Gonadal response after a single-dose stimulation test with recombinant human chorionic gonadotropin (rhCG) in patients with isolated prepubertal cryptorchidism
Oliveira et al. Basic and Clinical Andrology
Gonadal response after a single-dose stimulation test with recombinant human chorionic gonadotropin (rhCG) in patients with isolated prepubertal cryptorchidism
Leticia Ribeiro Oliveira 0
Thais Kataoka Homma 0
Renata Reis Woloszynek
Vinícius Nahime Brito
Carlos Alberto Longui 0
0 Pediatric Endocrinology Unit, Pediatrics Department, Irmandade da Santa Casa de Misericórdia de São Paulo, and Santa Casa de São Paulo School of Medical Sciences , Rua Dr. Cesário Mota Jr, 112, Vila Buarque, São Paulo CEP 01221-020 , Brazil
Background: The evaluation of prepubertal gonadal Leydig cells secretion requires gonadotropin stimulation. Urinary hCG (human chorionic gonadotropin) is currently unavailable in many countries, however, recombinant hCG (rhCG) can be used. Our aim was to evaluate rhCG-stimulated testicular hormones in a group of patients with cryptorchidism. Methods: We evaluated 31 prepubertal boys (age range, 0.75-9.0 years) presenting with unilateral (n = 24) or bilateral (n = 7) cryptorchidism. Patients with other genital abnormalities, previous use of hCG or testosterone or previous surgeries were excluded. Blood samples were obtained at baseline and 7 days after a single subcutaneous dose of rhCG (Ovidrel® 250 mcg) to measure the testosterone, DHT (dihydrotestosterone), AMH (anti-Mullerian hormone), and inhibin B levels. Results: rhCG stimulation significantly increased testosterone levels from 10 ng/dl to 247.8 ± 135.8 ng/dl, increased DHT levels from 4.6 ± 0.8 to 32.3 ± 18.0 ng/dl, and increased the T/DHT ratio from 2.2 ± 0.4 to 8.0 ± 3.5. There was also a significant increase in inhibin B (from 105.8 ± 65.2 to 132.4 ± 56.1 pg/ml; p < 0.05) and AMH levels (from 109.4 ± 52.6 to 152.9 ± 65.2 ng/ml; p < 0.01) after the rhCG stimulation. Conclusions: In this cohort, hormonal responses can be elicited after the rhCG stimulation test, suggesting that rhCG is a promising stimulation test to replace the urinary hCG test during the evaluation of gonadal Leydig cells function. The clinical applicability and adequate performance of rhCG testing must be investigated in future studies.
Human Chorionic Gonadotropin; Cryptorchidism; Testosterone; Anti-Mullerian hormone; Recombinant hCG
Contexte: L’évaluation de la sécrétion des cellules gonadiques de Leydig prépubères nécessite une stimulation par
les gonadotrophines. La gonadotrophine chorionique humaine (hCG) urinaire est actuellement indisponible dans
de nombreux pays; toutefois, l’hCG recombinante (rhCG) peut être utilisée. Notre objectif était d’évaluer les
hormones testiculaires sous stimulation par rhCG dans un groupe de patients qui présentaient une cryptorchidie.
Méthodes: Nous avons évalué 31 garçons prépubères (âgés de 0,75 à 9 ans) qui présentaient une cryptorchidie uni
(n=24) ou bilatérale (n=7). Ont été exclus les patients avec d’autres anomalies génitales, ceux qui avaient été traités
auparavant par hCG ou testostérone ou par chirurgie. Des échantillons sanguins ont été prélevés au départ et à 7
jours après une unique dose sous cutanée de rhCG (Ovidrel® 250 mcg) pour les mesures des taux de testostérone,
dihydrotestostérone (DHT), hormone antimüllérienne (AMH) et inhibine B.
Résultats: La stimulation par rhCG augmente de façon significative les taux de testostérone de 10 ng/dl à 247.8
±135.8 ng/dl, de DHT de 4.6±0.8 à 32.3±18.0 ng/dl, et le ratio T/DHT de 2.2±0.4 à 8.0±3.5. Les taux d’inhibine B (de
105.8±65.2 à 132.4±56.1 pg/ml; p< 0.05) et d’AMH (de 109.4±52.6 to 152.9±65.2 ng/ml; p< 0.01) ont aussi été
significativement augmentés après stimulation par rhCG.
