ABNORMAL SPERMATOGENESIS IN XYY MALES: A REPORT ON 4 CASES ASCERTAINED THROUGH A POPULATION STUDY*

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1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No.5, May 1973 Printed in U.S.A. ABNORMAL SPERMATOGENESIS IN XYY MALES: A REPORT ON 4 CASES ASCERTAINED THROUGH A POPULATION STUDY* NIELS E. SKAKKEBAEK, M.D., EVA ZEUTHEN, M.D., JOHANNES NIELSEN, M.D., AND HANS YDE, M.D. The Chromosome Laboratory, Department of Obstetrics and Gynaecology, Rigshospitalet, University of Copenhagen, the Cytogenetic Laboratory, Aarhus State Hospital, Risskov, Denmark, and Department of Medicine, M. the County Hospital, Randers, Denmark During the last couple of years several reports have indicated that spermatogenesis in XYY males may be impaired. I - IO Skakkebaek et al. 9 recently concluded that XYY males in general have a much higher risk of testicular failure than males in the general population, but also stated that most of the examined cases were found in institutions and therefore the findings might be biased. We shall here report on a quantitative, histologic analysis of the seminiferous epithelium in four XYY males found in a prevalence study of XYY males in the general population. All had abnormal seminiferous epithelium. MATERIALS AND METHODS Four 21-year-old men with the chromosome constitution 47, XYY were investigated. They were found in a prevalence study of chromosome abnormalities in the general population carried out by Eva Zeuthen and Johannes Nielsen from the Cytogenetic Laboratory, Aarhus State Hospital, Risskov (to be published). The four men are listed in Table 1. No evidence of chromosome mosaicism was found in any of them as judged from analysis of 30 or more cells from lymphocyte cultures. All four had normal secondary sex characteristics. The testes, measured by Prader's orchidometer, were of normal size in 2 cases. One (115) had Received October 12, * Supported by grants from the Danish Medical Research Council and the Danish National Board of Social Wellfare, Department of Mental Retardation. 390 large testes, each measuring approximately 40 cc. (normal range according to Prader ll cc.). One (105) had one testis below the normal range (6 cc.) and one of normal size (20 cc.). (The biopsy from the latter subject originated from the testis of normal size.) Unilateral testicular biopsies, removed under local anaesthesia, were fixed in Cleland's fixative, embedded in paraplast, and serially sectioned at 4 J.L according to a previously reported method. 12 The tubule method of Rowley and Heller 13 was used for quantitative analysis of the seminiferous epithelium. This differential count is based upon the description of the epithelium by Clermont 14 and Heller and Clermont. 15 All counts were performed by the first author. A differential count of germ cells and Sertoli cells was made on 30 randomly chosen tubule cross sections in the biopsy from Subject 115, which contained only one category of tubules. In the remaining subjects more than one category of tubules were present (e.g., tubules with complete spermatogenesis and tubules with spermatogenic arrest). In these cases separate counts were made on 30 tubules from each category. Tubules containing only Sertoli cells were excluded from the counts. The total number of counted germ cells of each of the different types was divided by the total number of Sertoli cells found in the same cross sections of tubules. The resultant values are referred to as the Sertoli cell ratio (SCR). In biopsies with a heterogeneous pic-..

