Highlights
· This study compares the gonadorelin stimulation test and triptorelin stimulation test to determine whether triptorelin can be used as a substitute when gonadorelin is not available. The results show that the triptorelin stimulation test showed similar diagnostic accuracy to the gonadorelin stimulation test.
Introduction
Puberty is a transitional period from childhood to adulthood, which begins with reactivation of the hypothalamic-pituitary-gonadal (HPG) axis [1]. Precocious puberty is defined as the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. Precocious puberty is classified into gonadotropin-releasing hormone (GnRH)-dependent precocious puberty and GnRH-independent precocious puberty depending on activation of the HPG axis. For many reasons, the prevalence of precocious puberty is increasing, especially among girls [2]. Thus, breast development is occurring at a younger age. As parents are becoming more aware of this phenomenon, the number of girls visiting pediatric endocrinologists for evaluation has increased [3].
Gonadotropin-dependent precocious puberty, or central precocious puberty (CPP), is caused by premature activation of the HPG axis and can be treated with GnRH agonists. Patient history, physical examination, basal gonadotropin levels, and laboratory tests can be used to diagnose CPP [4,5] The GnRH stimulation test is considered the gold standard for detecting activation of the HPG axis [6,7]. CPP is diagnosed when bone age is more than 1 year higher than chronological age and when the peak luteinizing hormone (LH) level is above 5 mIU/mL during the GnRH stimulation test.
The safety and effectiveness of GnRH agonists have been demonstrated over decades, and they are the treatment of choice for CPP [8]. GnRH agonists are agonists of the GnRH receptor, which normally stimulates release of LH and follicle-stimulating hormone (FSH) upon binding with GnRH. After the first phase of rapid GnRH release, continuous stimulation with GnRH agonists desensitizes the pituitary gland to GnRH, leading to GnRH receptor downregulation. As a result, the HPG axis is suppressed, which is a desirable effect when treating CPP.
For the stimulation test for diagnosing CPP, GnRH (gonadorelin) is not always readily available; hence, GnRH agonist (triptorelin acetate) has been suggested as a substitute for GnRH. Clinicians have focused on the acute stimulatory effect on gonadotropin release when administered as a single dose. Bhatia et al. demonstrated that the peak concentration of GnRH agonist was achieved 15–60 minutes after the injection, which is sufficient time to achieve gonadotropin secretion [9,10]. This first phase rapid release of GnRH makes it ideal for use as a GnRH substitute in the stimulation test.
This study evaluated the diagnostic validity of the triptorelin stimulation test compared with the gonadorelin stimulation test in the diagnosis of CPP. We also determined the optimal length of the stimulation test performed with triptorelin acetate.
Materials and methods
1. Study population
This retrospective study included girls who visited the Pediatric Endocrinology Clinic at Korea University Ansan Hospital between October 2021 and May 2022 for premature thelarche (PT) evaluation. A total of 100 girls aged 7–8 years was enrolled; 50 girls underwent a GnRH (gonadorelin) stimulation test and the remaining 50 underwent a GnRH agonist (triptorelin acetate) stimulation test. We first selected girls who underwent triptorelin stimulation test and satisfied the enrollment criteria. We then reviewed the girls who underwent gonadorelin stimulation test and selected participants with similar clinical characteristics such as age and body mass index (BMI). Participants with organic diseases were excluded from this study.
2. Clinical data collection
Patient data were obtained from the medical records through a retrospective review. Data on age, height, weight, BMI, parents' heights, breast Tanner stage, bone age at initial visit, and LH and FSH levels during stimulation tests were collected [11]. Height was measured to the nearest 0.1 cm using a rigid stadiometer (Seca, Hamburg, Germany), and weight was measured to the nearest 0.1 kg using a calibrated balance scale (Seca). The height standard deviation score (SDS), weight SDS, and BMI SDS were calculated using Cole's Lambda Mu Sigma method following the 2017 Korean National Growth Chart [12]. Bone age was measured by a single pediatric endocrinologist using the Greulich and Pyle method. To diagnose CPP, a gonadorelin stimulation test was performed by administering 0.1-mg gonadorelin (Relefact, Sanofi-Aventis, Gentilly, France) after collecting a baseline sample. Samples were collected at 30, 45, 60, and 90 minutes to measure LH and FSH levels. The triptoreline (Decapaptyl, Ferring, Saint Prex, Switzerland) stimulation test was performed similarly. Basal LH and FSH were sampled before subcutaneous injection of 0.1-mg triptorelin. LH and FSH samples were collected at 30, 60, 90, and 120 minutes after triptorelin injection. Serum LH and FSH levels were measured using electrochemiluminescence technology (Cobas 6000, Roche Diagnostics, Indianapolis, IN, USA).
3. Definition
CPP was diagnosed when breast Tanner stage was II or above in patients under the age of 8, when LH levels during the gonadorelin or triptorelin stimulation test peaked above 5 mIU/L, and when bone age was advanced over chronological age by more than 1 year before the age of 9.
4. Statistical analyses
Data are expressed as mean±standard deviation. Shapiro-Wilk normality test was conducted and paired t-tests were used to compare the clinical characteristics and laboratory results from 2 stimulation tests. P-values < 0.05 were considered statistically significant. To compare the sensitivity and specificity of these 2 stimulation tests, receiver operating characteristic (ROC) curve analysis was performed. IBM SPSS Statistics version 20.0 (IBM Co., Armonk, NY, USA) was used to conduct statistical analyses.
