A biomarker for diagnosing central precocious puberty in women and its application

Abstract

The present invention relates to the field of biomarker technology, and in particular to a biomarker for diagnosing central precocious puberty in women and its application. The present invention provides a biomarker for diagnosing central precocious puberty in women, wherein the marker is one, two or three of Leptin, RBP4 or Chemerin. The present invention has experimentally found that the peripheral blood Leptin, RBP4 and Chemerin levels of female children with CPP are significantly higher than those of healthy children, and the serum Leptin and RBP4 levels change in the early stages of CPP children, suggesting that these three fat factors may be involved in the onset of CPP.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarkers, and in particular to a biomarker for diagnosing central precocious puberty in women and an application thereof. Background Art

[0002] Central precocious puberty (CPP) is a common pediatric endocrine disease that refers to the early activation of the hypothalamus-pituitary-gonadal axis (HPG axis), which leads to rapid development of internal and external reproductive organs and the appearance of secondary sexual characteristics before the age of 7.5 in girls and 9 in boys. This disease has a huge impact on the adult height and psychological quality of children. However, the pathogenesis of CPP is still unclear. It is currently believed that the factors that cause the early activation of the HPG axis in puberty include genetics, nutrition, and environmental endocrine disruptors. With the improvement of living standards, the proportion of obese children has also increased year by year. The early onset of puberty is closely related to nutrition and body fat. Adipose tissue is an important endocrine organ that can secrete a variety of active mediators to play an endocrine metabolic and regulatory role. These active mediators are adipose factors. Therefore, obesity leads to dysregulation of the secretion of adipose factors, which in turn leads to the early activation of the HPG axis, which may play an important role in the occurrence of central precocious puberty.

[0003] Currently, the gold standard for diagnosing CPP is the GnRH stimulation test, but the test is cumbersome to operate, requires repeated blood sampling, and children with peripheral precocious puberty may be at risk of developing central precocious puberty.

[0004] In recent years, literature reports at home and abroad have shown that there is a connection between adipose factors and CPP. Leptin is the earliest, most widely studied and most complete adipose factor in CPP. It is secreted by white adipose tissue and has the functions of regulating diet, energy consumption and reproduction. The production and secretion of Leptin is positively correlated with the body fat content. Increased serum Leptin levels have been observed in obese women and CPP women. The pulsed secretion of GnRH by GnRH neurons in the hypothalamus acts on the pituitary gland, thereby initiating the HPG axis to start 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 neuronal cells expressing kisspeptin, stimulating the KISS-1 gene to overexpress kisspeptin. At the pituitary level, high concentrations of serum Leptin can also stimulate the upregulation of GnRH receptor expression, increase the sensitivity of gonadotropin to GnRH pulses, and promote the connection between hypothalamic GnRH neurons and the pituitary gland, resulting in the synchronization of Leptin-LH pulses and ovulation. Therefore, obesity leads to a large amount of fat accumulation in the body. Fat cells can produce and secrete Leptin, but obese people have leptin resistance in their bodies, which leads to hyperleptinemia. Leptin can pass through the blood-brain barrier. When its accumulation reaches a certain threshold, it binds to the hypothalamic GnRH neurons and LepR on the pituitary gland to play a regulatory role, causing the premature activation of the HPG axis, which may be one of the pathogenesis of CPP.

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

[0006] RBP4 is mainly secreted by the liver and adipose tissue, and is responsible for binding and transporting retinol in the blood. Serum RBP4 levels are positively correlated with visceral fat. In mouse ovaries, it was found that FSH can affect transcription factors HMGA1, SF-1, and LRH-1 through the 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 the liver, and increase the bioavailability of sex hormones. Decreased insulin sensitivity and reduced SHBG levels have also been observed in women with CPP. However, there are currently no studies at home and abroad exploring the relationship between RBP4 and CPP. Summary of the invention

[0007] The purpose of the present invention is to provide a biomarker for diagnosing central precocious puberty in women and its application. The present invention explores the influence of adipose factors on the HPG axis, selects three adipose factors that may be associated with CPP, namely leptin, retinol binding protein 4 (RBP4), and chemerin, detects the concentrations of adipose factors Leptin, RBP4, and Chemerin in peripheral blood, and performs correlation analysis with clinical indicators of children with CPP, such as sex hormone levels, bone age, BMI, etc., in order to discover CPP, especially early warning indicators of CPP in obese girls, and provide a basis for early diagnosis and prevention of CPP in women.