2 ~ May 1973 ABNORMAL SPERMATOGENESIS IN XYY MALES 391 TABLE 1. Case Age Marital Sperm Leydig Children status' count cellst Tubules with:j: C A S SG+S mill./cc. % S 143 N S 13 N M One son 13 N S N ~ * S, single; M, married. t C, complete spermatogenesis; A, spermatogenic arrest; S, Sertoli cells only; SG + S, spermatogonia and Sertoli cells. tn, normal. ture, an estimate of the distribution of tubules of each category, e.g., tubules with spermatogenic arrest and Sertoli-cell-only tubules, was obtained by classification of all tubules in one randomly chosen section (Table 1). The findings were compared with similar studies in a control group of 21 men with normal chromosome complements and normal sperm counts. 16 The semen specimens were examined by the method of Hammen. 17 Further details on the four men will be published separately (Eva Zeuthen and Johannes Nielsen.) RESULTS TABLE 2. Sertoli Cell Ratios from X Y Men and a Control Group* Case All four men had impaired spermatogenesis. In Subject 115 only one category of tubules was found. All types of germ cells were present. The number of late spermatids was, however, slightly reduced (Tables 1 and 2). A heterogeneous picture was seen in Subjects 275, 113, and 105 (Figs. 1 and 2 and Tables 1 and 2). Subject 275 had four categories of tubules: tubules with complete spermatogenesis (40%); tubules with arrest at spermatocyte level (37%); tubules containing spermatogonia and Sertoli cells (13%); and tubules containing Sertoli cells only (10%). Subject 113 showed three categories of tubules: tubules with complete spermatogenesis (14%); tubules with arrest at the sper- Spermatocytes Spermatids Spermatogonia PLT Liz + P Sa + 1 Sc + Sb Sd A. Tubules from a homogeneous biopsy No. of Sertoli cells B. Tubules from heterogeneous biopsies Tubules with spermatogenic arrest Tubules with complete spermatogenesis C. Tubules from a control group Mean Range { * PL, preleptotene; L, leptotene; Z, zygotene; and P, pachytene spermatocytes. Sa, Sb, Sc, and Sd are consecutive steps in the maturation process of spermatids. matocyte level (59%); and tubules containing Sertoli cells only (27%). Subject 105 had three categories of tubules: tubules with complete spermatogenesis (38%); tubules with arrest at spermatocyte level (50%); and tubules containing spermatogonia and Sertoli cells only (12%).

3 392 SKAKKEBAEK ET AL. Vol. 24 FIG. 1. Section of a testicular biopsy from a male with the 47, XYY chromosome complement (Case 113). x 250. N, tubule containing all types of germ cells. SA, tubule with a complete arrest of spermatogenesis at primary spermatocyte level. SS, tubule containing only Sertoli cells and a few spermatogonia. FIG. 2. Same as Fig. 1. High power magnification of a tubule with arrest of spermatogenesis at primary spermatocyte level. x Note the degenerated germ cell (D).

4 , May 1973 ABNORMAL SPERMATOGENESIS IN XYY MALES 393 The results from the differential counts of germ cells are presented in Table 2. The tubules with complete spermatogenesis contained a reduced number of late spermatids in Subject 275. On the other hand, in Subjects 113 and 105 all types of germ cells were found to be within normal limits in the category of tubules with complete spermatogenesis. No attempt was made to count the cell content of tubules containing spermatogonia and Sertoli cells, because the biopsies were too small for a statistical evaluation. All biopsies appeared to have normal numbers of Leydig cells. Actual counts were not made. The semen analysis in Subject 115 showed 143 million spermatozoa/mi., 49% motile cells with fair motility and 24% morphologically abnormal cells. The semen specimen in Subject 275 showed 13 million/mi., 21% motile cells with poor motility and 82% morphologically abnormal cells, and the spermiogram of Subject 113 had the following characteristics: 13 million cells/mi., 60% motile cells with fair motility and 48% morphologically abnormal cells. The motility was examined from 21/4-4 hr. after ejaculation due to transport of the specimens to the laboratory. No specimen could be obtained from Subject 105, and it was not possible to perform repeated analysis in any of the cases. DISCUSSION The present study