Chromatin-Positive Klinefelter's Syndrome

Transcription

1 Chromatin-Positive Klinefelter's Syndrome A Quantitative Analysis of Spermatogonial Deficiency at 3, 4, and 12 Months of Age K. MIKAMO, D.Se., M. AGUERCIF, M.D., P. HAZEGHI, M.D., and R. MARTIN-Du PAN, M.D. LSTICULAR DYSGENESIS in the chromatin-positive syndrome of Klinefelter has been described as occurring long before puberty. Ten-month-old patients were reported by Lanman et al. and by Lawrence and Yuceoglu, and 8 patients from 7 to 12 years of age were reported by Ferguson-Smith. 4 A common abnormality of the testes of prepubertal patients is the relative reduction in the number of spermatogonia. Both for elucidation of the causes determining the morphologic character of the syndrome and the physiology of the abnormal testes, it should be of interest to determine the earliest stages in neonatal or in embryonic development at which this germcell deficiency might be detectable. Recently we have had an opportunity to study testicular histology in 3 infant patients with 47 jxxy karyotype syndrome in whom testicular biopsies were performed at 3, 4, and 12 months of age, respectively. MATERIALS AND METHODS The patients reported in the paper are 3 of 4 cases of chromatin-positive males found in an investigation of nuclear sex determination in a nonselected newborn population (2649 males and 2403 females) delivered at our hospital. The nuclear sexes of all 5052 babies were studied through the use of fetal membranes that had been preserved in 95% alcohol immediately after From the Department of Gynecology and Obstetrics and the Department of Pediatrics, University of Geneva, Geneva, Switzerland. Supported by the Ford Foundation Population Program, Grant 64-4,11. We express our thanks to Prof. E. Witschi, Anatomy Institute, University of Basel, for his many important sug,;estions and for revising the paper. We are indebted to Prof. H. de Watteville and Dr. R. Borth of the senior author's. department for their interest and help in!nany ways, especially for providing clinical and laboratory accommodation. Our thanks arc also due to Drs. A. Cuendet and G. Stern who operated on the patients. 731

2 732 MIKAMO ET AL. FERTILITY & STERILITY the infant's birth and stained with acetic orcein. Usually epithelial and connective tissue of amnion were utilized, but in cases with advanced degeneration of these tissues, chorionic connective tissue or buccal mucosa were used. When chromatin-positive male babies were thus found, further confirmation of the nuclear sex was sought through the study of the infants' buccal mucosa, and karyotype analysis was performed with cultured leukocytes. Testicular biopsies were performed at 90 days (Case 1), 120 days (Case 2), and 12 months (Case 3). Immediately after the operation, biopsy specimens were preserved for 5 hr. in Zenker's fluid. The tissues were sectioned at 10 JL and stained with Harris' hematoxylin and eosin, or with Heidenhain's azan. The cellular elements of the seminiferous tubules were classified and analyzed quantitatively by the method of Mancini et al. The relative numbers of spermatogonial cells and undifferentiated Sertoli cells were compared with results the authors obtained in examining scrotal testes in normal control populations of approximately the same age. OBSERVATIONS Case 1. The patient was born as the second child of a 27-year-old mother who at the age of 22 had given live birth to a normal female and had at the age of 23 recorded a spontaneous abortion in the first trimester. In the present instance, during pregnancy the mother had monosymptomatic toxicosis. The breech delivery was accomplished by Bracht's maneuver. The child weighed 3690 gm. at birth. Shortly after birth, bilateral hydrocele was noticed. Pallor, sudation, and unconsciousness were observed at 5 months of age. Blood pressure was found to be 11 cm. Hg (flush) and 12.5/8 (manometer). However, renal functions (urea and creatinine clearances) and morphology (intravenous pyelogram) were normal, and the blood electrolytes, plasma renin activity, and urinary excretion of 17-ketosteroids, 17-hydroxy corticosteroids, aldosterone, ca techolamines, and vanillylmandelic acid were found also to be within normal range. No heart disease could be diagnosed. Several EEG recordings were considered to be within normal limits. Pneumoencephalography revealed the presence of an interventricular cistern. Somatic growth was in the normal range. In cytogenetic study, a single chromatin body had been found at high frequency in the fetal membranes obtained at birth. After birth the buccal mucosa, as well as the testicular tissue, were chromatin positive. The karyotype in leukocytes was found to be 47/X.XY. At the time of biopsy, an accumulation of fluid between tunica vllginalis and tunica albuginea was observed, which had made the size of scrotum (Fig. la)

