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Hospital for Children and Adolescents (A.M.W., T.R., S.W., L.D.), University of Helsinki, 00029 Helsinki, Finland; Biomedicum Helsinki, Institute of Biomedicine/Physiology (T.R.), University of Helsinki, 00014 Helsinki, Finland; Kindertagesklinik (F.H.), 4410 Liestal, Switzerland; and The Family Federation of Finland (T.T.), 00101 Helsinki, Finland
Address all correspondence and requests for reprints to: Leo Dunkel, Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, 00029 Helsinki, Finland. E-mail: leo.dunkel{at}hus.fi.
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Abstract |
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Fourteen nonmosaic 47,XXY boys (aged 10–14 yr) were enrolled. Every fourth month their puberty was staged, and serum was obtained for hormone analyses. Each boy underwent a single testicular biopsy. Biopsy specimens of seven peripubertal boys (testicular volume < 2.0 ml) had spermatogonia of adult type, whereas older boys with larger testes (> 2.0 ml) exhibited no germ cells. No meiotic germ cells were detectable in any of these subjects. Depletion of germ cells was associated with an increase in testicular volume but was not immediately reflected in levels of serum gonadotropin, inhibin B, or anti-Müllerian hormone. In contrast, hypergonadotropism and suppression of serum inhibin B and anti-Müllerian hormone developed later, during midpuberty, after an unequivocal increase in serum testosterone (>2.5 nmol/liter) levels and degeneration of Sertoli cells.
In conclusion, these prepubertal and early pubertal boys with KS had diploid germ cells that vanished in early puberty when testicular volume increased, whereas serum gonadotropin and inhibin B levels displayed pathological changes later during midpuberty.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Normally the estimated onset of release of spermatozoa (spermarche) occurs at a median age of 13.4 yr, at Tanner stage P2–3, and between testicular volumes of 4.7 and 19.5 ml (8, 9). Healthy boys may have their spermarche as early as at Tanner stage G2 (10). In boys with KS, testicular volume peaks at midpuberty, and thereafter testicular growth ceases, simultaneously with development of the hypergonadotropism typical of adult KS patients (11, 12). It is thus possible that at the time that healthy boys experience spermarche, testicular function in boys with KS is relatively normal. We investigated testicular biopsies of adolescent boys with KS for the presence of germ cells. Our aim was to evaluate whether germ cells for future use in ICSI can be obtained in early adolescence. In addition, we characterized how the morphological changes in the testis were reflected in pituitary-gonadal axis activation and especially in peripheral levels of the testicular-specific markers inhibin B and anti-Müllerian hormone (AMH).
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Subjects and Methods |
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Fourteen subjects with nonmosaic karyotype 47,XXY were enrolled from the outpatient clinic at the Hospital for Children and Adolescents, University of Helsinki. One boy (patient 14, Tables 1
and 2) was diagnosed by an amniocentesis. The other 13 boys were diagnosed between 5 and 10.5 yr of age by child neurologists. Karyotype analyses were performed for nonendocrinological indications (problems with speech, learning, and/or behavior). Before the start of systematic prospective surveillance, these patients had been followed up irregularly with visit intervals of 6–24 months in the same clinic. These visits included routine hormone analyses (measurements of gonadotropin and testosterone levels), physical examination with measurement of testicular size (width and length of testes measured with a ruler to the nearest millimeter), and assessment of Tanner pubertal stage (8). None of these patients had a history of previous cryptorchidism, nor were any on androgen therapy. Data from these routine clinical surveillance visits were collected from patient records and merged with data obtained during the prospective part of the study.
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The parents of each boy gave their informed consent for participation in this study that had been approved by the research ethics committee of the hospital.
Assays
The blood samples were drawn between 0830 and 1530 h. After clotting, the serum was separated by centrifugation and stored at –20 C until required. Serum FSH and LH levels were measured by ultrasensitive immunofluorometric assays, as previously described (15). FSH and LH concentrations of less than 0.1 IU/liter were treated as 0.1 IU/liter. The interassay coefficient of variation (CV) for FSH was less than 3.3% and for LH less than 4.4%. The intraassay CV for FSH was less than 4.4% and for LH less than 4.1%. Serum testosterone concentrations were measured by a RIA after separation of steroid fractions on Lipidx-5000 microcolumns (16, 17). The detection limit for testosterone was 0.1 nmol/liter. The interassay CV was less than 15% and the intraassay CV was less than 9%. Serum inhibin B (Serotec, Oxford, UK) and AMH (Immunotech-Coulter, Marseille, France) levels were measured by commercially available immunoenzymometric assays according to manufacturers’ instructions. The detection limit for inhibin B was 15.6 pg/ml. The interassay CV was less than 15% and the intraassay CV less than 5%. The detection limit for AMH was 5.5 pmol/liter, and the interassay CV was 13.4%.
