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Relation Between Testicular Cytology, Ketosteroids, Androgen, and FSH
Relation Between Testicular Cytology., l{etosteroids., Androgen., and FSH Jack Hyman, M.D.
Tms
consists in a correlation of the clinical and laboratory data of 7 cases of testicular dysfunction. It is written in an effort to clarify our knowledge of the physiology of the human testicle. An evaluation of total testicular function and testicular pituitary interrelationship is presently based upon several diagnostic procedures. The complete semen analysis remains an important measure of exocrine function. Testicular biopsy, as popularized by Charny, gives an accurate histologic impression of spermatogenic activity, but has unfortunately been of much less assistance in evaluating androgenic failure. Goldzieher and Hamblen, in their summary of the present status of the histopathology of androgenic failure, emphasized that there are no specific histochemical technics for measurement of hormone production in the Leydig cells. The cytology of Leydig cells, even when a variety of special stains is used, has failed to be too informative. Assavs and determinations of urinary excretion of 17-ketosteroids, andro" . gen, and gonadotropin provide an index of hormonal activity and must be carried out for diagnostic and prognostic evaluation. Clinical studies of patients with testicular failure have been grouped to form several syndromes described by different investigators. In 1943, Heller, Nelson, and Roth described a syndrome which they designated "functional PRESENTATION
Abstract of a thesis submitted to the Graduate School of Tulane University of Louisiana in partial fulfillment of the requirements for the degree of Master of Science. Studies completed while Assistant in Urology, Ochsner Clinic, New Orleans, La. 245
246
HYMAN
[Fertility & Sterility
prepuberal castration." The testes were absent in these patients and only Wolffian duct structures were found by scrotal exploration. Gynecomastia was present in half of the cases and a eunuchoid skeleton in some of the patients. Gonadotropin was increased in the urine. Excretion of 17-ketosteroids was normal or low nom1al and failed to increase after treatment with chorionic gonadotropin. It was believed that absence of the testis was either congenital or due to prepuberal atrophy. The patients responded well to testosterone, with considerable improvement in the eunuchoidism. In 1942, Klinefelter, Reinfenstein and Albright described a syndrome characterized by gynecomastia and aspermatogenesis, with normal Leydig cell function and increased excretion of follicular-stimulating hormone. Testicular biopsy showed hyalinization of seminiferous tubules with no peritubular fibrosis. Less advanced lesions showed hyalinization of the basement membrane and tunica propria of the tubules with impaired spermatogenesis. There was a relative increase in interstitial cells. In 1945, Heller and Nelson extended this syndrome to include patients without gynecomastia and with reduced Leydig cell function. There was constant azoospermia in their group, testicular biopsy revealing the similar hyalinization of basement membrane of the tubules with only Sertoli cells present. They were not able to find any correlation between the height of the urinary gonadotropin, histologic appearance of the Leydig cells, clinical evidence of androgenic failure, or the 17-ketosteroid excretion. Del Castillo and associates described what they considered to be a different syndrome which possessed all the features of the aforementioned cases except for normal tubular basement membranes and only Sertoli cells in the tubules. Hypoleydigism was absent. Excretion of 17-ketosteroid was normal or decreased; excretion of follicular-stimulating hormone was also nom1al. However, one gains the impression that these patients fall into the same general category as the Klinefelter, Albright, and Heller syndrome. Goldzieher and Hamblen reported 8 cases of testicular failure with a critical review of each case in an effort to correlate clinical and laboratory observations. They observed no quantitative correlation between the elevation of gonadotropic excretion and 17-ketosteroid excretion and the histology of the Leydig cells at testicular biopsy. In short, they concluded that the 8 cases illustrated "notable discrepancies between clinical evidence of androgenic activity, urinary hormone excretion and testicular histology."
Vol. 1, No. 3, 1950]
TESTICULAR CYTOLOGY AND HORMONES
247
PRESENTATION OF CASES*
Urinary gonadotropin was assayed by the method of Klinefelter, Albright and Griswald, the normal range being 6.6 to 52 mouse units per twenty-four hours. Excretion of 17-ketosteroids was determined by the Segaloff modification of the Zimmerman Reaction, the normal mean being 15.9 mg. with a range of 7.2 to 27.2 mg. per 24 hours of transdehydronandrosterone. Androgen excretion was assayed by the method of Hollander, Klempler and Franks. Normal values for our laboratory have not yet been completely determined. All urine was collected after dehydration of the patient and stored under refrigeration. The verity of collection was checked by determination of concomitant creatin excretion.