Conclusion: Dans cette cohorte, des réponses hormonales ont été obtenues après le test de stimulation par rhCG;
ceci suggère que rhCG est un test de stimulation prometteur pour remplacer le test par l’hCG urinaire lors de
l’évaluation de la fonction gonadique des cellules de Leydig. L’applicabilité clinique et l’évaluation adéquate de la
performance du test par rhCG doivent être étudiées dans de futures études.
Mots-clés: Gonadotrophine chorionique humaine, Cryptorchidie, Testostérone, Hormone antimüllérienne, rhCG
The hypothalamic-pituitary axis is inactivated during
childhood, and the testes are functionally quiescent regarding
androgen production by Leydig cells. Therefore, during
the prepubertal phase, testicular steroidogenesis cannot
be evaluated by measuring basal steroid levels; testicular
steroidogenesis evaluation can be performed only after
recombinant luteinizing hormone (LH) and/or gonadotropin
chorionic hormone (hCG) stimulation [1–3].
hCG stimulates the androgen secretion of Leydig cells,
allowing the identification of any intra-abdominal testicular
tissue in patients with true bilateral cryptorchidism. The
hCG test is also used to differentiate between
constitutional pubertal delay and hypogonadotropic hypogonadism,
as well as to investigate patients with sex differentiation
disorders (i.e., by recognising the presence of testicular
tissue and enzymatic defects) .
Stimulatory tests using chorionic gonadotropin
extracted from the urine of pregnant women (uhCG)
have long been used to characterise the pattern of
gonadal steroid production. At least three test-protocols
using intramuscular doses of 100 IU/Kg (maximum of
2000 IU) have been well described: one daily dose for
five consecutive days, one dose every 4 days (a total
of four injections), and one injection every week for
six consecutive weeks [1, 4]; in these protocols a
testosterone response higher than 140 ng/dl is
considered satisfactory. As in many other countries,
uhCG is currently unavailable in Brazil, and it has been
replaced by recombinant human chorionic gonadotropin
(rhCG) [5–7]. To the best of our knowledge, no
previous study of the use of rhCG in children has been
In this study, we used rhCG stimulation testing to
evaluate gonadal function in a cohort of patients with
This prospective study included cryptorchid boys during
4 years, evaluating 31 prepubertal boys (mean
chronological age, 3.3 years; SD, 2.7 years; range, 0.75–9.0 years)
with unilateral (n = 24) or bilateral (n = 7) cryptorchidism.
Patients with any other genital abnormality, previous
use of hCG or testosterone or previous surgeries were
excluded. After receiving approval from the Institutional
Ethics Committee, informed written consent was obtained
for each patient from the parents or legal guardians.
Blood samples for the hormonal measurements were
obtained at baseline and 7 days after a single
subcutaneous dose of rhCG (Ovidrel® 250 mcg).
The following hormonal concentrations were measured at
both time points: testosterone (T) (chemiluminescent assay,
L2KTW2, Gwynedd, England), dihydrotestosterone (DHT)
(radioimmunoassay post-extraction KIPI9900, Louvain La
Neuv, Belgium) androstenedione (radioimmunoassay,
DSL3800, Prague, Czech Republic), 17-hydroxyprogesterone
(17OHP) (radioimmunoassay, KIP1409, Louvain-la-Neuve,
Belgium), inhibin B and anti-Mullerian hormone (AMH)
(ELISA, Gen II Beckman Coulter Company, TX, USA).
Beta-hCG was measured using a post-hCG sample
(chemiluminescent assay, Beckman Coulter, Inc. 4300 N.
Harbor Blvd., Fullerton, CA, USA). The lower limits of
detection for testosterone, DHT, 17OHP, AMH, inhibin B and
beta-hCG were 15 ng/dl, 3 ng/dl, 0.11 ng/ml, 4.8 pg/ml,
0.2 ng/ml, and 2.7 mUI/ml, respectively. The intra-assay
coefficient of variation (CV) for testosterone, DHT, 17OHP,
beta-hCG were 2.0 %, 4.8 %, 5 %, and 1.6 %, respectively,
and the limits varied from 2.4 to 2.9 % and 5.2 to 9.0 % for
inhibin B and AMH, respectively. The inter-assay CV for
DHT was 15 %, and it varied from 4.6 to 7.8 % for AMH.