Testicular biopsies
An open-knife testicular biopsy was taken under general anesthesia from each subject. The major portion of the specimen was cryopreserved for possible further use in ICSI in adulthood (see below). A piece was fixed in glutaraldehyde and then further subdivided and embedded in Epon, sectioned at 1.0 µm, and stained with toluidine blue. Histomorphometric analysis was performed by light microscopy at a total magnification of x400. The Leydig cells were morphologically classed as fetal, juvenile, or adult types by the following criteria: Leydig cells were regarded as fetal if they had an extrinsically located large nucleus with two or more nucleoli, juvenile if they had an irregular nucleus and dark cytoplasm, and adult if they were large cells with a round nucleus and a cytoplasm containing crystalloid and lipid droplets. The Sertoli cells were morphologically classed as Sa, Sb, and Sc types and were regarded as Sa type if they were round with scant cytoplasm and had a round nucleus, and Sb if they were oval and larger and had an irregular oval nucleus and cytoplasm with recognizable structures. They were classified as Sc or adult if they were large and had a nucleus displaying one or more deep invaginations (18, 19).
For all specimens containing germ cells, germ cell counts (number of adult type pale spermatogonia per seminiferous tubule, Ap/tubule, and number of adult type dark spermatogonia, Ad/tubule) were calculated per cross-section of tubule. Spermatogonia were regarded as type A if they were of an irregular shape with a round nucleus. Furthermore, they were regarded as Ap type if the nucleus had one or two nucleoli and as Ad if the nucleus had one or two pale round areas (19). All tubules from an average of nine sections (range 6–12) per biopsy specimen were studied. The average number of tubules studied per biopsy specimen was 130 (range 71–177).
Germ cell numbers were quantitatively compared with those of available normal testicular biopsies. Eleven identically prepared testicular specimens of adolescent boys were analyzed (11 yr, n = 5; 13 yr, n = 4; 15 yr, n = 1; and 19 yr, n = 1). Indications for these biopsies had been scrotal pain (n = 5), hydatid torsion (n = 3), retractile testis (n = 1), and paratesticular fibrosis (n = 1); one specimen was taken postmortem (accidental death).
For electron microscopy, one Epon-embedded specimen was sectioned at 50 nm with a Reichert E ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a JEOLJEM 1200 EX transmission electron microscope (JEOL, Tokyo, Japan).
Cryopreservation of testicular tissue
Testicular tissue was cut into small pieces in in vitro fertilization-Universal medium (Medicult, Copenhagen, Denmark) and diluted slowly drop by drop with equal volume of freezing medium (Irvine Scientific, Santa Ana, CA). The samples were frozen in 0.5-ml straws by exposure of the straws first for 15 min to + 4 C, then 15 min to –20 C, and for 45 min in nitrogen vapor before placing them into liquid nitrogen. Two to seven vials were created per patient.
Statistical analyses
Descriptive data are reported as medians and ranges or means. In those cases with no laboratory analyses at the time of testicular biopsy, values were interpolated from data obtained before and after biopsy with the assumption that changes between the two time points had been linear. The unpaired two-tailed Student’s t test was used for comparisons between groups; P < 0.05 was considered significant.
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Results |
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. These biopsies were obtained at a median age of 11.8 yr (range 10.1–14.0). Representative specimens from five KS boys and one boy with a normal karyotype are shown in Fig. 1. The subjects were divided into two groups according to the presence (group I) or absence (group II) of germ cells in the biopsies.
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Spermatogonia of Ap type were found in seven of 14 and of Ad type in six of 14 biopsy specimens (Table 1). In the specimens with germ cells present, the average number of Ap spermatogonia per seminiferous tubule was reduced; mean number of Ap spermatogonia was 0.77 (range 0.04–1.17), and average number of Ad spermatogonia per tubule was 0.01, whereas all normal controls had consistently more than 0.46 (mean 1.5) Ad spermatogonia per tubule (P < 0.001). No meiotically dividing germ cells (spermatocytes) or postmeiotic spermatids appeared in any of the biopsy specimens of the KS subjects. Furthermore, at the time of cryopreservation of testicular tissue, no spermatozoa were seen in any of the specimens. The parents and the older boys have received this information, and we have thoroughly discussed these results with them.