Case 1 R. W. E., a 34-year-old Hondurian plantation foreman, had had bilateral mumps orchitis with subsequent atrophy at the age of 20 years. Libido and sexual performance were normal. General examination revealed no abnormalities except for the testicles, which were about 2 em. in diameter. Semen analysis was as follows: Volume, 2 cc.; count of undiluted specimen, 1 sperm per red cell field; motility, the few sperms seen were all motile; morphology, few sperms seen were morphologically normal. A testicular biopsy revealed almost complete hyalinization of the seminiferous tubules and an occasional tubule with incomplete spermatogenesis. The Leydig cells appeared to be normal and relatively increased in number. The 17-ketosteroid excretion was 18.5 mg. in twenty-four hours. Urinary gonadotropins were greater than 96 mouse units and less than 192 mouse units in twenty-four hours, and androgen excretion was 6.4 mg. of androsterone in twenty-four hours. Comment: It is noteworthy that the few spermatozoa found in the semen had normal characteristics. Increased urinary gonadotropin excretion indicates pituitary hyperactivity in the face of testicular destruction. Normal Leydig cells associated with normal ketosteroid and androgen, along with no evidence of clinical hypoleydigism, are correlative observations of note. The few sperm found in the semen indicate that isolated tubules must be functioning; however, the biopsy failed to include these.
Case2 E. C., a 13-year-old boy, had congenital, bilateral cryptorchidism. Neither testis was found in the scrotum, and when he was first seen in the Department of Endocrinology, it was questionable whether they were palpable at all. The penis • Assay of urinary hormone excretion was made through the courtesy of Dr. Albert Segaloff, of the Endocrine Research Laboratory of the Alton Ochsner Medical Foundation.
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[Fertility & Sterility
was small, prepubescent in type. The 17-ketosteroid excretion varied from 0 to 3.6 mg. in twenty-four hours. No gonadotropins were detected in twenty-four hours' urine assay. The patient was given 500 international units of chorionic gonadotropin daily for two months. The testicles failed to descend into the scrotum; however, the penis increased in size and pubic hair appeared: When he was examined in the Department of Urology in March, 1947, testicles about 1 em. in diameter could be palpated in the inguinal canals. A Torek type of orchidopexy was performed and a testicular biopsy was done. Sections of the testicle revealed immature seminiferous tubules filled with undifferentiated cells; the Leydig cells were clumped and appeared to be normal (Fig. 1). Two months after operation,
FIGURE
1.
Case 2, Cryptorchid. Undifferentiated tubular cells.
the 17-ketosteroid excretion had risen to 9.6 mg. for twenty-four hours. About six months later the 17-ketosteroid excretion was 4.7 mg. for twenty-four hours, and the testes were about 1.5 em. in diameter, soft in consistency.
Comment: This case illustrates the hormonal imbalance . in . bilateral cryptorchidism. Pituitary gonadotropic failure is apparent from the absence of urinary gonadotropins. It illustrates further the failure of descent after large doses of chorionic gonadotropin. The presence of Leydig cells in the biopsy makes one wonder if they were present before the chorionic gonadotropin was administered. An additional finding of interest is the increase in 17-ketosteroid excretion, shortly after the testes were removed from the inguinal canals.
Case3
C. J., a 23-year-old medical student, had bilateral cryptorchidism until the age of 9 years, when orchidopexy was done. The right testis returned to the inguinal canal shortly after operation. The left testis remained in the scrotum for eighteen
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months and then migrated to the external inguinal ring. Physical examination revealed a normal-appearing male except for the external genitalia. A testis 2 em. in diameter was palpated in the right inguinal canal and a normal-sized testis was palpated on the left at the external inguinal ring. Semen analysis, on two occasions, failed to reveal any spermatozoa. The 17-ketosteroid excretion was 21.4 mg. for twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 288 mouse units and less than 384 mouse units. A Torek type of orchidopexy was done and biopsy of the left testis was obtained at this time. The testicular biopsy revealed seminiferous tubules containing no spermatogenic cells beyond the stage of primary spermatocytes; Leydig cells appeared normal and were relatively increased in number. The testes have remained anchored to the thigh thus far.
