Biomarker for diagnosing female central precocious puberty and use thereof

Abstract

A biomarker for diagnosing female central precocious puberty and the use thereof. The marker is one, two or three of Leptin, RBP4, or Chemerin. Experiments indicate that peripheral blood levels of Leptin, RBP4 and Chemerin in female children with CPP are significantly higher than those in healthy children. Furthermore, the serum levels of Leptin and RBP4 are already altered in the early stage of CPP in child patients, suggesting potential involvement of these three adipokines in the onset of CPP.

Description

A biomarker for diagnosing central precocious puberty in females and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biomarkers, in particular to a biomarker for diagnosing central precocious puberty in females and application thereof. BACKGROUND

[0002] Central precocious puberty (CPP) is a common pediatric endocrine disease caused by the premature activation of the hypothalamic-pituitary-gonadal axis (HPG axis), which leads to the rapid development of internal and external genital organs and the appearance of secondary sexual characteristics in girls before the age of 7.5 and in boys before the age of 9. The incidence of this disease is about 1 / 5000-1 / 10000, and the incidence in girls is about 5-10 times that in boys. This disease has a significant impact on the adult height and psychological quality of the children. Early menarche is considered to be one of the high-risk factors for breast cancer, and is also associated with high blood pressure, type 2 diabetes, stroke, estrogen-dependent cancer, increased cardiovascular disease mortality. However, the pathogenesis of CPP is not clear, and it is currently believed that factors that cause the premature activation of the HPG axis include genetics, nutrition, environmental endocrine disruptors, etc. With the improvement of living standards, the proportion of obese children has been increasing year by year, and the advancement of puberty is closely related to nutrition and body fat. Adipose tissue is an important endocrine organ that can secrete various active mediators to play endocrine metabolic and regulatory roles. These active mediators are adipokines, so obesity can lead to the disordered secretion of adipokines, which in turn can cause the premature activation of the HPG axis and may play an important role in the occurrence of central precocious puberty.

[0003] The current gold standard for diagnosing CPP is the GnRH stimulation test, but this test is complicated to operate, requires repeated blood sampling, and children with peripheral precocious puberty may have the risk of transforming into central precocious puberty.

[0004] In recent years, there are reports of the relationship between adipokines and CPP, Leptin is the earliest, most complete one of the adipokines in CPP, which is secreted by white adipose tissue, and has the functions of regulating diet, energy consumption, reproduction, etc. The production and secretion of Leptin are positively correlated with the body fat content, and the serum Leptin level is elevated in obese women and CPP women. The pulsatile secretion of GnRH in hypothalamic GnRH neurons acts on the pituitary, thereby starting the HPG axis to begin puberty. The main regulatory center of GnRH neurons is the hypothalamic neurons expressing kisspeptin and neurokinin B. Leptin can pass through the blood-brain barrier and bind to the Leptin receptor (LepR) on the kisspeptin-expressing neuron cells, stimulate the overexpression of Kisspeptin by KISS-1 gene. At the pituitary level, high concentrations of serum Leptin can also stimulate the up-regulation of GnRH receptor expression, increase the sensitivity of gonadotropins to GnRH pulses, and promote the connection between hypothalamic GnRH neurons and the pituitary, thereby leading to Leptin-LH pulse and ovulation synchronization. Therefore, obesity leads to a large accumulation of fat in the body, and adipocytes can produce and secrete Leptin, but obese people have leptin resistance, leading to hyperleptinemia. Leptin can pass through the blood-brain barrier and bind to the LepR on the hypothalamic GnRH neurons and pituitary to play a regulatory role when it accumulates to a certain threshold, which may be one of the mechanisms of CPP.

