ORIGINAL PAPER

Jpn J Reprod Endocrinol ⓒ2003 by
Japan Society of Reproductive Endocrinology

The Usefulness of GnRH and hCG Testing forthe Differential Diagnosis of Delayed Puberty andHypogonadotropic Hypogonadism in Prepubertal Boys
Naoko SATO1), Noriyuki KATSUMATA1), Reiko HORIKAWA2) and Toshiaki TANAKA2)
1) Department of Growth and Puberty, National Research Institute for Child Health and Development, Tokyo 154-8567, Japan
2) Division of Endocrinology and Metabolism, National Center for Child Health and Development, Tokyo 157-8535, Japan

Abstract. [Objective] Discrimination between hypogonadotropic hypogonadism (HH) and constitutional delayed
puberty (DP) is difficult in prepubertal patients. We retrospectively analyzed the usefulness of the gonadotropin-releasing hormone (GnRH) test using time-resolved fluoroimmunoassay (TR-FIA) for the measurement of gonadotropins and the human chorionic gonadotropin (hCG) test to discriminate between HH and DP during the prepubertal
period.
[Method] Gonadal function was evaluated by the hCG test and the GnRH test using TR-FIA in 16 prepubertal boys
who were suspected of having HH, of whom after long-term observation 11 boys were finally diagnosed with HH and5 boys with DP.
[Results] In the GnRH test, peak LH levels were below the normal range in all HH patients and within the normal
range in 4 of the 5 DP patients. Peak FSH levels were within the normal range in 4 of the 5 DP patients and below
the normal range in 9 of the 11 HH patients. In the hCG test, testosterone levels after 3-day administration of hCG
were below 50ng/ml in all HH patients except one 6-month-old infant and above 50ng/dl in all DP patients.
[Conclusion] GnRH testing using the TR-FIA assay is useful for diagnosis of HH during the prepubertal period. The
combination of GnRH and hCG tests might improve diagnostic accuracy.

Key words: Hypogonadotropic hypogonadism, GnRH test, hCG test, Delayed pubert, TR-FIA

Introduction

though sensitivity differed significantly between these tworeports, both suggested that the GnRH and hCG tests

Differentiation between hypogonadotropic hypogonad-

were of limited value in the diagnosis of HH during theism (HH) and constitutional delayed puberty (DP) in the

prepubertal period. In more recent reports, more sensitive

prepubertal period remains difficult despite advances in

immunoradiometric assays (IRMA) [4, 5], instead of theendocrinological techniques. Accurate diagnosis makes it

standard RIA assay and TR-FIA, (Delfia FSH, LH, Pharma-

possible to induce puberty with appropriate timing and to

cia-Upjohn) have been recently used as an ultra-sensitive

improve quality of life in HH patients. We previously re-

non-RIA assay for LH and FSH [6]. To clarify the useful-ported the usefulness of the LHRH test relying on a stan-ness of TR-FIA and the discriminative power of GnRH anddard RIA assay [1] for the diagnosis of HH, but the sensi-

hCG testing, we evaluated gonadal function by GnRH test-

tivity and specificity of plasma LH measurement in theing using TR-FIA and hCG testing in 16 prepubertal boys

GnRH test are unsatisfactory. Dunkel et al. [2] and Job [3]with suspected HH.

reported that the hCG test was more reliable than the
GnRH test for diagnosis of gonadotropin deficiency. Al-

Accepted: August 4, 2003
Correspondence: Toshiaki TANAKA, Division of Endocrinology and Metabolism, National Center for Child Health andDevelopment, 2-10-1, Ohkura, Setagaya-ku, Tokyo 157- 8535,
Japan
TEL: +81-3-3414-8121
FAX: +81-3-5494-7136
E-mail: tanaka-t@ncchd.go.jp

Subjects
Sixteen Japanese prepubertal boys suspected during
the prepubertal period of having HH were studied. After
long-term observation 11 boys were diagnosed with HH
and 5 boys with DP. Those diagnosed with HH included
seven with Kallmann syndrome (KS), one with DAX-1
gene mutation, one with idiopathic isolated HH, one withJapanese Journal of Reproductive Endocrinology (2003) 8 : 49-53

49

Naoko SATO et al.

Table 1. Clinical features in hypogonadotropic hypogonadism and delayed puberty

panhypopituitarism, and one with CHARGE association.

