Application of sulfated progesterone metabolite in diagnosis of intrahepatic cholestasis in gestation period

Abstract

The invention provides an application of a sulfated progesterone metabolite in diagnosis of intrahepatic cholestasis in a gestation period, and particularly, the sulfated progesterone metabolite provided by the invention comprises one or more of Preg17olS, PM6S, PregS, PMS and PM3S < + >. A diagnostic model established on the basis of the sulfonated progesterone metabolite shows high diagnostic performance, and is a diagnostic marker for intrahepatic cholestasis in the gestation period.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to the application of sulfonated progesterone metabolites in the diagnosis of intrahepatic cholestasis of pregnancy. Background Art

[0002] Intrahepatic cholestasis of pregnancy (ICP) is one of the liver diseases unique to pregnancy and is a common cause of pregnancy jaundice second only to viral hepatitis. Its incidence shows significant regional and ethnic differences, affecting 0.2% to 28% of pregnancies globally. There is currently no exact epidemiological data in China. The etiology and pathogenesis of ICP are not clear. Genetic factors and non-genetic factors such as estrogen, progesterone, immunity, and environment act together. Its main clinical features include skin pruritus and abnormal biochemical indicators such as bile acids.

[0003] Despite the high prevalence and impact of ICP, research on its early diagnosis and effective treatment remains limited, mainly due to insufficient understanding of its underlying mechanisms. In addition, there are also challenges in the diagnostic criteria for ICP. Currently, the diagnosis of ICP is mainly based on pruritus during pregnancy and elevated levels of total serum bile acids (TBA) and alanine aminotransferase (ALT). Although TBA is the most commonly used biomarker, elevated TBA levels also occur in asymptomatic hypercholesterolemia and 10% of healthy pregnant women, complicating the diagnosis of ICP. The diagnostic sensitivity and specificity of ALT and aspartate aminotransferase (AST) are also relatively low, and less than 70% of ICP patients have elevated ALT and AST. These limitations highlight the urgent need for reliable biomarkers to achieve early diagnosis of ICP and improve diagnostic criteria. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention has conducted research. By high-throughput screening of 1490 compounds for those that can activate the putative pruritus receptor hMRGPRX4 (abbreviated as hX4), several sulfonated progesterone metabolites were finally identified as effective activators of hX4. Utilizing the significant difference in sulfonated progesterone levels between ICP patients and healthy pregnant women, the present invention developed a computational model for diagnosing intrahepatic cholestasis of pregnancy according to different gestational stages. These models can accurately diagnose ICP and can predict the risk of its onset several months before the clinical symptoms appear.

[0005] The research results of the present invention not only provide convincing evidence that sulfonated progesterone can activate hX4 to induce ICP-related pruritus symptoms, but also can diagnose and early predict the onset risk of ICP, providing an important tool for the early intervention and treatment of the disease.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, there is provided an application of sulfonated progesterone metabolites in the diagnosis of intrahepatic cholestasis of pregnancy, and the sulfonated progesterone metabolites include one or more of Preg17olS (fully named 17-hydroxypregnenolonesulfate), PM6S (fully named (3α, 5β)-pregnanolone sulfate), PM3S+, PregS (fully named Pregnenolone sulfate), and PMS.

[0008] Further, the sulfonated progesterone metabolites are a combination of Preg17olS, PM6S, PM3S+, PregS, and PMS.

[0009] Further, the PMS includes a combination of three isomers, namely PM4S (fully named (3α, 5α)-pregnanolone sulfate), PM5S (fully named (3β, 5α)-pregnanolone sulfate), and PM7S (fully named (3β, 5β)-pregnanolone sulfate).

[0010] Further, the PM3S+ includes a combination of two isomers, namely PM3S (fully named (3α, 5β)-pregnanediol sulfate) and 3β5α-diolS (fully named (3β, 5α)-pregnanediol sulfate).

[0011] Specifically, the diagnosis means that when the level of sulfonated progesterone metabolites in a subject's sample is up-regulated, the subject has ICP or is at risk of developing ICP.

[0012] In the present invention, the term "up-regulated" refers to an increase in the absolute or relative amount of the metabolite. In the case of obtaining a reference result from a subject or group known not to have ICP, the disease or susceptibility can be diagnosed based on the difference between the test result obtained from the sample and the above reference result, that is, based on the difference in the qualitative or quantitative composition of at least one metabolite. In some embodiments, the relative or absolute amount difference is significant, that is, outside the reference value range of the 45th to 55th percentile, 40th to 60th percentile, 30th to 70th percentile, 20th to 80th percentile, 10th to 90th percentile, 5th to 95th percentile.

