of ribonucleic and desoxyribonucleic acid and amino acids. Assuredly future studies on spermatozoa must extend beyond their span of life in the testis and include the changes in physiologic maturation which occur during their sojourn in the epididymis. Such studies as those of Lardy on sperm regulator mechanisms seem destined to have a very important bearing on our understanding of
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the processes which combine to provide the capacity of the sperm to move and to fertilize an ovum. Further detailed studies of the spermatogenic process in men whose infertility is associated with such patterns as germinal cell arrest and aberrant mitotic activity seem likely to indicate the existence of profound disturbances in chromosomal behavior. It is possible, too, that study of the testes of men whose wives tend to abort early fetuses will reveal the cause of fetal maldevelopment as related, in many instances, to faults of sperm formation.
SUMMARY Three types of testicular patterns which have been seen in association with 126 cases of eunuchoidism are described and discussed from the standpoint of differentiation and therapeutic procedures. The testicular patterns which have been observed in 119 cases of azoospermia and 378 cases of oligospermia or abnormal spermatozoa are considered and discussed. Attention is called to the directions in which future observations and utilization of available procedures may be expected to improve our understanding of infertility in the male. This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service.
REFERENCES I. Berthrong, M., Goodwin, W. E., and Scott, W. W.: J. Clin. Endocrinol. 9:579, 1949. 2. Charney, C. W.: The testicular biopsy. A five year survey. In Diagnosis in Sterility. Edited by E. Engle. Springfield, Ill., Charles C Thomas, 1946. 3. Del Castillo, E. B., Trabucco, A., and de la Baize, F. A.: J. Clin. Endocrinol. 7:493, 1947. 4. Elftman, H.: Anat. Rec. 106:381, 1950. 5. Engle, E. T., and Southam, A.: Endocrine aspects of infertility in the male. In Progress in Clinical Endocrinology. Edited by S. Soskin. New York, Grune and Stratton, 1950. 6. Heller, C. G., and Nelson, W. 0.: Recent Progress in Hormone Research 3:229, 1948. 7. Heller, C. G., and Nelson, W. 0.: J. Clin. Investigation 25:915, 1946. 8. Heller, C. G., and Nelson, W. 0.: J. Clin. Endocrinol. 7:345, 1948. 9. Heller, C. G., Nelson, W. 0., Hill, I. C., Henderson, E., Maddock, W. 0., and Jungck, E. C.: Proc. A. Study Internal Secretions, p. 27, 1950. 10. Heller, C. G. Nelson, W. 0., and Roth, A.: J. Clin. Endocrinol. 3:573, 1943. 11. Hotchkiss, R. S.: New York State J. Med. 41:564, 1941. 12. Hotchkiss, R. S.: Fertility in Men. Philadelphia, J. B. Lippincott Co., 1944.
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13. Howard, R. P., Sniffen, R. C., and Simmons, F. A.: Proc. A. Study of Internal Secretions, p. 35, 1948. 14. Howard, R. P., Sniffen, R. C., Simmons, F. A., and Albright, F.: J. Clin. Endocrinol. 10:121, 1950. 15. Hurxthal, L. M., Bruns, H. J., and Musulin, N.: J. Clin. Endocrinol. 9:1245, 1949. 16. Jungck, E. C., Maddock, W. 0., Heller, C. G., and Nelson, W. 0.: J. Clin. Endocrinol. 9:355, 1949. 17. Kinsell, I. W.: J. Clin. Endocrinol. 7:781, 1947. 18. Lardy, H. A.: The metabolic regulator in mammalian spermatozoa. New York, Conference on Human Reproduction of the National Research Council Committee on Maternal Health, 1950. 19. McCullagh, E. P.: Recent Progress in Hormone Research 2:295, 1948. 20. Nelson, W. 0.: M. Clin. North America, January: 97, 1948. 21. Nelson, W. 0.: Hypogonadism in the male. In Progress in Clinical Endocrinology. Edited by S. Hoskins. New York, Grune and Stratton, 1950. 22. Nelson, W. 0.: Spermatogenesis in testes of men with blocked or absent efferent ducts. New York, Conference on Human Reproduction of the National Research Council Committee on Maternal Health, 1950. 23. Nelson, W. 0., and Heller, C. G.: J. Clin. Endocrinol. 5:1, 1945. 24. Nelson, W. 0., and McEnery, W. B., Jr.: Endocrinology (in press). 25. Simmons, F. A.: Clinical interpretation of the semen analysis. In Diagnosis in Sterility. Edited by E. Engle. Springfield, Ill., Charles C Thomas, 1946. 26. Werner, S.C.: Am. J. M. Sc. 3:52, 1947.