Comment: This case is of interest from the point of increased gonadotropin excretion, which probably indicates pituitary activity on testes which are refractive by virtue of position. The 17-ketosteroid excretion and the Leydig
FIGURE
2.
Case 4, Germinal Aplasia. Sertoli cells present.
cells correlate with the clinically normal androgen activity. It will be interesting to see what effect surgical descent of the testes will have on the histology of the seminiferous tubules. Testicular biopsy is contemplated when the second stage of the Torek procedure is done.
Case4 L. A. S., a 26-year-old naval pilot, had been married for four years without producing conception. Libido and sexual performance were normal. Physical examination revealed no abnormalities, except for the testes, which were slightly smaller
250
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[Fertility & Sterility
than normal. Semen analyses on several occasions showed only an occasional spermatozoa, too few to count; 50 per cent of those seen were actively motile. The 17-ketosteroid excretion varied from 17 to 20 mg. for twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 6.6 mouse units and less than 13.2 mouse units. Androgen excretion in twenty-four hours was 2.2 mg. of androsterone. A biopsy was obtained from both testes (Fig. 2), and sections from both testes showed the same microscopic picture. The seminiferous tubules showed no evidence of spermatogenesis; only Sertoli cells were evident. There was slight thickening of the basement membrane of the seminiferous tubules. The Leydig cells appeared to be nonnal. The patient was given large doses of equine gonadotropin with no change in the picture. The wife was subsequently impregnated by artificial insemination of donor semen.
Comment: This patient's syndrome seems to be the same as that described by Del Castillo and associates. The remarkable finding of spermatozoa in the semen with the tubular histology seen at testicular biopsy can be reconciled only by assuming the pathologic alteration was not universally present throughout the testes. Here again, androgen activity, ketosteroids, urinary androgens, and Leydig cells correlate fairly well.
Case5
R. J. W., a 23-year-old medical student, had failed to have testicular growth and the development of secondary sexual characteristics at puberty. For the last two years, the patient had been taking 25 mg. of testosterone in oil, three times each week. There was definite improvement in the secondary sexual characteristics. Examination revealed a fairly muscular male with slightly accentuated hips, slightly hypertrophied breasts, a penis smaller than normal, and both testes about 2 em. in diameter in the scrotum. The prostate was small; prostatic and vesicular secretion obtained by massage revealed no sperm. Since the patient had stopped taking testosterone to obtain basal studies at examination, an ejaculate could not be obtained. The 17-ketosteroid excretion varied from 8.3 to 11 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was less than 6.6 mouse units. Testicular biopsy revealed immature seminiferous tubules filled with undifferentiated cells. No Leydig cells were apparent; only fibroblasts were seen in the interstitial spaces. The patient was given 1000 R.U. of chorionic gonadotropin daily and was to return for subsequent study.
Case6
F. L., a 34-year-old male, was obviously eunuchoid in stature, had slight gynecomastia, prepubescent external genitalia, and scant axillary and pubic hair. Puberty had never occurred. Ejaculations had not been possible. The 17-ketosteroid excretion varied from 5.4 to 6.8 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was less than 6.6 mouse units. The excretion
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of androgen in twenty-four hours was 0.85 mg. of androsterone. Testicular biopsy revealed (Fig. 3) immature seminiferous tubules filled with undifferentiated cells. There was no thickening or hyalinization of the basement membrane, or FIGURE
FIGURE
4.
3.
Case 6, Pretreatment. Leydig cells absent.
Case 6, Posttreatment. Leydig cells apparent. Tubules differentiating.
peritubular fibrosis. Leydig cells were not seen in the interstitial spaces. The patient was given large doses of chorionic gonadotropin ( 1000 international units daily), and the 17-ketosteroid excretion rose to 12.5 mg. in twenty-four hours. The penis and testes increased in size. The patient began to have ejaculations. A recently examined ejaculate consisting of 1 cc. of semen did not contain sperma-
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tozoa. After six months of treatment, a second testicular biopsy was done (Fig. 4). The remarkable demonstration of Leydig cells, where none were before, was seen in these sections. The cells had less than the normal cytoplasm, but contained secretory granules. They appeared in groups. The seminiferous tubules likewise appeared to be changing, with a tendency to differentiation of its cells, which were so completely undifferentiated in the previous biopsy.
