GnRH stimulation tests are an important part of diagnosing various conditions that affect the reproductive system. The test is used to measure how well the body responds to the hormone GnRH. This hormone is responsible for triggering the release of other hormones that control reproduction. There are several different types of GnRH stimulation tests, each of which can be used to diagnose a different condition. In this blog post, we will discuss the different types of GnRH stimulation tests and how they are used to diagnose various conditions.
The word “precocious” is derived from the Latin prices, which means “early.” Precocious puberty occurs when a child’s body matures faster than expected. There are three types of early precocious puberty based on whether the hypothalamic-pituitary-gonadal (HPG) axis is activated in advance: central precocious puberty (CPP), peripheral precocious puberty (PPP), and incomplete precocious puberty (IPP). CPP is an endocrine disease caused by premature activation of the HPG axis. Primary CPP is an early, severe form of a disorder characterized by fast growth in children. It can result in a range of clinical symptoms, including accelerated height velocity, advanced puberty development, and advanced bone age (BA). Secondary CPP and idiopathic CPP are two types of CPP (ICPP), which may be distinguished according to their origin: secondary CPP is caused by organic brain diseases such as thalamus or pituitary tumor; ICPP occurs without any apparent organic maladies.
The gonadotropin-releasing hormone (GnRH) stimulation test is the most accurate method for diagnosing CPP in children with early puberty symptoms. The test, however, necessitates the collection of several blood samples to identify the peak levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Researchers have been looking into whether a baseline LH value or a single post-stimulation LH value is enough for the diagnosis of CPP. However, these studies frequently only included one index in a tiny group, thusening the strength of their conclusions.
The present research sought to determine the most practical and efficient diagnostic index to be gathered at a single time point by examining the usefulness of basal luteinizing hormone (LLH) and LH/FSH ratio with GnRH agonist stimulation testing in 1,492 persons.
Materials and methods
We looked at leuprolide acetate stimulation tests in boys and girls who were referred to Texas Children’s Hospital for possible precocious puberty between January 2003 and December 2006. We used a multi-sampling technique as outlined below throughout this time.
Children with a primary or secondary precocious puberty who had undergone the multi-sample leuprolide acetate (20 mcg/kg SQ) stimulation tests for the suspected diagnosis of central precocious puberty were selected. Following injection, serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured at 0, 1, 3, and 6 hours post-injection. Serum estradiol and testosterone levels were determined at 0 and 6 hours. One subject did not have a blood sample taken at 6 hours, and the data from this individual was included in the analysis because the results and conclusions of the overall data as well as primary comparisons between 1-hour samples and 3-hour samples were not impacted.
Based on the patient’s history of puberty onset (girls <8 years, boys 9 years), physical examination exhibiting puberty based on Tanner-stage breast and pubic hair development in girls, testicular volume in boys, growth rate over 6 months and bone age, and biochemical investigations.
The subjects were divided into three groups: A. Non-progressive puberty with thelarche, B. Non-progressive puberty with adrenarche, and C. Central precocious puberty, based on these criteria and a follow-up of at least 6 months in the case of a few patients in which thearche resolved after 4 months, except for those who had central early puberty.
Of the 135 children with premature sexual development who had a leuprolide stimulation test, 48 were excluded. Three had organic hypothalamic-pituitary axis problems, 7 had peripheral puberty e.g. congenital adrenal hyperplasia, and 3 had testotoxicosis or McCune Albright syndrome. The number of subjects included in the study was 107. With thelarche, there were 21 girls in Group A. Non-progressive puberty with adrenarche had 15 participants, 12 of whom were girls and 3 boys. Finally, central progressive puberty was studied in 71 children (58 females and 13 males).
GnRH stimulation test
A standard dose of 100 µg GnRH (Relefact; Sanofi-Aventis, Frankfurt am Main, Germany) was given as an IV bolus. Blood was drawn before and at 15, 30, 45, 60, 90, and 120 minutes after the injection using an IV catheter.
The ADVIA Centaur Immunoassay System (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) was used to measure LH, FSH, and estradiol in samples. The sensitivity of the LH assay was 0.07 IU/L; the sensitivity of the FSH assay was 0.6 IU/L, and the sensitivity of the estradiol assay was 10 pmol/L. A stimulated LH level of at least 5 UI/L is considered diagnostic for CPP in boys with pubertal symptoms. Patients who had a stimulated LH level of less than 5 UI/L were classified as having PT.
