Evaluation of growth hormone provocative tests in Egyptian children with growth hormone-related short stature

Abstract

BACKGROUND

Diagnosing growth hormone deficiency (GHD) in children remains challenging because of the considerable variability in the results of growth hormone (GH) provocative tests.

AIM

To evaluate the validity of different GH provocative tests, determine the sampling time associated with the peak GH response, and identify optimal GH cut-off values for excluding GHD.

METHODS

This cohort study included 50 children aged 5-16 years presenting with proportionate short stature. GH secretion was stimulated using the insulin tolerance test, clonidine test, and levodopa (L-DOPA) test. Serum GH and insulin-like growth factor-1 levels were measured using commercially available enzyme-linked immunosorbent assay kits.

RESULTS

Among the studied participants, 48% were diagnosed with GHD, while 52% were classified as having idiopathic short stature. The clonidine stimulation test showed the highest median GH level at 90 minutes, followed by the insulin-induced peak at 45 minutes and the L-DOPA peak at 60 minutes. A statistically significant positive correlation was observed between the insulin-like growth factor-1 Z-score and the GH level measured at 90 minutes during the clonidine test. Receiver operating characteristic curve analysis demonstrated optimal GH cut-off values of 7.54 ng/mL for the insulin test, 8.48 ng/mL for the clonidine test, and 4.63 ng/mL for the L-DOPA test. These cut-offs showed sensitivities of 59.3%, 77.8%, and 80%, and specificities of 91.3%, 82.6%, and 70%, respectively.

CONCLUSION

Oral clonidine and L-DOPA stimulation tests appear to be useful and safe alternatives for diagnosing GHD, avoiding the risk of hypoglycemia associated with the insulin tolerance test. Lower GH peak cut-off values may improve the diagnostic performance of these provocative tests.

Key Words: Short stature; Growth hormone; Provocative tests; Children; Insulin-induced peak

Core Tip: Growth hormone (GH) provocative testing in children demonstrates considerable variability, which can complicate the accurate diagnosis of GH deficiency. This study emphasizes that oral clonidine and levodopa stimulation tests represent safer, more practical, and reliable alternatives to the insulin tolerance test. Additionally, adopting lower, optimized cut-off values for GH levels enhances diagnostic precision and reduces the risk of misclassification in pediatric patients undergoing evaluation.

INTRODUCTION

Normal linear growth reflects a child’s overall health and is regulated by endocrine signalling, nutritional status, and inflammatory mediators[1,2]. Short stature is a frequent cause of pediatric endocrine referral, yet the diagnosis of growth hormone deficiency (GHD) remains complex[3,4]. According to El-Shafie et al[5], 17% of 33150 Egyptian children between the ages of 6 and 11 who attended 59 primary schools in various regions of Egypt had small stature. According to a study by Ramadan et al[6], 5% of 419 school children and adolescents in Tripoli, Libya, in 2009-2010 had short stature[6]. Between 1990 and 2010, the prevalence of stunting in children under five fell from 39.7% to 26.7% worldwide (the rate in North Africa was 21.9% in 2010)[7].

Growth hormone (GH) is secreted in a pulsatile pattern, rendering random serum measurements unreliable[8]. Consequently, pharmacologic provocative stimulation tests remain the cornerstone of GHD diagnosis. However, significant inter-test variability and lack of assay-standardized cut-off values compromise diagnostic precision[9,10]. Common stimulatory agents include insulin-induced hypoglycemia, clonidine, and levodopa (L-DOPA). Hypoglycemia induced by insulin stimulates GH release; by suppressing somatostatin, which inhibits GH release, and stimulates adrenergic receptors, which promote GH release[11]. Clonidine is an α2-adrenergic agonist that induces GH-releasing hormone secretion and inhibits somatostatin release from the hypothalamus[12], while L-DOPA administration indirectly induces GH secretion from the pituitary[13]. Constitutional delay, chronic systemic disease, nutritional inadequacy, hypothyroidism, genetic disorders, skeletal dysplasia, and psychosocial deprivation are the main differential diagnoses for short stature and probable GH deficiency[14]. The absence of a universal gold standard for the diagnosis of GH deficiency in children has led to inconsistent GH thresholds across studies and clinical guidelines[15]. This study aimed to compare the diagnostic validity of three GH provocative tests (Insulin, L-DOPA, and Clonidine), identify optimal sampling times, and propose assay-specific GH cut-off values for normal GH response.

