Hormonal changes throughout puberty in congenital hypogonadotropic hypogonadism and Pump indication

Background It is difficult to capture the exact time of pubertal initiation in normal children, including the detailed patterns of physical development, the cut-off values of hormone changing at pubertal initiation and maturation. Method Patients diagnosed with CHH were included in to prospectively investigate the hormones changes by GnRH pulsed pump therapy. We investigate testis volume and the hormones of HPG axis at basal and LHRH stimulated at 0 and the end of week 1, 4, and 12. Receiver operating characteristic (ROC) curve was plotted to determine the cut-off values of pubertal hormones. Results: Twenty-four CHH patients received pulse LHRH therapy were rolled in this study. ①stimulated FSH reached peak and LH increased significantly at week4. At this time point we workup the cut-off hormones values for pubertal initiation by ROC. They are: basal LH 1.32 IU/L, LH/FSH 0.34, the stimulated LH 4.45 IU/L and LH/FSH 0.54 for initiation. ②Basal INH-b elevated at week1, followed by T at week4, then AMH decreased at week12 coupled with testicle and penis growth. ③the pituitary response of CHH is normal when LH in the range of 1.81-3.17 IU/L and the LH / FSH in the range of 0.57-0.87. Conclusion ① we got the cut-off values of puberty initiation: basal LH 1.32 IU/L, stimulated LH 4.45 and LH/FSH 0.54 signed HPG axis activated. ②we got the cut-off hormones values of normal pituitary response: stimulated LH in the range of 1.81-3.17 IU/L and LH / FSH in the range of 0.57-0.87.


Introduction
It is difficult to capture the exact time of initiation of puberty in normal children, such as when breasts in girls and testicles in boys begin to grow, especially in boys. As a result, research on the detailed patterns of physical development during puberty and the cut-off values and changes of hormones at initiation of puberty and developmental maturation is rare. It is generally believed that hormonal changes during puberty are a direct result of the stimulating effect of luteinizing hormone (LH) on initiation of puberty (1; 2); thus, LH is widely accepted as an indicator to evaluate initiation of puberty. However, researchers haven't yet to reach an agreement on the cut-off value of LH, especially in boys. The previous reference value was based on inquiries regarding signs and symptoms and laboratory tests on blood samples. For example, information on when breasts begin to develop in girls (at approximately 10 years of age), when testicle volume begins to increase in boys (at 11.5 years of age), and the age of initial menstruation in girls and seminal emission in boys are obtained through inquiry instead of observation. Hence, the data provide a time frame (time interval), rather than a time point, and it is difficult to quantify the time frame. Therefore, the exact time of initiation of puberty is uncertain when it is determined based on the initial onset of secondary sexual characteristics or growth spurts or serum hormone levels (3). As a result, it is difficult to determine precise cut-off values for initiation of puberty from previous studies (4,5). Moreover, the reference cut-off values for initiation of puberty vary greatly between boys and girls (6), theoretically, the values of boys and girls should be similar. Is this difference due to gender differences? Or is it because the onset of puberty in boys is more difficult to detect, leading to the delay of the measured time point?
Although it is not easy to detect initiation of puberty in boys, it is easy and precise to determine testicle volume and penis length (7). Testosterone (T) level is usually concordant with changes in secondary sexual characteristics in boys, whereas the link between oestrogen level and physical changes is weak in girls. It is more reliable and practical to investigate Tanner staging and T levels to determine and identify puberty in boys (7). Some recent studies have shown that besides LH and follicle-stimulating hormone (FSH), anti-Mullerian hormone (AMH) and inhibin B (INH-B) are also involved in the initiation of puberty (11; 12).
Hence, a series of hormonal changes are responsible for initiating puberty; however, systematic research on these changes is lacking. Therefore, in this study, we recruited congenital hypogonadotropic hypogonadism (CHH) patients to be a model to prospectively study the initiation and developing of their puberty with exogenous GnRH, getting the physical characteristics and hormones changes during the induction of puberty and to determine the cut-off values of hormones in the HPG axis during three stages: initiation, development, and maturation. GnRH pump therapy is safe and effective; but it is important to determine whether pump therapy is indicated for patients with pituitary disorders. We further discussed the pump treatment indication of CHH.

Clinical data:
(1) They were examined at baseline and followed-up at weeks 1 ± 1 day, 4 ± 2 days, and 12 ± 2 days after treatment, then followed up every 3 months till 12 months. An increased Testosterone when testicle volume was greater than 3 ml was used as the indicator for initiation of puberty. (2) Data collection: height, weight, body mass index (BMI), PL and testicle volume, biochemistry, hormones; (4) LHRH stimulation test(15); 2.
1.4 Protocol for GnRH pump pulse therapy (13):8-10∝ g of GnRH (200∝ g/mL) was injected every 90 minutes with a pump. If any side effects occurred or T > 500 ng/dl, the dose of GnRH was reduced accordingly. 3.