[0005] Chemerin is mainly secreted by white adipose tissue, liver and placenta, etc. in the body, and is an important regulator of blood pressure, immune system, angiogenesis and inflammation, which is positively correlated with obesity and insulin resistance. It has the function of regulating progesterone secretion in mouse follicles, can inhibit FSH-induced steroid hormone production in PCOS rat models, can inhibit testosterone (T) production stimulated by hCG in rat Leydig cells, and can reduce IGF-1-induced secretion of progesterone and estradiol (E2) in in vitro cultured human primary granulosa cells. Due to the negative feedback mechanism of HPG axis, the decrease of sex hormone level can negatively regulate the increase of GnRH, FSH and LH secretion in hypothalamus and pituitary, which may be one of the reasons for the acceleration of HPG axis activation in children with CPP leading to CPP.

[0006] RBP4 is mainly secreted by liver and adipose tissue, responsible for binding and transporting retinol in blood, and serum RBP4 level is positively correlated with visceral fat. In Korean population, serum RBP4 level of adolescents in normal puberty is found to be elevated, while in healthy adult female population in the United States, RBP4 is found to be significantly positively correlated with FSH and LH, and negatively correlated with E2. In mouse ovary, FSH is found to affect transcription factors HMGA1, SF-1 and LRH-1 through cAMP-PKA pathway to regulate the dynamic expression of RBP4. In addition, overexpression of RBP4 can cause insulin resistance in mice, inhibit the production of SHBG in liver, and increase the bioavailability of sex hormones. In CPP women, insulin sensitivity and SHBG level are also observed to be decreased. However, there is no research on the relationship between RBP4 and CPP at home and abroad. SUMMARY

[0007] The purpose of the present application is to provide a biomarker for diagnosing central precocious puberty in females and its application, the present application explores the effect of adipokines on HPG axis, selects three adipokines leptin, retinol binding protein 4 (RBP4) and chemerin which may be associated with CPP, detects the concentration of adipokines leptin, RBP4 and chemerin in peripheral blood, and carries out correlation analysis with clinical indicators such as sex hormone level, bone age and BMI of CPP children, in order to find early warning indicators of CPP, especially obese girls with CPP, and provide basis for early diagnosis and prevention of female CPP.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009] The present application provides a biomarker for diagnosing central precocious puberty in females, the marker being one, two or three of leptin, RBP4 or chemerin.

[0010] The present application provides the use of the biomarker in preparing a reagent for diagnosing central precocious puberty in females.

[0011] The present application provides a kit for detecting central precocious puberty in females, the kit comprising a reagent containing the biomarker.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] 1. The peripheral blood Leptin, RBP4 and Chemerin levels of CPP female children are significantly higher than those of healthy children, and the serum Leptin and RBP4 levels change in the early stage of CPP children, which indicates that the three kinds of adipokines may be involved in the pathogenesis of CPP.

[0014] 2. Bone age, the difference between bone age and age, T, the length of uterus measured by B-ultrasound and serum RBP4 level can be used as the influencing factors and evaluation indexes of CPP in female children.

[0015] 3. The diagnostic value of combined detection of peripheral blood RBP4 and Chemerin in CPP is higher than that of independent detection of Leptin, RBP4 and Chemerin, and has potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 (a) Comparison of serum Leptin levels between CPP group and control group; (b) Comparison of serum RBP4 levels between CPP group and control group; (c) Comparison of serum Chemerin levels between CPP group and control group (Note: * represents P<0.05, and ** represents P<0.01).

[0017] Figure 2 (a) Comparison of serum Leptin levels between NM group and M group; (b) Comparison of serum RBP4 levels between NM group and M group; (c) Comparison of serum Chemerin levels between NM group and M group (Note: * represents P<0.05, and ns represents P>0.05).

[0018] Figure 3 Correlation heat map of Leptin, Chemerin and RBP4 with clinical data.

[0019] Figure 4 Correlation analysis heat map of Leptin, Chemerin and RBP4.

[0020] Figure 5 ROC curve of Leptin, Chemerin and RBP4 detected alone.