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

[0009] The present invention provides a biomarker for diagnosing central precocious puberty in women, wherein the biomarker is one, two or three of Leptin, RBP4 or Chemerin.

[0010] The present invention provides the use of the biomarker in preparing a diagnostic reagent for female central precocious puberty.

[0011] The present invention provides a kit for detecting central precocious puberty in women, wherein the kit comprises a reagent containing the biomarker.

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

[0013] 1. The peripheral blood Leptin, RBP4, and Chemerin levels of female children with CPP were significantly higher than those of healthy children, and the serum Leptin and RBP4 levels changed in the early stages of CPP children, suggesting that these three adipokines may be involved in the pathogenesis of CPP.

[0014] 2. Bone age, the 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 indicators for the occurrence of CPP in female children.

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

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

[0018] Figure 3 Heat map of the correlation between Leptin, Chemerin, RBP4 and clinical data.

[0019] Figure 4 Heat map of correlation analysis among Leptin, Chemerin and RBP4.

[0020] Figure 5 ROC curves of Leptin, Chemerin and RBP4 detected separately.

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

[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] Experimental Example 1

[0024] 1. Research materials and equipment

[0025] 1.1 Research subjects

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

[0027] Inclusion criteria:

[0028] 1. All confirmed cases are in accordance with the consensus on diagnosis and treatment of central precocious puberty (2015):

[0029] 1) Girls develop breasts before 8 years old or menarche before 10.0 years old;

[0030] 2) Gonadal enlargement, i.e., pelvic ultrasound shows that the uterus is 3.4-4.0 cm long, the ovarian volume is 1-3 ml (ovarian volume = length × width × thickness × 0.5233), and multiple follicles with a diameter of ≥4 mm can be seen, with the uterine body length >3.2 cm as the cutoff value for diagnosing CPP;

[0031] 3) Serum gonadotropin and sex hormones reach puberty levels, LH basal value> 0.2U / L; LH basal value < 0.2U / L but sex hormone stimulation test is performed, LH peak ≥ 5.0U / L and the ratio of LH peak to FSH peak ≥ 0.6 indicates the activation of the gonadal axis;

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

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

[0034] 2. Aged less than 10 years at the time of consultation;

[0035] Exclusion criteria:

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

[0037] 2. Patients with intracranial lesions indicated by cranial imaging examinations, such as tumors with endocrine function or other space-occupying lesions; and central nervous system abnormalities caused by acquired injuries, such as trauma, postoperative surgery, radiotherapy and chemotherapy, etc.;

[0038] 3. Those suffering from other congenital or hereditary diseases;

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

[0040] (2) Control group: 50 healthy female children of the same age range who underwent puberty physical examinations in the pediatric outpatient clinic of this hospital were selected as the control group.

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

[0042] 2.1.2 Main reagents and instruments

[0043] Table 1 Main reagents

[0044]

[0045] Table 2 Main instruments

[0046]

[0047] 2. Experimental process and methods

[0048] 2.1 Clinical data and blood sample collection

[0049] Collect and record the general condition and medical history of all patients in detail, including age, height, weight, and calculate the BMI index (BMI = weight in kg / height in m2). 2 ), Tanner staging, and breast staging. Color Doppler ultrasound was used to detect the development of the uterus and ovaries of the patients. The sex hormone series (FSH, LH, progesterone, T, E2, and pituitary prolactin) were measured by the biochemical laboratory of the laboratory department of our hospital and collected through the HIS system. All children underwent left wrist X-ray anteroposterior films, and bone age was calculated according to the "Bone Age Atlas 2016 Edition".