ROC curve:
Receiver operating characteristic (ROC) curve was used to determine the cut-off hormones value at the initiation of puberty, which were used to evaluate the time points of initiation, development, maturation. The area under the curve (AUC) was used to identify the cut-off value. A cut-off value of hormone had a diagnostic value when the AUC was greater than 0.8 and p < 0.05. A value with a certain level of sensitivity and specificity was used as the cut-off value. HPG axis initiation was considered when a relationship between LH and T was established, so the ROC curve plotted when approximately 50% of the patients showed an HPG initiating. Maturation was considered when feedback of the HPG axis was established and GnRH reached the peak and no longer rising.

Statistical analysis
This was a prospective cohort study in which sex hormones levels were compared before and after continuous LHRH pulse therapy. GraphPad Prism v5 was used to plot curves. SPSS v17.0 was used for statistical analysis. Repeated measures analysis of variance was performed to analyse hormonal changes at different time points before and after treatment in the same patient.

Determination of hormone levels
A MAGLUMI® 2000 instrument was used to perform radioimmunoassay to determine the levels of T.
A Roche e601 instrument was used to perform chemiluminescence assays to determine the levels of LH, FSH, AMH and INH-B.
1.8. The study was approved by the ethics committee of Beijing Children's Hospital, which acts in compliance with ethical standards defined by the Declaration of Helsinki. Written consent for research purposes was provided by all participants and their parents or legal guardians.

1.
General information A total of 24 patients with HH (KS17) were included in this study. The average age at first visit was 14.01 ± 1.40 years; the bone age was ≥ 12, at Tanner I. Fifteen patients (62.5%) had a small penis, others had small penis and cryptorchidism. All 24 patients completed 4 weeks and 18 patients completed 12 weeks study.

Basal hormones profiles
After treatment, FSH and INH-B began to rise at week 1, with no changes in T, AMH levels or testicle volume. LH started increasing with a significant increase combined with T level at the end of week4, but no changes in testicle volume or AMH. At W12, LH and INH-B continued to rise, but FSH stopped increasing at a high level, testosterone could be detected and the testicle volume was greater than 3 ml since then, while, AMH began to decrease (Detailed values in Table 1 and figure 1). (15) LH peaked at 30 minutes and FSH at 60 minutes after LHRH stimulation. We take values at these 2 points respectively to study ROC.

Stimulated Hormones Profiles
Stimulated LH did not rise until week 4, but FSH increased significantly at week 1 and stabilized by week 4. LH/FSH ratio decreased at week 1, began to increase at week 4, and stabilized at week12.

Discussion
Many factors are involved in the initiation of puberty, which is a result of the interaction between multiple neural nuclei in the brain and hypothalamic neuroendocrine cells and is subject to the effects of environmental and metabolic factors (8-10; 13; 16). It is a challenge to investigate puberty because it is not easy to capture the exact puberty initiation time individually. Continuous LHRH pituitary pulse therapy of CHH patients mimics the process of puberty. This prospective study with highly homogeneous patients, regularly sample and physical data taking, assured the results reliable.
We got the initiation-developing-maturation anthropometry and cut-off values of each phase provided a useful reference for the diagnosis of early and delayed puberty.

The hormones basal and stimulated profiles and the cut-off values of initiation of the HPG axis and puberty
At week 1, INH-B rose and followed by LH and FSH (Fig. 1). LH/FSH decreased first then climbed up until testosterone increase at last means puberty ignition. Therefore, the decrease in stimulated Some studies on hormone levels at different Tanner stages have shown somewhat different values from this study (17)(18)(19). We consider the previous studies were cross-sectional descriptive study, whereas this was a prospective longitudinal study of hormone levels, which reflected dynamic changes in hormone levels. Therefore, the results of this study are more precise and reliable.
The cut-off values in this study were similar to the reference values of girls (6) The authors have no acknowledgements.

Author's contributions
Chunxiu Gong performed clinical examination, diagnosis and treatment for this patient, also proposed views and revised the whole paper. Xiaoya Ren performed the collection and analysis of the clinical data and wrote the whole paper. YuanyuanTian, Guoshuang Feng, Yi Wang, Beibei Zhang, Bingyan Cao and Jiajia Chen contributed clinical thought to the part of discussion.

Funding
The research was supported by Beijing Municipal Science and Technology Funding (No.

Z151100003915103) and Beijing Municipal Administration of Hospital Clinical Medicine Development
of Special Funding Support (NO. ZYLX201821).

Availability of data and materials
Data are available in a public, open access repository. All data relevant to the study are included in the article or uploaded as supplementary information.

Ethics approval and consent to participate
The research protocol was approved by the ethics committee of Beijing Children's Hospital, Capital Medical University (ID2012-28), and written informed consent was received from all patients or legal guardians.

Consent for publication
Consent was obtained from all patients for publication Figure 1 Hormone changes in CHH

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