[0013] In a second aspect of the present invention, there is provided an application of a reagent for detecting the level of the sulfonated progesterone metabolites described in the first aspect of the present invention in a sample in the preparation of a product for diagnosing intrahepatic cholestasis of pregnancy.

[0014] Further, the product includes a kit, a chip, a test strip, high-throughput sequencing, a system, a device, an apparatus.

[0015] In the present invention, the product may include a solid substrate such as a chip, a glass slide, an array, etc., which has reagents capable of detecting and / or quantifying one or more blood metabolites or metabolite sources from other samples at predetermined positions fixed on the substrate. As an illustrative example, reagents fixed at discrete predetermined positions may be provided to the chip for detecting and quantifying metabolite markers in a blood sample, the concentration of any number or any combination thereof.

[0016] Further, the reagent detects the level of metabolite markers in the sample by one or several of targeted or non-targeted nuclear magnetic resonance method, chromatography, spectroscopy, mass spectrometry, chromatography-mass spectrometry.

[0017] Further, the reagent detects the level of metabolite markers in the sample by chromatography-mass spectrometry.

[0018] In the present invention, the sample for detecting the metabolite markers or the sample to be tested refers to a composition obtained from or derived from a subject (such as an individual of interest), comprising cell entities and / or other molecular entities characterized and / or identified based on physical, biochemical, chemical, and / or physiological characteristics.

[0019] Further, the subject refers to any individual of interest. Preferably, the subject refers to a living organism suffering from or suspected of suffering from intrahepatic cholestasis of pregnancy, including humans, other mammals, preferably primates, and particularly preferably humans.

[0020] Further, the sample is a biological sample. Samples of biological origin (i.e., biological samples) usually contain a variety of metabolites. Preferred experimental samples to be used in the method of the present invention are samples from body fluids, preferably samples from blood, plasma, serum, feces, lymph, sweat, saliva, tears, semen, vaginal fluid, urine, or cerebrospinal fluid, or samples from cells, tissues, or organs obtained by, for example, dissection. This also includes samples containing subcellular compartments or organelles (such as mitochondria, Golgi networks, or peroxisomes). In addition, biological samples also include gas samples, such as volatiles of an organism. The biological sample is from a subject specifically described elsewhere herein. Techniques for obtaining the above different types of biological samples are well known in the art. For example, a blood sample is obtained by blood collection, a urine sample is obtained by urine collection, and a fecal sample is obtained by fecal collection.

[0021] Further, the sample is selected from blood, serum, plasma.

[0022] Further, the sample is plasma.

[0023] Further, the product further includes a reagent for processing the sample.

[0024] Further, the sample is pretreated before being used in the detection of the present invention. The pretreatment may include processes required for releasing or separating compounds, or removing excess substances or wastes. Suitable techniques include centrifugation, extraction, fractionation, purification, and / or enrichment of compounds. In addition, other pretreatments are performed to provide the compounds in a form or concentration suitable for compound analysis. For example, if gas chromatography coupled with mass spectrometry is used in the method of the present invention, it will be necessary to derivatize the compounds before the gas chromatography. The suitable and necessary pretreatment depends on the tool for performing the method of the present invention and is well known to those skilled in the art. The pretreated sample as described above is also included in the term "sample" as used in the present invention.

[0025] Further, the product further includes an instruction manual, which should clearly describe how to use the product to evaluate whether a subject has ICP or is at risk of having ICP.

[0026] In a third aspect of the present invention, there is provided a product for diagnosing intrahepatic cholestasis of pregnancy, the product including a reagent for detecting the level of the sulfonated progesterone metabolite described in the first aspect of the present invention in a sample.

[0027] In a fourth aspect of the present invention, there is provided the use of the sulfonated progesterone metabolite described in the first aspect of the present invention in constructing a diagnostic model for intrahepatic cholestasis of pregnancy.

[0028] In a fifth aspect of the present invention, there is provided a method for constructing a diagnostic model for intrahepatic cholestasis of pregnancy, the steps of the method including obtaining data on the level of the sulfonated progesterone metabolite described in the first aspect of the present invention and clinical characteristic data in a sample, and inputting the data into a machine learning algorithm to construct a diagnostic model.

[0029] Further, the steps of the method include: dividing the data on the level of the sulfonated progesterone metabolite into a training set and a test set, extracting the data on the expression level of gene markers in the training set and inputting it into a machine learning algorithm to construct a prediction model, and validating it through the test set to evaluate the model efficacy.