Ortho Award The American Society for the Study of Sterility offers an annual award of $1000.00 known as the Ortho Award, for an outstanding contribution to the subject of infertility and sterility. Competition is open to those in clinical practice as well as individuals whose work is restricted to research in the basic sciences. Essays submitted for the 1951 award must be received not later than March 1, 1951. The Prize Essay will appear on the program of the 1951 meeting of the Society. The winner of the 1950 award was Dr. Min Chueh Chang of the Worcester Foundation for Experimental Biology. The prize-winning paper was entitled "Fertility and Sterility as Revealed in the Study of Fertilization and Development of the Rabbit Egg." For particulars concerning the 1951 contests, address the American Society for the Study of Sterility, 20 Magnolia Terrace, Springfield, Mass.
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BIOPSY of the human testis is rapidly attaining a role in the diagnosis of defects of male gonadal function which parallels that served by endometrial biopsy in the female. 2 • 11 • 12 • 20 • 21 • 25 Indeed, considerably more information can be obtained by careful study of the testicular biopsy than is possible from endometrial tissue, since the latter simply reflects the character of ovarian endocrine function without revealing defects of gametogenesis other than the failure of ovulation. With technics now available it is possible to determine, in the case of the testicular biopsy, not only the morphologic characteristics of endocrine and gametogenic defects but also the lipid, carbohydrate, and protein changes which are associated with testicular function. As investigations in these areas progress it is not too much to expect that at least some of the defects of reproduction in the male will be sufficiently understood to permit intelligent and efficacious treatment. In pursuing this thought it may be of interest to note the nature, distribution, and frequency of defects which are encountered in studies on biopsies of the human testis and to indicate the areas in which future studies may aid our understanding and treatment of these defects. With this in mind it is proposed to discuss briefly the morphologic findings in 623 men whose testicular biopsies have been studied in considerable detail. Included in this summary are patients whom we have studied as well as numerous cases in which biopsy material was referred to us for diagnostic opinion. A number of cases are not included because of insufficient data or because of unsatisfactory biopsy preparations. From the College of Medicine, State University of Iowa, Iowa City, Iowa. 477
478
[Fertility & Sterility
NELSON
Patients who present themselves for biopsy fall into two major groups, men whose primary complaint is deficient production of androgenic hormone and men who are infertile. These two principal categories, eunuchoidism and infertility, will serve as a basis for discussing the most common types of testicular abnormalities which are encountered.
EUNUCHOIDISM Although most eunuchoidal patients present very similar pictures in so far as body configurations, lack of secondary sexual characters, etc., are concerned, the histologic picture of biopsy specimens may show several different conditions. The scrotal contents may lack testicular tissue entirely or show the scarcely recognizable atrophic elements found in men whose testes either failed to develop or atrophied very early in life. 10 More commonly testes can be identified, but show severe sclerosis of the tubules and poorly formed interstitial elements as in the eunuchoidal or moderately eunuchoidal types 23 of the Klinefelter-Albright-Reifenstein syndrome. The testicular condition which is most commonly encountered in eunuchoidal men is failure of tubular and Leydig cell maturation due to lack of adequate stimulation by hypophysial gonadotrophins. Table 1 shows the incidence of these three types of testicular conditions in 126 eunuchoidal men, and lists briefly the histologic characteristics which TABLE 1. Condition Testicular aplasia (early atrophy) Klinefelter (eunuchoidal and moderately eunuchoidal types) H ypogonadotrophic eunuchoidism
Testicular Conditions Associated with Eunuchoidism No. Cases
Tubules
15
None
28
Usually only remnants Infantile
83
Peritubular Conn. Tiss.
