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Longitudinal Studies of Inhibin B Levels in Boys and Young Adults with Klinefelter Syndrome

Department of Growth and Reproduction, Rigshospitalet, DK-2100 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Anna-Maria Andersson, Department of Growth and Reproduction, Rigshospitalet, GR-5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: anna{at}rh.dk.

	Abstract

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The aim of the study was to investigate the longitudinal changes of inhibin B in a group of patients with Klinefelter syndrome (KS; karyotype 47,XXY) progressing through puberty and to compare them to a group of age- and puberty-matched controls.

Seven boys with nonmosaic KS (karyotype 47,XXY) and 11 controls were followed with longitudinal serum inhibin B measurements every 3–12 months as they approached and entered puberty. None of the boys had significant bone age delay, and all entered puberty at the normal time and progressed through it at the expected time. In addition, 15 young adults with KS, aged 16.7–29.5 yr, were studied.

We found normal levels of inhibin B in prepubertal boys with KS and controls. In patients with KS as well as controls, inhibin B increased progressively before clinical pubertal onset. However, during late puberty inhibin B levels decreased gradually to the low/unmeasurable levels observed later in adult KS, while remaining unchanged in the controls.

	Introduction

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TESTICULAR HISTOLOGY IN adult men with Klinefelter syndrome (KS) is characterized by degenerative changes in the seminiferous epithelium accompanied by hyalinization and fibrosis of the seminiferous tubules and hyperplasia of the Leydig cells (1, 2). As a consequence of these degenerative processes, Klinefelter patients have small firm testes ranging from 3–5 ml (3). However, the natural history of the development of the atrophic testes in adults with KS is only partly known. The few studies that have tried to elucidate this development in prepubertal XXY males have been complicated by problems of early diagnosis and tissue sampling resulting in a small sample size, which has contributed to the difficulties in interpreting the results. Müller et al. (4), using a stereological technique, investigated the testicular lesions in prepubertal boys with karyotype XXY and observed that germ cell number was normal in infants, whereas no germ cells were observed in boys older than 2 yr, suggesting that the degenerative changes occur early in life. In contrast, Ferguson-Smith et al. (5) noted the presence of germ cells, although in a significantly reduced number, in seven of eight KS patients aged 7–12 yr. With the intensified use of amniocentesis, early diagnosis has resulted in a more detailed description of germ cell development in KS. However, these data are also equivocal, ranging from a normal testis histology pattern (6) to changes similar to those observed in the adult KS (7).

Even less is known about the Sertoli cell number and function in KS during childhood. The gonadal peptide hormone inhibin B is secreted by Sertoli cells in pre- and peripubertal boys, and the serum level of inhibin B is considered to reflect Sertoli cell function (8, 9). The onset of male puberty is associated with increasing serum concentration of inhibin B, and already by pubertal stage II the adult serum level of inhibin B has been reached (10). During puberty, as the Sertoli cells maturate, a change in the regulation of inhibin B production takes place, going from being solely Sertoli cell-dependent to being highly dependent on spermatogenesis (11). Thus, in adult men, the serum inhibin B level is a marker of spermatogenesis rather than a marker of Sertoli cell per se, and men with testicular dysfunction including adult patients with KS syndrome have very low or undetectable levels of inhibin B (9, 12, 13). To our knowledge, no studies exist of inhibin B during childhood and puberty in patients with KS. We report here a longitudinal inhibin B study on a group of patients with KS (karyotype 47,XXY) progressing through puberty and compare them to a group of age- and puberty-matched controls.