MATERIALS AND METHODS

A prospective cohort study that was carried out with 50 children and adolescents of both sexes presented to the endocrinology clinic, Faculty of Medicine, South Valley University, Qena, with proportionate short stature. They were aged 5 to 16 years. The study was performed according to the instructions presented in the Declaration of Helsinki, during the study period from February 2021 to December 2022. This study was approved by the local Ethics Committee of the Faculty of Medicine, Qena University, Qena, Egypt (Ethical approval code: No. SVU-MED-PED025-1-20-12-109). Informed written consent was taken from parents or caregivers of the included participants for participation in the study and publication.

Cases with familial or constitutional short stature were excluded. Cases associated with bony dysplasia, chronic diseases, malnutrition, other endocrine disorders, and chromosomal or genetic disorders were also excluded. A child is considered short when his or her height fall by and deviates from the mean for children of that sex and age by at least two standard deviations[16], and the proportionality of short stature was assessed by measuring the upper body/lower body segment ratio (US/LS ratio) and measuring arm span[17]. The mid-parental height was calculated as the mother’s height plus the father’s height divided by 2 ± 6.5 cm, and the target height was then calculated as mid-parental height ± 10 cm[18].

Cases were considered to have idiopathic short stature (ISS) when they matched the definition of short stature with normal GH stimulation tests in the absence of endocrine, systemic, or chromosomal abnormalities[19,20]. Traditionally, cases are identified as GHD when serum peak GH levels are < 10 ng/mL on two provocative tests[21]. Detailed medical history was taken with a special focus on the nutritional history, family history suggesting a familial or constitutional delay of growth, history of low birth weight, history of admission to the Neonatal Intensive Care Unit, history of any associated endocrine disorders, history suggestive of chronic diseases, and drug therapy.

Careful general examination with anthropometric measurement was carried out; weight/kg and height/cm were measured using an electronic scale and a stadiometer, respectively. Body mass index was calculated, and all measurement results were compared to World Health Organization reference values[22]. Anthropometric measurements were expressed as Z scores standard deviation[23]. Precise systematic examinations were done to exclude chronic diseases, disproportionate short stature, dysmorphic syndrome, other endocrine disorders, chromosomal disorders, and genetic disorders.

A - provocative GH tests

All provocative tests were accomplished early in the morning hours after 8 hours of overnight fasting. Tests were supplied by (TOSOH Bioscience, Inc., 6000 Shoreline Ct, Suite 101, South San Francisco, CA 94080, United States, No. 0025266).

Insulin tolerance test: Intravenous insulin dose equivalent to 0.1 unit/kg in children aged more than four years old. An adequate, valid test is one that shows a decrease in blood glucose by 44%-50% of its initial level or becomes lower than 40 mg/dL[11,24].

Clonidine test: An average dose of clonidine (0.15 mg/m² with a maximum dose of 0.25 mg) was given with follow-up for the patients for possible side effects such as drowsiness, hypotension, nausea, and vomiting[13].

L-DOPA test: L-DOPA is administered orally; 125 mg for young patients with a weight of less than 15 kg, 250 mg for weights from 15 kg to 30 kg, and 500 mg for higher weights[25].

In each test, blood samples were drawn at baseline, 30, 45, 60, 90, and 120 minutes; blood was gathered in 6 separate plain tubes. Serum was separated by centrifugation at 3000 rpm for ten minutes after the blood was left to clot for 15 minutes at 37 °C. The collected serum should be clear and non-haemolyzed.

B-insulin-like growth factor-1

Was also measured in the collected baseline blood samples using a commercially available enzyme-linked immunosorbent assay kit supplied by Elabscience Biotechnology Inc., United States, with catalogue No. E-EL-H0086, using a microplate enzyme-linked immunosorbent assay kit reader (EMR 500, United States). Bone age was determined by the radiograph of the left hand and wrist according to the method of Greulich and Pyle[26].