0.05mIU/ml for FSH. Testosterone was measured by RIA

Table 1 shows the final diagnosis, clinical features, and(Diagnostic Products Co., USA).

age at examination.

Results
Methods

１）Clinical features

All patients underwent both hCG and GnRH testing.

All KS patients had cryptorchidism or migrating testes.

The GnRH stimulation test was performed by intravenous

All KS patients except Cases 3 and 7 had hyposmia or an-

injection of gonadorelin acetate (Tanabe), at a dose of 100

osmia. Cases 2 and 3 were KS brothers with an identical2µg/m followed by blood sample collections for LH and

R631X nonsense mutation of the KAL1 gene, but they had

FSH measurements at 0, 30, 60, 90 and 120 min after

significantly different phenotypes of olfactory sensitivity

injection. The hCG (Teikokuzouki) was injected intra-

and renal abnormalities. Case 2 (the elder brother) was2

muscularly at a dose of 3,000IU/m once a day for threeanosmic and had two normal kidneys, while Case 3 (thedays, and blood sampling for testosterone was done daily

younger brother) had normal olfactory sensitivity andfrom day 1 to day 4. Serum LH and FSH levels were mea-

right renal agenesis. Case 7 had a normal sense of smell,

sured by TR-FIA (Pharmacia). The intra-assay coefficientalthough he carried a splice mutation in intron 4 of theof variation ranged from 4.21% to 6.85% and the inter-assay

KAL1 gene. Gene analysis was performed because he hadcoefficient of variation from 1.99% to 7.00%. The limit ofright renal aplasia, which suggested X-linked KS. Case 9

sensitivity of the assay was 0.08mIU/ml for LH andshowed high general hyperpigmentation at initial observa-

50Japanese Journal of Reproductive Endocrinology Vol. 8 2003

GnRH and hCG Testing in Delayed Puberty and Hypogonadotropic Hypogonadism during Prepuberty

Fig.1 The normal range of peak testosterone response tohCG stimulation is more than 150ng/dl [1]. Peak testosterone levels were less than 50ng/dl in all HH patients(KS, IHH, MPHD and CHARGE association) except Case
6. The peak testosterone level in Case 6 was 237ng/dl at
initial examination at age 6 months, but it had decreased
to below 50ng/ml at the second examination at 7 years.
The peak testosterone levels ranged from 50 to 150ng/dl
Fig. 1. Peak testosterone responses to the hCG test ( ■―■ , ○―○ the hCG test was repeated at intervals). Wide solid line
shows the lower limit of normal response. Dotted line shows
the lower limit of subnormal response.

in four DP patients. Only one patient with DP (Case 16)
responded with a level greater than 150ng/dl. Case 12(DP) was tested three times by stimulation with hCG, towhich the testosterone response gradually improved fromtion at 6 years and was subsequently found to have alow (101ng/dl at 10 years and 123ng/dl at 11.3 years) to

DAX-1 mutation. Case 10 had a history of complicated de-the normal range (423ng/dl at 12.4 years).

livery with breech presentation and asphyxia; he was diag-

３）GnRH test

nosed at age 11 years with growth hormone deficiency
characterized by short stature and was later found to haveFig.2(a) shows the peak LH response to GnRH. Peak

HH. Case 11 was diagnosed as CHARGE syndrome by as-

LH levels were below the normal range in all HH patients,sociation with deafness, corneal dysplasia, and deviation

but within the normal range in all DP patients except Caseof aortic valve on initial consultation; HH was detected

14. In Case 14, peak LH was below the normal range at

during the observation period.

12.3 years but rose to 8.2mU/ml (normal pubertal re-

Five patients with delayed puberty were also suspectedsponse) at age 13.5 years. His testis volume increased andof having HH at an earlier diagnosis because of the pres-growth spurt occurred after administration of anabolic ster-ence of cryptorchidism, migrating testes, micropenis or

oid at 13.6 years. We conclude that administration of ana-

small testis, but they were eventually diagnosed with de-bolic steroid induced puberty in Case 14.

layed puberty due to development of spontaneous puber-

Fig.2(b) shows the distribution of peak FSH levels in

ty. Case 14, who lacked clinical features of HH, was diag-

the GnRH test. Peak FSH levels were within the normal

nosed at initial observation with HH because of his low

range in two HH patients but below the normal range inpeak LH response to GnRH, but he later developed spon-

the remaining nine HH patients. Only one of the DP pa-

taneous puberty.

tients showed a low FSH response, but his peak LH level
was within the normal range at age 10 years.