supports the assumption that the seminiferous epithelium is impaired in the majority of XYY men. Including the present study, the spermatogenesis has been studied in a total of 28 men with a 47, XYY karyotype and only 5 of these were reported to have normal spermatogenesis The majority of previously investigated XYY men constitutes a selected group since they have been discovered through surveys in institutions for mentally retarded or criminals. However, it appears that XYY men ascertained in the general population may have a similar pathologic picture of the seminiferous epithelium. All of the four men in the present study showed abnormalities of the seminiferous epithelium. Three of the men had very severe changes with arrest of spermatogenesis at spermatocyte level and tubules containing Sertoli cells only. Tubules containing spermatogonia and Sertoli cells were also seen. It appears to be characteristic for XYY men to have a considerable variation within the same testis, where the different categories of pathologic tubules are often found together or in combination with tubules containing all types of germ cells. Thus, tubules with spermatogenic arrest at spermatocyte level have been found in more than 50% of all XYY men and Sertoli-cell-only tubules have been found in approximately 30%. These findings are, however, not pathognomonic for XYY men, since the same variation within a testis may be found in infertile 46, XY men, although apparently with a lower frequency. 18 Men with autosomal abnormalities may also show such abnormalities. 19 An apparent discrepancy between the sperm count and the testicular histology was found in one of the men, who showed a slight inhibition of spermatogenesis in spite of a normal sperm count. This subject (115) had, however, unusually large testes and it is possible that this may compensate for the moderate impairment of spermatogenesis. The pathogenesis of the testicular failure in XYY men is not known. The possibility that inhibition at the spermatid level is caused by less viability of germ cells with unbalanced genetic material should be considered. However, unbalanced male gametes may participate in formation of the zygote. Furthermore, meiotic studies on XYY men have shown an apparent absence of the second Y

5 394 SKAKKEBAEK ET AL. Vol. 24 chromosome in the majority of primary spermatocytes.3. 4, 7, It may be that the function of the Leydig cells is abnormal, although this remains to be shown. The concentration of luteinizing hormone, which stimulates the Leydig cells was reported to be increased in plasma of some XYY males Other investigators could, however, not confirm these findings One of the men of the present study (Case 113) had a son, although severe changes of the seminiferous epithelium were present. Several other XYY men have produced offspring.27 This does not, however, exclude that XYY men in general are subfertile. Few of these men apparently seek advice in fertility clinics The reason may be that only a few are married and desire to have children. In conclusion, the present study supported the assumption that the seminiferous epithelium is impaired in the majority ofxyy men. Furthermore, preliminary evidence is provided that the histology of the seminiferous epithelium of XYY men in the general population is similar to that of previously investigated XYY men, the majority of whom were found in institutions for mentally handicapped or criminals. SUMMARY Four XYY men, ascertained through a population study, were investigated by a quantitative, histologic analysis of the seminiferous epithelium. All had abnormal seminiferous epithelium. Three of the cases showed severe impairment including arrest at the primary spermatocyte level, tubules containing Sertoli cells only and tubules containing Sertoli cells and spermatogonia. One of the subjects showed a slight reduction in the number of late spermatids, although all types of germ cells were present. The results correlated well with sperm counts, except in the last case with extremely large testes and a normal sperm count in spite of the slight impairment of spermatogenesis. All biopsies appeared to have normal numbers of Leydig cells. The investigation supported the assumption that the seminiferous epithelium is impaired in the majority of XYY men. REFERENCES 1. NIELSEN, J., CHRISTENSEN, A.