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 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. 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 study.