Marked increase Variable, tends to increase with age
Intertubular Cells
Ganadotrophins
None
High
Few or no Leydig cells Mesenchymal cells
High
Low
are seen in biopsy tissue. It will be noted that the levels of gonadotrophin (urinary) are high in the first two groups and low in the third group. In the case of testicular aplasia or atrophy or of the Klinefelter syndrome the defect is primary to the testis and the two conditions may be grouped together as primary or hypergonadotrophic hypogonadism. In the third group the defect is secondary to the testis and may be designated as secondary or hypo-
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gonadotrophic hypogonadism. Only in the latter group may treatment with gonadotrophic hormones be expected to induce improvement in the eunuchoidal condition.
INFERTILITY The types of testicular defects which are found in infertile men are complex. It is advantageous to divide them in two groups on the basis of whether or not the seminal specimens contain sperm. In men whose semen samples lack spermatozoa one of four more or less typical pictures can be expected. These conditions which cause azoospermia, in the order in which the 119 cases reported here have been observed, are germinal cell aplasia ( 46 cases), germinal cell arrest ( 27 cases), generalized fibrosis ( 24 cases), and occlusion or absence of efferent ducts ( 22 cases). The testes of men whose azoospermia is due to germinal cell aplasia usually are approximately normal in size and the biopsy picture presents a remarkably uniform appearance. The tubules usually are uniform, although slightly reduced, in size and show very little peritubular fibrosis. The striking feature is the complete lack of germ cells, the tubules being populated by Sertoli cells only. In occasional cases a few tubules may show a sprinkling of germ cells and even spermatogenesis, but the number of sperm produced is so small that they escape detection in the ejaculate. The Leydig cells usually are normal in appearance and numbers, but occasionally show an apparent increase. The condition is presumed to be due either to failure of the primitive germ cells to migrate into the developing testes 3 or to very early death of the primitive germ cells. Germinal cell arrest is a curious condition in which the spermatogenic process fails to progress beyond one of the early stages of sperm formation. The testes usually are somewhat reduced in size and the tubules reflect this reduction. As a rule the spermatogonia are normal in numbers and apparent behavior, proceeding to the formation of primary spermatocytes. At this point, in most cases of this type, the process appears to break down. As a result the number of primary spermatocytes is increased, but few or no advanced stages are seen. Less frequently arrested development occurs at the secondary spermatocyte or spermatid stage. In any event the result is the same-very few or no sperm are produced. The arrested cells, whatever their stage of development, undergo fragmentation and slough into the tubular lumina. The cause of germinal cell arrest is unknown, but may
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represent one or more defects in the mechanisms of chromosomal synapse and division. Careful study of the abortive attempts of primary spermatocytes to carry out the complicated processes of the first maturation division is strongly suggestive of a serious disturbance in chromosomal behavior. Other possible causes of germinal cell arrest are failure of the Sertoli cells to provide the nutritive environment necessary for spermatogenesis or a defective action of the gonadotrophic hormones. The testes of men whose azoospermia is due to generalized peritubular fibrosis are smaller than normal and present a histologic picture of thickened peritubular connective tissues. A few tubules may be fairly large and contain the various stages of spermatogenesis, but the great majority are small and show only the earlier stages or are completely replaced by connective tissue. As a consequence either no sperm are produced or the number is so small as to escape detection in the ejaculate. Leydig cells show considerable variability, in some instances their numbers appear to be normal while in other cases they may be reduced or show an apparent increase. Peritubular fibrosis is a progressive process and appears to continue until all germ cells are eliminated and the tubules completely sclerosed. Its fundamental cause is unknown, but probably many factors may lead to initiation of the process. It is seen in men with histories of testicular trauma, orchitis, severe infections, and reduced vascular supply to the testes. The testes of men with the noneunuchoidal type of the Klinefelter syndrome show the picture of generalized fibrosis. In other cases no pertinent basis for the condition can be found. In about one of five men with azoospermia the principal reason for the condition is found to be bilateral absence or obstruction of the epididymis or vas deferens. In the majority of such cases the testicular condition as revealed by biopsy is such that fertility would be expected. 22 In some instances spermatogenesis is impaired, but the types of defects are similar to those seen in men with intact ducts and it seems probable that the lack of a complete and patent efferent duct system has no specific effect on spermatogenesis. In most instances the biopsy picture is quite normal even in men who have never possessed passages for their sperm. The condition is caused by congenital absence of some part of the duct system or by infection or trauma of the vas deferens or epididymis. Table 2 presents data on the incidence of these defects and summarizes the principal histologic features of each. It should be emphasized that some
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variation from the usual picture may be expected, but the large majority of cases of each category will correspond to the description. It is worth noting further that probably in none of these groups does the condition of azoospermia have an endocrine basis. The one possible exception is germinal cell arrest, but this defect probably will be found to have its basis in aberrations of chromosomal activity. Two of the conditions, generalized fibrosis and TABLE 2.