Comment: Cases 5 and 6, because of their similarity, will be discussed together. Patient No. 5, having had the advantage of substitution therapy, presented a less extreme clinical picture. Apparently, pituitary hypofunction, as evidenced by less than normal urinary gonadotropins, is the underlying cause. The 17-ketosteroid excretion was low normal in both cases; however, the correlation between clinical hypoleydigism and absence of Leydig cells is good. Low androgen excretion also correlates much better with the foregoing than the 17-ketosteroid excretion. In case 6, the pronounced rise in 17-ketosteroid excretion and obvious appearance of Leydig cells in the testes, under the influence of luteinizing hormone, is of considerable interest.
Case7
J. M., a 32-year-old streetcar conductor, had been married four years without producing conception. A semen examination two years previously was reported to have shown many nonmotile sperm. A semen study was made on three occasions, and each time no spermatozoa were found. The 17-ketosteroid excretion was 15.6 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 6.6 and less than 13.2 mouse units. A testicular biopsy showed an apparent reduced rate of spermatogenesis in the seminiferous tubules and no spermatogenic cell form beyond the secondary spermatocyte. The basement membranes of the tubules were normal. The few Leydig cells seen in these sections appeared to be normal. Shortly after this, the patient's wife became pregnantmuch to the consternation of the investigators-and all further investigation ceased. Unfortunately, the wife delivered an eight-month stillborn, and two years later the patient presented himself for further investigation. A semen analysis on three occasions revealed: volume, 6.5 cc.; count, 6 million per cubic centimeter; motility, 40 per cent; morphology, 69 per cent oval forms, 20 per cent oval forms with cytoplasmic appendages, 5 per cent tapered forms, 5 per cent amorphous forms, and 1 per cent twin-tailed. The 17-ketosteroid excretion was 13.1 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 13.2 and less than 26.4 mouse units. Biopsy of both testicles showed fairly active spermatogenesis with mature forms being formed in the tubules. The basement membranes of the tubules were nonnal. Leydig cells appeared to be normal. The patient was given large doses of equine gonadotropin with the thought that
Vol. 1, No.3, 1950)
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further stimulation of spermatogenesis might occur. After six months of intermittent therapy, his wife became pregnant. Semen analysis revealed: concentration, 10 million per cubic centimeter; motility, 80 per cent; morphology, 84 per cent, apparently normal spermatozoa. Comment: This patient illustrates the remarkable variations in physiologic
activity of which the human is capable. When the patient was first seen, the semen was definitely that of azoospermia. The seminal findings correlated exactly with the testicular biopsy, which disclosed incomplete maturation of spermatogenic cells in the tubules. The conception of his wife, so difficult to reconcile at the time, is more readily accepted in view of our recent studies of the patient. The change in the semen and testicular histology revealed in recent studies has occurred spontaneously. Had treatment been pursued in the beginning, these changes most certainly would have been ascribed to the treatment. This emphasizes the care one must exercise in evaluating the therapeutic response observed in treating patients with seminal deficiency.
SUMMARY 1. A brief review is presented of testicular dysfunction as manifested in several clinical syndromes. 2. Seven cases of testicular dysfunction are reported in which "complete" diagnostic studies were done. 3. An effort has been made to correlate testicular cytology with the excretion of ketosteroids, androgen, and FSH.
REFERENCES 1. Charny, C. W., and Meranze, D. R.: Surg., Gynec. & Obst. 74:836-842, 1942. 2. Del Castillo, E. B., Trabucco, A., and de la Baize, F. A.: J. Clin. Endocrinol. 7:493-502, 1947. 3. Goldzieher, J. W., and Hamblen, E. C.: Surg., Gynec. & Obst. 85:583-596, 1947. 4. Heller, C. G., and Nelson, W. 0.: J. Clin. Endocrinol. 5:1-2, 1945. 5. Heller, C. G., Nelson, W. 0., and Roth, A. A.: J. Clin. Endocrinol. 3:573-588, 1943. 6. Hollander, F., Klempner, E., and Frank, R. T.: Proc. Soc. Exper. Bioi. & Med. 46:1-5, 1941. 7. Klinefelter, H. F., Jr., Albright, F., and Griswold, G. C.: J. Clin. Endocrinol. 3:529-544, 1943. 8. Klinefelter, H. F., Jr., Reifenstein, E. C., Jr., and Albright, F.: J. Clin. Endocrinol. 2:615-627, 1942. 9. Segaloff, A.: Personal Communication.