	Materials and Methods

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Longitudinal study of peripubertal boys

Eleven boys were followed longitudinally with clinical controls, including blood sampling for routine hormone measurements every 3–12 months as part of their clinical surveillance program, as they approached and entered puberty. For several years measurement of inhibin B levels has been part of the routine clinical care of patients with growth or reproduction problems attending our clinic, and thus longitudinal inhibin B levels were available. The median (range) age of the boys at the first measurement was 12.2 yr (10.3–13.1 yr), and the median (range) age for the last measurement was 14.2 yr (11.8–15.8 yr). None of the boys suffered from any genitourinary disorders, such as cryptorchidism or hypospadias. None had significant bone age delay; all entered puberty at the normal time and progressed through it at the expected time. One patient had isolated GH deficiency and received GH treatment. Four patients had skeletal dysplasias, and two of those received GH treatment. Four patients had familial short stature, and one of those received GH treatment. One patient had acquired hypothyreosis and received treatment with L-thyroxine. One patient had a pituitary incidentaloma and received no treatment. All of the boys, except the one with GH deficiency, had normal GH response to a challenge test. All of the boys, except the one with hypothyreosis, had normal basal thyroid function. All boys had a normal pituitary/gonadal function.

Longitudinal study of prepubertal boys with KS

Seven boys with KS (karyotype 47,XXY) were followed longitudinally with clinical controls and blood sampling for routine hormone measurements (including inhibin B) every 3–6 months as part of their clinical surveillance program as they approached and entered puberty. The median (range) age of the boys at the first measurement was 11.0 yr (8.9–12.6 yr), and the median (range) age for the last measurement was 13.7 yr (12.6–14.5 yr). In addition, longitudinal serum inhibin B levels were obtained from 15 young men with KS, aged 16.7–29.5 yr. In three patients, treatment with testosterone undecenoate (TU; 40 mg three times per week) was initiated before clinical onset of puberty. In one patient, treatment with TU was stopped approximately 2 yr after clinical onset of puberty and was followed by im injections of testosterone enanthate. The boys were otherwise healthy, without evidence of other endocrine or systemic disease.

Testicular size was determined using a Prader orchidometer. A testicular volume greater than 3 ml was taken as a definite sign of puberty. The onset of puberty was taken as the midpoint between the age of two adjacent dates with testicular volumes of 3 and 4 ml, respectively.

Cross-sectional inhibin B levels in normal 16- to 30-yr-old men used as reference material were obtained in two different projects approved by the local ethical committees and have been published in part previously (10, 14).

Hormone analyses

Samples were collected between midmorning and late afternoon. Inhibin B was determined by a double antibody enzyme immunometric assay (Oxford Bio-Innovation Ltd., Oxford, UK) using a monoclonal antibody raised against the inhibin ß-B subunit in combination with a labeled antibody raised against the -subunit as previously described (15). The detection limit was 20 pg/ml, and intra- and interassay coefficients of variation were 15% and 18%, respectively. FSH and LH were measured by time-resolved immunofluorometric assay (DELFIA, Wallac, Inc. Turku, Finland) with detection limits of 0.06 and 0.05 U/liter, respectively. Intra- and interassay coefficients of variation were both less than 8% in the FSH and LH assays. Normal ranges for inhibin B, FSH, and LH levels in normal prepubertal boys have been published previously (10).

Statistics

If more than one blood sample from the same time period was available for the individual patients, the median value was calculated. Nonparametric statistics were used because data did not have a Gaussian distribution. Descriptive data were expressed as medians and ranges. Within-individual changes between successive time periods were tested by Wilcoxon signed-rank test. Any differences in inhibin B comparing same time periods between controls and KS was tested by the Mann-Whitney U test.

	Results

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Figure 1 shows serial changes in median inhibin B levels in 7 boys with KS and 11 control boys over 1–2 yr preceding entry into clinical puberty and 2–3 yr after pubertal onset. In addition, the longitudinal inhibin B levels in 15 young adults with KS are shown in relation to a 95% prediction interval of healthy normal men (10, 14).

View larger version (15K): [in this window] [in a new window]   Figure 1. Left, Longitudinal changes in inhibin B levels in 11 control boys and 7 boys with KS undergoing serial blood sampling as they went through puberty. Samples were classified into time periods before and after the onset of puberty, defined as a testicular volume larger than 3 ml (prepuberty, 12–24, 6–12, and 0–6 months; and postpuberty, 0–6, 6–12, 12–24, and 24–36 months postpuberty). Right, Longitudinal inhibin B levels in 15 young adults with KS are shown in relation to a 95% prediction interval of healthy normal men. Data are expressed as medians, with error bars representing the range.

