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

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