Condition
No. Cases
Testicular Conditions in Men with Azoospermia (Leydig cells usually in normal range)
Spermatogonia
Germinal cell aplasia Germinal cell arrest
46
None
27
Numerous
Generalized fibrosis Obstructed or absent ducts
24
Reduced numbers Numerous
22
Spermatocytes
Spermatids Peritubular and Sperm Conn. Tiss.
Ganadotrophins
None
None
High
Increased primary; usually few secondary Reduced numbers Usually numerous
Few
Few Usually numerous
Usually normal Usually normal
Low normal to normal
Marked increase Variable
Usually high Normal
germinal cell aplasia, are associated with high urinary levels of gonadotrophins, but the latter is a secondary effect which is explained best by taking into account the failure of the germinal epithelium to utilize gonadotrophic hormone. Although Del Castillo, Trabucco, and de la Baize; and Howard, et al. 13 claimed that men with germinal cell aplasia have normal gonadotrophins it has been shown by Heller and Nelson 6 that such men have definitely elevated urinary levels of gonadotrophin. Apparently Howard, Sniffen, Simmons and Albright14 have more recently reassessed their patients in this category since they now report that men with germinal cell aplasia have elevated gonadotrophins. The earlier argument-which was advanced on the basis of the occurrence of normal levels of gonadotrophin as evidence in support of the theory that the Sertoli cells produce some pituitary-inhibiting substance (variously designated as inhibin, estrin or "X" hormone)would appear to require modification. Probably a great deal of the confusion which has existed in these cases stems from the variable application of the "too little" and "too much" method used in assaying gonadotrophins and the indecision regarding whether the method assays only FSH or a combination of gonadotrophins as other related procedures do. The misunderstandings which arise in evaluating various methods for assay of gonadotrophins are apparent in such publications as the recent one of Engle and Southam.
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The most complex and most poorly characterized pictures seen in testicular biopsies are those found in infertile men whose semen specimens contain spermatozoa in numbers which are usually below the range commonly regarded as characteristic of fertility. It should be emphasized that it is not possible at this time to set up clearly delineated standards and measurements which separate the infertile from the fertile male. The factors which must be considered are numerous, variable, and not always subject to control. However, in general it may be said that men whose sperm counts are regularly less than 50 to 60 million per cc. are likely to be infertile and may be expected to show recognizable defects in their testes. These may be present singly, but more frequently some combination of them is present. They are: regional peritubular fibrosis, disorganized spermatogenesis, sloughing of immature cells, incomplete germinal cell arrest, and germinal cell atrophy. An additional condition which may exist in the testes of men with oligospermia in combination with other defects is abnormalities of mitotic activity. This defect is seen also in men with higher sperm counts and in either case is always predictable on the basis of the percentage of abnormal sperm in the seminal specimen. Table 3 summarizes 314 cases in each of which the sperm count averaged less than 68 million per cc. and 64 cases in which the sperm count ranged TABLE 3. Testicular Conditions in Men with Oligospermia (Leydig cells and gonadotrophins usually in normal range) No.
Spermatogonia
Condition
Cases
Sloughing and disorganization
152
Numerous
Incomplete germinal cell arrest
74
Numerous
Regional fibrosis
58
Germinal cell atrophy Abnormal mitoses
30
Numerous except in regions of fibrosis Reduced numbers Numerous
64
Spermatocytes Numerous, much sloughing Increased primary; numerous or reduced secondary Numerous except in regions of fibrosis Reduced numbers Usually numerous, but many abnormal
Spermatids and Sperm
Peritubular Conn. Tiss.