２）hCG test
The results of the hCG stimulation test are shown inabFig. 2 (a). Peak LH levels by the GnRH test. Dotted lines show the upper and the lower limits of normal response for age group.
(b). Peak FSH levels by the GnRH test. Dotted lines show the upper and the lower limits of normal response for age
group.

ORIGINAL PAPER

51

Naoko SATO et al.

abFig. 3 (a). Distribution of peak LH levels by the GnRH test and peak testosterone levels by the hCG test in subjects aged 10
years and older. The rectangular open box indicates subnormal levels.
(b). Distribution of peak FSH level by the GnRH test and peak testosterone by the hCG test in subjects aged 10 years
and older. The rectangular open box indicates subnormal levels.

These reports suggested that a single GnRH test using４）Combination of GnRH and hCG testing in subjects aged 10 years and older

RIA assay was insufficient to discriminate between HH
and DP and that the GnRH test should be repeated to diagnose HH.

Fig.3(a) shows the distribution of peak LH levels in the

Immunoradiometric assay (IRMA) provides improved

GnRH test and the testosterone peak levels in the hCG

diagnostic potential and sensitivity for LH in the diagnosis

test in patients at age 10 years and older. The rectangularof puberty and delayed pubertal development [4, 5]. TR-

box indicates peak LH levels less than 2.0mIU/ml andFIA, which is a non-RIA assay, has higher sensitivity thanpeak testosterone levels less than 50ng/dl. All HH pa-

IRMA [7]. Fujiwara et al. [7] showed a strong correlation

tients’ levels were within the rectangular box, while all DP

between IRMA and TR-FIA in LH and FSH measurements

patients’ levels were outside. The rectangular box in(LH: r=0.953, n=371 and FSH: r=0.991, n=399). The limit ofFig.3(b) indicates peak FSH levels below the normal

sensitivity of this TR-FIA assay was 0.08mIU/ml for LH

range (FSH<4.38mIU/ml) coupled with peak testosterone

and 0.05mIU/ml for FSH; that of IRMA was 0.15mIU/ml

levels less than 50ng/dl. Most HH patients and all DP pa-

for both LH and FSH [8].

tients showed the same pattern as the LH/hCG peak com-

In the present study, the false normal peak LH response

bination. Only one HH ( isolated GnD) patient had a nor-rate was 0% in the patients with HH (0/11); the false low

mal FSH with low testosterone response. Two HH

response rate was 16.7% in DP patients (1/6). Therefore,

patients under 10 years old exhibited low peak LH andthe sensitivity and the specificity of peak LH levels in the

low peak FSH levels, while one DP patient under 10 years

diagnosis of HH were 100% and 83.3%, respectively. The

showed normal peak levels (not shown).

peak LH level in a repeated GnRH test was normal in one
DP patient with a false low LH peak; in this patient, theDiscussion

peak testosterone level in the hCG test was within thenormal range. The sensitivity and the specificity of peak

The hCG test has been reported to diagnose HH before

FSH levels in the diagnosis of HH were 81.8% and 83.3%,

puberty [1]. However, the GnRH test using RIA assay wasrespectively. Dunkel et al. [9] suggested that peak LH lev-

noted to be of limited value in predicting sexual matura-els were more reliable than peak FSH levels in the diagno-tion in boys with HH. False low peak LH in the GnRH test

sis of HH. The present study produced comparable re-was found in 18% of patients with isolated GHD who com-sults.

pleted sexual maturation later. A normal peak LH re-

The hCG test results can also compensate for errone-sponse was found in 16 of 39 GHD patients (41%) in whom

ous results of a GnRH stimulation test. According to Tana-sexual maturation did not occur spontaneously [1]. Dun-ka et al. [1], the normal peak testosterone level after ankel et al. reported that peak LH levels in GnRH tests werehCG stimulation test is greater than 150ng/dl. The sensi-

subnormal in only 65% (15/23) of patients with HH [2].

tivity and the specificity of the hCG test in the diagnosis of52

Japanese Journal of Reproductive Endocrinology Vol. 8 2003

GnRH and hCG Testing in Delayed Puberty and Hypogonadotropic Hypogonadism during Prepuberty

HH were 91.7% and 100%, respectively, when the cut-off

grateful to Dr. John Parks and Milton Brown (Emory Universi-

value was 50ng/dl. However, a diagnosis of HH is difficultty) for their special advice. This study was supported by thein an infant less than 6 months old. Case 6 had an initial

grant from Ministry of Health, Labor and Welfare.