[0021] Figure 6 ROC curve of combined detection of RBP4 and Chemerin. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0023] Experimental Example 1

[0024] 1. Research data and instruments and equipment

[0025] 1.1 Research object

[0026] (1) CPP group: 50 cases of female children diagnosed with central precocious puberty in the Department of Pediatrics of Nantong University Affiliated Hospital from June 2021 to November 2022 were collected as research objects, including 27 cases without menstrual onset and 23 cases with menarche.

[0027] Inclusion criteria:

[0028] 1. All diagnosed cases met the diagnosis and treatment consensus (2015) for central precocious puberty:

[0029] 1) Breast development in girls before the age of 8 or menarche before the age of 10.0;

[0030] 2) Enlargement of the gonads, i.e. pelvic ultrasound showed uterine length 3.4-4.0 cm, ovarian volume 1-3 ml (ovarian volume = length x width x thickness x 0.5233), and multiple follicles with diameter ≥4 mm were visible, and the length of the uterus was >3.2 cm as the diagnostic CPP value;

[0031] 3) Serum gonadotropin and sex hormone reached puberty level, LH basic value >0.2 U / L; LH basic value <0.2 U / L but progressive hormone stimulation test, LH peak value ≥5.0 U / L and the ratio of LH peak value to FSH peak value ≥0.6 prompted the start of the gonadal axis;

[0032] 4) Bone age is more than actual age ≥1 year;

[0033] 5) Linear growth acceleration, annual growth rate higher than that of healthy children of the same age.

[0034] 2, the age is less than 10 years old;

[0035] Exclusion criteria:

[0036] 1. Precocious puberty secondary to the following diseases: congenital adrenal hyperplasia, McCune-Albright syndrome, primary hypothyroidism;

[0037] 2. Head imaging examination suggests intracranial lesions, such as endocrine function tumors or other space-occupying lesions; and acquired damage leading to central nervous system abnormalities, such as trauma, postoperative, radiotherapy, etc.;

[0038] 3. Patients with other congenital and genetic diseases;

[0039] 4. Combined with other related endocrine diseases: diabetes, hyperthyroidism, hypothyroidism, dwarfism, giantism, etc.

[0040] (2) Control group: 50 cases of healthy female children of the same age range for physical examination in the Department of Pediatrics of our hospital were selected as the control group.

[0041] This project was recorded and approved by the Ethics Committee of Nantong University Affiliated Hospital, with the ethics review number 2022-K028-01. All experimental subjects were informed of the relevant information and signed the informed consent form.

[0042] 2.1.2 Main reagents and instruments

[0043] Table 1 Main reagents

[0044] Table 2 Main instruments

[0045] 2. Experimental process and method

[0046] 2.1 Clinical data and blood sample collection

[0047] The general condition and medical history of all patients were collected and recorded in detail, including age, height, weight, calculation of BMI index (BMI = weight kg / height square m 2 ), Tanner stage, breast stage. Color Doppler ultrasound was used to detect the development of the uterus and ovaries in children. Sex hormone series (FSH, LH, progesterone, T, E2, pituitary prolactin) were measured by the biochemical laboratory of the inspection department of our hospital, and collected by the HIS system. All children received left wrist X-ray anteroposterior film, and the bone age was calculated according to the "Bone Age Atlas 2016 Edition".

[0048] Under the informed consent, 5ml of peripheral venous blood of all CPP children and control group children was collected in a coagulation biochemical tube in the morning on an empty stomach, centrifuged at 3500r / min for 10min, 200μL of the upper serum was taken with a pipette and divided into 3 dry sterile enzyme-free centrifuge tubes, sealed and checked the information label, and stored in the biological sample bank of our hospital -80℃ refrigerator.

[0049] 2.2 ELISA method for detecting the concentration of Leptin in serum samples

[0050] (1) Take the serum sample from the -80℃ refrigerator, place it at room temperature to dissolve, and take the ELISA kit from the 4℃ refrigerator, place it at room temperature for 30 minutes.

