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Testicular Dysgenesis as an Etiologic Factor in Cryptorchidism
THE JOURNAL OF UROLOGY
Vol. 72, No. 4, October 1954 Printed in U.S.A.
TESTICULAR DYSGENESIS AS AN ETIOLOGIC FACTOR IN CRYPTORCHIDISM ARTHUR R. SOHVAL From the Endocrine Laboratory and Clinic of the Department of Medicine, the Mount Sinai Hospital, New York, N. Y.
It has long been conjectured that inherent disorders of the testis may be related to or responsible for maldescent. The pioneer observations of John Hunter (1728-1793) led him to suspect that congenital testicular imperfection prevents .descent and interferes with normal function. 1 In 1910 Bland-Sutton2 revived /the concept that a testis is retained because it is imperfect. Subsequently, other workers 3 • 4 • 6 • 6 subscribed to the belief that testicular "infantilism", "undevel'opment" or "hypoplasia" may play a role in the etiology of cryptorchidism. N elson7 has recently stated the view that in certain cases undescended testes may be an expression of defective development. The indefinite state of our knowledge of this problem is reflected in the fact that various urological texttJooks8-11 and review articles12 • 13 fail to mention it. In an effort to shed further light on the relationship between testicular mald . relopment and maldescent, a series of cryptorchid glands was subjected to 0\ m!!-tic histologic analysis. The purpose of the present communication is to sys e 1 the results of this investigation in which microscopic evidence of testicurecorc . d . h . .fi f s· h fi d" Iar d YSl ~enesis was .encountere wit s1g111 cant requency. mce t ese n mgs t observed m a control group of normal scrotal testes from correspond. 1og1c . importance . were no no . d s 1t . 1s . fe1t t h at t h ey may represent f actors of et10 . mg age pe. . . in some ca&'es of cryptorch1d1sm. MATERIAL AND METHOD
T bl 1 enun 1erates the pertinent data of 42 patients whose imperfectly descen~e~ gonads w~re studied: The age range ~s from prematuri!y (7 months) to 78 ears. The tm,~+,1cular spemmens were obtamed at necropsy m 7 cases. In 35 patknts fresh testic ular tissue was provided surgically, 6 by biopsy and 29 by 0
ablation. A ted for publicatio11 September 10, 1953. , H~Eter, J.: A descriptie'n of the situation of ~he testis in t!ie foetus, with its descent into the scrotum. In The w 01 ·ks of John Hunter with Notes. Edited by Palmer, J. F., Lonn Rees Orme r3rown, Green and Longman. 1837, vol. 4 pp. 15, 17. d .L on· ongma , ' '· · oner 84 · 19-30 1910 2 Bland-Su~ton,_J.: Practiti_ 1. Chir:, 100: 1151-ilSO, 1913. ,4 E Uffreduzz1 0 .. Arch. f. kln · · 1 d man. of H obd ay, F . : C ryptorc h'd' 1 ism 1n amma s an cc l es, w'. McA . .. Discussi ~n ,. 923 Proc. Roy. Soc. Med., 17: 3-17," · . 5 Staemmler, M.: Arch. f. path. _Ana~., 245. 304-321, 1923. . . H R and Slotopolsky,, B .. Deutsche Ztschr. f. Chir., 188. 76-100, 1924. • S h' 7 Nc 1mz, w· o ·. Recent Progress ,;n Hormone Research, 6: 29-56, 1951. s A·.; Urological Path?Jogy. Philadelphia: Le.a & Febiger, 1952, vol. 2. 9 C bell M . Clinical Pediatrw Urology. Philadelphia: W. B. Saunders Co., 1951. 10 Rcl~ck, 'H. The Practice of Urology. Philadelphia: J. B. Lippincott Co., 1949,
n:rbi~t p:
·c.:
voli, iowsley,
o. s. and Kirwin, T. J.: 'Clinical Urology.
Baltimore: Williams and Wilkins
Coif ~ 4~by W C . New Eng. J. Med., 2t'.12: 701-704, 1945. 1s L~~is, t. G.: "j. Urol., 60: 345-356, H}48. 693
694
ARTHUR R. SOHVAL
TABLE
Case No.
Age
1. Perti"nent data on 42 patients with undescended gonads Sizet (cm.)
Side*
I
Location
Method of Obtaining Specimen
-~ R
0.9
Canal
2
Premature (7 mos.) 2 days
R
0.9
Abdomen
3
2 mos.
rL
-
Canal
Biopsy
4
4
"
RI
1
Autopsy
5
6
"
RI
1.1
Abdomen (R) Canal (L) Abdomen
6
7
"
RI
1.3
Abdomen
7 yrs. 10 "
RI L
1 1.1
l
7
10 12
"
L
10
"
R
1.3 2
Canal Canal (mid) Canal Abdomen
11
13
"
RI
1.5
Abdomen
12
13 13
"
L RL
-
Canal (mid) Abdomen
8 9
13
"
1 x 0.7 x 0.5 (R) 1.5 x 1.2 x 0.8 (L)
-
15
15 17
" "
L L
2.5
X
16
18
"
L
3
2.5
17 18
21 23
"
R
3.5
"
RI
19
27
"
Rl
20
28
"
L
2
21
28
"
R
asmall"
22 23
29 30
"
"
R L
24
30
"
R
25
30
"
R
26
30
"
R
Tumor 140 g.
27
31
"
R
3
28
" "
L R
"fair 11
29
33 35
Tumor9x7x6
30
36
"
R
4X 2X 1
Canal (high) Abdomen Outside ext. ring Canal
31
36
"
rL
"fair"
Canal
14
X
X
1.5
X
1
2
X
2.5 X 2.5 0.5
X
"
" Ablation
" Biopsy
Germinal aplasia Immature tubules (majority) Immature tubules (about 50 per cent)
Shortness stature
r .)