Sertoli cells, Leydig cells, and interstitial tissue
In the biopsy specimens of patients 1–10, the Sertoli cells were of Sa and Sb type and exhibited relatively normal appearance (Table 1, and Figs. 1, A and B, and 2). However, marked degeneration of Sertoli cells was evident in patients 11–14 (Table 1, Fig. 1, C and D). Boys 1–10 had Leydig cells of juvenile type that showed none or only moderate hyperplasia, whereas the older subjects 11–14 had huge hyperplastic Leydig cells (Table 1, Fig. 1, C and D). Group II could thus be subdivided into two groups based on absence (group IIA) or presence (group IIB) of Sertoli cell degeneration and Leydig cell hyperplasia. Fibrosis and hyalinization of the interstitium and peritubular connective tissue were visible in all groups; in addition, these signs of degeneration increased with age (Table 1). Figure 1, A–D, show the differing degrees of the degeneration process. The degeneration was not uniformly detectable throughout the relatively small biopsy specimens, whereas we found within the same biopsies areas with marked degeneration and areas with only moderate changes (Fig. 1E).
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Patients in group I (n = 7) (spermatogonia present in biopsy specimen) showed no signs of hypergonadotropic hypogonadism (Table 2). There occurred, however, an overlap in serum FSH, LH, testosterone, inhibin B, and AMH levels with group II (no spermatogonia in the specimen). Subjects in group IIA (n = 3) maintained normal gonadotropin, inhibin B, and AMH levels, whereas those in more advanced puberty (group IIB, n = 4) had clear hypergonadotropism and low levels of circulating Sertoli cell markers, inhibin B, and AMH (Table 2). Testicular volume was therefore the only statistically significant variable that could fully differentiate between groups I and II (Fig. 3A). Although differences in FSH and testosterone levels were not statistically significant between these two groups, levels were higher in group II (Fig. 3A and Table 2). Furthermore, all subjects with FSH and LH concentrations above 7 IU/liter showed depletion of germ cells and clear degeneration of Sertoli cells (i.e. findings consistent with group IIB) (Tables 1 and 2).
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Serum inhibin B increased in early puberty, but this initial rise was followed by a rapid suppression accompanied by a simultaneous increase in serum testosterone (Figs. 4 and 5). A strong, inverse nonlinear correlation existed between serum inhibin B and testosterone levels (Fig. 4B).
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and 5). There also existed a strong, inverse nonlinear relationship between serum AMH and testosterone levels (Figs. 4 and 5). Before testosterone had reached 2.5 nmol/liter, a marked drop in serum AMH had already occurred (Fig. 4B). |
Discussion |
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We found, surprisingly, that in early adolescence as many as 50% of the boys with KS had germ cells in their testes and that the depletion of these cells accelerated at the onset of puberty. This presence of germ cells during peripuberty contrasts with results of a report by Müller et al. (5), in which no germ cells were detectable in KS boys beyond the age of 2 yr. A probable explanation is that all boys in Müller’s publication were cryptorchid, whereas none in our group had any history of testicular maldescendent. Cryptorchidism is known to cause marked decrease in germ cell numbers also in boys with a normal karyotype (21, 22).
Ad spermatogonia that develop postnatally from fetal spermatogonia under gonadotropin and testosterone stimulation are of fundamental importance for the development of male fertility (23). In the boys with KS, the fact that the number of these cells was markedly reduced indicates a severely impaired fertility potential even in early puberty. Furthermore, we observed only type A spermatogonia in our boys with KS, which suggests an arrest in germ cell development at the stage of A spermatogonia. Normally, type A spermatogonia transform into type B spermatogonia and further to the first primary spermatocytes at the age of 5 yr (18, 24). At present, whether this defect in spermatogenesis in the XXY testis is intrinsic to germ cells or due to the inability of the Sertoli cells to support normal germ cell development is unknown. A defect in Sertoli and germ cell communication in the differentiating XXY testis has been suggested (25).
Activation of the hypothalamic pituitary testicular axis was associated with depletion of germ cells. A strong correlation appeared between an increasing testosterone level and suppression of the Sertoli cell markers inhibin B and AMH. It is possible that the molecular mechanisms induced by the altered dosage of X-encoded genes in testicular cells may accelerate loss of germ cells. For example, the androgen receptor gene is located on the X chromosome (26).
The testicular volume of KS boys is already below normal during childhood (1.0–1.5 ml; normally 1.8 ml) (27, 28). During puberty their testes grow (11, 12), which was also observed in the current work (Figs. 3 and 5). In healthy boys, the proliferation of germ cells is predominantly responsible for the pubertal growth of the testes (29, 30). Because we could detect no spermatogenesis beyond the development of type A spermatogonia and the number of these cells was low, the pubertal testicular growth in KS boys was due almost solely to the proliferation of Sertoli cells and interstitial cells. This might suggest that the immature Sertoli cells of boys with KS are responding to the pubertal increase in FSH stimulation, as seen in boys with a normal karyotype. On the other hand, the prepubertal Sa and Sb cells were not capable of transforming into the adult Sc cell type, and the degeneration of Sertoli cells increased markedly during puberty. It is therefore tempting to hypothesize that the regression of the testes in KS includes Sertoli cell degeneration. Consistently, electron microscopy revealed degenerating Sertoli cells, a rare finding in testes of healthy early pubertal boys.