Results
Clinical characteristics of age, height, weight, BMI, and breast Tanner stage were similar between the 2 groups (Table 1). Basal LH, FSH, and estradiol levels also did not show differences between the 2 groups (Table 1).
We examined changes in LH levels during the stimulation tests. The trend of LH change was similar in the groups, and the highest LH level was seen 60 minutes after stimulation (Table 2). Approximately 20% of the gonadorelin stimulation test group and 24% of the underwent triptorelin stimulation test group were diagnosed with CPP (Table 1).
Comparison of the LH levels between the CPP group and non-CPP group showed that basal LH was below 0.2 mIU/mL in both groups. Girls diagnosed with CPP showed the highest LH level at 60 minutes, and the mean concentrations were 8.15 mIU/mL and 9.73 mIU/mL for the gonadorelin and triptorelin group, respectively. The non-CPP group showed the highest LH concentration at 90 minutes in the gonadorelin group and at 120 minutes in the triptorelin group (Table 3).
In ROC curve analysis, we observed similar sensitivity and specificity between the 2 groups; area under the curve was 0.99 for the triptorelin group and 0.97 for the gonadorelin group (Fig. 1).
Discussion
The incidence of CPP is increasing. As breast development is occurring at earlier ages for various reasons, it is important to evaluate whether the reason for PT is CPP [13]. Breast Tanner stage, accelerated growth, bone age advancement, and uterine and ovarian enlargements are some helpful parameters when diagnosing CPP. However, these are not absolute indications, and not all features must be present in a patient. Evaluating gonadotropin levels is the key to diagnosis, and the gonadorelin stimulation test is the gold standard for CPP diagnosis [14]. Gonadotropin level measured after GnRH stimulation allows the differentiation of self-limited PT from thelarche due to CPP.
GnRH is secreted from hypothalamus and activates the GnRH receptor, which is expressed on the surface of pituitary gonadotropic cells, to stimulate the synthesis of FSH and LH. GnRH agonist is an agonist for the GnRH receptor. These synthetic molecules show higher affinity to the receptors and release more gonadotrophs compared with natural GnRH [15]. When bound to the GnRH receptor, GnRH agonists result in strong stimulation and release of FSH and LH. A single injection can produce an initial flare response and increases in FSH and LH, which allows it to be used during the stimulation test instead of GnRH. However, continued stimulation desensitizes the pituitary gland to GnRH. Thus, FSH and LH levels decrease, leading to a hypogonadotropic and hypogonadal state [16]. This is why GnRH agonists are used as treatment for hormone-sensitive situations, such as precocious puberty, in vitro fertilization, and prostate and breast cancers [17]. These 2 opposing features of GnRH agonist allow them to be used as a stimulant and depressor of gonadotrophs. The safety and effectiveness of GnRH agonist (GnRHa) as a treatment option for CPP has been well established. However, few studies have examined the effectiveness of triptorelin, a GnRHa, as a substitute for GnRH when diagnosing CPP.
Previous studies have shown that the triptorelin stimulation test can substitute for the gonadorelin stimulation test with high diagnostic accuracy [18,19]. However, the reported optimal duration of stimulation test differs among studies. Some reports state that analysis of an LH sample 30 minutes after triptorelin injection is sufficient, and some state that continuation until 180 minutes after triptorelin injection gives a higher accuracy [20,21]. Our study evaluated the efficacy of the triptorelin stimulation test and appropriate length of study time. Clinical characteristics of age, height, weight, and BMI were similar between the 2 groups. Both groups showed the highest LH level at 60 minutes after the stimulation. Approximately 20% of the girls who underwent gonadorelin stimulation test and 24% who underwent the triptorelin stimulation test were diagnosed with CPP. Of the girls who were diagnosed, peak LH was seen at 60 minutes after the stimulation. Our data suggest that the triptorelin stimulation test showed similar trends of LH elevation and diagnostic rate compared with the classic gonadorelin stimulation test in diagnosing CPP. In ROC curve analysis, we observed similar sensitivity and specificity between the 2 groups, and the AUC values were similar, which indicates similar performance of the 2 tests. These results suggest that the triptorelin test may be a valid alternative to the traditional gonadorelin test when differentiating CPP from self-limiting PT. Our study also showed that the triptorelin stimulation test up to 120 minutes was sufficient to make a diagnosis of CPP.
One limitation of our study was that we did not perform gonadorelin and triptorelin stimulation tests on the same subject. Unlike previous studies that performed both tests on a single subject, we performed the 2 tests on 2 groups of girls. We tried to compensate by selecting girls with similar clinical characteristics such as age, height, and weight. Despite our efforts to establish groups with similar characteristics, the mean bone age for the gonadorelin group was 8.63±0.41 years compared to 8.87±0.29 years for the triptorelin group. While the difference was not significant (P=0.08), we speculated an effect on the results since advanced bone age is an important factor when diagnosing CPP. Another factor that could have affected the result was different sampling intervals. The gonadorelin stimulation test was performed by collecting hormone samples at baseline and 30, 45, 60, and 90 minutes after gonadorelin injection, while the triptorelin stimulation test collected samples at baseline and 30, 60, 90, and 120 minutes after triptorelin injection. We only used baseline, 30, 60, 90 minutes values when comparing the 2 groups.
In conclusion, the triptorelin stimulation test showed similar diagnostic accuracy to the gonadorelin stimulation test. These results indicate that GnRH agonist can not only be used as a treatment modality for CPP, but can also be used as a reliable alternative to the traditional gonadorelin stimulation test.
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