" Ablation
Biopsy Ablation
Canal
"
Canal Canal (high)
"
Germinal aplasia. Immature tubules (rare small foci)
Hermaphro, lite
Immature tubules (many small foci) Immature tubules (single large focus)
"
Canal
"
Canal Canal
"
2.5
Abdomen
"
3
Outside ext. ring Abdomen
"
1.5
Multiple congenital anomalies Congenital en· cephalopathy'·
"
Ablation
"smalF 1
Remarks
Multiple congenital anomalies Pseudohermaphrodite Multiple congenital anomalies
"
Canal
1
1.8
2
"
Biopsy
X
Evidence of Dysgenesis
Autopsy
Outside ext. ring
"normal"
X
Canal (low) Abdomen
I
"
"
" "
" BlOpsy
Absence of spermatogonia, Bertoli and Ley dig cells
Immature tut ,ules (few small f~ ;ci) Immature tubules (about 4'J per cent)
Prepuberal hypogonadism Pseudohermaph rodite Dwarfism
Immatt/ue tubules (mea~hy small foci) Imr 11'ature tubules (o· ccasional small foci)
Immature tubules (rare large com pact groups) Immature tubules (many small foci)
Immature tubules (many small foci)
Seminoma
Malignant teratoma
695
CRYPTORCHIDISM TABLE
Case No.
Age
-
Sizet
Side•
(cm.)
1.-Continued
Location
Method of Obtaining Specimen
Evidence of Dysgenesis
Canal (low)
Ablation
Immature tubules (few small foci) Immature tubules (about 20 per cent)
Remarks
-32
38 yrs.
33
39
"
R
34 35 36 37 38
39 41 42 42 48
" " " "
L
39
50
40
52
41
52
42
78
L
''atrophic''
Abdomen
"
"
R R R R
4.5 X 3 X 2.5 3 X 0.5 3.5 X 2.5 X 1 3.5 X 2 X 2 3.5 X 1.5 X 1.2
Abdomen Abdomen Canal (low) Canal Canal
" " " " "
"
R
3 X 2.5
"
"
R
Canal (high) Abdomen
"
R
3.5 X 3 X 1.8
"
2.9
Outside ext. ring Canal (low)
"
R
2. 7
X
1.5
2.5
X
2 X 1.9
"
Seminoma (early)
tubules (single small focus)
Immature
Immature tubules (few small foci)
"
• Where bilateral cryptorchidism existed the examined gonad is indicated by the capital letter. t In the absence of exact measurements the available.surgical or pathological description is given.
The majority of the patients showed no evidence of constitutional or endocrine disease. Associated congenital anomalies were present in 4 infants (cases 2, 4, 5 and 6). Two individuals aged 2 months and 27 years (cases 3 and 19) were male pseudohermaphrodites. A 13 year old patient (case 13) was a true hermaphrodite. The gonads are included in the present study because they were composed almost entirely of testicular tissue. Prepuberal hypogonadism was present in a 23 year old man (case 18). Involvement by malignant tumor occurred in 3 instances (cases 26, 29 and 33). A group of 64 normal scrotal testes derived from 59 patients served as controls. These ranged in age from prematurity (5½ months) to 59 years. Testicular tissue from 32 patients up to the age of 17 years was obtained at autopsy. The remainder was provided by open biopsy or ablation. Specimens which showed gross or microscopic evidence of disease, atrophy or fibrosis were excluded. Despite the limitations imposed on precise cytological analysis by postmortem examination, especially in persons succumbing to a fatal illness, the material obtained from this source compared favorably with the surgical preparations for the purposes of this investigation. The testicular specimens were fixed in 10 per cent formaldehyde in most of the cases. A few of those obtained by biopsy were fixed in Bouin's solution. RESULTS
After the prepuberal period. Areas of seminiferous tubules of a prepuberal type (containing undifferentiated rather than Sertoli cells) were identified in the undescended gonads of 14 out of 28 individuals aged 17 years or more and in 3 out of 5 patients ranging between 12 and 15 years of age. As observed in 17 cryptorchid patients beyond the prepuberal era, the number and distribution of immature tubules displayed considerable variation. In
696
ARTHUR R. SOHVAL
the majority of cases they were found singly or in small groups consisting of 3 to 20 tubules lying closely together (fig. 1, A). Although the immature tubules were always subnormal in size for the age of the individual their actual diameters exhibited considerable variation from case to case. Varying degrees of tubular immaturity were manifested. Marked immaturity was characterized by smaller solid tubules. Their cell population was usually quite dense causing the tubules to appear filled with nuclei (fig. 1, B). Somewhat less immaturity was manifested by tubules which tended to be larger, slightly tortuous and to contain relatively fewer cells. The latter were usually present in layers of one to three cell thickness around the periphery of the tubule often with their long axes perpendicular to the basement membrane. Gradual lessening in the degree of immaturity was characterized by further increase in tubular diameter and tortuosity and progressive changes in the undifferentiated nuclei. In addition to an overall increase in size, the nuclei were more often oval than round. However, they were occasionally quite narrow and elongated with a pronounced tendency to be arranged at right angles to the enveloping basement membrane. This created, at times, a striking palisade-effect. The focal grouping of immature tubules presented a distinctive variation in 3 patients (cases 20, 26, 30). In contrast with the other cases showing discrete circular or oval cross sections of tubules lying singly or in islands, the tubules in these cases were closely set in compact, sharply circumscribed units with a nodular or lobulated appearance (fig. 2). The nuclei of the undifferentiated cells were numerous and exhibited considerable pleomorphism. Palisading of narrow elongated nuclei was especially conspicuous in the narrower elongated tubules. The presence of spermatogonia could be established with certainty in the immature tubules in but one patient, a 12 year old boy (case 10). In no instance were definite lumens found in immature tubules. In approximately one-half of the cases one or more immature tubules contained eosinophilic, colloid-like bodies (fig. 2, A). A unique finding was present in the immature tubules of case 26 (fig. 2, B). Most of this intra-abdominal testis was destroyed by seminoma. In the grossly and microscopically uninvolved portion near the surface there were several groups of nodular units of compact immature tubules. Occupying the central portion of many of these tubules were large oval, fairly homogeneous masses. Although the partially eosinophilic character of these masses created a superficial resemblance to the colloid-like bodies they were not identical. In addition to being larger and exerting pressure on adjacent cells, they had the morphologic and staining characteristics of calcium. An outstanding feature of the immature tubules was the comparative freedom of their basement membranes from significant fibrotic thickening. This was in marked contrast to the sclerosis of mature tubules in the same section of most adult cryptorchid testes. Sharing this partial immunity to the deleterious effects of cryptorchidism were the undifferentiated cells of the immature tubules. In general their nuclear integrity was well preserved. This was in decided contrast to the findings in most mature tubules. In addition to progressive fibrosis the
CRYPTORCHIDISM
697