Statistical analysis

Statistics Package for Social Sciences (SPSS) version 26 was utilized for analyzing the included data. The Kolmogorov-Smirnov test and the Shapiro-Wilk test were used to check normal distribution. Categorical variables were described by n (%) and evaluated by the χ² test and Fisher’s exact test, while continuous data were expressed as mean ± SD for normally distributed data/or median and interquartile range (IQR) (median for non-normal distributed data). The student t-test was used for normally distributed parameters when comparing two groups, while in comparing two and three groups with non-normally distributed data; The Mann-Whitney test and the Kruskal-Wallis test were utilized. Spearman’s correlation coefficient was used for correlating non-normally distributed data. Receiver operating characteristic curve (ROC curve): Was used to detect the cut-off value, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Probability (P-value) was treated as insignificant if its value is more than 0.05 and significant if its value is less than 0.05.

RESULTS

The current study included 50 children and adolescents with proportionate short stature; their mean age was 10.3 ± 2.91 years. Females represented the highest percentage of cases (66%). The studied cases had a height of more than, with a mean height Z score of -2.77 ± 0.61. The mean IGF-1 was 169.77 ± 38.39 ng/mL, and a median IGF-Z score of -0.87 and an IQR of -1.4 to 0.53. GHD was detected in 24 cases (48%) and ISS in 26 cases (52%). Comparing the GHD group and the ISS group regarding demographic and clinical data showed no statistically significant differences between the two groups as regards age, sex, anthropometric measurement, mid-parental height, IGF-1, and IGF-1-Z score, with a P-value > 0.05 (Table 1).

Table 1 Demographic data of the studied cases.

Variables	Total cases, n = 50	Patients’ subgroups		P value
		GHD group (n = 24)	ISS (n = 26)
Age/years				0.324
mean ± SD	10.3 ± 2.91	9.83 ± 3.37	10.73 ± 2.39
Sex, n (%)				0.616
Male	17 (34)	9 (37.5)	8 (30.8)
Females	33 (66)	15 (62.5)	18 (69.2)
Weight/kg				0.099
mean ± SD	26.67 ± 9.72	25.08 ± 9.8	28.13 ± 9.6
Weight Z score				0.207
Median (IQR)	-1.7 (-2.8 to -1.29)	-1.835 (-3.2 to -1.35)	-1.55 (-2.02 to -1.26)
Height/cm				0.180
mean ± SD	121.86 ± 14.79	118.46 ± 16.94	125 ± 11.98
Height Z score				0.129
mean ± SD	-2.77 ± 0.61	-2.88 ± 0.59	-2.68 ± 0.63
BMI				0.831
mean ± SD	17.37 ± 3.73	17.49 ± 3.86	17.26 ± 3.68
BMI Z score				0.977
Median (IQR)	-0.16 (-1.12 to 0.29)	-0.12 (-1.4 to 0.25)	-0.235 (-0.8 to 0.39)
Mid parenteral height				0.846
mean ± SD	165.93 ± 7.51	165.77 ± 7.31	166.08 ± 7.83
IGF (ng/mL)				0.662
mean ± SD	169.77 ± 38.39	165.73 ± 39.71	173.49 ± 37.52
IGF Z score				0.727
Median (IQR)	-0.87 (-1.4 to 0.53)	0.53 (-0.39 to 1.44)	0.9 (0.46 to 1.54)
Chronological age - bone age/years				0.537
mean ± SD	2.49 ± 0.88	2.54 ± 0.59	2.44 ± 1.1

IQR: Interquartile range; BMI: Body mass index; IGF: Insulin-like growth factor; GHD: Growth hormone deficiency; ISS: Idiopathic short stature.

Peak GH levels with the insulin tolerance test (ITT) were detected at 45 minutes with a median of 4.53 ng/mL and IQR of 1.52-6.96 ng/mL, while the clonidine test showed a Peak level at 90 minutes with a median of 5.62 ng/mL and IQR of 3.36-8.47 ng/mL, and the L-DOPA test peak was detected at 60 minutes with a median of 4.64 ng/mL and IQR of 1.9-7.25 ng/mL. Comparing the levels of the 3 provocative tests showed a statistically significant difference in the 5^th sample at 90 minutes. This was done with a P-value of 0.004 (Figure 1A).

Figure 1 Comparing growth hormone levels. A: Comparing growth hormone (GH) levels by different provocative tests (n = 50); B: Cases with GH levels ≥ 10 ng/mL in at least one test sample. GH provocation test by insulin (n = 14); GH provocation test by clonidine (n = 20); GH provocation test by levodopa (n = 19); C: Comparing GH response by different provocation tests in cases with GH levels < 10 ng/mL. GH provocation test by insulin (n = 36); GH provocation test by clonidine (n = 30); GH provocation test by levodopa (n = 31). ^aP < 0.05 vs the corresponding insulin tolerance test. GH: Growth hormone; L-DOPA: Levodopa.