Within-individual pair-wise comparisons between successive time periods indicated that inhibin B levels increased progressively before puberty: from 12–24 month to 0–6 month (P = 0.03; n = 6). Inhibin B increased further during the 18-month period spanning the onset of puberty from 6–12 months before to 6–12 months after puberty onset (P < 0.05; n = 6), after which no further increase was found. Within-individual pair-wise comparisons between successive time periods indicated that FSH also increased progressively before puberty from 1.97 IU/liter (at 12–24 months before pubertal onset) to 2.60 IU/liter (at 0–6 months before pubertal onset; P < 0.02; n = 7). FSH further increased during the 0–6 month period after the onset of puberty to 3.37 IU/liter (P < 0.05; n = 6), after which no further increase was found.

Klinefelter

Age at onset of puberty did not differ in controls and KS, with median ages of 12.9 (10.4–13.8) and 12.4 (11.6–13.7) yr, respectively. Within-individual pair-wise comparisons between successive time periods showed no significant differences in inhibin B before pubertal onset. After the onset of puberty, inhibin B decreased, but did not reach statistical significance (P = 0.11), probably due to the low number of data points. Before onset of puberty, no significant difference in inhibin B levels could be found between KS boys and the controls comparing same time periods. However, after the onset of puberty (from 0–6 months to 12–24 months), inhibin B levels were significantly lower in boys with KS than in controls (P = 0.03).

Within-individual pair-wise comparisons between successive time periods indicated that FSH increased during the 12-month period spanning the onset of puberty from 1.23 to 1.33 IU/liter (P < 0.05; n = 6). Later in puberty, FSH seemed to increase further, but due to the low number of data points, this increase did not reach statistical significance. Comparing the same time periods before onset of puberty, no significant difference in FSH levels could be found between KS boys and the controls. After the onset of puberty, FSH levels tended to be higher in boys with KS compared with controls, although this difference was not statistically significant, possibly due to the low number of data points.

In the group of adolescent and young adult men with KS followed longitudinally [median (range), age of sampling, 20.6 (16.7–29.5) yr], only 3 of 15 patients had at a single occasion an inhibin B measurement just above the detection limit of the assay.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here, we present what we believe is the first longitudinal study of inhibin B during prepuberty and puberty in patients with KS. We found normal levels of inhibin B in prepubertal boys with KS, compared with a group of control boys. However, during late puberty inhibin B levels decreased in boys with KS and were significantly reduced compared with the group of controls. The normal levels of inhibin B in prepubertal boys with KS suggest that their Sertoli cell function was relatively normal during childhood and early puberty. This was not surprising, because it is known that the Sertoli cells are the most resistant cells of the seminiferous epithelium (16, 17), which is also indicated by the fact that inhibin B is normal in prepubertal boys with unilateral and bilateral cryptorchidism (18) and that prepubertal boys with Sertoli cell only pattern exhibited normal inhibin B levels (11). The initial rise in inhibin B levels in KS and controls during prepuberty and early puberty is presumably reflecting a FSH-stimulated increase in inhibin B production by immature Sertoli cells. During pubertal maturation of the human male, inhibin B production becomes germ cell-dependent and thus no longer is a marker of Sertoli cell function alone. In the adult KS testes, virtually all germ cells and the majority of the Sertoli cells disappear, which could explain why inhibin B was undetectable in our adult KS patients and as reported previously in adults with KS (9, 12, 13). However, occasionally single foci of spermatogenesis do exist in the testes of KS (19), explaining why a few of our patients with KS occasionally had low measurable inhibin B levels and why patients with KS may sire offspring with or without assisted reproduction (20).

In the control boys followed longitudinally, inhibin B increased progressively over the 2-yr period before onset of clinical puberty, with a further increase over the period spanning the onset of puberty. This is in agreement with the findings of Crofton et al. (21). No significant difference in FSH levels could be found between KS boys and the controls in prepuberty and early puberty. This is in line with the observation that significantly elevated levels of FSH only are observed in 16- to 18-yr-old KS several years after onset of puberty (3).