Fm. 1. A, nest of immature seminiferous tubules in intra-abdominal testis of a 52 year old man (case 40). Marked immaturity is characterized by very small solid tubules densely packed with undifferentiated cells. Spermatogonia are absent and there is negligible thickening of basement membranes. Below are a few markedly fibrotic and hyalinized tubules showing no or little residual lumen. (X 200) B, Large number of closely set immature tubules in the canalicular testis of a 28 year old man (case 21). Tubules are small, solid and densely packed with undifferentiated cells having. ovoid or elongated nuclei. Their basement membranes are slightly thickened. Immediately adjacent on left is a group of tubules which had previously matured to extent that they contain differentiated Sertoli cells and spermatogonia. These tubules are considerably larger, have lumens, contain relatively fewer cells and show moderate fibrosis of basement membranes and peritubular connective tissue. Leydig cells are conspicuously grouped in dense collagenous intertubular tissue. Latter tends to penetrate adjacent mass of immature tubules. (X 100)
698
ARTHUR R. SOHVAL
Frn. 2. A, sharply circumscribed unit of compact immature tubules in inguinal testis of a 36 year old man (case 30). Basement membranes of individual tubules are not always distinct. Intratubular epithelial cells are numerous and exclusively of undifferentiated type. They exhibit considerable pleomorphism and occasional palisading. Central colloidlike bodies are present. Adjacent tubules show marked fibrosis and hyalinization. (X 200) B, sharply circumscribed units of compact immature tubules in intra-abdominal testis of a 30 year old man (case 26). Most of this gonad was destroyed by seminoma but this section is from uninvolved portion near surface. Large calcifi.c concretions have formed in several tubules causing pressure on adjacent undifferentiated cells. Fragmentation is an artefact. Spermatogonia cannot be identified with certainty. Basement membranes of component tubules are indistinct in some places and slightly thickened in others. (X 200)
CRYPTORCHIDISM
699
latter usually showed a significant decrease in cell population and residual Sertoli cells were often in a state of degeneration. Prepuberal period. The retained testicles of 7 boys ranging in age from prematurity to 7 years showed little, if any, deviation from the histologic pattern of normal scrotal glands of corresponding ages. In two boys of late prepuberal age, however, notable differences were encountered. The canalicular testis of a 10 year old boy (case 8) was approximately twothirds the size of its scrotal mate. Although there was histologic evidence of early maturation in both testicles this was decidedly retarded in the retained gonad where spermatogonia were also less numerous. In another 10 year old boy (case 9) the tubules of the inguinal testis were completely lacking in spermatogonia. The tubules were tightly packed with numerous undifferentiated cells, some of which were enlarging. However, none showed plasmosomes indicative of Bertoli cell differentiation. DISCUSSION
The recognition of testicular dysgenesis depends upon two major considerations. Firstly, seminiferous tubules of the prepuberal type (containing undifferentiated rather than Sertoli cells) are not encountered in the normal scrotal testicle beyond the prepuberal age. Their presence in the gonad of an adult is, therefore, an indication of immaturity. In the absence of a recognizable constitutional, metabolic or endocrine disorder this finding denotes an inherent developmental defect of a congenital nature. This conclusion is equally valid for the cryptorchid testis where tubular immaturity is readily distinguishable from tubular regression, atrophy and fibrosis. The latter are degenerative changes secondary to maldescent. Although it is difficult to prove that tubular immaturity in the undescended testis is not a consequence of cryptorchidism per se the weight of the evidence suggests that the converse is true. Equally essential for the identification of certain types of testicular dysgenesis is the verified absence of cytological components such as spermatogonia, Sertoli or Leydig cells. This must not be the result of postnatal local or systemic noxious influences. Furthermore, the age of the patient with respect to puberty is of utmost importance. Before puberty, spermatogonia are present in the tubules of the normal scrotal as well as the cryptorchid testis. Their total absence in normally or abnormally situated gonads in this age period is pathological and, therefore, congenital in origin. After the completion of puberty, Sertoli and Leydig cells are found in scrotal and retained glands although their number and distribution may be disturbed in the latter. The partial or complete absence of Sertoli cells or the total absence of Leydig cells from the adult testis (under the conditions stated above) is indicative of an inherent congenital defect. In the present study of cryptorchid testes three varieties of testicular dysgenesis were recognizable histologically. In descending order of frequency these were: I) Various degrees of tubular immaturity occurring after the prepuberal age. 2) Total absence of spermatogonia just before and during the puberal period. 3) Complete absence of spermatogonia, Sertoli and Leydig cells in an adult.
700
ARTHUR R. SOHVAL
There was no definite correlation between the type or extent of defective gonadogenesis and the degree of maldescent. Dysgenetic testicular lesions were approximately evenly divided among abdominal and canalicular glands. Seminijerous tubular immaturity. This was the most frequent type of congenital testicular defect encountered in this series. Seminal tubules of a prepuberal type were identified in the retained gonads in 17 out of 33 individuals beyond the age of 10 years. The high incidence of this type of tubular immaturity and its absence from a control group of 39 normal scrotal testes from 34 patients of corresponding ages emphasize the relationship between testicular maldevelopment and maldescent. The presence of immature seminiferous tubules in the adult cryptorchid testis has been mentioned previously by Uffreduzzi3 and by Sniffen. 