[0030] Further, the diagnostic model obtains a classification result through the following criteria: when the level of one or more of the sulfonated progesterone metabolites described in the first aspect of the present invention is higher than the optimal cut-off value, a classification result that the subject has intrahepatic cholestasis of pregnancy or is at risk of having intrahepatic cholestasis of pregnancy is obtained; if the level of the sulfonated progesterone metabolite described in the first aspect of the present invention is lower than the optimal cut-off value, a classification result that the subject does not have intrahepatic cholestasis of pregnancy is obtained.

[0031] In the present invention, the term "optimal cut-off value" refers to a value that is statistically relevant to a specific outcome when compared with the analysis result. In a preferred embodiment, the optimal cut-off value is determined based on the statistical conclusion of a study comparing ICP patients and healthy pregnant women. Some such studies are shown in the example section herein, but studies from the literature and the experience of users of the methods described herein can also be used to generate or adjust the optimal cut-off value. The optimal cut-off value can also be determined by considering the circumstances and results of the patient's genetic background, clinical characteristics, working environment, and other relevant factors.

[0032] Furthermore, the clinical characteristic data includes whether the subject has intrahepatic cholestasis of pregnancy and the gestational stage at which the subject is located.

[0033] In some embodiments of the present invention, the method for constructing the diagnostic model belongs to those known to those skilled in the art and can be implemented and achieved in different ways to associate the gene marker expression level with a certain possibility or risk. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined, and the combined value is associated with the underlying question of whether the subject has the disease or is at risk of having the disease. The measured combination of the marker values can be combined by any suitable existing mathematical method, and a prediction model can be constructed through a machine learning algorithm.

[0034] Furthermore, the machine learning algorithm includes algorithm models developed using various development tools.

[0035] Furthermore, the development tools include TensorFlow, Scikit Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, Spell.

[0036] Furthermore, the algorithm models include generalized linear models, principal component analysis, logistic regression analysis, LASSO regression analysis, nearest neighbor analysis, support vector machines, neural network models, random forest models.

[0037] The sixth aspect of the present invention provides a diagnostic system for intrahepatic cholestasis of pregnancy, the system including a data classification unit, which is used to substitute the sulfonated progesterone metabolite level data into the diagnostic model constructed according to the method described in the fifth aspect of the present invention to obtain a classification result of whether the sample has intrahepatic cholestasis of pregnancy or is at risk of having intrahepatic cholestasis of pregnancy.

[0038] Further, the system further includes a data acquisition unit, which is used to acquire the data of the sulfonated progesterone metabolite level described in the first aspect of the present invention in the sample.

[0039] Further, the system further includes an output unit, which is used to output the classification result.

[0040] The seventh aspect of the present invention provides a diagnosis device for intrahepatic cholestasis of pregnancy, and the diagnosis device includes a memory and a processor.

[0041] The memory is used to store program instructions.

[0042] The processor is used to execute the program instructions. When the program instructions are executed, the following operations are performed: acquiring the data of the sulfonated progesterone metabolite level described in the first aspect of the present invention in the sample, and inputting the data of the sulfonated progesterone metabolite level into the diagnosis model constructed based on the method described in the fifth aspect of the present invention to obtain a classification result of whether the sample has intrahepatic cholestasis of pregnancy or has a risk of suffering from intrahepatic cholestasis of pregnancy.

[0043] To provide interaction with the user, the device may be a computer, which has: a display device for displaying information to the user; and a keyboard and a pointing device (such as a mouse) through which the user can provide input to the computer. Other types of devices may also be used to provide interaction with the user; for example, the feedback provided to the user may be any form of sensory feedback (such as visual feedback, auditory feedback, or tactile feedback); and the input from the user may be received in any form (including voice input, speech input, or tactile input).

[0044] Further, the sample is selected from blood, serum, and plasma.

[0045] Further, the sample is plasma.

[0046] The eighth aspect of the present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the following method is implemented: acquiring the data of the sulfonated progesterone metabolite level described in the first aspect of the present invention in the sample, and inputting the data of the sulfonated progesterone metabolite level into the diagnosis model constructed based on the method described in the fifth aspect of the present invention to obtain a classification result of whether the sample has intrahepatic cholestasis of pregnancy or has a risk of suffering from intrahepatic cholestasis of pregnancy.

[0047] Any combination of one or more computer-readable media may be employed. The computer-readable media may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the foregoing. In the present invention, the computer-readable storage medium may be any tangible medium that contains or stores a program, which can be used by or in conjunction with an instruction execution system, apparatus, or device. In some embodiments, more specific examples of the computer-readable storage medium include, but are not limited to: an electrical connection having one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing.