Reduced numbers
Variable
Reduced numbers
Usually normal
Numerous except in regions of fibrosis Reduced numbers Reduced normal cells; increased abnormal forms
Marked increase in some regions Variable Variable
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up to 110 million per cc., but in which at least 23 per cent of the sperm was abnormal. In classifying these cases for purposes of tabulation a given case was placed in one or another of the categories on the basis of the principal defect. Frequently more than·one histologic disturbance could be identified, for example, increased peritubular connective tissue commonly coexisted with disorganization and sloughing and abnormal patterns of mitosis were frequently seen in cases of incomplete germinal cell arrest. Two of the categories, incomplete germinal cell arrest and regional fibrosis, present similar pictures to the more severe variety of these conditions described in men with azoospermia. The pathologic changes involve lesser amounts of the sperm producing elements and consequently the effects are less complete. In many cases, at least, the process of regional fibrosis is a progressive one and eventually may be expected to involve larger numbers of tubules, as has been shown by serial biopsies. 23 Although peritubular fibrosis has commonly been regarded as irreversible 5 it has been shown recently by Heller, et aP that severe fibrosis which occurred in testes of men receiving testosterone propionate disappeared after cessation of treatment. Although further study may reveal the existence of different types of peritubular fibrosis it is clear that this serious defect is not necessarily irreversible. The most common histologic pattern in the testes of oligospermic men is one of disorganized spermatogenesis with sloughing of immature cells. In these cases the orderly pattern of spermatogenesis is severely disturbed and immature forms (particularly spermatocytes) slough into the tubular lumina. These defects give the seminiferous epithelium a jumbled appearance and many tubules appear to be plugged with sloughed cells. Some degree of peritubular fibrosis is encountered in many of these cases. Germinal cell atrophy is a condition seen occasionally in oligospermic men. It is characterized by a general reduction in the number of germ cells of all stages. The impression is strong that the spermatogonia are unable to propagate at a normal rate. As a result the numbers of more mature cells are greatly reduced and the germinal epithelium presents a very loose, poorly populated appearance. In the testes of men whose seminal specimens show unusually large numbers of abnormal cells it is possible regularly to observe striking aberrations of cell division and sperm maturation. These disturbances include aberrant chromosomal behavior, large numbers of multinucleated cells, and
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alterations in size of spermatozoa and are seen in the testes of men with normal as well as low sperm counts. DISCUSSION By way of commenting upon the various histologic patterns which are encountered in the testes of eunuchoidal and infertile men it may be of interest to indicate the direction in which further studies may solve some of the problems which now confront us. In eunuchoidal men the testicular patterns are well defined and once established indicate clearly both the nature of the defect and the course of treatment. In men with testicular aplasia and in cases of the Klinefelter syndrome the testes are beyond salvage and only such benefits can be expected as may accrue to the eunuchoidal state from treatment with androgen. On the other hand, the infantile testes of hypogonadotrophic eunuchoidism may be expected to undergo maturation when the missing factors, the gonadotrophic hormones, are supplied. It has been shown by Heller and Nelson7 • 8 that chorionic gonadotrophin will stimulate the development and function of Leydig cells and that follicle-stimulating hormone will initiate spermatogenesis in such testes. No other course of treatment has been found to produce these results in proved cases of hypogonadotrophic eunuchoidism. The scattered cases in which the use of testosterone has been said to result in the initiation of spermatogenesis15 • 17 • 26 all are too incompletely documented to permit proper evaluation. Unless such cases are proved to belong in the category of hypogonadotrophic eunuchoidism by testicular biopsy prior to treatment and unless treatment with testosterone is uncomplicated by other procedures it is impossible to determine the actual effect of testosterone on the prepuberal type of testis. In proved cases of this type which we have observed before and following treatment with testosterone or chorionic gonadotrophin it has been noted that these procedures do cause an increase in testicular size, an effect which is reflected in an enlargement of the seminiferous tubules and an apparent increase in the differentiation of Sertoli cells. However, the spermatogenic process has not appeared to have been initiated unless FSH also was given. Admittedly this point needs more study, but it is important to establish definitely the pretreatment condition of the testes if the effect of any measure is to be properly evaluated. It is worth noting briefly that in men with hypogonadotrophic eunuchoid-