14 Immature tubules have also been described16 in the descended testes of certain men with the Klinefelter-Reifenstein-Albright syndrome16 and in other forms of hypogonadism.17 These observations indicate that a testis may descend into the scrotum despite its congenital defectiveness. The finding of colloid-like bodies centrally situated in some immature tubules is consistent with their prepuberal character. These structures are apparently identical with those first described in the tubules of prepuberal normal scrotal testes by Blumensaat.18 The significance of the intratubular calcific concretions in case 26 remains unclear. They seem to correspond to the "stones" first reported in postpuberal testicular tubules by Oiye. 19 It is particularly noteworthy that the immature tubules appear to exhibit considerable resistance to the sclerosing and destructive effects of cryptorchidism. This contrasts sharply with the fibrotic obliteration and epithelial degeneration of tubules which had previously matured. The reason for this relative immunity is not clear but the fact itself is important with respect to the cause of the immaturity. Tubules, like other body structures, fail to develop because they are inherently incompetent or because they are inhibited or unstimulated. The injurious effects of cryptorchidism on the prepuberal testis are. meager and difficult to recognize while those seen after puberty are numerous and conspicuous. It is, therefore, evident that the deleterious effects occur principally after the tubules have matured. For this reason it is most unlikely that cryptorchidism inhibits maturation of prepuberal tubules to the extent of preventing transformation of their undifferentiated cells into Sertoli cells. If this were not true one would expect at least as much destruction of immature as of mature tubules. In the absence of other local or constitutional factors the conclusion is inescap~ able that tubular immaturity in the postpuberal testis is due to an intrinsic congenital defect. Further support to this concept is given by the great paucity of germinal cells Sniffen, R. C.: Ann. N. Y. Acad. Sci., 56: 609-618, 1952. Sniffen, R. C., Howard, R. P., and Simmons, F. A.: A. M.A. Arch. Path., 61: 293311, 1951. 16 Klinefelter, H.F., Jr., Reifenstein, E. C., Jr. and Albright, F.: J. Clin. Endocrin., 2: 615--627, 1942. 17 Sohval, A. R. and Soffer, L. J.: J. Clin. Endocrin. & Metab., 13: 408-414, 1953. 18 Blumensaat, C.: Virch. Arch. f. path. Anat. u Physiol., 273: 51-61, 1929. 19 Oiye, T.: Beitr. z. path. Anat. u.z. allg. Path., 80: 479-495, 1928. 14
15
CRYPTORCHIDISM
701
within the immature seminal tubules. Here, again, this observation cannot be ascribed to the injurious effects of cryptorchidism since residual spermatogonia were readily found elsewhere in the same sections in sclerotic tubules which had previously matured. It seems reasonable to conclude that the absence of germinal cells in these immature tubules is additional evidence of their primordial defectiveness. The sharply circumscribed units of compact immature tubules noted in cases 20, 26 and 30 merit special comment. Under low-power magnification the microscopic appearance of these nodular structures bears a striking resemblance to that of the testicular adenomas described and illustrated by Pick20 and Lecene and Chevassu21 in undescended testes. However, it is evid.ent that these 2 types of nodular formations in cryptorchid testes are unrelated. The apparent similarity of the nodular lesions in the present cases to newgrowth formation is underscored by a report in the literature. 22 In this instance it appears that characteristic microscopic foci of immature tubules were interpreted as early adenocarcinoma in retained testes. In another report, Scully and Parham23 describe and illustrate a lesion in a cryptorchid testis as a testicular adenoma, with the reservation that it is probably not a true tumor. In this instance, too, the lesion appears to be a characteristic circumscribed focus of immature tubules. Absence of specific cytological components. Additional manifestations of testicular dysgenesis were encountered in 3 patients whose undescended gonads were completely lacking in one or more types of specific cellular elements. Spermatogonia could not be identified in any of the tubules of the inguinal testis of a 10 year old boy (case 9). While it is not uncommon to find a diminished number of germ cells in cryptorchid testes in the late prepuberal or early adolescent era,7• 24 their complete disappearance as a result of malposition is not definitely known to occur before late adolescence. At the age of 10 years, therefore, it is most unlikely that the absence of spermatogonia is attributable to cryptorchidism per se. Since there was no history or evidence of local or systemic disease which might destroy germinal cells, their absence in this case is presumably due to a congenital defect. This type of lesion was first described in otherwise normal scrotal testes by Engle25 who later26 termed it germinal aplasia. Engle 27 and others28 , 29 • 30 believe it to be of congenital origin when known germ cell antagonists can be excluded. Pick, L.: Arch. f. Gynak., 76: 191-281, 1905. Lecene, P. and Chevassu, M.: Rev. de Chir., Paris, 35: 234-244, 1907. 22 Pace, J.M. and Cabot, H.: Surg., Gynec. & Obst., 63: 16-22, 1936. 23 Scully, R. E. and Parham, A. R.: Arch. Path., 46: 229-242, 1948. 24 Cooper, E. R. A.: J. Anat., 64: 5-27, 1929. 26 Engle, E. T.: The testis and hormones. In Problems of Ageing, ed. 2. Baltimore: Williams and Wilkins Co., 1942, pp. 475-494. 26 Engle, E.T.: J. Urol., 57: 789-798, 1947. 27 Engle, E.T. and Southam, A.: Endocrine aspects of infertility in the male. In Progress in Clinical Endocrinology, ed. by Soskin, S., New York: Grune & Stratton, 1950, pp. 455461. 28 Trabucco, A.: Medicina, Buenos Aires, 5: 369-387, 1945. 29 del Castillo, E. B., Trabucco, A. and de la Balze, F. A.: J. Clin. Endocrin., 7: 493-502, 1947. 80 Sohval, A. R. and Soffer, L. J.: Am. J. Med., 14: 328-348, 1953. 20 21
702
ARTHUR R, SOHVAL
Absence of spermatogonia was also established in both abdominal gonads of a 13 year old hermaphrodite (case 13). The sex glands of this patient were included in the study because they consisted preponderantly of testicular tissue. However, a few scattered microscopic foci of typical ovarian stroma lacking in ova or follicles indicate that the gonads are actually ovotestes. The absence of spermatogonia is regarded as due to germinal aplasia for the same reasons that applied in the previous case. An inherent defect in gonadogenesis readily explains the multiplicity of congenital lesions in this patient (germinal aplasia, focal tubular immaturity and ovarian stroma). Absence of spermatogonia, Sertoli and Leydig cells in a 23 year old man with prepuberal hypogonadism (case 18) indicated the most marked degree of testicular dysgenesis encountered in this series. This patient has been described in detail elsewhere.81 SUMMARY AND CONCLUSIONS
The incompletely descended gonads of 42 patients were studied in an e:ffort to elucidate the relationship between maldevelopment and maldescent of the testicle. Special attention was devoted to the detection of histologic evidence of defective gonadogenesis (testicular dysgenesis). Three varieties of testicular dysgenesis were encountered. The most frequent type was seminiferous tubular immaturity occurring after the prepuberal age. This was noted in approximately one-half of the cases. Much less frequent was another type of congenital lesion characterized by complete absence of spermatogonia (germinal aplasia) just before and during the puberal period. The least frequent and most profound developmental defect was found in the rudimentary testis of an hypogonadal adult. In this instance germinal cells, Sertoli and Leydig cells were absent. The high incidence of dysgenetic testicular lesions in this series of undescended testes supports the view that testicular imperfection is of etiologic significance in some cases of cryptorchidism. I am grateful to Dr. Sadao Otani who kindly placed the microscopic sections at my disposal.
1155 Park Ave., New York, N. Y. 31
Sohval, A. R. and Soffer, L. J.: J. Olin. Endocrin. & Metab., 12: 1229-1238, 1952.
