Paediatric
Ad SPERMATOGONIA AND SECRETORY CAPACITY OF LEYDIG CELLS IN CRYPTORCHIDISM
ZIVKOVIC
et al.
In a paper from Switzerland, the
authors describe the relationship
between adult dark spermatogonia
and the secretory capacity of
Leydig cells in cryptorchidism.
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JOURNAL COMPILATION
Relationship between adult dark
spermatogonia and secretory capacity
of Leydig cells in cryptorchidism
Dragana Zivkovic, Domingos T.G. Bica* and
Faruk Hadziselimovic
Institute for Child and Youth Health Care, Paediatric Urology, Novi Sad, Serbia
and Montenegro, *Clinica Uroped, Paediatric Urology, Rio de Janeiro, Brazil, and
Kindertagesklinik, Paediatric Andrology, Liestal, Switzerland
Accepted for publication 9 March 2007
OBJECTIVE
To examine whether hormonal therapy before
orchidopexy affects the histology of the testis
and to assess the responsiveness of the Leydig
cells, as it has been shown that although basal
plasma testosterone levels are within the
normal range in cryptorchid boys there is an
insufficient increase of testosterone after a
human chorionic gonadotrophin (hCG)
stimulation in 30% of cryptorchid boys.
PATIENTS AND METHODS
In all, 55 boys (aged 17 years) with a
unilateral undescended testis were included
in the study and divided into two groups.
Group I (32 boys) received hormonal therapy
before orchidopexy; 17 boys received a
long-acting LHRH analogue (buserelin)
administered as a nasal spray in doses of
20 g/day for 28 days, followed by 1500 IU
hCG intramuscularly (i.m.) once a week for
3 weeks, and the remaining 15 received
1500 IU hCG i.m. once a week for 3 weeks.
Group II (33 boys) had orchidopexy alone.
During orchidopexy biopsies were taken from
the undescended and contralateral descended
testes of the boys in both groups for
histological analyses. Variations in the
number of adult dark (Ad) spermatogonia per
tubule (Ad/T) were assessed and testosterone
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levels were measured during the course of the
hormonal therapy (before treatment, 14 days
after initiation of buserelin administration,
24 h after each hCG injection, and 3 months
after cessation of therapy).
RESULTS
In group I, 17 boys (53%) had a normal Ad/T
after hormonal treatment vs only six (18%) in
group II after orchidopexy alone (P = 0.019).
In the hormonally treated boys (group I) we
compared the testosterone values 24 h after
the second injection of hCG (when the
response was most pronounced). Those with a
normal Ad/T had a mean (SD) testosterone
level of 199.5 (97.6) ng/dL vs 99.6 (85) ng/dL
in those with an inadequate Ad/T response to
hormonal therapy (P < 0.003).
CONCLUSION
We have confirmed that there are two
subgroups of cryptorchid boys. Patients with
a sufficient Leydig cell secretory capacity will
have normal testicular histology and Ad
spermatogonia count after hormonal
treatment. While those with a suboptimal
Leydig cell capacity will have a low Ad
spermatogonia count and consequently poor
prognosis for future fertility, despite
successful surgery. As to whether different
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Z I V K O V I C ET AL.
types and durations of the hormonal therapy
in patients with impaired Leydig cell response
could lead to improved testicular histology
and consequently improved prognosis for
future fertility, remains to be answered.
KEYWORDS
cryptorchidism, hormonal therapy, Leydig
cells, testosterone, child
INTRODUCTION
Maturation of germ cells starts with the
transformation of gonocytes into adult
spermatogonia, and it is usually completed by
the age of 6 months. At the age of 3 months,
adult dark (Ad) spermatogonia start to appear
and steadily increase in number [1]. The
number of Ad spermatogonia is reduced
in the testes of cryptorchid boys, indicating
that there is a failure in the maturation
process of the germ cells. This reduction
is more severe than the reduction in
the number of total germ cells. This
disproportionate reduction in Ad
spermatogonia is correlated directly to
future spermiograms and represents the
fundamental abnormality in germ cell
development in cryptorchidism [2].