[0050] With informed consent, 5 ml of peripheral venous blood was collected from all CPP children and children in the control group in the early morning in the fasting state in a procoagulant biochemical tube, centrifuged at 3500 r / min for 10 min, and 200 μL of the upper serum was aspirated with a pipette into 3 dry sterilized enzyme-free centrifuge tubes, which were sealed and the information labels were checked and stored in the -80°C refrigerator of our hospital's biological sample bank.

[0051] 2.2 ELISA method to detect Leptin concentration in serum samples

[0052] (1) Take out the serum sample from the -80°C refrigerator and place it at room temperature until it dissolves. Take out the ELISA kit from the 4°C refrigerator and place it at room temperature for 30 minutes.

[0053] (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 using a low-speed centrifuge, and then use a pipette to transfer 120μL to the second tube. Repeat this process to make multiple dilutions. The concentrations of the final 5 tubes of standard are 40μg / L, 20μg / L, 10μg / L, 5μg / L, and 2.5μg / L.

[0054] (3) Sample addition: 1) Standard well: add 50 μL of standard and 50 μL of streptavidin-HRP; 2) Blank well: do not add anything; 3) Sample well: 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.

[0055] (4) Prepare washing solution: Dilute the 30-fold concentrated washing solution 30 times with distilled water and set aside.

[0056] (5) Washing: Remove the sealing film, discard the liquid in the wells, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat 5 times and pat dry on filter paper.

[0057] (6) Color development: Add 50 μL of color developer A and 50 μL of color developer B to each well, cover with a new sealing film, gently shake to mix, and place in a 37°C water bath in the dark for 15 minutes.

[0058] (7) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0059] (8) Determination: The assay should be performed within 10 minutes after the addition of the stop solution. The blank well should be used as the zero value, and the absorbance (OD value) of each well should be measured in sequence at a wavelength of 450 nm.

[0060] (9) Calculate concentration: Use EXCEL to calculate the regression equation (R) of the standard curve based on the concentration of the standard and the corresponding OD value. 2 >0.92), and the corresponding Leptin concentration of the sample was calculated based on the regression equation according to the OD value of the sample.

[0061] 2.3 ELISA to detect RBP4 concentration in serum samples

[0062] (1) All serum samples were diluted 1:5 with PBS before measurement. 10 μL of sample was added to 40 μL of PBS and mixed using a low-speed centrifuge.

[0063] (2) The dilution concentrations of the standard samples were 40 μg / L, 20 μg / L, 10 μg / L, 5 μg / L, and 2.5 μg / L, respectively.

[0064] The remaining steps are the same as those for detecting Leptin.

[0065] 2.4 ELISA method for detecting Chemerin concentration in serum samples

[0066] The detection method is the same as RBP4.

[0067] The dilution concentrations of the standard samples were 48ng / L, 24ng / L, 12ng / L, 6ng / L, and 3ng / L, respectively.

[0068] 2.5 Statistical analysis

[0069] SPSS 24.0 and Graphpad Prism 9.3.0 were used for data collation and analysis. All data were subjected to Kolmogorov-Smirnov normality test. The measured data that met the normal distribution were expressed as mean ± standard deviation. The data were expressed as P25, P75, and the comparison between the two groups was performed by independent sample t test. The non-normal distribution of quantitative data was expressed as M (P25, P75), and the comparison between the two groups was performed by Mann-Whitney U test. The relationship between Leptin, RBP4, Chemerin and clinical data such as height, weight, BMI, sex hormones, bone age, the difference between bone age and age, and uterine length was analyzed by Spearman correlation analysis. All statistical tests were tested with α = 0.05 as the test standard, and P < 0.05 was considered statistically significant. Logistic regression was used to analyze the predictive indicators of CPP in female children. The receiver operating characteristic curve (ROC) curve was used to analyze the diagnostic value of Leptin, RBP4, and Chemerin alone and in combination for CPP. The area under the curve (AUC), 95% CI, CUT OFF value, P value, sensitivity, and specificity were used to evaluate the diagnostic value.