The concept of Sertoli cells in boys with KS regressing after the initial phase of Sertoli cell proliferation during early puberty is in agreement with changes observed in serum levels of inhibin B (31). This glycoprotein is thought to reflect Sertoli cell function during prepuberty and become germ cell dependent during midpuberty (32). In our cohort of boys with KS, serum inhibin B levels during prepuberty and early puberty were normal (33, 34, 35), and measurable inhibin B levels did not fully correlate with the presence of germ cells in the testes. This suggests that during early puberty, serum inhibin B levels in boys with KS reflect the integrity or number of Sertoli cells or both. A normal inhibin B level and a low testosterone level were also shown to be inconsistent with the presence of spermatogonia (group IIA in Tables 1 and 2 and Fig. 4A).
AMH or Müllerian-inhibiting substance, a glycoprotein dimer of the TGFß family, is expressed in prepubertal but not adult Sertoli cells (36). Serum AMH levels remain high throughout childhood and wane at puberty (37). We observed high AMH values in KS boys during prepuberty and early puberty, followed by a decline as serum testosterone increased: the timing and magnitude of this decrease was similar to that in normal boys (37, 38) and in agreement with the inverse correlation between serum AMH and testosterone during normal puberty (38). It has been proposed that the dramatic suppression of AMH production requires meiotic entry in spermatogenesis (39). Rajpert-De Myets et al. (40) in their 1999 study noted, however, that the switch in AMH expression occurred also in adolescent boys without germ cells. Our observations, being consistent with these findings, suggest that meiotic activity is unnecessary for down regulation of AMH expression (Table 2). The decrease in serum AMH levels thus cannot serve as an indicator of spermatogenetic activity in KS boys because the biopsy specimens of the boys with low AMH values showed no spermatocytes (group IIB, Tables 1 and 2).
We obtained only a single biopsy from each subject, but even in these very small specimens, we could demonstrate the focal nature of the seminiferous epithelium degeneration (Fig. 1E), perhaps explaining the fact that some adult men with KS display areas of spermatogenesis. It is possible that in our work such areas or their predecessors may have escaped detection. In adult subjects with KS, the success rate for obtaining spermatozoa with sperm-containing tissue is currently at least 50% (41), but multiple testicular biopsies are always required for successful sperm recovery (42). We therefore hypothesize that if several biopsies had been performed, more germ cells would have been found in our subjects. The diagnosis of infertility in KS boys cannot be made on the basis of only one biopsy. These issues were thoroughly discussed with the boys and their parents.
Only a minority of subjects with KS is diagnosed before puberty (2). Later the condition may come to attention during evaluation of hypogonadism and infertility. In the current work, all boys were diagnosed prepubertally, and 13 of 14 patients initially presented with language and behavioral problems. We cannot formally exclude the possibility that these boys differ from other KS subjects in terms of fertility prognosis. However, to our knowledge there is no evidence in the literature indicating that early neurological problems in KS are related to testicular function.
The possible future use of the cryopreserved testicular samples of KS patients in infertility treatments most probably requires in vitro maturation of spermatogonia into mature spermatozoa or at least into late/elongated spermatids. Recent studies (43, 44) indicate that human testicular tissue can be cultured for at least up to 3 wk without essential loss of spermatogonia. Early results also suggest that meiosis and spermatogenesis may resume under culture conditions, yielding normal spermatids with some fertilizing potential (44).
In conclusion, we investigated whether early adolescence is a suitable time period in life for obtaining germ cells for future infertility treatment in subjects with KS. Our results show that early adolescent boys with KS have testicular germ cells that display a maturational arrest at the level of type A spermatogonia. No meiotic cells were detected in any of the biopsy specimens, and onset of puberty was associated with depletion of spermatogonia. Based on these results, it seems that early puberty does not provide an unique window of opportunity to increase fertility potential of subjects with KS. Future research is thus required to elucidate the mechanisms activated at puberty that ultimately lead to hypogonadism characteristic for the syndrome.
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Footnotes |
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Abbreviations: Ad, Spermatogonia of adult dark type; AMH, anti-Müllerian hormone; Ap, spermatogonia of adult pale type; CV, coefficient of variation; ICSI, intracytoplasmic sperm injection; KS, Klinefelter syndrome.
Received October 3, 2003.
Accepted February 10, 2004.
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References |
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-hydroxyprogesterone in human plasma by radioimmunoassay after separation on hydroxyalkoxypropyl sephadex. J Steroid Biochem 5:155–162[CrossRef][Medline]
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