testosterone level by the hCG test of 237.5ng/dl at age 6months, but his peak testosterone fell to below 50ng/dl at
7 years. Forest [10] also reported age-related changes inpost-hCG testosterone levels in the first six months of lifeand suggested that peak testosterone levels by the hCG
test are useful in the diagnosis of hypogonadism in boys
older than 6 months old. A testosterone value of 50ng/dl
in response to hCG stimulation could be used as a cut-off
value in the diagnosis of HH and serve to distinguish between HH and DP. When both GnRH and hCG testing are
performed in suspected patients aged 10 years and older,both the peak LH level by the GnRH test and the testosterone level by the hCG test have excellent diagnostic value.
Androgen deficiency causes the absence of sexual development, regression of sexual function and spermatogenesis, anemia, muscle atrophy, decrease in bone mineral density, increase in fat mass, and behavioral and moodalternations. In particular, males who do not produce sufficient testosterone at the appropriate adolescent age may
experience behavioral and cognitive impairments. Testosterone deficiency may negatively affect personal andworkplace relationships [11]. Correct diagnosis is required for appropriate timing of sex steroid supplement
and improvement of quality of life in HH patients.
In conclusion, the diagnostic value of GnRH for diagnosis of HH is improved by using TR-FIA to measure LH and
FSH. When used in combination with the hCG test, theGnRH test using TR-FIA could reduce HH diagnostic errors in boys.
Acknowledgements
We thank Mitsuko Sakai for assaying LH, FSH, and testos-

References
1. Tanaka T, Hibi I, Tanae A (1992) Predictability of sexual maturation by human chorionic gonadotropin (hCG) test and gonadotropin-releasing hormone (GnRH) test in prepubertal boys with
idiopathic growth hormone deficiency (GHD): Diagnosis of combined gonadotropin deficiency (GnD) before puberty. Clin Pediatr Endocrinol 1, 21-25.
2. Dunkel L, Perheentupa J, Virtanen M, Maenaa J (1985) Gonadotropin-releasing hormone test and human chorionic gonadotropin test in the diagnosis of gonadotropin deficiency in prepubertal boys. J Pediatr 107, 388-392.
3. Job JC, Canlorbe P (1981) The sex gland. In: J-C Job, M Pierson(eds) Pediatric endocrinology. John Wiley & Sons, New York,
pp.378-387.
4. Yoshizawa A, Tanaka T, Horikawa R, Miki Y, Junko I, Tanaka M,
Tanae A, Hibi I, Sakai M (1990) Clinical study on the immunoradiometric assay of luteinizing hormone and follicle stimulating
hormone in children. Clin Endocrinol (Tokyo) 38, 217-221.
5. Cavallo A, Zhou XH (1994) LHRH test in the assessment of puberty in normal children. Horm Res 41, 10-15.
6. Takeshita E, Ito S, Masumoto M, Ohta A, Noji S, Uchida M,
Kikuchi H, Iri H, Tanabe K, Nozawa S (1995) Fundamental andclinical evaluation of DELFIA LH, FSH kit. Med Pharm 33,
69-977.
7. Fujiwara H, Shimoshikiryo K, Okamoto H, Murata K (1989) Determination of gonadotropin by time-resolved fluoroimmunoassay. Clin Endocrinol (Tokyo) 22, 1547-1559.
8. Wennink JM, Delemarre-van de Waal HA, Schoemaker R,
Schoemaker H, Schoemaker J (1989) Luteinizing hormone andfollicle stimulating hormone secretion pattern in boys throughout puberty measured using highly sensitive immunoradiometricassays. Clin Endocrinol (Oxford) 31, 551-564.
9. Dunkel L, Perheentupa J, Virtanen M, Mäenää J (1985) GnRH
and hCG test are both necessary in differential diagnosis of maledelayed puberty. Am J Dis Child 139, 494-498.
10. Forest M (1979) Pattern of the response of testosterone and its
precursors to human chorionic gonadotropin stimulation in relation to age in infant and children. J Clin Endocrinol Metab 49,
132-137.
11. Zitzmann M, Nieschlag E (2000) Hormone substitution in male
hypogonadism. Mol Cell Endocrinol 161, 73-88.

terone and Seiko Honjyo for a great deal of help. We are also

ORIGINAL PAPER

53