Statistical analyses were performed using SigmaStat
for Windows (version 3.5, SPSS Inc., San Jose, CA, USA).
Descriptive results were presented as the means ±
standard deviation score (SDS) and percentile. To compare the
quantitative variables before and after the rhCG
stimulation, a paired t-test or Wilcoxon signed-rank test was used
according to the distribution patterns. To establish
correlations between variables, Pearson’s or Spearman’s
coefficient of correlation was calculated. Statistical significance
was set at p <0.05.
The 31 prepubertal boys had normal phallus lengths
(according to Lee et al. ), and no syndromic facies or genital
abnormalities. When testosterone levels from bilateral
cryptorchid boys (basal testosterone: 10 ± 0.0 ng/dl,
rhCGstimulated testosterone: 253.6 ± 128.0) were compared with
testosterone from unilateral cryptorchid boys (basal
testosterone: 10 ± 0.0 ng/dl, rhCG-stimulated testosterone: 228.0
± 169.9), no significant difference was observed (p = 0.669).
Therefore, both groups are presented as a single group.
Hormonal concentrations were assessed at baseline
and 7 days after the rhCG stimulation (Table 1) and
(Additional file 1). As expected, we observed normal
basal concentrations for all hormone levels in the group
of cryptorchidism patients with any other genital
abnormality or complications (Table 1).
A significant negative correlation was identified when
peak testosterone after rhCG was correlated to the age in
which stimulation test was performed (p = 0.035; r = 0.38).
Beta hCG levels were detectable during the stimulation
test, indicating that the drug was adequately injected.
Stimulation with a single subcutaneous dose of rhCG
significantly increased testosterone production (paired t-test,
p < 0.001) from 10 ng/dl at baseline to 247.8 ± 135.8 ng/dl
post stimulation (Table 1). The testosterone percentile
values (ng/dl) were p5(62.9), p10(106.2), p25(131.5),
p50(200.0), p75(317.2), p90(436.6) and p95(520.8).
The mean DHT values were 4.6 ± 0.8 ng/dl at baseline
and 32.3 ± 18.0 ng/dl after the rhCG stimulation (paired
t-test, p < 0.001) (Table 1). After the hCG stimulation,
the T/DHT ratio (Fig. 1) also increased significantly
from 2.2 ± 0.4 to 8.0 ± 3.5 (paired t-test, p < 0.001). Based
on the 10th percentile of our results, after 7 days of rhCG
stimulation, we defined the following normal inferior
response limits for the testosterone levels and T/DHT ratio:
106.2 ng/dl and 4.0, respectively.
There was also a significant increase in inhibin B (from
105.8 ± 65.2 to 132.4 ± 56.1 pg/ml; p < 0.05) and AMH
levels (from 109.4 ± 52.6 to 152.9 ± 65.2 ng/ml; p < 0.01)
after the rhCG stimulation. Based on the 10th percentile of
our results, we defined the normal lower limits of
detection after rhCG stimulation as 63.7 pg/ml and 76 ng/ml
for inhibin B and AMH, respectively. There was no
significant variation in the steroid precursors, such as the
17OHP and androstenedione levels.
The correlation between variables revealed positive and
significant correlations for the following comparisons: basal
AMH and basal inhibin B levels (r = 0.742, p < 0.001), basal
AMH and 7-day post-rhCG testosterone levels (r = 0.55,
p = 0.001) basal inhibin B and 7-day post-rhCG
testosterone levels (r = 0.64, p < 0.001), 7-day post-rhCG
inhibin and 7-day post-rhCG testosterone levels (r = 0.65,
p < 0.001), and 7-day post-rhCG AMH and 7-day
postrhCG inhibin B levels (r = 0.6, p = 0.03). No significant
correlation was observed when the 7-day post-rhCG
AMH level was compared with the 7-day post-rhCG
testosterone level (r = 0.287, p = 0.14) (Fig. 2).