Tms
consists in a correlation of the clinical and laboratory data of 7 cases of testicular dysfunction. It is written in an effort to clarify our knowledge of the physiology of the human testicle. An evaluation of total testicular function and testicular pituitary interrelationship is presently based upon several diagnostic procedures. The complete semen analysis remains an important measure of exocrine function. Testicular biopsy, as popularized by Charny, gives an accurate histologic impression of spermatogenic activity, but has unfortunately been of much less assistance in evaluating androgenic failure. Goldzieher and Hamblen, in their summary of the present status of the histopathology of androgenic failure, emphasized that there are no specific histochemical technics for measurement of hormone production in the Leydig cells. The cytology of Leydig cells, even when a variety of special stains is used, has failed to be too informative. Assavs and determinations of urinary excretion of 17-ketosteroids, andro" . gen, and gonadotropin provide an index of hormonal activity and must be carried out for diagnostic and prognostic evaluation. Clinical studies of patients with testicular failure have been grouped to form several syndromes described by different investigators. In 1943, Heller, Nelson, and Roth described a syndrome which they designated "functional PRESENTATION
Abstract of a thesis submitted to the Graduate School of Tulane University of Louisiana in partial fulfillment of the requirements for the degree of Master of Science. Studies completed while Assistant in Urology, Ochsner Clinic, New Orleans, La. 245
246
HYMAN
[Fertility & Sterility
prepuberal castration." The testes were absent in these patients and only Wolffian duct structures were found by scrotal exploration. Gynecomastia was present in half of the cases and a eunuchoid skeleton in some of the patients. Gonadotropin was increased in the urine. Excretion of 17-ketosteroids was normal or low nom1al and failed to increase after treatment with chorionic gonadotropin. It was believed that absence of the testis was either congenital or due to prepuberal atrophy. The patients responded well to testosterone, with considerable improvement in the eunuchoidism. In 1942, Klinefelter, Reinfenstein and Albright described a syndrome characterized by gynecomastia and aspermatogenesis, with normal Leydig cell function and increased excretion of follicular-stimulating hormone. Testicular biopsy showed hyalinization of seminiferous tubules with no peritubular fibrosis. Less advanced lesions showed hyalinization of the basement membrane and tunica propria of the tubules with impaired spermatogenesis. There was a relative increase in interstitial cells. In 1945, Heller and Nelson extended this syndrome to include patients without gynecomastia and with reduced Leydig cell function. There was constant azoospermia in their group, testicular biopsy revealing the similar hyalinization of basement membrane of the tubules with only Sertoli cells present. They were not able to find any correlation between the height of the urinary gonadotropin, histologic appearance of the Leydig cells, clinical evidence of androgenic failure, or the 17-ketosteroid excretion. Del Castillo and associates described what they considered to be a different syndrome which possessed all the features of the aforementioned cases except for normal tubular basement membranes and only Sertoli cells in the tubules. Hypoleydigism was absent. Excretion of 17-ketosteroid was normal or decreased; excretion of follicular-stimulating hormone was also nom1al. However, one gains the impression that these patients fall into the same general category as the Klinefelter, Albright, and Heller syndrome. Goldzieher and Hamblen reported 8 cases of testicular failure with a critical review of each case in an effort to correlate clinical and laboratory observations. They observed no quantitative correlation between the elevation of gonadotropic excretion and 17-ketosteroid excretion and the histology of the Leydig cells at testicular biopsy. In short, they concluded that the 8 cases illustrated "notable discrepancies between clinical evidence of androgenic activity, urinary hormone excretion and testicular histology."