The clonidine test had the highest number of cases with sufficient GH peak ≥ 10 ng/mL in at least one test sample, presented in 20 cases (40%), compared to 19 cases (38%) in L-DOPA and 14 cases (22%) in ITT. Comparing the GH peak in the three provocative tests in cases with GH levels ≥ 10 ng/mL in at least one test sample figured out that ITT had a median (IQR) peak of 11.22 (10.54-12.71) ng/mL with a range of 10.01-16.94 ng/mL, vs 11.65 (10.42-14.9) ng/mL in clonidine with a range of 10.31-28.87 ng/mL, and 10.27 (10.12-10.52) ng/mL in L-DOPA test with a range of 10.05-14.07 ng/mL without a significant difference.

While comparing the median and IQR levels for each sample separately of the three provocative tests in cases with optimal GH levels ≥ 10 ng/mL in at least one test sample showed that the clonidine test had the highest median level of GH levels of 10.48 ng/mL and IQR of 7.32-11.67 ng/mL at 90 minutes samples vs 6.55 (5.03-9.62) ng/mL in insulin and 7.24 (2.4-8.7) ng/mL in L-DOPA test with a statistically significant difference and a P-value of 0.006 (Figure 1B).

Comparing the GH peak in the three provocative tests in cases with GH levels < 10 ng/mL in all test samples figured out that ITT had a median (IQR) peak of 5.16 (4.13-6.82) ng/mL with a range of 1.86-9.61 ng/mL, vs 6.46 (3.47-8.31) ng/mL in clonidine with a range of 1.6-9.87 ng/mL, and 6.48 (4.12-8.1) ng/mL in L-DOPA test with a range of 1.55-9.36 ng/mL without also a significant difference. Also, comparing the median and IQR levels for each sample separately of the three provocative tests in cases with GH levels < 10 ng/mL in all test samples; 36 cases in ITT, 30 cases in the clonidine test, and 31 cases in the L-DOPA test showed no statistical differences in all provocative tests (Figure 1C).

Comparing the percentage of cases with optimal GH response in different provocative tests in each test sample showed that the highest number of cases were diagnosed with clonidine at 90 minutes, followed by the L-DOPA test at 60 minutes, and lastly by insulin at 45 minutes. There was also a statistically significant difference between the percentage of cases in the 3 tests in the 5^th sample according to GH secretion, with a P-value < 0.05 (Table 2). A significant positive correlation between the IGF-1 Z score and the sample test for clonidine at 90 minutes (r = 0.359 and P-value = 0.010) was detected (Figure 2).

Figure 2 Positive correlation between insulin-like growth factor-1 Z score and clonidine test peak sample (at 90 minutes). ^aMeans significant positive correlation (r = 0.359, P = 0.010). GH: Growth hormone; IGF-1: Insulin-like growth factor-1.

Table 2 Comparing the percentage of cases with optimal growth hormone response (≥ 10 ng/mL) in different provocative tests in each test sample, n (%).

Sampling time	Insulin tolerance test (n = 50)	Clonidine test (n = 50)	L-DOPA test (n = 50)	P value
Basal sample	2 (4)	1 (2)	1 (2)	0.773
Sample 2 (30 minutes)	5 (10)	3 (6)	5 (10)	0.714
Sample 3 (45 minutes)	6 (12)	6 (12)	1 (2)	0.121
Sample 4 (60 minutes)	3 (6)	4 (8)	7 (14)	0.359
Sample 5 (90 minutes)	1 (2)	11 (22)	3 (6)	0.001a
Sample 6 (120 minutes)	3 (6)	7 (14)	3 (6)	0.259

^aP value < 0.05 is considered significant. Cases may have more than one sample with growth hormone ≥ 10 ng/mL.

GH: Growth hormone; L-DOPA: Levodopa.