The hCG stimulation test is useful for several clinical
conditions, such as the investigation of a potential androgen
insufficiency, detection of testicular tissue undetectable
during physical examination, and identification of
enzymatic defects of testicular steroidogenesis . In addition,
analysing the peak testosterone and DHT secretion levels
Table 1 Hormonal concentrations at baseline and 7 days after a
single subcutaneous dose of human chorionic gonadotropin
(rhCG, Ovidrel® 250 mcg)
hCG human chorionic gonadotropin, rhCG recombinant human chorionic
gonadotropin, DHT dihydrotestosterone hormone, T/DHT Testosterone/DHT
ratio, AMH Anti-Mullerian hormone, 17OHP 17-hidroxi-progesterone hormone,
FSH Follicle stimulanting hormone, LH Luteinizing hormone, SD
Fig. 1 Individual responses of cryptorchid patients (n:31) before and
after the stimulatory test with a single subcutaneous dose of
recombinant human chorionic gonadotropin (rhCG, Ovidrel® 250
mcg). rhCG: recombinant human chorionic gonadotropin; DHT:
after hCG, as well its ratio, represent an important tool in
the study of patients with abnormal sex differentiation.
The hypothalamic-pituitary-Leydig cells axis is not
activated in the prepubertal phase, therefore, the evaluation of
Leydig cells function requires pharmacological stimulation
with gonadotropins [1, 2]. Until recently, the stimulation
test was performed using urinary hCG, but the withdrawal
of the medication from the markets in many countries
and the availability of the recombinant formulation
indicates a need for this new test standardisation.
Isolated cryptorchidism is a condition in which
testicular lesion is predominantly observed in tubular
Sertoli cells with little or absent involvement of
interstitial testosterone-producing Leydig cells. Therefore, at a
prepubertal stage, this group of patients would present a
potentially normal testosterone response and can be
useful as a control group of studies performed in children
aiming to investigate abnormalities of sexual differentiation
[10–12]. By studying prepubertal cryptorchid patients, we
could rule out severe testicular damage at the same time
we could recognize potentially adequate values for this
specific age, therefore avoiding the ethical issues of performing
a testicular stimulation test in normal children.
In this study, by using rhCG (Ovidrel®, 250 mcg,
approximately equivalent to 6500 UI), we were able to identify a
significant testicular response in prepubertal children after
a single subcutaneous dose. Previous standardization
protocols employed four to six small hCG doses of
1,000-1,500 UI. This is not possible when using the
rhCG as its commercial presentation is a 6500 UI diluted
in a reduced volume of 0.5 mL, making impracticable dose
fragmentation for this product. The interval of 7 days after
rhCG for hormonal assessment was chosen after a pilot
study of five individuals in which hormonal data was
measured at 3, 5, 7 and 10 days after rhCG; in this subjects
testosterone peak response was obtained after 7 days. A
previous study in adult healthy men found similar
testosterone peak after 250 mcg rhCG and uhCG 5000 UI .
In vitro fertilization studies analysing oocyte maturation
did not find significant differences in pharmacokinetics
between both compounds [5, 7].
Although our results cannot be used as normal
reference values, as the rhCG stimulation test was performed
in patients with isolated cryptorchidism and with no other
associated abnormalities, we suggest the use of our results
as an acceptable control response for this test. Our results,
which showed similarities in hormonal concentrations
between unilateral and bilateral cryptorchidism, align with
the results reported by Christiansen et al. , who also
compared unilateral and bilateral cryptorchid patients
with normal controls. An important observation is the
negative correlation between age and testosterone
during rhCG test. This may indicate a need to adjust the
expected testosterone peak response to age.
An increase in inhibin B, which was observed after the
hCG stimulation in this study, was also demonstrated in
a previous report  of cryptorchidism patients treated
with intramuscular uhCG in a 3-week stimulation protocol.
The authors found a peak inhibin B response of 147 pg/ml,
similar to that detected in our patients (129.7 pg/ml). This
response can be explained by the fact that in prepubertal
testes, Sertoli cells are able to synthesise both inhibin B
subunits (alpha and beta B) [13–15]. A different secretion
pattern is observed in the late pubertal and adult stages, in
which the beta subunit becomes a product of germinal
cells, while the alpha subunit is still secreted by Sertoli cells.