In controls and boys with KS, we defined a testicular volume greater than 3 ml as a definite sign of puberty and found no significant difference in age of onset of puberty between the two groups. This is in accordance with Ratcliffe et al. (22) who used the same definition of onset of puberty in KS. However, a testicular volume greater than 3 ml might not always indicate pubertal onset in KS, because serial measurements of prepubertal KS showed a reduced testicular size in several individuals (3). One might speculate that an increase in testicular volume from, e.g., 2–3 ml could indicate onset of puberty in some KS with a reduced prepubertal testicular volume. As a consequence, the age of onset of puberty in our boys with KS may in some cases have been overestimated, and this might explain the slightly higher prepubertal median level of inhibin B observed in our KS compared with the controls.

In several of our patients, the blood samples were collected during therapy with GH, L-thyroxine, or TU. Consideration must be given to whether this may have affected the results. However, it has been shown that therapy with either GH (21, 23) or L-thyroxine (23) has no significant effect on levels of inhibin B. Suppression of gonadotrophins with injections of testosterone (testosterone enanthate) was followed by a parallel decline in inhibin B levels in adult men (24). Little is known about the possible effect of TU on inhibin B levels. Brown et al. (25) in a double-blind, randomized trial found no significant effect of TU on basal or stimulated gonadotrophin levels during the 6-month treatment in boys with constitutional delay of growth. Similarly, a double-blind cross-over study in which men with hypo- and hypergonadotropic hypogonadism were treated with TU found no significant change in gonadotrophin concentration during treatment (26). Therefore, we do not expect that GH, L-thyroxine, or TU treatment significantly affected the observed inhibin B levels.

In conclusion, we found normal levels of inhibin B in prepubertal KS patients compared with a group of age- and puberty-matched controls, suggesting normal Sertoli cell function in prepubertal KS. During late puberty, inhibin B levels decreased gradually to the low/unmeasurable levels observed later in adult KS.

	Acknowledgments

 

	Footnotes

 
This work was supported by the Svend Andersen Foundation and the European Commission (Grant BMH4-CT98-9574).

Abbreviations: KS, Klinefelter syndrome; TU, testosterone undecenoate.

Received August 30, 2002.

Accepted October 31, 2002.