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ism initiation of testicular activity, androgen production in particular, by gonadotrophins is sometimes followed by spontaneous and continued testicular activity when treatment is withdrawn. Thus it is now our practice to recommend that a period of treatment (four to five months) be followed by a period of rest during which observations for continued improvement are made. Of interest, too, are rare instances of this syndrome in which treatment with large amounts of gonadotrophin does not produce evidences of testicular stiii?-ulation. It is presumed that these represent examples of failure of end-organs to respond to trophic stimuli. We have in one man observed the presence of one testis in which spermatogenesis was in progress while the opposite testis contained Leydig cells, but in other respects was similar to the testis of a hypogonadotrophic eunuchoid. This would appear to be a case of unilateral unresponsiveness of the tubules, but not of the Leydig cells. The testicular defects in which new approaches and new methods are most necessary are those conditions which are associated with infertility. It is clear that nothing can be done to salvage spermatogenesis in testes in which germ cells are absent due to congenital failure or to complete fibrosis. In men with defects of the efferent duct system fertility must depend primarily upon the possible establishment of patent ducts. However, men whose azoospermia is due to germinal cell arrest, or who are oligospermic, have testes which may be improved when the nature and cause of the defects are understood. At this juncture it is important to note that there is very little reason to believe that any deficiency of the known endocrine factors or disturbance of endocrine relationships plays a role in the vast majority of cases of men who are studied because of infertility. This statement, of course, excludes men whose testes do show marked disturbance which can be related to such endocrine diseases as tumors of the hypophysis. 1 The conditions of germinal cell atrophy and germinal cell arrest may have some endocrine basis, but this remains to be demonstrated. The histologic appearance of the biopsies suggests inadequate stimulation by gonadotrophic hormone, but the levels of gonadotrophin associated with these conditions are usually normal and the administration of gonadotrophins has thus far produced disappointing , responses. It is important, however, to remember that the available preparations of FSH are likely to produce antigonadotrophic hormones and thus sharply limit the gametogenic response. 16 It is clear that better preparations
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of FSH are highly desirable for use in conditions which may be due to deficient gonadotrophins. In all probability the recognized endocrine deficiencies are of less significance than disturbances which influence the behavior of the peritubular connective tissues and the function of the Sertoli cells. The tendency of the tissues making up the basement membrane and tunica propria of the human testicular tubule to undergo increased thickness and hyalinization presents a serious problem. Perhaps more refined cytochemical technics will reveal the nature of these changes and indicate the reason for their proliferation. As noted in an earlier section the recent demonstration that severe fibrosis need not be permanent lends encouragement to efforts directed toward halting the progression of the disturbance. It is probable that many instances of defective spermatogenesis may be due to disturbed function of the Sertoli cells. Although there has been a recent trend to ascribe the production of hormones ( inhibin, estrin, "X" hormone, etc.) to these cells 1 • 14 • 15 the point is far from proved that these cells do produce such hormones under normal conditions. Indeed, the array of evidence which can be marshaled against that probability at least equals the arguments in its favor. On the other hand there is ample and substantial evidence which indicates that the Sertoli cells are sustentacular and nutritive elements whose function it is to provide support and nourishment for the germinal elements as they proceed away from the basement membrane in their development. 4 • 24 Thus the form and structure of the Sertoli cell and the distribution of its lipids correspond closely to the cyclic waves of spermatozoon maturation. It seems likely that the further application of cytologic and cytochemical methods will reveal that some disturbances of spermatogenesis such as disorganization, premature sloughing, germinal cell arrest, and germinal cell atrophy may be due in part at least to defective Sertoli cell function. We may also expect revelations which will have an important bearing on our understanding of spermatogenesis as studies are made which employ methods for determining the distribution and utilization of ribonucleic and desoxyribonucleic acid and amino acids. Assuredly future studies on spermatozoa must extend beyond their span of life in the testis and include the changes in physiologic maturation which occur during their sojourn in the epididymis. Such studies as those of Lardy on sperm regulator mechanisms seem destined to have a very important bearing on our understanding of
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the processes which combine to provide the capacity of the sperm to move and to fertilize an ovum. Further detailed studies of the spermatogenic process in men whose infertility is associated with such patterns as germinal cell arrest and aberrant mitotic activity seem likely to indicate the existence of profound disturbances in chromosomal behavior. It is possible, too, that study of the testes of men whose wives tend to abort early fetuses will reveal the cause of fetal maldevelopment as related, in many instances, to faults of sperm formation.
SUMMARY Three types of testicular patterns which have been seen in association with 126 cases of eunuchoidism are described and discussed from the standpoint of differentiation and therapeutic procedures. The testicular patterns which have been observed in 119 cases of azoospermia and 378 cases of oligospermia or abnormal spermatozoa are considered and discussed. Attention is called to the directions in which future observations and utilization of available procedures may be expected to improve our understanding of infertility in the male. This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service.