[0070] 3. Results:

[0071] 3.1 Comparison of clinical data between the CPP group and the control group

[0072] According to the diagnostic criteria of the Consensus on Diagnosis and Treatment of Central Precocious Puberty (2015), a total of 50 CPP group samples and 50 control group samples were collected and included in this experiment.

[0073] After statistical analysis, there was no statistically significant difference 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 the CPP group were all higher than those in the control group, and the differences were statistically significant (P<0.05). See Table 3.

[0074] Table 3 Comparison of clinical data between CPP group and control group

[0075]

[0076] Note: BMI body mass index, FSH follicle stimulating hormone, LH luteinizing hormone, T testosterone, E2 estradiol, * indicates P < 0.05, ** indicates P < 0.01

[0077] 3.2 Determination and comparison of serum Leptin, RBP4, and Chemerin levels between the CPP group and the control group

[0078] The ELISA method was used to detect the levels of Leptin, RBP4, and Chemerin in the serum of all children. Statistical analysis showed that the levels of the three factors in the serum of the CPP group were significantly higher than those in the control group, and the difference was statistically significant (P < 0.05). See Table 4. Figure 1 .

[0079] Table 4 Comparison of serum Leptin, RBP4 and Chemerin levels between the two groups

[0080]

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

[0082] 3.3 Comparison between NM and M groups in children with CPP

[0083] According to clinical manifestations, the CPP group was divided into the non-menarche group (NM group) and the 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 the NM group and the M group (P < 0.05). The serum Leptin level in the NM group was higher than that in the M group, and the difference was statistically significant (P < 0.05); there was no statistical difference in RBP4 and Chemerin between the two groups (P > 0.05). See Table 5. Figure 2 .

[0084] Table 5 Comparison of clinical data and serum indicators between NM group and M group

[0085]

[0086] Note: * indicates P < 0.05, ** indicates P < 0.01

[0087] 3.4 Correlation analysis between serum Leptin, RBP4, Chemerin levels and clinical data

[0088] There was no significant correlation between serum Leptin and Chemerin levels and clinical data (P>0.05); serum RBP4 levels were negatively correlated with age, height, weight, breast stage, FSH, and LH (P<0.05). See Table 6, Figure 3 Serum Leptin level was positively correlated with serum Chemerin and RBP4 levels (P < 0.05). See Table 7. Figure 4 .

[0089] Table 6 Correlation analysis between serum Leptin, RBP4, Chemerin levels and clinical data

[0090]

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

[0092] Table 7 Correlation analysis among Leptin, Chemerin and RBP4

[0093]

[0094]

[0095] Note: ** indicates P < 0.01

[0096] 3.5 Analysis of influencing factors and evaluation indicators of CPP in female children

[0097] Through univariate analysis, it was found that height, weight, BMI, Tanner stage, breast stage, bone age, the 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 were all single factors affecting the occurrence of CPP in female children. According to statistical principles, 11 statistically significant factors, including BMI, Tanner stage, breast stage, bone age, the difference between bone age and age, T, E2, uterine length measured by B-ultrasound, serum Leptin level, serum Chemerin level, and serum RBP4 level, were used as independent variables for multivariate logistics regression analysis. The results showed that bone age, the 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 indicators for the occurrence of CPP in female children. See Table 8.

[0098] Table 8 Logistics regression analysis of factors affecting CPP in female children

[0099]

[0100] Note: * indicates P < 0.05, ** indicates P < 0.01

[0101] 3.6 Diagnostic value of serum Leptin, Chemerin, and RBP4 levels in CPP in female children Leptin, Chemerin, and RBP4 have an impact on each part of the hypothalamus-pituitary-gonadal axis, and therefore may play a role in the activation process of the gonadal axis, which may have important guiding significance for the early clinical diagnosis and prevention of CPP. The present invention explores the diagnostic value of Leptin, Chemerin, and RBP4 in girls with CPP, as well as the diagnostic value of the combined detection of Chemerin and RBP4 in girls with CPP.