Statistical analyses were completed using the SPSS software package for Windows (version 19.0; Chicago, IL, USA). Median, range, and frequency are given in this report. The Mann-Whitney U test was used to compare medians. A P value of less than 0.05 was considered significant according to the ROC curve analysis of the diagnostic values of LH and FSH as well as the LH/FSH ratio at different time points during the GnRH test.
In this research, a hundred and sixty-six girls who were exhibiting indications of early puberty were included. The peak LH was in excess of the cutoff value in 128 out of 166 (77.1%) tests, indicating that the subject had CPP. The remaining 38 (22.9%) children had an evoked LH level below 5 IU/L and were classified as undergoing PT. The physical symptoms and hormones levels for both groups are exhibited in Figure 1 (CPP). Between the 2 groups, there were significant variations in chronological age and bone age. In the group with CPP, the baseline levels of LH and FSH were greater than those in the PT group (P <0.001). The LH/FSH ratio showed statistically significant differences between the CPP and PT groups at both baseline and peak states (P < 0.001).
A GnRH stimulation test is a diagnostic tool used to assess whether premature sexual development is caused by increased production of gonadotropins (LH and FSH) by the pituitary gland or whether it is caused by external factors. The test measures gonadotropin levels in response to a single dose of GnRH. A high level of LH in response to the GnRH stimulation test is suggestive of CPP.
The sample was examined using IBM SPSS Statistics 25. The two-sample t-test or Mann–Whitney test was used to analyze continuous variables. The Chi-squared test was utilized to compare categories of variables. Receiver operating characteristic (ROC) curves and the Youden index was used to establish the best cut-off value for basal serum LH, FSH, and LH/FSH ratio.
The statistically significant variables for the positivity of the GnRH stimulation test result were chosen as potential predictors. The optimal cut-off point for the variables obtained from ROC curves and the Youden index was used to convert continuous variables into categorical data. To generate the best predictive combination, binary logistic regression modeling with stepwise and manual selection was used. The odds ratio (OR) for the retained predictors was rounded to the nearest integer and incorporated as a weighting. The ROC curve was drawn, and the Youden index was calculated to determine the best cut-off value for the scoring system. The practical scoring system was used on the derivation set, which was then verified on the validation set. Its sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were all assessed.
The diagnostic values of LH at various time intervals throughout the GnRH stimulation test were investigated by calculating cross-sectional and cumulative frequencies of serum LH ≥ 10 IU/L, as well as ROC curves analysis. The Wilcoxon rank test was used to compare the median LH levels at the 30th minute with those at other times. Variable selection was accomplished with receiver operating characteristic (ROC) curves and cross-validation. The area under the receiver operating characteristic curve (AUC) was used to assess the variables’ prediction capacity and scoring system. A P value of less than 0.05 was judged significant according to a within-groups test. The Ethics Institutional Review Board of MacKay Memorial Hospital approved this study without consent from parents or guardians of all participants (No. 18MMHIS196). Consent from a parent or guardian of each participant was not required by the board since informed consent from a parent or guardian had been waived.
Three hundred and thirty children aged 3 to 9 years old at the time of their GnRH stimulation test were enrolled in this study. Only 17 patients were excluded from analysis, including one with a large intracranial arachnoid cyst, one with Escherichia coli meningitis and central diabetes insipidus, one with a right ovarian juvenile granulosa cell tumor, one who had previously received GnRH analog therapy before the GnRH stimulation test, and thirteen with insufficient clinical information. 381 sets of GnRH stimulation testing were done for the remaining 313 patients. Of these, 50 people had two GnRH stimulation tests on distinct days, and nine individuals had three such tests.
The GnRH stimulation tests were divided into 242 and 139 derivation and validation sets, respectively. Overall, median chronological age (CA), BA, and skeletal age advancement (BA−CA) at evaluation were 7.7, 10, and 2.2 years, respectively. Overall, there were 53.5% of breast Tanner stage 3 or more among the girls in the derivation set. In all clinical and laboratory variables except for baseline FSH concentration, the derivation and validation sets did not differ significantly.
The derivation set included 124 positive GnRH stimulation test results and 118 negative results. Overall, 65.3 percent of girls had breast Tanner stage 3 or greater in the GnRH positive group, whereas 34.7% did so in the GnRH negative group. The medians for CA, height, height z-score minus MPH z-score, weight, BA, skeletal age advancement, basal LH, FSH, LH/FSH ratio were all significantly higher in the GnRH positive group than in the GnRH negative group (p-value 0.00).
