Klinefelter Syndrome in Adolescence - The Journal of Clinical ...

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(5):2263–2270
Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031725
Klinefelter Syndrome in Adolescence: Onset of Puberty
Is Associated with Accelerated Germ Cell Depletion
ANNE M. WIKSTRÖM, TANELI RAIVIO, FARUK HADZISELIMOVIC, SAKARI WIKSTRÖM,
TIMO TUURI, AND LEO DUNKEL
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
The process of germ cell depletion in patients with Klinefelter
syndrome (KS) is incompletely characterized. In the current
work, we evaluated the presence of germ cells in adolescent
boys with KS for possible future use in assisted reproduction
techniques.
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
IN 1942 HARRY KLINEFELTER described a syndrome
characterized by gynecomastia; small, firm testes with
hyalinization of the seminiferous tubules; elevated gonadotropins;
and azoospermia (1). With an estimated mean prevalence
of 152 per 100,000 males, Klinefelter syndrome (KS) is
one of the most common sex chromosome anomalies (2). It
is also the among most frequent genetic causes of human
infertility affecting approximately 11% of azoospermic and
4% of infertile men (3). In subjects with the karyotype
47,XXY, the process of germ cell depletion is incompletely
characterized. Some reports suggest that the degeneration of
germ cells starts in infancy, leading to the absence of or to a
significantly reduced number of germ cells even before puberty
(4–6). On the other hand, complete absence of germ
cells cannot be a uniform phenomenon because some adult,
nonmosaic 47,XXY men may father a child with the aid of
intracytoplasmic sperm injection (ICSI) (7).
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 hypergonadotro-
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.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine
community.
2263
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. (J Clin Endocrinol Metab 89: 2263–2270,
2004)
pism 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).
Subjects
Subjects and Methods
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.
At the start of the systematic follow-up, the median age of the subjects
was 11.5 yr (range 10.0–13.9). They were followed prospectively for 4–20
months (median 13 months). During the systematic surveillance, they
visited the Hospital for Children and Adolescents every fourth month.

2264 J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 Wikström et al. Testicular Degeneration in Adolescent KS Boys
TABLE 1. Histomorphometric analyses of testicular biopsies of 14 boys with KS, with key characteristics at the time of the biopsy
Patient
no.
At each visit, a physical examination was carried out as previously
described; testicular volume was calculated with the formula 0.52
length width (2), and converted to milliliters (13 and was expressed
as the mean volume of the left and right testis. Once a year skeletal age
was assessed according to the atlas of Greulich and Pyle (14). Sera were
obtained for FSH, LH, inhibin B, and AMH measurements every fourth
month and for testosterone, SHBG, and estradiol measurements at least
once a year.
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
Group
no.
Age
(yr)
Tanner
stage
Mean testicular
volume (ml)
Ap Ad
spermatogonia/
tubule
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
Ad
spermatogonia/
tubule
(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
Sertoli cells type
Leydig cell
hyperplasia
Interstitium and
peritubular
connective tissue
1 I 10.1 P1/G1 1.1 1.2 0.01 Sa/Sb fibr ()
2 I 10.1 P1/G1 1.3 0.04 0 Sa/Sb fibr , hyalin
3 I 10.3 P1/G1 0.8 0.8 0.006 Sa/Sb fibr ()
4 I 10.7 P1/G1 1.6 1.0 0.03 Sa/Sb fibr
5 I 11.6 P1/G1 1.0 0.1 0.01 Sa/Sb, pale fibr
6 I 11.9 P1/G1 1.8 1.2 0.01 Sa/Sb fibr , hyalin
7 I 12.5 P2/G2 1.7 1.2 0.03 Sa/Sb fibr , hyalin
8 IIA 11.7 P1/G2 2.0 0 0 Sa/Sb fibr ()
9 IIA 11.9 P1/G2 2.5 0 0 Sa/Sb, degen fibr
10 IIA 13.0 P1/G2 1.8 0 0 Sa/Sb, degen fibr , hyalin
11 IIB 11.8 P2/G2 3.9 0 0 Sa, pale, degen fibr , hyalin
12 IIB 13.7 P1/G2 3.4 0 0 pale, degen fibr
13 IIB 14.0 P2/G2 3.2 0 0 pale, degen fibr , hyalin
14 IIB 14.0 P3/G4 3.1 0 0 pale, degen, hyalin fibr , hyalin
The patients are divided into two groups according to presence or absence of spermatogonia. Group II is divided into subgroups A and B
according to presence or absence of hypergonadotropic hypogonadism (see Table 2). Puberty staged according to Tanner (8). Ap and Ad
spermatogonia counts per cross-section of seminiferous tubule. Sertoli cells staged as Sa, Sb, pale, and degenerating. The degree of Leydig cell
hyperplasia staged to . The degree of fibrosis of the interstitium are staged to and presence of hyalinization is marked hyalin.
TABLE 2. Laboratory parameters of 14 boys with KS at the time of testicular biopsy
Patient no. Group no.
FSH
(IU/liter)
S-LH
(IU/liter)
Testosterone
(nmol/liter)
inhibin B
(pg/ml)
AMH
(pmol/liter)
1 I 0.1 0.1 0.3 103 503
2 I 1.0 0.2 0.3 78 758
3 I 1.5 0.1 0.3 66 345
4 I 1.9 0.4 0.3 127 606
5 I 1.0 0.3 0.8 68 1156
6 I 1.5 0.5 0.8 75 803
7 I 1.1 0.5 1.1 69 1062
8 IIA 1.3 0.5 0.7 301 2658
9 IIA 0.5 0.2 0.3 166 1251
10 IIA 0.7 0.1 0.5 97 878
11 IIB 7.5 1.2 2.3 15.6 81
12 IIB 17.9 6.9 3.9 29 101
13 IIB 33.2 9.7 15.7 15.6 96
14 IIB 38.6 11.4 10.2 15.6 16
Patients are divided into two groups according to presence or absence of spermatogonia (see Table 1). Group II is divided into subgroups A
and B according to presence or absence of hypergonadotropic hypogonadism.
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 m, and stained with toluidine blue. Histomorphometric
analysis was performed by light microscopy at a total magnification
of 400. 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 classi-