[0102] The area under the ROC curve of RBP4 was slightly higher than that of Leptin, and the area under the ROC curve of Chemerin was slightly higher than that of RBP4 and Leptin; Leptin had a sensitivity of 72.0% and a specificity of 58.0%, RBP4 had a sensitivity of 46.0% and a specificity of 80.0%, and Chemerin had a sensitivity of 58.0% and a specificity of 72.0%. The area under the curve of the combined detection of RBP4 and Chemerin was 0.716, with a sensitivity of 66.0% and a specificity of 66.0%, and the diagnostic value was higher than that of the single detection. The ROC curve is shown in Figure 5 , Figure 6 , ROC curve related data are shown in Table 9.

[0103] Table 9 ROC related indicators

[0104]

[0105] In the present invention, by analyzing and comparing the clinical data of CPP children and healthy girls of the same age in the control group, we found that the height, weight and bone age of the children in the CPP group were significantly higher than those in 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 bones, the bone age was much greater than the actual age, resulting in premature closure of the epiphyseal end, which would affect the lifelong 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), and there was a risk of PCOS and estrogen-dependent diseases, so early diagnosis and prevention of CPP were very important. In addition, the BMI of the children in the CPP group was significantly higher than that of the control group (P < 0.05), and the BMI of the children in the menarche group (M group) in the CPP group was also significantly higher than that of the children in the non-menstrual group (NM group) (P < 0.05), indicating that obesity is related to the occurrence and development of CPP.

[0106] In the present invention, the serum Leptin level in the CPP group was higher than that in the control group (P < 0.05). The serum Leptin level in the NM group was higher than that in the M group (P < 0.05). The serum sOB-R level was not detected in the experiment, and it cannot be ruled out that the serum Leptin in the children in the M group combined with sOB-R, resulting in a decrease in the serum free Leptin level.

[0107] In the present invention, correlation analysis found that there is a correlation between Leptin and RBP4 and Chemerin. In addition, the present invention also suggests that the serum Leptin level is positively correlated with the serum Chemerin level (r=0.572, P<0.001). Leptin and Chemerin in serum are both secreted by adipose tissue, and there may be a mutual regulation between them.

[0108] RBP4 is mainly secreted by the liver and adipose tissue in the body. In the present invention, the serum RBP4 level of children in the CPP group was significantly higher than that of the normal control group (P < 0.05), and correlation analysis found that the serum RBP4 level of children in the CPP group was negatively correlated with age, height, weight, breast stage, FSH, and LH.

[0109] The results of our research in the present invention are the first to find that the expression of adipose factors RBP4 and Chemerin in the serum of female children with CPP is also upregulated. In addition, a significant increase in serum Leptin and RBP4 can be observed in the early stages of CPP, and correlation analysis suggests that Leptin may affect the expression levels of RBP4 and Chemerin. Further, through Logistics regression analysis, it was found that bone age, the difference between bone age and age, T, uterine length measured under B-ultrasound, and serum RBP4 levels can be used as predictive indicators for CPP in female children. The present invention combines the above indicators and may help in the early diagnosis of CPP in female children. The present invention suggests that there is a correlation between Leptin and RBP4 and Chemerin. Therefore, we explored the diagnostic value of Leptin, RBP4, Chemerin alone and the combined detection of RBP4 and Chemerin in female CPP. Combined with the area under the ROC curve AUC, the combined detection of RBP4 and Chemerin is higher than the independent detection of the three, and has a certain diagnostic value, providing a new, quick and convenient method for the early diagnosis of CPP in female children.

[0110] The above description is only the preferred embodiment of the present invention.

Claims

1. Application of RBP4 and Chemerin as biomarkers in the preparation of diagnostic reagents for central precocious puberty in women.

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