Vol. 1, No. 3, 1950]
TESTICULAR CYTOLOGY AND HORMONES
247
PRESENTATION OF CASES*
Urinary gonadotropin was assayed by the method of Klinefelter, Albright and Griswald, the normal range being 6.6 to 52 mouse units per twenty-four hours. Excretion of 17-ketosteroids was determined by the Segaloff modification of the Zimmerman Reaction, the normal mean being 15.9 mg. with a range of 7.2 to 27.2 mg. per 24 hours of transdehydronandrosterone. Androgen excretion was assayed by the method of Hollander, Klempler and Franks. Normal values for our laboratory have not yet been completely determined. All urine was collected after dehydration of the patient and stored under refrigeration. The verity of collection was checked by determination of concomitant creatin excretion.
Case 1 R. W. E., a 34-year-old Hondurian plantation foreman, had had bilateral mumps orchitis with subsequent atrophy at the age of 20 years. Libido and sexual performance were normal. General examination revealed no abnormalities except for the testicles, which were about 2 em. in diameter. Semen analysis was as follows: Volume, 2 cc.; count of undiluted specimen, 1 sperm per red cell field; motility, the few sperms seen were all motile; morphology, few sperms seen were morphologically normal. A testicular biopsy revealed almost complete hyalinization of the seminiferous tubules and an occasional tubule with incomplete spermatogenesis. The Leydig cells appeared to be normal and relatively increased in number. The 17-ketosteroid excretion was 18.5 mg. in twenty-four hours. Urinary gonadotropins were greater than 96 mouse units and less than 192 mouse units in twenty-four hours, and androgen excretion was 6.4 mg. of androsterone in twenty-four hours. Comment: It is noteworthy that the few spermatozoa found in the semen had normal characteristics. Increased urinary gonadotropin excretion indicates pituitary hyperactivity in the face of testicular destruction. Normal Leydig cells associated with normal ketosteroid and androgen, along with no evidence of clinical hypoleydigism, are correlative observations of note. The few sperm found in the semen indicate that isolated tubules must be functioning; however, the biopsy failed to include these.
Case2 E. C., a 13-year-old boy, had congenital, bilateral cryptorchidism. Neither testis was found in the scrotum, and when he was first seen in the Department of Endocrinology, it was questionable whether they were palpable at all. The penis • Assay of urinary hormone excretion was made through the courtesy of Dr. Albert Segaloff, of the Endocrine Research Laboratory of the Alton Ochsner Medical Foundation.
HYMAN
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[Fertility & Sterility
was small, prepubescent in type. The 17-ketosteroid excretion varied from 0 to 3.6 mg. in twenty-four hours. No gonadotropins were detected in twenty-four hours' urine assay. The patient was given 500 international units of chorionic gonadotropin daily for two months. The testicles failed to descend into the scrotum; however, the penis increased in size and pubic hair appeared: When he was examined in the Department of Urology in March, 1947, testicles about 1 em. in diameter could be palpated in the inguinal canals. A Torek type of orchidopexy was performed and a testicular biopsy was done. Sections of the testicle revealed immature seminiferous tubules filled with undifferentiated cells; the Leydig cells were clumped and appeared to be normal (Fig. 1). Two months after operation,
FIGURE
1.
Case 2, Cryptorchid. Undifferentiated tubular cells.
the 17-ketosteroid excretion had risen to 9.6 mg. for twenty-four hours. About six months later the 17-ketosteroid excretion was 4.7 mg. for twenty-four hours, and the testes were about 1.5 em. in diameter, soft in consistency.
Comment: This case illustrates the hormonal imbalance . in . bilateral cryptorchidism. Pituitary gonadotropic failure is apparent from the absence of urinary gonadotropins. It illustrates further the failure of descent after large doses of chorionic gonadotropin. The presence of Leydig cells in the biopsy makes one wonder if they were present before the chorionic gonadotropin was administered. An additional finding of interest is the increase in 17-ketosteroid excretion, shortly after the testes were removed from the inguinal canals.