ROC curves show that insulin had a cut-off value of 7.54 ng/mL with an area under the curve (AUC) of 0.764, a sensitivity of 59.3%, a specificity of 91.3%, a PPV was 65.6%, an NPV was 88.9%, and an accuracy of 74%. The clonidine test had a cut-off value of 8.48 ng/mL with an AUC of 0.844, a sensitivity of 77.8%, a specificity of 82.6%, a PPV was 76%, an NPV was 84%, and an accuracy of 80%. The L-DOPA had a cut-off value of 4.63 ng/mL with an AUC of 0.755, a sensitivity of 80%, a specificity of 70%, a PPV was 84.6%, an NPV was 66.7%, and an accuracy of 76%. Insulin had the highest specificity, clonidine had the highest AUC and accuracy, and L-DOPA had the highest sensitivity (Table 3 and Figure 3).

Figure 3 Performance characteristics of the optimal cut-off points of peak growth hormone responses to various provocative tests. A: Growth hormone (GH) provocation test by Insulin; B: GH provocation test by clonidine; C: GH provocation test by levodopa. L-DOPA: Levodopa.

Table 3 Optimal cut-off points of peak growth hormone responses to provocative tests, %.

Variables	AUC	Cut-off, ng/mL	Sensitivity	Specificity	PPV	NPV	Accuracy
GH provocation test by insulin	0.764	7.54	59.3	91.3	65.6	88.9	74
GH provocation test by clonidine	0.844	8.48	77.8	82.6	76	84	80
GH provocation test by L-DOPA	0.755	4.63	80	70	84.6	66.7	76

AUC: Area under curve; PPV: Positive predictive value; NPV: Negative predictive value; GH: Growth hormone; L-DOPA: Levodopa.

DISCUSSION

Different provocative tests have been reassessed to determine the most appropriate sampling time for detecting GHD, with the aim of reducing healthcare costs, minimizing patient burden, and decreasing the workload of medical staff[27]. The present study provides a comparative evaluation of three commonly used GH stimulation tests in children with short stature while also analyzing the optimal timing of peak GH responses. Additionally, the use of ROC curve analysis allowed the determination of test-specific diagnostic cut-off values, which may contribute to improving the diagnostic accuracy of GHD in clinical practice.

The present study included 50 proportionately short children and adolescents with a mean age of 10.3 ± 2.91 years. GHD was diagnosed in 24 cases (48%), while 26 cases (52%) were classified as ISS. Regarding the timing of peak GH response, the ITT demonstrated a peak GH level at 45 minutes after stimulation. This finding is consistent with the report by Rhee et al[25], who documented peak GH levels occurring approximately 45 minutes after insulin administration. However, other investigators, including Bozzola and Meazza[24], Vyas et al[28], and López Ruiz[29], reported that the GH peak during ITT may occur between 15 minutes and 30 minutes after the glucose nadir.

In the clonidine stimulation test, our findings demonstrated that the peak GH response occurred at 90 minutes. This observation agrees with the findings of Galluzzi et al[27], who reported that 92.1% of GH peaks occurred within 90 minutes after oral clonidine administration in children with short stature. Similarly, Borges et al[30] suggested that collecting blood samples at baseline, 60 minutes, and 90 minutes may be sufficient for performing the clonidine test. Gillis et al[31] also demonstrated that the highest GH levels are commonly observed at 30, 60, and 90 minutes following stimulation, supporting the feasibility of completing the clonidine test within 90 minutes. In contrast, Bozzola and Meazza[24] and López Ruiz[29] reported that the peak GH response following clonidine administration occurred earlier, at approximately 60 minutes.

For the L-DOPA stimulation test, the present study demonstrated a peak GH response at 60 minutes. In comparison, Bozzola and Meazza[24] and Rhee et al[25] reported an earlier peak at around 45 minutes. These variations in peak timing among studies may be attributed to differences in patient characteristics, stimulation protocols, and GH assay methodologies. In the present study, no significant differences were observed between the GHD and ISS groups regarding IGF-1 levels or IGF-1 Z-scores. These findings are consistent with previous studies[26,32,33] that demonstrated the limited ability of IGF-1 alone to differentiate between GHD and ISS, indicating its role as a supportive rather than definitive biomarker, confirming the importance of stimulation testing despite normal IGF-1 levels.