Fig. 2 Correlations between the hormonal concentrations in the cryptorchid patients (n:31) at baseline and after the recombinant human chorionic
gonadotropin test. rhCG: recombinant human chorionic gonadotropin; AMH: Anti-Mullerian hormone
Therefore, in contrast to observations in children, inhibin B
becomes a hormone of combined Sertoli-spermatocyte
origin in adults [14, 15]. This finding can explain why
the increment of inhibin B is only detected in
prepubertal children, such as those included in our study but
not in adults . An alternative explanation for the
detected inhibin B secretion during rhCG testing is the
direct effect of testosterone on prepubertal Sertoli cells.
High levels of hCG could bind to FSH receptors and
stimulate the hormonal production of Sertoli cells. The
possible promiscuous coupling versatility in signalling
associated with a single receptor seems to be an intrinsic
property of G-protein-coupled receptors [16, 17]. This
finding is reinforced by the positive and significant
correlation between the testosterone peak and inhibin B peak
responses after rhCG stimulation.
Regarding the AMH response, we detected a
significant increase in AMH values 7-days after the rhCG
application and a positive correlation between the
baseline and 7-day AMH levels and post-rhCG testosterone
levels. Our results can also be explained in a manner
similar to the observations for inhibin B (i.e., by the phase
of testicular development in which immature Sertoli cells
are stimulated by an acute single dose of rhCG). We
may conclude that a parallel increase in inhibin B and
AMH suggests that in the prepubertal stage, under
acute stimulation, the Sertoli cells retain the capability
of proportional secretion of both peptides.
One of the limitations of this study is the measurement
of steroid hormones through immunoassays. Recent
studies have shown that using mass spectrometry (MS) to
measure steroid hormones represents the gold standard
when used in the appropriate manner under highly
regulated conditions , and the immunoassays do not have
sufficient sensitivity to detect normal low levels in samples
of females and non-stimulated gonads of prepubertal
patients . The ability to correlate gonadal function after
an acute 7-day stimulation test with gonadal function in
adolescents during puberty or in adults (a longitudinal
follow-up of these patients is desirable) is not available at
this time, and it can represent another limitation of this
Although the protocol and dosage are different for
urinary and recombinant hCG we conclude that the 7-day
hormonal response after a single subcutaneous dose of
rhCG in outpatient clinics is a simple and promising
alternative to the urinary hCG test for acute evaluation of
prepubertal gonadal secretion. Besides, we have to
consider that urinary hCG is currently unavailable in many
countries. We show that recombinant hCG triggers a
response and provides a proof-of-concept for a future
validation of this test. Clinical applicability under distinct
conditions must be investigated in further studies.
17OHP: 17-hidroxi-progesterone hormone; AMH: Anti-Mullerian hormone;
betahCG: β-subunit of hCG; CV: Coefficient of variation; DHT: Dihydrotestosterone
hormone; FSH: Follicle stimulanting hormone; hCG: Human chorionic
gonadotropin; LH: Luteinizing hormone; P: Percentile; rhCG: Recombinant
human chorionic gonadotropin; T: Testosterone; T/DHT: Testosterone/DHT ratio;
uhCG: Urinary human chorionic gonadotropin
We wish to thank Pediatric Endocrinology Unit of Santa Casa de São Paulo
Medical School and Developmental Endocrinology Unit of Hospital das
Clínicas da Faculdade de Medicina da USP, São Paulo. We also thank to
Merck Serono Brasil for kindly donate the recombinant hCG for this study.
Availability of data and materials
We consider important to show as supplementary data all items analysed
from each patient and the hormonal measurements at each time of the
assessment: see Additional file 1: Table of information.
OLR, HTK were responsible for patients data capture and applying the test
using recombinant hCG. LCA carried out the immunoassays and performed
the statistical analysis. WRR and BVN contributed to hormone dosages and
participated in the design of the study. All of them helped to draft the
manuscript. All authors read and approved the final manuscript.
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