REFERENCES I. Berthrong, M., Goodwin, W. E., and Scott, W. W.: J. Clin. Endocrinol. 9:579, 1949. 2. Charney, C. W.: The testicular biopsy. A five year survey. In Diagnosis in Sterility. Edited by E. Engle. Springfield, Ill., Charles C Thomas, 1946. 3. Del Castillo, E. B., Trabucco, A., and de la Baize, F. A.: J. Clin. Endocrinol. 7:493, 1947. 4. Elftman, H.: Anat. Rec. 106:381, 1950. 5. Engle, E. T., and Southam, A.: Endocrine aspects of infertility in the male. In Progress in Clinical Endocrinology. Edited by S. Soskin. New York, Grune and Stratton, 1950. 6. Heller, C. G., and Nelson, W. 0.: Recent Progress in Hormone Research 3:229, 1948. 7. Heller, C. G., and Nelson, W. 0.: J. Clin. Investigation 25:915, 1946. 8. Heller, C. G., and Nelson, W. 0.: J. Clin. Endocrinol. 7:345, 1948. 9. Heller, C. G., Nelson, W. 0., Hill, I. C., Henderson, E., Maddock, W. 0., and Jungck, E. C.: Proc. A. Study Internal Secretions, p. 27, 1950. 10. Heller, C. G. Nelson, W. 0., and Roth, A.: J. Clin. Endocrinol. 3:573, 1943. 11. Hotchkiss, R. S.: New York State J. Med. 41:564, 1941. 12. Hotchkiss, R. S.: Fertility in Men. Philadelphia, J. B. Lippincott Co., 1944.
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13. Howard, R. P., Sniffen, R. C., and Simmons, F. A.: Proc. A. Study of Internal Secretions, p. 35, 1948. 14. Howard, R. P., Sniffen, R. C., Simmons, F. A., and Albright, F.: J. Clin. Endocrinol. 10:121, 1950. 15. Hurxthal, L. M., Bruns, H. J., and Musulin, N.: J. Clin. Endocrinol. 9:1245, 1949. 16. Jungck, E. C., Maddock, W. 0., Heller, C. G., and Nelson, W. 0.: J. Clin. Endocrinol. 9:355, 1949. 17. Kinsell, I. W.: J. Clin. Endocrinol. 7:781, 1947. 18. Lardy, H. A.: The metabolic regulator in mammalian spermatozoa. New York, Conference on Human Reproduction of the National Research Council Committee on Maternal Health, 1950. 19. McCullagh, E. P.: Recent Progress in Hormone Research 2:295, 1948. 20. Nelson, W. 0.: M. Clin. North America, January: 97, 1948. 21. Nelson, W. 0.: Hypogonadism in the male. In Progress in Clinical Endocrinology. Edited by S. Hoskins. New York, Grune and Stratton, 1950. 22. Nelson, W. 0.: Spermatogenesis in testes of men with blocked or absent efferent ducts. New York, Conference on Human Reproduction of the National Research Council Committee on Maternal Health, 1950. 23. Nelson, W. 0., and Heller, C. G.: J. Clin. Endocrinol. 5:1, 1945. 24. Nelson, W. 0., and McEnery, W. B., Jr.: Endocrinology (in press). 25. Simmons, F. A.: Clinical interpretation of the semen analysis. In Diagnosis in Sterility. Edited by E. Engle. Springfield, Ill., Charles C Thomas, 1946. 26. Werner, S.C.: Am. J. M. Sc. 3:52, 1947.
Ortho Award The American Society for the Study of Sterility offers an annual award of $1000.00 known as the Ortho Award, for an outstanding contribution to the subject of infertility and sterility. Competition is open to those in clinical practice as well as individuals whose work is restricted to research in the basic sciences. Essays submitted for the 1951 award must be received not later than March 1, 1951. The Prize Essay will appear on the program of the 1951 meeting of the Society. The winner of the 1950 award was Dr. Min Chueh Chang of the Worcester Foundation for Experimental Biology. The prize-winning paper was entitled "Fertility and Sterility as Revealed in the Study of Fertilization and Development of the Rabbit Egg." For particulars concerning the 1951 contests, address the American Society for the Study of Sterility, 20 Magnolia Terrace, Springfield, Mass.
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