Case3
C. J., a 23-year-old medical student, had bilateral cryptorchidism until the age of 9 years, when orchidopexy was done. The right testis returned to the inguinal canal shortly after operation. The left testis remained in the scrotum for eighteen
Vol. 1, No. 3, 1950]
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months and then migrated to the external inguinal ring. Physical examination revealed a normal-appearing male except for the external genitalia. A testis 2 em. in diameter was palpated in the right inguinal canal and a normal-sized testis was palpated on the left at the external inguinal ring. Semen analysis, on two occasions, failed to reveal any spermatozoa. The 17-ketosteroid excretion was 21.4 mg. for twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 288 mouse units and less than 384 mouse units. A Torek type of orchidopexy was done and biopsy of the left testis was obtained at this time. The testicular biopsy revealed seminiferous tubules containing no spermatogenic cells beyond the stage of primary spermatocytes; Leydig cells appeared normal and were relatively increased in number. The testes have remained anchored to the thigh thus far.
Comment: This case is of interest from the point of increased gonadotropin excretion, which probably indicates pituitary activity on testes which are refractive by virtue of position. The 17-ketosteroid excretion and the Leydig
FIGURE
2.
Case 4, Germinal Aplasia. Sertoli cells present.
cells correlate with the clinically normal androgen activity. It will be interesting to see what effect surgical descent of the testes will have on the histology of the seminiferous tubules. Testicular biopsy is contemplated when the second stage of the Torek procedure is done.
Case4 L. A. S., a 26-year-old naval pilot, had been married for four years without producing conception. Libido and sexual performance were normal. Physical examination revealed no abnormalities, except for the testes, which were slightly smaller
250
HYMAN
[Fertility & Sterility
than normal. Semen analyses on several occasions showed only an occasional spermatozoa, too few to count; 50 per cent of those seen were actively motile. The 17-ketosteroid excretion varied from 17 to 20 mg. for twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 6.6 mouse units and less than 13.2 mouse units. Androgen excretion in twenty-four hours was 2.2 mg. of androsterone. A biopsy was obtained from both testes (Fig. 2), and sections from both testes showed the same microscopic picture. The seminiferous tubules showed no evidence of spermatogenesis; only Sertoli cells were evident. There was slight thickening of the basement membrane of the seminiferous tubules. The Leydig cells appeared to be nonnal. The patient was given large doses of equine gonadotropin with no change in the picture. The wife was subsequently impregnated by artificial insemination of donor semen.
Comment: This patient's syndrome seems to be the same as that described by Del Castillo and associates. The remarkable finding of spermatozoa in the semen with the tubular histology seen at testicular biopsy can be reconciled only by assuming the pathologic alteration was not universally present throughout the testes. Here again, androgen activity, ketosteroids, urinary androgens, and Leydig cells correlate fairly well.
Case5
R. J. W., a 23-year-old medical student, had failed to have testicular growth and the development of secondary sexual characteristics at puberty. For the last two years, the patient had been taking 25 mg. of testosterone in oil, three times each week. There was definite improvement in the secondary sexual characteristics. Examination revealed a fairly muscular male with slightly accentuated hips, slightly hypertrophied breasts, a penis smaller than normal, and both testes about 2 em. in diameter in the scrotum. The prostate was small; prostatic and vesicular secretion obtained by massage revealed no sperm. Since the patient had stopped taking testosterone to obtain basal studies at examination, an ejaculate could not be obtained. The 17-ketosteroid excretion varied from 8.3 to 11 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was less than 6.6 mouse units. Testicular biopsy revealed immature seminiferous tubules filled with undifferentiated cells. No Leydig cells were apparent; only fibroblasts were seen in the interstitial spaces. The patient was given 1000 R.U. of chorionic gonadotropin daily and was to return for subsequent study.
Case6
F. L., a 34-year-old male, was obviously eunuchoid in stature, had slight gynecomastia, prepubescent external genitalia, and scant axillary and pubic hair. Puberty had never occurred. Ejaculations had not been possible. The 17-ketosteroid excretion varied from 5.4 to 6.8 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was less than 6.6 mouse units. The excretion
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of androgen in twenty-four hours was 0.85 mg. of androsterone. Testicular biopsy revealed (Fig. 3) immature seminiferous tubules filled with undifferentiated cells. There was no thickening or hyalinization of the basement membrane, or FIGURE
FIGURE
4.
3.
Case 6, Pretreatment. Leydig cells absent.