It has been shown that, although basal
plasma testosterone levels are normal in
cryptorchid boys, there is an insufficient
increase of testosterone after a hCG
stimulation, compared with the normal
response, in 30% of the cryptorchid
patients [3].
PATIENTS AND METHODS
Boys aged 17 years, with a unilateral
undescended testis were included in the
study. Boys with retractile testes were
excluded. Group I consisted of 32 boys treated
hormonally; 17 received a long-acting LHRH
analogue (buserelin) administered as a nasal
spray in doses of 20 g/day for 28 days,
followed by 1500 IU hCG i.m. once a week for
3 weeks, and 15 received 1500 IU hCG i.m.
once a week for 3 weeks. Even though there
were patients undergoing different protocols
of hormonal therapy, we have considered
them as one group, as it was shown that hCG
and LHRH seem to be equally effective in
treating cryptorchidism [4]. During the course
of the hormonal therapy, testosterone levels
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FIG. 1. The percentage of testicular biopsies with an
Ad/T > 0.1 in boys treated hormonally (group I) and
those treated with orchidopexy alone (group II).
% of patients with Ad/T > 0.1
60
50
40
Group I
30
Group II
20
10
0
p = 0.019
were measured before treatment, 14 days
after initiation of buserelin administration,
24 h after each hCG injection, and 3
months after cessation of therapy [5].
After the hormonal therapy all the boys
had orchidopexy. Patients that had achieved
a scrotal position of the testis after the
hormonal treatment had bilateral testicular
biopsy to eliminate potential risk of
microscopic damage of the testes. Group II
consisted of 33 boys who had orchidopexy
alone, with no previous hormonal therapy.
During orchidopexy biopsies were taken from
the undescended and contralateral descended
testes of boys in both groups.
Biopsies were fixed in 2% glutaraldehyde
in phosphate buffer, post-fixed in osmium-
tetroxide, embedded in EPON, and sectioned
at 0.51.0 m. The histological sections were
stained with toluidine blue, and examined by
light microscopy. The number of complete
tubules in the entire biopsy was estimated
(50 tubules). The Ad spermatogonia were
counted and expressed as the number of Ad
spermatogonia per tubule (Ad/T).
The boys were subdivided in two groups
according to their Ad/T value: Ad/T >0.1, those
with a normal number of Ad spermatogonia
[1] after treatment and Ad/T 0.1, those with
inadequate response to hormonal therapy.
The number of boys with a normal number of
Ad spermatogonia from group I and II was
compared to determine whether hormonal
stimulation had any effect on the histology of
the testis.
In group I, the response to the second
injection of hCG was compared between
the boys in two Ad/T subgroups of
group I.
Parental consent was obtained for all patients
for hormonal and surgical treatment. The
JOURNAL COMPILATION
FIG. 2. The plasma testosterone levels after the
second injection of hCG in boys that achieved a
normal Ad spermatogonia count after completion
of the treatment (Ad/T > 0.1) and in those that still
had a low Ad spermatogonia count after completion
of the hormonal treatment (Ad/T 0.1).
second injection of hCG, ng/dL
Testosterone level after the
300
250
200
Ad/T 0.1
150
Ad/T > 0.1
100
50
0
p < 0.003
Institutional Review Boards and Independent
Ethics Committees of University Childrens
Hospital Basel, and Kindertagesklinik Liestal,
in accordance with Helsinki declaration,
approved all aspects of this study.
Statistical analysis was done with the two-
tailed Fishers test.
RESULTS
In group 1 (hormonally treated), 17 boys
(53%) had an Ad/T > 0.1 and 15 (47%)
had an Ad/T 0.1. While in group II
(surgically treated), only six boys (18%)
had an Ad/T > 0.1 and 27 (82%) had an
Ad/T 0.1. There were significantly more boys
with an Ad/T > 0.1 (normal Ad spermatogonia
count) in the hormonally treated group than
those that had orchidopexy alone (P = 0.019)
(Fig. 1).