Vol. 72, No. 4, October 1954 Printed in U.S.A.
TESTICULAR DYSGENESIS AS AN ETIOLOGIC FACTOR IN CRYPTORCHIDISM ARTHUR R. SOHVAL From the Endocrine Laboratory and Clinic of the Department of Medicine, the Mount Sinai Hospital, New York, N. Y.
It has long been conjectured that inherent disorders of the testis may be related to or responsible for maldescent. The pioneer observations of John Hunter (1728-1793) led him to suspect that congenital testicular imperfection prevents .descent and interferes with normal function. 1 In 1910 Bland-Sutton2 revived /the concept that a testis is retained because it is imperfect. Subsequently, other workers 3 • 4 • 6 • 6 subscribed to the belief that testicular "infantilism", "undevel'opment" or "hypoplasia" may play a role in the etiology of cryptorchidism. N elson7 has recently stated the view that in certain cases undescended testes may be an expression of defective development. The indefinite state of our knowledge of this problem is reflected in the fact that various urological texttJooks8-11 and review articles12 • 13 fail to mention it. In an effort to shed further light on the relationship between testicular mald . relopment and maldescent, a series of cryptorchid glands was subjected to 0\ m!!-tic histologic analysis. The purpose of the present communication is to sys e 1 the results of this investigation in which microscopic evidence of testicurecorc . d . h . .fi f s· h fi d" Iar d YSl ~enesis was .encountere wit s1g111 cant requency. mce t ese n mgs t observed m a control group of normal scrotal testes from correspond. 1og1c . importance . were no no . d s 1t . 1s . fe1t t h at t h ey may represent f actors of et10 . mg age pe. . . in some ca&'es of cryptorch1d1sm. MATERIAL AND METHOD
T bl 1 enun 1erates the pertinent data of 42 patients whose imperfectly descen~e~ gonads w~re studied: The age range ~s from prematuri!y (7 months) to 78 ears. The tm,~+,1cular spemmens were obtamed at necropsy m 7 cases. In 35 patknts fresh testic ular tissue was provided surgically, 6 by biopsy and 29 by 0
ablation. A ted for publicatio11 September 10, 1953. , H~Eter, J.: A descriptie'n of the situation of ~he testis in t!ie foetus, with its descent into the scrotum. In The w 01 ·ks of John Hunter with Notes. Edited by Palmer, J. F., Lonn Rees Orme r3rown, Green and Longman. 1837, vol. 4 pp. 15, 17. d .L on· ongma , ' '· · oner 84 · 19-30 1910 2 Bland-Su~ton,_J.: Practiti_ 1. Chir:, 100: 1151-ilSO, 1913. ,4 E Uffreduzz1 0 .. Arch. f. kln · · 1 d man. of H obd ay, F . : C ryptorc h'd' 1 ism 1n amma s an cc l es, w'. McA . .. Discussi ~n ,. 923 Proc. Roy. Soc. Med., 17: 3-17," · . 5 Staemmler, M.: Arch. f. path. _Ana~., 245. 304-321, 1923. . . H R and Slotopolsky,, B .. Deutsche Ztschr. f. Chir., 188. 76-100, 1924. • S h' 7 Nc 1mz, w· o ·. Recent Progress ,;n Hormone Research, 6: 29-56, 1951. s A·.; Urological Path?Jogy. Philadelphia: Le.a & Febiger, 1952, vol. 2. 9 C bell M . Clinical Pediatrw Urology. Philadelphia: W. B. Saunders Co., 1951. 10 Rcl~ck, 'H. The Practice of Urology. Philadelphia: J. B. Lippincott Co., 1949,
n:rbi~t p:
·c.:
voli, iowsley,
o. s. and Kirwin, T. J.: 'Clinical Urology.
Baltimore: Williams and Wilkins
Coif ~ 4~by W C . New Eng. J. Med., 2t'.12: 701-704, 1945. 1s L~~is, t. G.: "j. Urol., 60: 345-356, H}48. 693
694
ARTHUR R. SOHVAL
TABLE
Case No.
Age
1. Perti"nent data on 42 patients with undescended gonads Sizet (cm.)
Side*
I
Location
Method of Obtaining Specimen
-~ R
0.9
Canal
2
Premature (7 mos.) 2 days
R
0.9
Abdomen
3
2 mos.
rL
-
Canal
Biopsy
4
4
"
RI
1
Autopsy
5
6
"
RI
1.1
Abdomen (R) Canal (L) Abdomen
6
7
"
RI
1.3
Abdomen
7 yrs. 10 "
RI L
1 1.1
l
7
10 12
"
L
10
"
R
1.3 2
Canal Canal (mid) Canal Abdomen
11
13
"
RI
1.5
Abdomen
12
13 13
"
L RL
-
Canal (mid) Abdomen
8 9
13
"
1 x 0.7 x 0.5 (R) 1.5 x 1.2 x 0.8 (L)
-
15
15 17
" "
L L
2.5
X
16
18
"
L
3
2.5
17 18
21 23
"
R
3.5
"
RI
19
27
"
Rl
20
28
"
L
2
21
28
"
R
asmall"
22 23
29 30
"
"
R L
24
30
"
R
25
30
"
R
26
30
"
R
Tumor 140 g.
27
31
"
R
3
28
" "
L R
"fair 11
29
33 35
Tumor9x7x6
30
36
"
R
4X 2X 1
Canal (high) Abdomen Outside ext. ring Canal
31
36
"
rL
"fair"
Canal
14
X
X
1.5
X
1
2
X
2.5 X 2.5 0.5
X
"
" Ablation
" Biopsy
Germinal aplasia Immature tubules (majority) Immature tubules (about 50 per cent)
Shortness stature
r .)
" Ablation
Biopsy Ablation
Canal
"
Canal Canal (high)
"
Germinal aplasia. Immature tubules (rare small foci)
Hermaphro, lite
Immature tubules (many small foci) Immature tubules (single large focus)
"
Canal
"
Canal Canal
"
2.5
Abdomen
"
3
Outside ext. ring Abdomen
"
1.5
Multiple congenital anomalies Congenital en· cephalopathy'·
"
Ablation
"smalF 1
Remarks
Multiple congenital anomalies Pseudohermaphrodite Multiple congenital anomalies
"
Canal
1
1.8
2
"
Biopsy
X
Evidence of Dysgenesis
Autopsy
Outside ext. ring
"normal"
X
Canal (low) Abdomen
I
"
"
" "
" BlOpsy
Absence of spermatogonia, Bertoli and Ley dig cells
Immature tut ,ules (few small f~ ;ci) Immature tubules (about 4'J per cent)
Prepuberal hypogonadism Pseudohermaph rodite Dwarfism
Immatt/ue tubules (mea~hy small foci) Imr 11'ature tubules (o· ccasional small foci)
Immature tubules (rare large com pact groups) Immature tubules (many small foci)
Immature tubules (many small foci)
Seminoma
Malignant teratoma
695
CRYPTORCHIDISM TABLE
Case No.