[0051] (2) Dilution of standard: take 5 1.5ml centrifuge tubes, add 120μL of standard diluent to each tube, add 120μL of original standard to the first tube, mix well with a low-speed centrifuge, and then take 120μL with a pipette and move it to the second tube. Repeat this process for multiple dilutions. The final concentrations of the 5 standard samples are 40μg / L, 20μg / L, 10μg / L, 5μg / L, and 2.5μg / L, respectively.

[0052] (3) Sample addition: 1) Standard wells: Add 50 μL of standard and 50 μL of streptavidin-HRP; 2) Blank wells: Add nothing; 3) Sample wells: Add 40 μL of sample, 10 μL of RBP4 antibody, and 50 μL of streptavidin-HRP. Cover with sealing film, gently shake to mix, and incubate in a 37°C water bath for 60 minutes.

[0053] (4) Prepare washing solution: Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside.

[0054] (5) Washing: Remove the sealing film, discard the liquid in the well, shake dry, fill each well with washing liquid, let stand for 30 seconds and then discard, repeat 5 times, and pat dry on filter paper.

[0055] (6) Color development: Add 50 μL of color developer A and color developer B to each well, cover with a new sealing film, gently shake to mix, and place in a 37°C water bath to develop color for 15 minutes in the dark.

[0056] (7) Termination: Add 50 μL of termination solution to each well to terminate the reaction.

[0057] (8) Measurement: The measurement was performed within 10 minutes after the stop solution was added. The blank well was zeroed and the absorbance (OD value) of each well was measured in sequence at a wavelength of 450 nm.

[0058] (9) Calculate concentration: Using an Excel spreadsheet, calculate the regression equation (R²) for the standard curve based on the concentration of the standard and the corresponding OD value. 2 (>0.92), the corresponding Leptin concentration of the sample is calculated on the regression equation based on the OD value of the sample.

[0059] 2.3 ELISA method for detecting RBP4 concentration in serum samples

[0060] (1) All serum samples were diluted 1:5 with PBS before testing. 10 μL of sample was added to 40 μL of PBS and mixed with a low-speed centrifuge.

[0061] (2) The standard concentrations were 40 μg / L, 20 μg / L, 10 μg / L, 5 μg / L and 2.5 μg / L.

[0062] The remaining steps are the same as for detecting leptin.

[0063] 2.4 Detection of Chemerin Concentration in Serum Samples by ELISA

[0064] The detection method is the same as that for RBP4.

[0065] The standard dilution concentrations were 48 ng / L, 24 ng / L, 12 ng / L, 6 ng / L, and 3 ng / L, respectively.

[0066] 2.5 Statistical analysis

[0067] Statistical software SPSS24.0 and Graphpad Prism 9.3.0 were used for data processing and analysis. The normality test of Kolmogorov-Smirnov was performed on all data. The measurement data with normal distribution were expressed as mean ± standard deviation (X ± s), and the independent sample t test was used for comparison between two groups. The measurement data with non-normal distribution were expressed as M (P25, P75), and the Mann-Whitney U test was used for comparison between two groups. Spearman correlation analysis was used to analyze the relationship between leptin, RBP4, chemerin and clinical data such as height, weight, BMI, sex hormone, bone age, difference between bone age and age, uterine length, etc. All statistical tests were set at α = 0.05 as the test standard, and P < 0.05 was considered statistically significant. Logistics regression analysis was used to analyze the predictive indicators of CPP in female children. The diagnostic value of single detection and combined detection of leptin, RBP4 and chemerin for CPP was analyzed by Receiver Operating Characteristic Curve (ROC) curve. The diagnostic value was evaluated by Area Under The Cure (AUC), 95% CI, CUT OFF value, P value, sensitivity and specificity, etc.

[0068] 3. Results:

[0069] 3.1 Comparison of clinical data between CPP group and control group

[0070] According to the diagnostic criteria of "Central Precocious Puberty Diagnosis and Treatment Consensus (2015)", a total of 50 samples of CPP group and 50 samples of control group were collected for this experiment.