	References

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1. Skakkebæk NE 1969 Two types of tubules containing only Sertoli cells in adults with Klinefelter’s syndrome. Nature 223:643–645[CrossRef][Medline]
2. Gomez-Acebo J, Parrilla R, Abrisqueta JA, Pozuelo V 1968 Fine structure of spermatogenesis in Klinefelter’s syndrome. J Clin Endocrinol Metab 28:1287–1294[Abstract/Free Full Text]
3. Ratcliffe SG 1982 The sexual development of boys with the chromosome constitution 47, XXY (Klinefelter’s syndrome). Clin Endocrinol Metab 11:703–716[CrossRef][Medline]
4. Müller J, Skakkebæk NE, Ratcliffe SG 1995 Quantified testicular histology in boys with sex chromosome abnormalities. Int J Androl 18:57–62[Medline]
5. Ferguson-Smith MA 1959 The prepubertal testicular lesion in chromatin positive Klinefelter’s syndrome (primary micro-orchidism) as seen in mentally handicapped children. Lancet i:219–222
6. Flannery DB, Brown JA, Redwine FO, Winter P, Nance WE 1984 Ante-natally detected Klinefelter’s syndrome in twins. Acta Gent Med Gemellol 33:51–56
7. Murken JR, Ruthkowski SS, Walther JV, Viestlenfelder SR, Remberger KH, Zornmer F 1974 Klinefelter’s syndrome in a fetus. Lancet ii:171
8. Andersson A-M, Skakkebæk NE 2001 Serum inhibin B levels during male childhood and puberty. Mol Cell Endocrinol 180:103–107[CrossRef][Medline]
9. Anawalt BD, Bebb RA, Matsumoto AM, Groome NP, Illingworth PJ, McNeilly AS, Bremner WJ 1996 Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 81:3341–3345[Abstract]
10. Andersson A-M, Juul A, Petersen JH, Müller J, Groome NP, Skakkebæk NE 1997 Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone and estradiol levels. J Clin Endocrinol Metab 82:3976–3982[Abstract/Free Full Text]
11. Andersson A-M, Müller J, Skakkebæk NE 1998 Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels. J Clin Endocrinol Metab 83:4451–4458[Abstract/Free Full Text]
12. Klingmüller D, Haidl G 1997 Inhibin B in men with normal and disturbed spermatogenesis. Hum Reprod 12:2376–2378[Abstract/Free Full Text]
13. Hipler UC, Hochheim B, Knoll B, Tittelbach J, Schreiber G 2001 Serum inhibin B as a marker for spermatogenesis. Arch Androl 46:217–222[CrossRef][Medline]
14. Jensen TK, Andersson A-M, Hjollund NHI, Scheike T, Kolstad H, Giwercman A, Henriksen TB, Ernst E, Bonde JP, Olsen J, McNeilly A, Groome NP, Skakkebæk NE 1997 Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone levels. A study of 349 Danish men. J Clin Endocrinol Metab 82:4059–4064[Abstract/Free Full Text]
15. Groome NP, Illingworth PJ, O’Brien M, Pai R, Rodger FE, Mather JP, McNeilly AS 1996 Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 81:1401–1405[Abstract]
16. Clermont Y, Perey B 1957 Quantitative study of the cell population of the seminiferous tubules in immature rats. Am J Anat 100:241–267[CrossRef][Medline]
17. Heller GV, O’Keefe KB, Heller CG 1968 Effects of follicle stimulating hormone (FSH) on Sertoli cells in the hypophysectomized rat. Clin Res 16:113
18. Christiansen P, Andersson A-M, Skakkebæk NE, Juul A 2002 Serum inhibin B, FSH, LH and testosterone levels before and after chorionic gonadotropin stimulation in prepubertal boys with cryptorchidism. Eur J Endocrinol 147:95–101[Abstract]
19. Skakkebæk NE, Philip J, Hammen R 1969 Meiotic chromosomes in Klinefelter’s syndrome. Nature 221:1075–1076[CrossRef][Medline]
20. Tournaye H, Staessen C, Liebaers I, Van Assche E, Devroey P, Bonduelle M, van Steirteghem A 1996 Testicular sperm recovery in nine 47, XXY Klinefelter patients. Hum Reprod 11:1644–1649[Abstract/Free Full Text]
21. Crofton PM, Evans AE, Groome NP, Taylor MR, Holland CV, Kelnar CJ 2002 Inhibin B in boys from birth to adulthood: relationship with age, pubertal stage, FSH and testosterone. Clin Endocrinol (Oxf) 56:215–221[CrossRef][Medline]
22. Ratcliffe SG, Butler GE, Jones M 1991 Edinburgh study of growth and development of children with sex chromosome abnormalities. IV. Birth Defects Orig Artic Ser 26:1–44
23. Juul A, Andersson A-M, Pedersen SA, Jørgensen JOL, Christiansen JS, Groome NP, Skakkebæk NE 1998 Effects of growth hormone replacement therapy on IGF-related parameters and on the pituitary-gonadal axis in GH-deficient males. Horm Res 49:269–278[CrossRef][Medline]
24. Anderson RA, Wallace EM, Groome NP, Bellis AJ, Wu FCW 1997 Physiological relationships between inhibin B, follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum Reprod 12:746–751[Abstract/Free Full Text]
25. Brown DC, Butler GE, Kelnar CJH, Wu FCW 1995 A double blind, placebo controlled study of the effects of low dose testosterone undecanoate on the growth of small for age, prepubertal boys. Arch Dis Child 73:131–135[Abstract/Free Full Text]
26. Skakkebæk NE, Bancroft J, Davidson DW, Warner P 1981 Androgen replacement with oral testosterone undecanoate in hypogonadal men: a double blind controlled study. Clin Endocrinol (Oxf) 14:49–61[Medline]

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Christiansen, P. Articles by Skakkebæk, N. E. Search for Related Content PubMed PubMed Citation Articles by Christiansen, P. Articles by Skakkebæk, N. E.