3 VOL. 19, No.5, 1968 SPERMATOGONIAL DEFICIENCY 733 considerably larger than that of normal individuals. Size and appearance of the testis itself on the operated side (right) seemed normal (Fig. IB). The testicular tissue appeared normal with regard to types of spermatogonia, undifferentiated Sertoli cells, and interstitial tissue (Fig. Ie). The quantitative study, however, revealed an abnormality. Spermatogonia and undifferentiated Sertoli cells were counted in 300 seminiferous tubules sectioned transversely (or nearly so). The number of germ cells in each tubule thus counted ranged from o to 7 (average, 3). When compared with the control study by Mancini et al. (Table 1), the relative number of spermatogonial cells was only 21% of that in scrotal testes of suckling babies up to 60 days old and 24% of that of infants over 60 days and up to 4 years. The count is significantly lower even taking into account the individual variation in their material (personal communication from Fig. IA. Genitalia of Case 1, go-day-old infant with 47 jxxy chromosomal constitution. Large scrotum and normal penis. (X 0.6) B. Exposed right testis after opening of tunica vaginalis and drainage of considerable fluid. Normal proportions. (X 0.6) C. Testicular biopsy. Relative scarcity of spermatogonia. Otherwise, no remarkable abnormality. (Zenker's fluid, H&E, 10 p., X 200)

4 734 MIKAMO ET AL. FERTILITY & STERILITY TABLE 1. Number of Spermatogonia and Undifferentiated Sertoli Cells per 50 Seminiferous Tubules in Infant Patients with 47/XXY Karyotype* Cases Postnatal (up to 60 days; avo of 7 control cases)9 Case 1: 90-day-old Case 2: 120-day-old Case 3: 12-month-old Infants up to 4 years (av. of 16 control cases)9 Spermatogonia Undifferentiated Sertoli cells Diameter of tulrnle (p.) * The tissue was sectioned at 5 p. in the material of Mancini et al. and at 10 p. in our Case~ 1, 2, and 3. The counts in the latter were divided by 2 to correct for the difference in thickne:,;s Dr. Mancini). Even if the numerical correction should not result in the precisely comparative values (Table 1), the low count in the present case seems significant. The number of undifferentiated Sertoli cells and the diameter of tubules (54 p.) are not strikingly different from those of the control. Case 2. The patient was born as the first child of a 22-year-old mother whose 2 previous pregnancies were interrupted in the :6.rst trimester by curettage (at ages 19 and 20 years). The present pregnancy occurred shortly after a treatment involving insumation of oviducts. The pregnancy and delivery progressed normally. The bhild weighed 2860 gm. at birth. Psychomotor development was recorded to be normal, as were physical findings-except for abnormal external genitalia (Fig. 2A).. The right testis had incompletely descended, and hydrocele of the left was found at biopsy. The penis was smaller than normal. Cytogenetic investigation revealed a single sex-chromatin body at high frequency in the fetal membranes, buccal mucosa, and testicular tissue. The karyotype was 47 jxxy in leukocytes and skin. The testicular biopsy was obtained from the descended left testis at the subject's age of 4 months. As to the external appearance, the testis was very similar to that of Case 1. A considerable amount of fluid had accumulated between tunica vaginalis and tunica albuginea (Fig. 2B). However, the testis itself appeared normal externally (Fig. 2C). No remarkable histologic abnormality was detected except for a reduced number of germ cells (Fig. 2D). Spermatogonia and undifferentiated Sertoli cells were counted in 300 seminiferous tubules in the same way as in the previous case. The number of germ cells in each tubule ranged from 0 to 8 (average, 2.3). Compared with result of control testes counted by Mancini et al. (Table 1), the relative number of the spermatogonia was only 16% of that of the suckling babies up to 60 days old and 18% of that of infants over 60 days and up to 4 years old. As was true of Case 1, there was no striking difference between Case 2 and the control cases in the number of undifferentiated Sertoli cells and in the diameter of seminiferous tubules (Table 1).