We compared the testosterone values 24 h
after the second injection of hCG (when the
response was most pronounced) in boys from
group I: the mean (SD) testosterone level in
boys with a Ad/T of >0.1 was 199.5 (97.6) ng/
dL vs 99.6 (85) ng/dL in those with an Ad/T
0.1 (P < 0.003) (Fig. 2).
DISCUSSION
The effects of hormonal therapy on the
contralateral descended testis have been
sporadically studied [6]. Bergada et al. [6]
found stimulated maturation of germ cells
in treated patients, which was directly
related to both the dose and duration of
the treatment. In the present study there
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 Ad SPERMATOGONIA AND SECRETORY CAPACITY OF LEYDIG CELLS IN CRYPTORCHIDISM
was a significant difference in the histology
of the testes of the boys who had orchidopexy
alone and those who received hormonal
therapy of any kind beforehand; the Ad/T was
significantly higher in the hormonally treated
boys. It has been shown that the number of
Ad spermatogonia is correlated to future
fertility [2]. The effects of testosterone on
the transformation of gonocytes into Ad
spermatogonia have not yet been analysed.
We have shown in the present study, for
the first time, that the transformation of
gonocytes into Ad spermatogonia is a
testosterone-dependent process. If an
adequate increase in plasma testosterone
follows hormonal stimulation, normal
germ-cell maturation occurs. Patients that
have an insufficient Leydig cell response
to hormonal stimulation, resulting in an
inadequate testosterone increase, will have
poor testicular histology and a low Ad
spermatogonia count.
In conclusion, from the present study there
appears to be two subgroups of cryptorchid
boys; those with a sufficient Leydig cell
secretory capacity and those with a
suboptimal Leydig cell secretory capacity. As
to whether different types and durations of
hormonal therapy in cryptorchid boys with
impaired Leydig cell response could lead
to improved testicular histology and
consequently improved prognosis for future
fertility, remains to be answered.
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CONFLICT OF INTEREST
None declared.
REFERENCES
1 Hadziselimovic F, Emmons LR, Buser M.
A diminished postnatal surge of Ad
spermatogonia in cryptorchid infants is
additional evidence for hypogonadotropic
hypogonadism. Swiss Med Wkly 2004;
134: 3814
2 Hadziselimovic F, Herzog B. The
importance of both an early orchidopexy
and germ cell maturation for fertility.
Lancet 2001; 358: 11567
3 Forest MG. Pattern of the response
to hCG stimulation in prepubertal
cryptorchid boys. In Job CL ed.,
Cryptorchidism, Diagnosis and Treatment.
Pediatr Adolesc Endoc. Basel: Karger,
1979: 10820
4 Esposito C, De Lucia A, Palmieri A et al.
Comparison of five different hormonal
treatment protocols for children with
cryptorchidism. Scand J Urol Nephrol
2003; 37: 2469
5 Bica DT, Hadziselimovic F. Buserelin
treatment of cryptorchidism: a
randomized, double-blind, placebo-
controlled study. J Urol 1992; 148:
61721
6 Bergada C, Mancini RE. Effects of
gonadotropins in the induction of
spermatogenesis in human prepubertal
testis. J Clin Endocrinol Metab 1973; 37:
93543
Correspondence: Faruk Hadziselimovic,
Kindertagesklinik, Oristalstrasse 87a, CH-4410
Liestal, Switzerland.
e-mail: faruk@magnet.ch;
zdragana@eunet.yu
Abbreviations: Ad, adult dark; Ad/T, Ad
spermatogonia per tubule.
EDITORIAL COMMENT
This interesting and significant paper from a
group who has pioneered our understanding
of the endocrinopathy causing
cryptorchidism and its effect on fertility adds
further insight into this problem. This is new
data that appears especially relevant in an era
when male adult fertility appears to be
decreasing. This paper deserves careful study
and should pave the way toward a better
understanding of both cryptorchidism and
fertility.
Howard M. Snyder III, MD,
Division of Urology, Department of Surgery,
The Childrens Hospital of Philadelphia,
Philadelphia, PA, USA
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