Case 6, Posttreatment. Leydig cells apparent. Tubules differentiating.
peritubular fibrosis. Leydig cells were not seen in the interstitial spaces. The patient was given large doses of chorionic gonadotropin ( 1000 international units daily), and the 17-ketosteroid excretion rose to 12.5 mg. in twenty-four hours. The penis and testes increased in size. The patient began to have ejaculations. A recently examined ejaculate consisting of 1 cc. of semen did not contain sperma-
252
HYMAN
[Fertility & Sterility
tozoa. After six months of treatment, a second testicular biopsy was done (Fig. 4). The remarkable demonstration of Leydig cells, where none were before, was seen in these sections. The cells had less than the normal cytoplasm, but contained secretory granules. They appeared in groups. The seminiferous tubules likewise appeared to be changing, with a tendency to differentiation of its cells, which were so completely undifferentiated in the previous biopsy.
Comment: Cases 5 and 6, because of their similarity, will be discussed together. Patient No. 5, having had the advantage of substitution therapy, presented a less extreme clinical picture. Apparently, pituitary hypofunction, as evidenced by less than normal urinary gonadotropins, is the underlying cause. The 17-ketosteroid excretion was low normal in both cases; however, the correlation between clinical hypoleydigism and absence of Leydig cells is good. Low androgen excretion also correlates much better with the foregoing than the 17-ketosteroid excretion. In case 6, the pronounced rise in 17-ketosteroid excretion and obvious appearance of Leydig cells in the testes, under the influence of luteinizing hormone, is of considerable interest.
Case7
J. M., a 32-year-old streetcar conductor, had been married four years without producing conception. A semen examination two years previously was reported to have shown many nonmotile sperm. A semen study was made on three occasions, and each time no spermatozoa were found. The 17-ketosteroid excretion was 15.6 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 6.6 and less than 13.2 mouse units. A testicular biopsy showed an apparent reduced rate of spermatogenesis in the seminiferous tubules and no spermatogenic cell form beyond the secondary spermatocyte. The basement membranes of the tubules were normal. The few Leydig cells seen in these sections appeared to be normal. Shortly after this, the patient's wife became pregnantmuch to the consternation of the investigators-and all further investigation ceased. Unfortunately, the wife delivered an eight-month stillborn, and two years later the patient presented himself for further investigation. A semen analysis on three occasions revealed: volume, 6.5 cc.; count, 6 million per cubic centimeter; motility, 40 per cent; morphology, 69 per cent oval forms, 20 per cent oval forms with cytoplasmic appendages, 5 per cent tapered forms, 5 per cent amorphous forms, and 1 per cent twin-tailed. The 17-ketosteroid excretion was 13.1 mg. in twenty-four hours. The excretion of gonadotropin in twenty-four hours was greater than 13.2 and less than 26.4 mouse units. Biopsy of both testicles showed fairly active spermatogenesis with mature forms being formed in the tubules. The basement membranes of the tubules were nonnal. Leydig cells appeared to be normal. The patient was given large doses of equine gonadotropin with the thought that
Vol. 1, No.3, 1950)
TESTICULAR CYTOLOGY AND HORMONES
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further stimulation of spermatogenesis might occur. After six months of intermittent therapy, his wife became pregnant. Semen analysis revealed: concentration, 10 million per cubic centimeter; motility, 80 per cent; morphology, 84 per cent, apparently normal spermatozoa. Comment: This patient illustrates the remarkable variations in physiologic
activity of which the human is capable. When the patient was first seen, the semen was definitely that of azoospermia. The seminal findings correlated exactly with the testicular biopsy, which disclosed incomplete maturation of spermatogenic cells in the tubules. The conception of his wife, so difficult to reconcile at the time, is more readily accepted in view of our recent studies of the patient. The change in the semen and testicular histology revealed in recent studies has occurred spontaneously. Had treatment been pursued in the beginning, these changes most certainly would have been ascribed to the treatment. This emphasizes the care one must exercise in evaluating the therapeutic response observed in treating patients with seminal deficiency.
SUMMARY 1. A brief review is presented of testicular dysfunction as manifested in several clinical syndromes. 2. Seven cases of testicular dysfunction are reported in which "complete" diagnostic studies were done. 3. An effort has been made to correlate testicular cytology with the excretion of ketosteroids, androgen, and FSH.
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