5 Vo.L. 19, No.. 5, 1968 SPERMATo.Go.NIAL DEFICIENCY' 735 Case 3. This was the second child born to a 27-year-old mother who recorded 2 previous pregnancies at 23 and 25 years; the first had resulted in a liveborn normal male but the second was terminated in a spontaneous abortion in the first trimester. Delivery in the present case was accomplished by cesarean section because of fetal distress following early rupture of the fetal membranes. The child was externally normal and weighed 3140 gm.. After birth, no abnormality was found in physical and psychomotor development. A single chromatin body was found at high frequency in the fetal membranes, buccal mucosa, and testicular tissue. The karyotype in leukocytes was 47/XXY. Fig. 2A. Genitalia of Case 2, 120-day-old infant with 47/XXY chromosomal constitution. Unilaterally descended testicles and small penis. (X 0.6) B. Left testis before opening of tunica vaginalis shows considerable enlargement caused by accumulation of fluid between tunica vaginalis and albuginea. (X 0.6) C. Mter opening of tunica vaginalis. Normal size. (X 0.6) D. Testicular biopsy. Relative scarcity of spermatogonia. Otherwise, no remarkable abnormality. (Zenker's fluid, H&E, 10 /L, X 2(0)

6 736 MIKAMO ET AL. FERTILITY & STERILITY Both testes were descended normally. The operated testis (right) appeared externally normal. The deficiency of spermatogonia, however, was much more marked than in the previous cases. Spermatogonia were completely absent in the majority of the' sections through seminiferous tubules (Fig. 3). In 250 sections Fig. 3. Testicular biopsy of Case 3, 12-monthold infant with 47/XXY chromosomal constitution. Marked scarcity of spermatogonia. Area selected to demonstrate spermatogonia (arrows). (Zenker's fluid, H&E, 10 p., X 200) of tubules selected at random, there were found only 2 spermatogonia. The count was not more than 0.1% of that in the normal testes (Table 1). This unequivocal deficiency is similar to the findings previously reported in some cases 4, 7, 8 of chromatin-positive male children. The diameter of the tubules ( 43 p.) was smaller than in the normal individuals of about the same age (53 p. in the specimens from the studies of Mancini et al.). The number of undifferentiated Sertoli cells and the aspect of the interstitial tissue seemed normal. DISCUSSION Owing to the difficulty of collecting a sufficient number of specimens with adequate quality, the normal postnatal development of the human testis has not been well established. Reports so far published are mainly based on the specimens obtained at autopsy or by biopsy of testes of children whose causes of death or disease are not sufficiently clear, In this relative uncertainty there are several control studies,1, 3, 9, 15 although some do not provide a description of the quantity and morphologic criteria of spermatogonial cells during the first few years of life, The study by Mancini et al. was chosen to evaluate our cases because of the applied quantitative method of analysis based on careful classification of types of spermatogonia, and because of' a relatively large number olspecimens from young children,

7 VOL. 19, No.5, 1968 SPERMATOGONIAL DEFICIENCY 737 In the normal development of the human testes, the relative number of germ cells remains approximately stationary from a fetal phase through the postnatal life before puberty. 9 Other investigators have noted almost unchanged or very little progress of tubular development during the first 4 or 5 years-though the reports do not supply any clear documentation regarding actual or relative number of germ cells. 1, 3, 15 Because in our cases reduction of spermatogonia was noticeable as early as 3 and 4 months after birth, prenatal initiation of germ-cell deficiency seems not unlikely. We noted no indication {)f a particular factor (or factors) that may have caused drastic reduction in the number of spermatogonia following birth. When the 3 cases are compared regarding the spermatogonial deficiency, the question arises whether the difference between them should be attributed to the subjects' age difference alone; this is not necessarily so, since great variation is known to exist even between cases of similar ages. 4,13 Several factors may be responsible for the wide variation in testicular histology. These factors may include the following: 1. Hereditary control of germ-cell development, such as individual differences in the initial number of primordial germ cells migrating into the germinal ridge, and frequency of mitotic divisions and degree of degeneration during testicular development. 2. Differences in sex chromosome constitution, such as XXY, XXYY, XXj XXY, XY jxxjxxy, XY jxxy jxxyy, and XXY jxxyy, to mention only patterns with a single chromatin body. 3. Combination with autosomal aberration, such as in mongolism Overripeness of ova-as suggested by Witschi 16 from experimental evidence in amphibians. In the zygotes derived from overripe eggs, destructive effects on embryogenesis were seen which resulted in various developmental abnormalities and a germ-cell deficiency ranging from moderate to complete sterility. Effects on meiotic as well as on cleavage spindles were found to cause nondisjunction of chromosomes. 2, 10, 11, 17 Klinefelter's syndrome with a normal male karyotype (chromatin-negative Klinefelter's syndrome) is an interesting subject in relation to the effects of overripeness of ova because the deficiency of germ cells in such individuals may be caused by the same effects without being accompanied by chromosome aberration. Chromatin-negative Klinefelter's syndrome is not too rare an abnormality. Nelson found about 20% in 107 cases of clinically chosen patients with Klinefelter's syndrome. He implied that the primary cause of the testicular defect is a reduction in the germ-cell population'during embryonic life. The syndrome was suggested to be related to a high maternal age by Ferguson-Smith etal. 6 Effect of a high maternal age has been known in chromatin-positive Klinefelter syndrome and, more