Age
-
Sizet
Side•
(cm.)
1.-Continued
Location
Method of Obtaining Specimen
Evidence of Dysgenesis
Canal (low)
Ablation
Immature tubules (few small foci) Immature tubules (about 20 per cent)
Remarks
-32
38 yrs.
33
39
"
R
34 35 36 37 38
39 41 42 42 48
" " " "
L
39
50
40
52
41
52
42
78
L
''atrophic''
Abdomen
"
"
R R R R
4.5 X 3 X 2.5 3 X 0.5 3.5 X 2.5 X 1 3.5 X 2 X 2 3.5 X 1.5 X 1.2
Abdomen Abdomen Canal (low) Canal Canal
" " " " "
"
R
3 X 2.5
"
"
R
Canal (high) Abdomen
"
R
3.5 X 3 X 1.8
"
2.9
Outside ext. ring Canal (low)
"
R
2. 7
X
1.5
2.5
X
2 X 1.9
"
Seminoma (early)
tubules (single small focus)
Immature
Immature tubules (few small foci)
"
• Where bilateral cryptorchidism existed the examined gonad is indicated by the capital letter. t In the absence of exact measurements the available.surgical or pathological description is given.
The majority of the patients showed no evidence of constitutional or endocrine disease. Associated congenital anomalies were present in 4 infants (cases 2, 4, 5 and 6). Two individuals aged 2 months and 27 years (cases 3 and 19) were male pseudohermaphrodites. A 13 year old patient (case 13) was a true hermaphrodite. The gonads are included in the present study because they were composed almost entirely of testicular tissue. Prepuberal hypogonadism was present in a 23 year old man (case 18). Involvement by malignant tumor occurred in 3 instances (cases 26, 29 and 33). A group of 64 normal scrotal testes derived from 59 patients served as controls. These ranged in age from prematurity (5½ months) to 59 years. Testicular tissue from 32 patients up to the age of 17 years was obtained at autopsy. The remainder was provided by open biopsy or ablation. Specimens which showed gross or microscopic evidence of disease, atrophy or fibrosis were excluded. Despite the limitations imposed on precise cytological analysis by postmortem examination, especially in persons succumbing to a fatal illness, the material obtained from this source compared favorably with the surgical preparations for the purposes of this investigation. The testicular specimens were fixed in 10 per cent formaldehyde in most of the cases. A few of those obtained by biopsy were fixed in Bouin's solution. RESULTS
After the prepuberal period. Areas of seminiferous tubules of a prepuberal type (containing undifferentiated rather than Sertoli cells) were identified in the undescended gonads of 14 out of 28 individuals aged 17 years or more and in 3 out of 5 patients ranging between 12 and 15 years of age. As observed in 17 cryptorchid patients beyond the prepuberal era, the number and distribution of immature tubules displayed considerable variation. In
696
ARTHUR R. SOHVAL
the majority of cases they were found singly or in small groups consisting of 3 to 20 tubules lying closely together (fig. 1, A). Although the immature tubules were always subnormal in size for the age of the individual their actual diameters exhibited considerable variation from case to case. Varying degrees of tubular immaturity were manifested. Marked immaturity was characterized by smaller solid tubules. Their cell population was usually quite dense causing the tubules to appear filled with nuclei (fig. 1, B). Somewhat less immaturity was manifested by tubules which tended to be larger, slightly tortuous and to contain relatively fewer cells. The latter were usually present in layers of one to three cell thickness around the periphery of the tubule often with their long axes perpendicular to the basement membrane. Gradual lessening in the degree of immaturity was characterized by further increase in tubular diameter and tortuosity and progressive changes in the undifferentiated nuclei. In addition to an overall increase in size, the nuclei were more often oval than round. However, they were occasionally quite narrow and elongated with a pronounced tendency to be arranged at right angles to the enveloping basement membrane. This created, at times, a striking palisade-effect. The focal grouping of immature tubules presented a distinctive variation in 3 patients (cases 20, 26, 30). In contrast with the other cases showing discrete circular or oval cross sections of tubules lying singly or in islands, the tubules in these cases were closely set in compact, sharply circumscribed units with a nodular or lobulated appearance (fig. 2). The nuclei of the undifferentiated cells were numerous and exhibited considerable pleomorphism. Palisading of narrow elongated nuclei was especially conspicuous in the narrower elongated tubules. The presence of spermatogonia could be established with certainty in the immature tubules in but one patient, a 12 year old boy (case 10). In no instance were definite lumens found in immature tubules. In approximately one-half of the cases one or more immature tubules contained eosinophilic, colloid-like bodies (fig. 2, A). A unique finding was present in the immature tubules of case 26 (fig. 2, B). Most of this intra-abdominal testis was destroyed by seminoma. In the grossly and microscopically uninvolved portion near the surface there were several groups of nodular units of compact immature tubules. Occupying the central portion of many of these tubules were large oval, fairly homogeneous masses. Although the partially eosinophilic character of these masses created a superficial resemblance to the colloid-like bodies they were not identical. In addition to being larger and exerting pressure on adjacent cells, they had the morphologic and staining characteristics of calcium. An outstanding feature of the immature tubules was the comparative freedom of their basement membranes from significant fibrotic thickening. This was in marked contrast to the sclerosis of mature tubules in the same section of most adult cryptorchid testes. Sharing this partial immunity to the deleterious effects of cryptorchidism were the undifferentiated cells of the immature tubules. In general their nuclear integrity was well preserved. This was in decided contrast to the findings in most mature tubules. In addition to progressive fibrosis the
CRYPTORCHIDISM
697
Fm. 1. A, nest of immature seminiferous tubules in intra-abdominal testis of a 52 year old man (case 40). Marked immaturity is characterized by very small solid tubules densely packed with undifferentiated cells. Spermatogonia are absent and there is negligible thickening of basement membranes. Below are a few markedly fibrotic and hyalinized tubules showing no or little residual lumen. (X 200) B, Large number of closely set immature tubules in the canalicular testis of a 28 year old man (case 21). Tubules are small, solid and densely packed with undifferentiated cells having. ovoid or elongated nuclei. Their basement membranes are slightly thickened. Immediately adjacent on left is a group of tubules which had previously matured to extent that they contain differentiated Sertoli cells and spermatogonia. These tubules are considerably larger, have lumens, contain relatively fewer cells and show moderate fibrosis of basement membranes and peritubular connective tissue. Leydig cells are conspicuously grouped in dense collagenous intertubular tissue. Latter tends to penetrate adjacent mass of immature tubules. (X 100)