-L., JOHNSEN, S. Go, AND FR0LAND, A. Psychopathology and testis histology in a patient with the XYY syndrome. Acta Med Scand 180:747, BALODIMOS, M. C., LISCO, H., IRWIN, I., MERRILL, W., AND DINGMAN, J. F. XYY karyotype in a case of familial hypogonadism. J Clin Endocr 26:443, TETTENBORN, U., SCHWINGER, E., AND GROPP, A. Testicular function in men with XYY chromosomes. Deutsch Med Wschr. 95:158, : HULT~N, M. Meiosis in XYY men. Lancet 1:7l7, SKAKKEBAEK, N. E., PHILIP, J., MIKKELSEN, M., HAMMEN, R., NIELSEN, J., PERB0LL, 0., AND YDE, H. Studies on spermatogenesis meiotic chromosomes and sperm morphology in two males with a 47, XYY chromosome complement. Fertil Steril 21:645, 1970.' 6. SHAPIRO, L. R. Hormones and the XYY male. Lancet 1:1347, TETTENBORN, U., GROPP, A., MURKEN, J.-D., TINNEFELD, W., FUHRMANN, W., AND SCHWINGER, E. Meiosis and testicular histology in XYY males. Lancet 2:267, SANTEN, J. R., DE KRETSER, D. M., PAULSEN, C. A., AND VERHEES, J. Gonadotrophins and testosterone in the XYY syndrome. Lancet 2:371, SKAKKEBAEK, N. E., HULT~N, M., JACOBSEN, P., AND MIKKELSEN, M. Quantification of human seminiferous epithelium. II. Histological studies in eight 47, XYY men. J Reprod Fertil 32:391, BENEZECH, M., ROBERT, G., LUCIANI, J.-M., AND NOEL, B. Etude somatique, psychiatrique et hormonale avec histologie testiculaire de 6 psychopathes XYY. Inform Psychiat 48:175, PRADER, A. Testicular size: Assessment and clinical importance. Triangle 7:240, ROWLEY, M. J., AND HELLER, C. G. The testicular biopsy: surgical procedure, fixation and staining technics. Fertil Steril 17:177, ROWLEY, M. J., AND HELLER, Co G. Quantification of the cells of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant. Z Zellforsch 115:461,

6 May 1973 ABNORMAL SPERMATOGENESIS IN XYY MALES CLERMONT, Y. The cycle of the seminiferous epithelium in man. Amer J Anat 112:35, HELLER, C. G., AND CLERMONT, Y. Kinetics of the germinal epithelium in man. Recent Progr Hormone Res 20:545, SKAKKEBAEK, N. E., AND HELLER, C. G. Quantification of human seminiferous epithelium. I. Histological studies in 21 fertile men with normal chromosome complements. J Reprod Fertil 32: 379, HAMMEN, R. Impaired Fertility in Man with Special Reference to the Male. Munksgaard, Copenhagen, SKAKKEBAEK, N. E., HAMMEN, R., PHILIP, J., AND REBBE, H. Quantification of human seminiferous epithelium. III. Histological studies in 44 infertile men with normal chromosome complements. Acta Path Microbiol Scand 81:97, SKAKKEBAEK, N. E., HUL'nfN, M., AND PHILIP, J. Quantification of human seminiferous epithelium. IV. Histological studies in 17 men with numerical and structural autosomal aberrations. Acta Path Microbiol Scand 81:112, THOMPSON, H., MELNYK, J., AND HECHT, F. Reproduction and meiosis in XYY. Lancet 2:831, MELNYK, J., THOMPSON, H., RUCCI, A. J., VANASEK, F., AND HAYES, S. Failure of transmission of the extra chromosome in subjects with 47, XYY karyotype. Lancet 2:797, Hsu, L. Y., SHAPIRO, L. R., AND HIRSCHHORN, K. Meiosis in an XYY male. Lancet 1:1173, HULTtN, M., AND PEARSON, P. L. Fluorescent evidence for spermatocytes with two Y chromosomes in an XYY male. Ann Hum Genet 34:273, PAPANICOLAOU, A. D., KIRKHAM, K. E., AND LORAINE, J. A. Abnormalities in urinary gonadotrophin excretion in men with a 47, XYY sex chromosome constitution. Lancet 2:608, PARKER, C. E. Luteinizing hormone in XYY men. Lancet 1:1101, HUDSON, B., BURGER, H., WIENER, S., SUTHER LAND, G., AND BARTHOLOMEW, A. A. Plasma testosterone and luteinizing hormone in XYY men. Lancet 2:699, COURT BROWN, W. M. Males with an XYY sex chromosome complement. J Med Genet 5:341, KJESSLER, B. Karyotype, Meiosis and Spermatogenesis in a Sample of Men Attending an Infertility Clinic. S. Karger, Basel, PHILIP, J., SKAKKEBAEK, N. E., HAMMEN, R., JOHNSEN, S. G., AND REBBE, H. Cytogenetic investigations in male infertility. Acta Obstet Gynec Scand 49:235, KJESSLER, B. "Chromosomes and Gametic Output in 1000 Infertile Males." In Excerpta Med. Int. Congr. Ser. 234b VII World Congress on Fertility and Sterility. Tokyo, Kyoto, October, 1971, p. 141.