8 738 MIKAMO ET AL. FERTILITY & STERILITY strikingly, in Klinefelter-mongol patients. 5 Biologically it can be supposed that aged women may increasingly develop mechanisms to produce both intrafollicular (preovulatory) and intratubal (postovulatory) overripeness of ova. Contemplated studies of embryonic stages may reveal whether the diminution starts at early fetal stages because of reduced viability of XXY germ cells or is also influenced by the same causes that bring about the abnormal chromosome constitution. In this aspect, a recent publication of Singh and Carr on histologic study of ovaries in XO human embryos and fetuses is particularly interesting. These workers seem to have discovered deficiency of follicle cells in specimens that were more than 75 mm. in crown-to-rump length. SUMMARY Three cases of chromatin-positive male infants (47 jxxy, Klinefelter's syndrome) were studied with biopsies of scrotal testes obtained at 90 days, 120 days, and 12 months of age, respectively. The relative number of spermatogonia in the first case was 24%, in the second 18%, and in the third 0.1% of that found in testes of a normal control population of similar age range (over 60 days and up to 4 years old) as reported by Mancini et al. Because of the early age of the patients-2 being younger than any so far reported-the possibility is considered that diminution of germ cells in the syndrome might start in a prenatal stage. The individual differences in relative spermatogonial depletion probably reflect quantitative variations in the physiologic factors that also bring about the chromosomal aberrations. Degrees of follicular and postovulatory overripeness of the eggs are proposed as the most likely causative factors. Lahoratoire d'hormonologie et d'emhryologie, Maternite 1211 Geneve 4, Switzerland REFERENCES 1. ALBERT, A., UNDERDAHL, L. 0., GREENE, L. F., and LORENZ, N. Male hypogonadism: I. The normal testis. Proc Mayo Clin 28:409, BUTCHER, R. L., and FUGo, N. W. Overripeness and the mammalian ova: II. Delayed ovulation and chromosome anomalies. Fertil Steril18:297, CHARNY, C. W., CONSTON, A. S., and MERANZE, D. R. Testicular developmental histology. Ann NY Acad Sci 55:597, FERGUSON-SMITH, M. A. The prepubertal testicular lesion in chromatin-positive Klinefelter's syndrome (primary micro-orchidism) as seen in mentally handicapped children. Lancet 1:219, FERGUSON-SMITH, M. A. "Sex Chromatin, Klinefelter's Syndrome and Mental Deficiency." In The Sex Chromatin, Moore, K. L., Ed. Saunders, Philadelphia, 1966, p.277.

9 VOL. 19, No.5, 1968 SPERMATOGONIAL DEFICIENCY FERGUSON-SMITH, M. A., MACK, W. S., ELLIS, P. M., and DICKSON, M. Chromosome analysis and parental age in the Del Castillo syndrome. Lancet 2: 1121, LANMAN, J. T., SKLARIN, B. S., COOPER, H. L., and HIRSCHHORN, K. Klinefelter's syndrome in a ten-month-old mongolian idiot: Report of a case with chromosome analysis. New Eng J Med 26.3:887, LAWRENCE, R, and YUCEOGLU, A. M. Seminiferous tubule dysgenesis: A case of Klinefelter's syndrome in a ten-month-old child. Arner J Dis Child 101:635, MANCINI, R E., NARBAITZ, R, and LAVIERI, J. C. Origin and development of germinative epithelium and Sertoli cells in the human testis: Cytological, cytochemical, and quantitative study. Anat Rec 1.36:477, MIKAMO, K. Overripeness of the eggs in Xenopus laevis Daudin. Arner Zool 2: 541, MIKAMO, K. Intrafollicular overripeness and teratologic development. Cytogenetics 7:212, NELSON, W. O. The Klinefelter syndrome. Fertil SteriI8:535, OVERZIER, C. "The So-called True Klinefelter's Syndrome." In 1ntersexuality, Overzier, c., Ed. Acad. Press, New York, 1963, p SINGH, R P., and CARR, D. H. The anatomy and histology of XO human embryos and fetuses. Anat Rec 155:369, SNIFFEN, R C. The testis: I. The normal testis. Arch Path (Chicago) 50:259, WITscm, E. Genetic and postgenetic sex determination. Experientia 16:274, WITSCHI, E., and LAGUENS, R Chromosomal aberraticns in embryos from overripe eggs. Develop BioI 7:605, 1963.