698
ARTHUR R. SOHVAL
Frn. 2. A, sharply circumscribed unit of compact immature tubules in inguinal testis of a 36 year old man (case 30). Basement membranes of individual tubules are not always distinct. Intratubular epithelial cells are numerous and exclusively of undifferentiated type. They exhibit considerable pleomorphism and occasional palisading. Central colloidlike bodies are present. Adjacent tubules show marked fibrosis and hyalinization. (X 200) B, sharply circumscribed units of compact immature tubules in intra-abdominal testis of a 30 year old man (case 26). Most of this gonad was destroyed by seminoma but this section is from uninvolved portion near surface. Large calcifi.c concretions have formed in several tubules causing pressure on adjacent undifferentiated cells. Fragmentation is an artefact. Spermatogonia cannot be identified with certainty. Basement membranes of component tubules are indistinct in some places and slightly thickened in others. (X 200)
CRYPTORCHIDISM
699
latter usually showed a significant decrease in cell population and residual Sertoli cells were often in a state of degeneration. Prepuberal period. The retained testicles of 7 boys ranging in age from prematurity to 7 years showed little, if any, deviation from the histologic pattern of normal scrotal glands of corresponding ages. In two boys of late prepuberal age, however, notable differences were encountered. The canalicular testis of a 10 year old boy (case 8) was approximately twothirds the size of its scrotal mate. Although there was histologic evidence of early maturation in both testicles this was decidedly retarded in the retained gonad where spermatogonia were also less numerous. In another 10 year old boy (case 9) the tubules of the inguinal testis were completely lacking in spermatogonia. The tubules were tightly packed with numerous undifferentiated cells, some of which were enlarging. However, none showed plasmosomes indicative of Bertoli cell differentiation. DISCUSSION
The recognition of testicular dysgenesis depends upon two major considerations. Firstly, seminiferous tubules of the prepuberal type (containing undifferentiated rather than Sertoli cells) are not encountered in the normal scrotal testicle beyond the prepuberal age. Their presence in the gonad of an adult is, therefore, an indication of immaturity. In the absence of a recognizable constitutional, metabolic or endocrine disorder this finding denotes an inherent developmental defect of a congenital nature. This conclusion is equally valid for the cryptorchid testis where tubular immaturity is readily distinguishable from tubular regression, atrophy and fibrosis. The latter are degenerative changes secondary to maldescent. Although it is difficult to prove that tubular immaturity in the undescended testis is not a consequence of cryptorchidism per se the weight of the evidence suggests that the converse is true. Equally essential for the identification of certain types of testicular dysgenesis is the verified absence of cytological components such as spermatogonia, Sertoli or Leydig cells. This must not be the result of postnatal local or systemic noxious influences. Furthermore, the age of the patient with respect to puberty is of utmost importance. Before puberty, spermatogonia are present in the tubules of the normal scrotal as well as the cryptorchid testis. Their total absence in normally or abnormally situated gonads in this age period is pathological and, therefore, congenital in origin. After the completion of puberty, Sertoli and Leydig cells are found in scrotal and retained glands although their number and distribution may be disturbed in the latter. The partial or complete absence of Sertoli cells or the total absence of Leydig cells from the adult testis (under the conditions stated above) is indicative of an inherent congenital defect. In the present study of cryptorchid testes three varieties of testicular dysgenesis were recognizable histologically. In descending order of frequency these were: I) Various degrees of tubular immaturity occurring after the prepuberal age. 2) Total absence of spermatogonia just before and during the puberal period. 3) Complete absence of spermatogonia, Sertoli and Leydig cells in an adult.
700
ARTHUR R. SOHVAL
There was no definite correlation between the type or extent of defective gonadogenesis and the degree of maldescent. Dysgenetic testicular lesions were approximately evenly divided among abdominal and canalicular glands. Seminijerous tubular immaturity. This was the most frequent type of congenital testicular defect encountered in this series. Seminal tubules of a prepuberal type were identified in the retained gonads in 17 out of 33 individuals beyond the age of 10 years. The high incidence of this type of tubular immaturity and its absence from a control group of 39 normal scrotal testes from 34 patients of corresponding ages emphasize the relationship between testicular maldevelopment and maldescent. The presence of immature seminiferous tubules in the adult cryptorchid testis has been mentioned previously by Uffreduzzi3 and by Sniffen. 14 Immature tubules have also been described16 in the descended testes of certain men with the Klinefelter-Reifenstein-Albright syndrome16 and in other forms of hypogonadism.17 These observations indicate that a testis may descend into the scrotum despite its congenital defectiveness. The finding of colloid-like bodies centrally situated in some immature tubules is consistent with their prepuberal character. These structures are apparently identical with those first described in the tubules of prepuberal normal scrotal testes by Blumensaat.18 The significance of the intratubular calcific concretions in case 26 remains unclear. They seem to correspond to the "stones" first reported in postpuberal testicular tubules by Oiye. 19 It is particularly noteworthy that the immature tubules appear to exhibit considerable resistance to the sclerosing and destructive effects of cryptorchidism. This contrasts sharply with the fibrotic obliteration and epithelial degeneration of tubules which had previously matured. The reason for this relative immunity is not clear but the fact itself is important with respect to the cause of the immaturity. Tubules, like other body structures, fail to develop because they are inherently incompetent or because they are inhibited or unstimulated. The injurious effects of cryptorchidism on the prepuberal testis are. meager and difficult to recognize while those seen after puberty are numerous and conspicuous. It is, therefore, evident that the deleterious effects occur principally after the tubules have matured. For this reason it is most unlikely that cryptorchidism inhibits maturation of prepuberal tubules to the extent of preventing transformation of their undifferentiated cells into Sertoli cells. If this were not true one would expect at least as much destruction of immature as of mature tubules. In the absence of other local or constitutional factors the conclusion is inescap~ able that tubular immaturity in the postpuberal testis is due to an intrinsic congenital defect. Further support to this concept is given by the great paucity of germinal cells Sniffen, R. C.: Ann. N. Y. Acad. Sci., 56: 609-618, 1952. Sniffen, R. C., Howard, R. P., and Simmons, F. A.: A. M.A. Arch. Path., 61: 293311, 1951. 