[0071] After statistical analysis, there was no statistical significance in age distribution, progesterone and pituitary prolactin between the two groups (P > 0.05); the height, weight, BMI, Tanner stage, breast stage, bone age, difference between bone age and age, FSH, LH, T, E2, and uterine length measured by B ultrasound in CPP group were all higher than those in control group, and the difference was statistically significant (P < 0.05). See Table 3.

[0072] Table 3 Comparison of clinical data between CPP group and control group

[0073] Note: BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; T, testosterone; E2, estradiol; * represents P < 0.05, ** represents P < 0.01

[0074] 3.2 Comparison of serum Leptin, RBP4 and Chemerin levels between CPP group and control group

[0075] The serum levels of Leptin, RBP4 and Chemerin were detected by ELISA in all children. Statistical analysis showed that the serum levels of the three factors in CPP group were significantly higher than those in control group (P<0.05). See Table 4 and Fig. 1.

[0076] Table 4 Comparison of serum Leptin, RBP4 and Chemerin levels between two groups

[0077] Note: *P<0.05, **P<0.01

[0078] 3.3 Comparison between NM group and M group in CPP children

[0079] According to the clinical manifestations, the CPP group was divided into non-menarche group (NM group) and menarche group (M group). Statistical analysis between the two groups showed that there were significant differences in age distribution, height, weight, BMI, Tanner stage, breast stage, bone age, difference between bone age and age, FSH, LH, T, E2, and uterine length measured by B ultrasound between NM group and M group (P<0.05). The serum Leptin level in NM group was higher than that in M group (P<0.05), and there was no significant difference in RBP4 and Chemerin between the two groups (P>0.05). See Table 5 and Fig. 2.

[0080] Table 5 Comparison of clinical data and serum indicators between NM group and M group

[0081] Note: *P<0.05, **P<0.01

[0082] 3.4 Correlation analysis between serum Leptin, RBP4, Chemerin levels and clinical data

[0083] There was no significant correlation between serum Leptin, Chemerin levels and clinical data (P>0.05). The serum RBP4 level was negatively correlated with age, height, weight, breast stage, FSH and LH (P<0.05). See Table 6 and Fig. 3. The serum Leptin level was positively correlated with serum Chemerin and RBP4 levels (P<0.05). See Table 7 and Fig. 4.

[0084] Table 6 Correlation analysis of serum Leptin, RBP4, Chemerin levels and clinical data

[0085] Note: * indicates P < 0.05, ** indicates P < 0.01

[0086] Table 7 Correlation analysis of Leptin, Chemerin, RBP4

[0087] Note: ** indicates P < 0.01

[0088] 3.5 Analysis of influencing factors and evaluation indexes of CPP in female children

[0089] Through single factor analysis, it is found that height, weight, BMI, Tanner stage, breast stage, bone age, difference between bone age and age, FSH, LH, T, E2, uterine length measured by B-ultrasound, serum Leptin level, serum Chemerin level and serum RBP4 level are single factors affecting the occurrence of CPP in female children. According to the statistical principle, 11 statistically significant factors including BMI, Tanner stage, breast stage, bone age, difference between bone age and age, T, E2, uterine length measured by B-ultrasound, serum Leptin level, serum Chemerin level and serum RBP4 level are used as independent variables for multiple factor logistics regression analysis, and the results show that bone age, difference between bone age and age, T, uterine length measured by B-ultrasound and serum RBP4 level can be used as influencing factors and evaluation indexes of CPP in female children. See Table 8.

[0090] Table 8 Logistics regression analysis of influencing factors of CPP in female children

[0091] Note: * indicates P < 0.05, ** indicates P < 0.01

[0092] 3.6 Diagnostic value of serum Leptin, Chemerin and RBP4 levels in CPP in female children

[0093] Leptin, Chemerin and RBP4 have influence on each part of hypothalamic-pituitary-gonadal axis, so they may play a role in the starting process of gonadal axis, and may have important guiding significance for early clinical diagnosis and prevention of CPP. The diagnostic value of Leptin, Chemerin and RBP4 in CPP in female children, and the diagnostic value of Chemerin and RBP4 combined detection in CPP in female children are explored.