Nevertheless, a significant positive correlation was observed between the IGF-1 Z-score and the peak GH level during the clonidine stimulation test at 90 minutes, suggesting that this time point may represent one of the most informative measurements for detecting the peak GH response during clonidine stimulation testing. Clinically, this finding indicates that the 90-minute sampling point could play an important role in improving the diagnostic performance of the test and may contribute to optimizing the sampling schedule, potentially reducing unnecessary blood sampling while maintaining diagnostic accuracy in the evaluation of GHD. ROC curve analysis in the current study demonstrated varying diagnostic performances among the stimulation tests. The ITT showed a cut-off value of 7.54 ng/mL with a sensitivity of 59.3% and specificity of 91.3%. The clonidine stimulation test showed a cut-off value of 8.48 ng/mL with a sensitivity of 77.8% and specificity of 82.6%, whereas the L-DOPA test demonstrated a cut-off value of 4.63 ng/mL with a sensitivity of 80% and specificity of 70%.

The optimal GH cut-off value for diagnosing GHD remains controversial. Previous studies have reported a wide range of cut-off values, typically between 3 μg/L and 10 μg/L[29,34]. Paula and Czepielewski[35] suggested that two GH stimulation tests with peak GH responses below 5 μg/L are required to confirm the diagnosis of GHD. One of the major challenges in establishing a universal cut-off value is the absence of a true gold standard diagnostic test, together with the variability among GH assay methods[36]. Consequently, several studies have emphasized that GH cut-off levels should be interpreted according to the specific assay used for hormone measurement[24,37-41].

For instance, Wagner et al[37] evaluated seven different GH assays and reported clinically relevant cut-off limits ranging from 4.32 ng/mL to 7.77 ng/mL. Similarly, Guzzetti et al[40] reported cut-off values of 5.1 μg/L for ITT and 6.8 μg/L for the clonidine test in patients with organic GHD, achieving sensitivities of 94.4% and 88.5% and specificities of 89.6% and 97.3%, respectively. Felício et al[42] reported a cut-off value of 4.515 μg/L for ITT with 75.5% sensitivity and 45.5% specificity, whereas a clonidine cut-off value of 4.095 μg/L demonstrated 54.5% sensitivity and 52.6% specificity. Additionally, Kamoun et al[12] suggested that GH levels ≥ 7 ng/mL during fasting or ITT are strongly indicative of normal GH secretion, whereas levels between 3 ng/mL and 5 ng/mL may indicate possible deficiency. Regarding comparative diagnostic performance, our study supports clonidine's performance (highest AUC, good sensitivity/specificity) as a viable and safer (non-hypoglycemic) oral alternative to ITT. Petriczko et al[43] suggested that the L-DOPA stimulation test may better predict pituitary GH reserve in certain cases of GHD. Similarly, Bolu et al[44] reported an L-DOPA cut-off value of 8.93 ng/mL with 100% specificity and 50% sensitivity, whereas a clonidine stimulation test cut-off value of 9.76 ng/mL achieved 94% specificity and 88% sensitivity.

Study’s limitations

Despite these findings, several limitations should be considered. First, the relatively small sample size may limit the generalizability of the results. Second, the study was conducted in a single center, which may introduce selection bias. Furthermore, variations in GH assay methodologies and physiological factors influencing GH secretion may affect the reproducibility of stimulation test results. Regarding pubertal status, Tanner staging was not systematically recorded in the included participants at recruitment, which is also another limitation, given its known impact on GH secretion and IGF-1 interpretation. Additionally, parental consanguinity was not systematically collected, and this is another limitation due to its potential relevance in genetic causes of short stature. Another limitation is the cross-sectional nature of our study, with the absence of longitudinal treatment or growth outcome data.

CONCLUSION

Overall, the present findings contribute to the ongoing effort to optimize GH stimulation testing in children with short stature. The results highlight the variability in peak GH timing and diagnostic cut-off values among commonly used provocative tests (insulin, clonidine, and L-DOPA), emphasizing the need for careful interpretation of results in the clinical context. Our data suggest that specific sampling times, particularly 45 minutes for the ITT, 90 minutes for the clonidine test, and 60 minutes for the L-DOPA test, may capture the peak GH response in a large proportion of patients. Normal GH levels can be considered by ITT, clonidine test, and L-DOPA test, with cut-off values of ≥ 7.54 ng/mL, 8.48 ng/mL, and 4.63 ng/mL, respectively. These findings support the potential for simplifying stimulation test protocols while maintaining diagnostic reliability. Nevertheless, the variability in GH assay methods and the absence of a universally accepted diagnostic threshold underscore the importance of further large-scale, multicenter studies to establish standardized testing protocols and assay-specific cut-off values for the accurate diagnosis of GHD.