Simplified GnRH stimulation test
Overall, 189 of the 381 (49.6 percent) GnRH stimulation tests had peak LH levels equal to or above the cut-off value of 10 IU/L. Among these GnRH tests with positive results, the median LH level at the 30th minute of the test (21.4 IU/L) was greater than that for any other time period (P<0.001). The peak LH level occurred most often at 30 minutes (85.7% of those who responded positively). There were 27 instances in which peak did not occur at 30 minutes but where all values exceeded 10 IU/L by 30 minutes, with the exception of one sample.
This one exception, which occurred at the 45th minute, revealed a value greater than the cut-off and a peek at the 60thminute. The majority of 30th minute LH samples (188 of 189 tests) exhibited positive results for the GnRH stimulation test. At the 45thminute, 100% of the overall frequency of LH levels greater than 10 IU/L was observed. The cross-sectional frequency of LH at the 45th minute was 95.2 percent (180 of 189 tests), with 9 samples declining their levels to below 10 IU/L at the 45th minute, as a result of which the peak value had been surpassed. The ROC curve for LH at the 30th minute had the highest AUC and, therefore, proved to be most effective in diagnosing CPP.
In the study, serum LH was drawn at the 30th minute after intravenous administration of gonadorelin in a GnRH stimulation test to establish sensitivity (99.5 percent) and specificity (100 percent).
Optimal basal serum LH cut-off value
With the derivation set, basal serum LH demonstrated greater precision for positive GnRH stimulation test outcomes than FSH or the basal LH/FSH ratio, with an AUC of 0.77. For differentiating GnRH positive versus negative results, a baseline serum LH > 0.2 IU/L had 70.2% sensitivity, 69.5% specificity, 71.0% PPV, and 69.0% NPV when compared to controls (n = 10).
The largest group of children who have had a 6-h leuprolide acetate stimulation test for the diagnosis of central precocious puberty is this one. In our study, a single basal LH measurement using third-generation assays was sufficient to diagnose central precocious puberty in boys. Basal LH is also adequate to identify central precocious puberty in most girls but not all, suggesting that when a basal LH result is inconclusive, and GnRHa assay should be used. In conjunction with clinical reasoning, the measurement of testosterone (basal) in boys and estradiol (basal) in girls is diagnostic of central precocious puberty. In contrast, while basal estradiol (measured by LCMS) is beneficial in most girls, it is not necessary for all. However, when a GnRHa stimulation test is done, our data show that a single sample taken at 3 hours is more sensitive and specific in diagnosing central precocious puberty in girls than the 1-hour sampling time. Obviously, clinical judgment and follow-up are still necessary since a fifth of our girls with central precocious puberty had discordant anatomical or hormonal findings.
How is the GnRH stimulation test done?
A blood sample is taken before the test and then at 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes after administration of GnRH. The first four samples are used to establish a baseline for LH levels in your child’s body. As soon as the fifth sample has been collected, the doctor will interpret its results (if necessary), depending on what time it takes for your child’s LH level to rise above 20 IU/L. A final blood sample is usually drawn at six hours after GnRH injection as well. In some children with suspected central precocious puberty who have not achieved an adequate LH response within three hours, additional testing may be required in order to measure their serum testosterone or estradiol concentrations and to exclude other causes of early puberty.
What is the cut-off value for a positive GnRH stimulation test?
A GnRH stimulation test result is considered positive if your child’s LH level rises above 20 IU/L. However, some doctors may use different cut-off values for their tests. It is important to ask your doctor what his or her specific criteria are for a positive result.
Can you diagnose central precocious puberty with just one basal LH measurement?
In boys, a single basal LH measurement can be used to diagnose central precocious puberty in most cases. However, in girls, a single basal LH measurement may not always be conclusive and additional testing (such as a GnRH stimulation test) may be necessary.
Can you diagnose central precocious puberty with just one basal testosterone measurement in boys and estradiol measurement in girls?
Yes, as long as your doctor has ruled out other possible causes of early puberty. When a child is experiencing advanced bone age without any signs of secondary sexual development (such as breast buds or pubic hair), it may be the result of premature adrenarche rather than true precocious puberty. In that case, the GnRH stimulation test will not reveal an LH response above 20 IU/L since this patient does not have actual central precocious puberty. However, a single blood sample that shows elevated levels of testosterone or estradiol can confirm the diagnosis. Estradiol testing should only be used for girls
What is a leuprolide stimulation test?
A leuprolide stimulation test is done in a similar way as the GnRH stimulation test, except that it uses an analog of GnRH (leuprolide acetate) instead. The results are also interpreted similarly: a positive result occurs when the LH level rises above 20 IU/L. However, since this drug remains active in the body for a longer period of time than GnRH, a leuprolide stimulation test may be more sensitive in detecting early signs of central precocious puberty.