[0094] The area under the ROC curve of RBP4 is slightly higher than that of Leptin, and the area under the ROC curve of Chemerin is slightly higher than that of RBP4 and Leptin; the sensitivity of Leptin is 72.0%, the specificity is 58.0%, the sensitivity of RBP4 is 46.0%, the specificity is 80.0%, and the sensitivity of Chemerin is 58.0%, the specificity is 72.0%. The area under the curve of combined detection of RBP4 and Chemerin is 0.716, the sensitivity is 66.0%, and the specificity is 66.0%, which is higher than that of single detection. The ROC curve is shown in Figures 5 and 6, and the related data of the ROC curve is shown in Table 9.

[0095] Table 9: Related indicators of ROC

[0096] In the present application, by analyzing and comparing the clinical data of CPP children and healthy control group girls of the same age, we found that the height, weight and bone age of the CPP group children were significantly higher than those of the control group (P<0.05), indicating that the growth and development speed of CPP children was significantly faster than that of healthy children of the same age. Due to the rapid growth of the skeleton, the bone age was much larger than the actual age, which would affect the final height of the children. On the other hand, the T and E2 levels of CPP children were also higher than those of the control group (P<0.05), which had the risk of developing PCOS and estrogen-dependent diseases, so early diagnosis and prevention of CPP were very important. In addition, the BMI of the CPP group children was significantly higher than that of the control group (P<0.05), and the BMI of the children in the menarche group (M group) was significantly higher than that of the children in the non-menarche group (NM group) (P<0.05), indicating that obesity was related to the occurrence and development of CPP.

[0097] In the present application, the serum Leptin level of the CPP group was higher than that of the control group (P<0.05). The serum Leptin level of the NM group was higher than that of the M group (P<0.05). In the experiment, the serum sOB-R level was not detected, which could not rule out the possibility that the combination of serum Leptin and sOB-R in the M group children led to the decrease of serum free Leptin level.

[0098] In the present application, the correlation analysis found that there was a correlation between Leptin and RBP4, Chemerin, respectively. In addition, the study of the present application also suggested that the serum Leptin level was positively correlated with the serum Chemerin level (r=0.572, P<0.001). Both Leptin and Chemerin in serum were secreted by adipose tissue, and they might have mutual regulation effect.

[0099] RBP4 is mainly secreted by liver and adipose tissue in vivo. In the present application, the serum RBP4 level of the CPP group was significantly higher than that of the normal control group (P<0.05), and the correlation analysis found that the serum RBP4 level of the CPP children was negatively correlated with age, height, weight, breast stage, FSH and LH.

[0100] Our research results first found that the expression of adipokines RBP4 and Chemerin in the serum of female CPP children was also up-regulated. And the significant increase of serum Leptin and RBP4 can be observed in the early stage of CPP disease, and the correlation analysis suggests that Leptin may affect the expression level of RBP4 and Chemerin. Further, through Logistics regression analysis, it is found that bone age, the difference between bone age and age, T, the length of uterus measured by B-ultrasound and serum RBP4 level can be used as the prediction index of female CPP children, and the combination of the above indexes may be helpful for the early diagnosis of female CPP children. The present application suggests that there is a correlation between Leptin and RBP4 and Chemerin. Therefore, we respectively explored the diagnostic value of Leptin, RBP4 and Chemerin detection alone and RBP4 and Chemerin combined detection in female CPP. Combined with the area AUC under the ROC curve, the combined detection of RBP4 and Chemerin is higher than the independent detection of the three, and has a certain diagnostic value, which provides a new, fast and convenient method for the early diagnosis of female CPP children.

[0101] The above only describes the preferred embodiments of the present application.

Claims

1. Use of RBP4 and Chemerin as biomarkers in the preparation of a diagnostic reagent for central precocious puberty in females.

Images