Wikström et al. Testicular Degeneration in Adolescent KS Boys J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 2265
fied 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.
Results
Results of histomorphometric analyses of the biopsy specimens,
as well as the key characteristics of the 14 patients at
the time of testicular biopsy are presented in Table 1. 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.
Germ cells
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).
Testicular morphology reflected in hormone levels
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).
Longitudinal changes in serum hormone levels
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).
During prepuberty and early puberty, serum AMH levels
were high, but with advancing puberty, AMH was suppressed
simultaneously with inhibin B (Figs. 4 and 5). There
also existed a strong, inverse nonlinear relationship between
serum AMH and testosterone levels (Figs. 4 and 5). Before

2266 J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 Wikström et al. Testicular Degeneration in Adolescent KS Boys
FIG. 1. Testicular biopsies of five adolescent boys with KS from list in Table 1 and one normal boy. Stain, Toluidine blue. A, Patient 4, two
types of seminiferous tubules identifiable: those with no spermatogonia on the right and those with spermatogonia of adult pale type (Ap) on
the left. Ad, Spermatogonia of adult dark type. Sertoli cells, Sa and Sb type; Leydig cells, juvenile type, some hyperplasia; interstitium, increased
fibrosis (magnification, 400). B, Patient 8, no spermatogonia. Sertoli cells, Sa and Sb type; Leydig cells, juvenile type. Interstitial compartment
appears normal (magnification, 400). C, Patient 12, no spermatogonia. Sertoli cells, Sa and Sb type, pale and degenerative. Interstitial
compartment has increased volume, hyperplastic Leydig cells, and some fibrosis (magnification, 400). D, Patient 14, seminiferous tubules
completely hyalinized. Interstitial compartment has hyperplastic Leydig cells and extensive fibrosis (magnification, 400). E, Patient 7,
centrally seminiferous tubules with Ap and/or Ad spermatogonia. This area is surrounded by tubules in which the degeneration process is
evident: tubules with pale degenerative Sertoli cells and small tubules with dark small apoptotic cells (magnification, 200). F, Normal control,
age 11 yr, testicular biopsy taken at surgical exploration because of scrotal pain (magnification, 200).

Wikström et al. Testicular Degeneration in Adolescent KS Boys J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 2267
FIG. 2. Patient 2, group I (Table 1). Electron micrograph of testicular
biopsy of boy with KS at age 10.1 yr and Tanner stage P1G1. Undifferentiated
Sertoli cells resembling Sa type with a round nucleus with
a small nucleolus lacking lamellar body and with crystalloids from
Charcot-Bötscher. In the center, one degenerating Sertoli cell
(magnification, 2500).
testosterone had reached 2.5 nmol/liter, a marked drop in
serum AMH had already occurred (Fig. 4B).
Discussion
With the aid of modern infertility treatment technologies
such as ICSI, some men with KS may father children. However,
in adulthood, when these treatments come into consideration,
the testes of most KS men are completely atrophied.
In the current work we investigated whether
adolescent patients with KS have germ cells and evaluated
whether early puberty is the optimal period for retrieving
germ cells for further use in ICSI. Although this concept has
gained further actuality with the recent report of a successful
extraction of sperm-containing tissue from a 15-yr-old boy
with KS (20), it has not been previously investigated
systematically.
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 (33–35), 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 TGF family, is expressed in prepubertal but not
adult Sertoli cells (36). Serum AMH levels remain high

2268 J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 Wikström et al. Testicular Degeneration in Adolescent KS Boys
FIG. 3. A, Testicular volumes and serum FSH,
testosterone, inhibin B, and AMH levels in 14
boys with KS with spermatogonia present or
absent in the biopsy specimen (n 7, both
groups). Horizontal lines, 10th, 25th, 50th,
75th, and 90th percentiles, and extreme values
are shown separately. B, Longitudinal changes
in individual testis volumes. Black circles,
group I, white circles, group II (see Table 1).
FIG. 4. A, Longitudinal changes in inhibin
B, testosterone, and AMH concentrations
in 14 boys with KS. B, Correlations between
inhibin B and testosterone and AMH
and testosterone. Dashed lines, serum testosterone
2.5 nmol/liter. Black circles,
group I; white circles, group II (see Table 1).

Wikström et al. Testicular Degeneration in Adolescent KS Boys J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 2269
FIG. 5. Distributions of serum FSH,
LH, testosterone, inhibin B, and AMH
concentrations and testicular volume in
14 boys with KS followed up during puberty.
Tanner G stages. There were
22–32 observations at stage G1, 13–21
observations at G2, 14–16 observations
at G3, and five to six observations at G4.
Horizontal lines, 10th, 25th, 50th, 75th,
and 90th percentiles, and extreme values
are shown separately (white circles).
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.
Acknowledgments
Received October 3, 2003. Accepted February 10, 2004.
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@hus.fi.
This work was supported by grants from the Medical Society of
Finland (Finska Läkaresällskapet) (to A.M.W.), the Mjölbolsta Founda-

2270 J Clin Endocrinol Metab, May 2004, 89(5):2263–2270 Wikström et al. Testicular Degeneration in Adolescent KS Boys
tion (to A.M.W.), the Foundation for Pediatric Research (to T.R.), and
Helsinki University Central Hospital.
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