16 Klinefelter, H.F., Jr., Reifenstein, E. C., Jr. and Albright, F.: J. Clin. Endocrin., 2: 615--627, 1942. 17 Sohval, A. R. and Soffer, L. J.: J. Clin. Endocrin. & Metab., 13: 408-414, 1953. 18 Blumensaat, C.: Virch. Arch. f. path. Anat. u Physiol., 273: 51-61, 1929. 19 Oiye, T.: Beitr. z. path. Anat. u.z. allg. Path., 80: 479-495, 1928. 14
15
CRYPTORCHIDISM
701
within the immature seminal tubules. Here, again, this observation cannot be ascribed to the injurious effects of cryptorchidism since residual spermatogonia were readily found elsewhere in the same sections in sclerotic tubules which had previously matured. It seems reasonable to conclude that the absence of germinal cells in these immature tubules is additional evidence of their primordial defectiveness. The sharply circumscribed units of compact immature tubules noted in cases 20, 26 and 30 merit special comment. Under low-power magnification the microscopic appearance of these nodular structures bears a striking resemblance to that of the testicular adenomas described and illustrated by Pick20 and Lecene and Chevassu21 in undescended testes. However, it is evid.ent that these 2 types of nodular formations in cryptorchid testes are unrelated. The apparent similarity of the nodular lesions in the present cases to newgrowth formation is underscored by a report in the literature. 22 In this instance it appears that characteristic microscopic foci of immature tubules were interpreted as early adenocarcinoma in retained testes. In another report, Scully and Parham23 describe and illustrate a lesion in a cryptorchid testis as a testicular adenoma, with the reservation that it is probably not a true tumor. In this instance, too, the lesion appears to be a characteristic circumscribed focus of immature tubules. Absence of specific cytological components. Additional manifestations of testicular dysgenesis were encountered in 3 patients whose undescended gonads were completely lacking in one or more types of specific cellular elements. Spermatogonia could not be identified in any of the tubules of the inguinal testis of a 10 year old boy (case 9). While it is not uncommon to find a diminished number of germ cells in cryptorchid testes in the late prepuberal or early adolescent era,7• 24 their complete disappearance as a result of malposition is not definitely known to occur before late adolescence. At the age of 10 years, therefore, it is most unlikely that the absence of spermatogonia is attributable to cryptorchidism per se. Since there was no history or evidence of local or systemic disease which might destroy germinal cells, their absence in this case is presumably due to a congenital defect. This type of lesion was first described in otherwise normal scrotal testes by Engle25 who later26 termed it germinal aplasia. Engle 27 and others28 , 29 • 30 believe it to be of congenital origin when known germ cell antagonists can be excluded. Pick, L.: Arch. f. Gynak., 76: 191-281, 1905. Lecene, P. and Chevassu, M.: Rev. de Chir., Paris, 35: 234-244, 1907. 22 Pace, J.M. and Cabot, H.: Surg., Gynec. & Obst., 63: 16-22, 1936. 23 Scully, R. E. and Parham, A. R.: Arch. Path., 46: 229-242, 1948. 24 Cooper, E. R. A.: J. Anat., 64: 5-27, 1929. 26 Engle, E. T.: The testis and hormones. In Problems of Ageing, ed. 2. Baltimore: Williams and Wilkins Co., 1942, pp. 475-494. 26 Engle, E.T.: J. Urol., 57: 789-798, 1947. 27 Engle, E.T. and Southam, A.: Endocrine aspects of infertility in the male. In Progress in Clinical Endocrinology, ed. by Soskin, S., New York: Grune & Stratton, 1950, pp. 455461. 28 Trabucco, A.: Medicina, Buenos Aires, 5: 369-387, 1945. 29 del Castillo, E. B., Trabucco, A. and de la Balze, F. A.: J. Clin. Endocrin., 7: 493-502, 1947. 80 Sohval, A. R. and Soffer, L. J.: Am. J. Med., 14: 328-348, 1953. 20 21
702
ARTHUR R, SOHVAL
Absence of spermatogonia was also established in both abdominal gonads of a 13 year old hermaphrodite (case 13). The sex glands of this patient were included in the study because they consisted preponderantly of testicular tissue. However, a few scattered microscopic foci of typical ovarian stroma lacking in ova or follicles indicate that the gonads are actually ovotestes. The absence of spermatogonia is regarded as due to germinal aplasia for the same reasons that applied in the previous case. An inherent defect in gonadogenesis readily explains the multiplicity of congenital lesions in this patient (germinal aplasia, focal tubular immaturity and ovarian stroma). Absence of spermatogonia, Sertoli and Leydig cells in a 23 year old man with prepuberal hypogonadism (case 18) indicated the most marked degree of testicular dysgenesis encountered in this series. This patient has been described in detail elsewhere.81 SUMMARY AND CONCLUSIONS
The incompletely descended gonads of 42 patients were studied in an e:ffort to elucidate the relationship between maldevelopment and maldescent of the testicle. Special attention was devoted to the detection of histologic evidence of defective gonadogenesis (testicular dysgenesis). Three varieties of testicular dysgenesis were encountered. The most frequent type was seminiferous tubular immaturity occurring after the prepuberal age. This was noted in approximately one-half of the cases. Much less frequent was another type of congenital lesion characterized by complete absence of spermatogonia (germinal aplasia) just before and during the puberal period. The least frequent and most profound developmental defect was found in the rudimentary testis of an hypogonadal adult. In this instance germinal cells, Sertoli and Leydig cells were absent. The high incidence of dysgenetic testicular lesions in this series of undescended testes supports the view that testicular imperfection is of etiologic significance in some cases of cryptorchidism. I am grateful to Dr. Sadao Otani who kindly placed the microscopic sections at my disposal.
1155 Park Ave., New York, N. Y. 31
Sohval, A. R. and Soffer, L. J.: J. Olin. Endocrin. & Metab., 12: 1229-1238, 1952.