[0048] Further, the sample is selected from blood, serum, and plasma.

[0049] Further, the sample is plasma.

[0050] Advantages and beneficial effects of the present invention: The present invention provides sulfonated progesterone metabolites that can be used for diagnosing intrahepatic cholestasis of pregnancy, and provides an ICP diagnosis model constructed based on metabolite levels at different pregnancy stages, which can accurately diagnose ICP and can predict the risk of ICP occurrence at an early stage. The median time of successful prediction is about the 14th week, significantly earlier than the median time of clinical diagnosis, which is about 163 days, providing an important tool for early intervention and treatment of the disease. Description of the Drawings

[0051] Figure 1 Graphs for determining the results of sulfonated progesterone activating the hMRGPRX4 receptor by high-throughput screening, where A is a flow chart for determining candidate pruritogens through the hX4 receptor; B is a schematic diagram of high-throughput screening of pruritogens and structural diagrams of nine best pruritogenic metabolites; in C, the left graph is a schematic diagram of the sulfonation effect of SULT enzyme in the liver, and the right graph is a schematic diagram of the relative activation of the hX4 receptor by sulfonated progesterone and its corresponding non-sulfonated precursor; D is a dose-response curve of DCA, PM5S, PM3S, and PregS and their corresponding non-sulfonated precursors activating hX4; E is a schematic diagram of the relative activation of the hX4, hMRGPRX (abbreviated as hX1), hMRGPRX2 (abbreviated as hX2), and hMRGPRX3 (abbreviated as hX3) receptors induced by the shown compounds (100 μM).

[0052] Figure 2Results of sulfonated progesterone activating DRG neurons expressing hX4 and inducing pruritus. Among them, A is a schematic diagram of the process of culturing, transfecting WT DRG neurons and performing calcium imaging; B-D show the calcium imaging results of PM5S, PM3S and PM5S with hX4 antagonist (1-55). From left to right are the representative images, representative fluorescence and dose-response curves of hX4-P2A-RFP (hX4 + ), or control (hX4 - ) in DRG neurons; E is a schematic diagram of the construction process of hX4 humanized rats; F is a quantification diagram of scratching episodes induced by subcutaneous injection of PM5S (left) or PM3S (right) at a specified dose (dissolved in 50 μl solvent) into the nape of the neck of hX4 humanized rats; G is a quantification diagram of scratching episodes induced by subcutaneous injection of 300 μg PM5S (left) or PM3S (right) into the nape of the neck of hX4 humanized rats; H is a quantification diagram of scratching episodes induced by 300 μg PM5S, 300 μg PM3S, and 500 μg 5-HT in WT rats.

[0053] Figure 3 Results of sulfonated progesterone causing pruritus in humans. Among them, A is a schematic diagram of the process of a double-blind study on the ability of various compounds (injecting 25 μL of solvent into each arm) to induce pruritus in human subjects; B is a time-course diagram of the perceived itching intensity induced by PM5S or vehicle; C is a violin plot showing the peak of the perceived itching intensity induced by PM5S or vehicle at different time stages in Figure B; D is a summary diagram of the area under the curve (AUC) of the 30-min trajectory curves obtained from individual subjects; E-G are similar to B-D, but distinguish the results of male and female subjects; H is a representative image of the injection site and a statistical chart of the erythema area measured 30 min after injection; I-J are the results of the time-course (I) and AUC (J) of the perceived itching intensity induced by PM5S or vehicle after pretreatment with a topical antihistamine.

[0054] Figure 4 Results showing the correlation between sulfonated progesterone levels and the intensity of ICP pruritus. Among them, A is a schematic diagram of the strategy for detecting the effect of sulfonated progesterone in ICP-related pruritus; In the upper part of Figure B is a representative total ion chromatogram of various sulfonated progesterones separated and identified by HPLC-MS / MS, and in the lower part is a summary diagram of different sulfonated progesterone levels at specified time points in 35 ICP patients and 27 control subjects; C is an analysis diagram of the correlation between the itching intensity and blood drug concentration in ICP patients in the late pregnancy and control subjects.

[0055] Figure 5Diagnostic result chart of sulfonated progesterone levels for ICP, where A is a schematic diagram of the ICP diagnostic model scheme generated by the generalized linear model (GLM) based on sulfonated progesterone levels; B is a heat map of the plasma sulfonated progesterone curves of individuals measured in the third trimester of pregnancy in the cohort; C is an ROC curve chart for diagnosing ICP with a single sulfonated progesterone metabolite; D is an ROC curve chart for diagnosing ICP with a combination of sulfonated progesterone metabolites (PS model); E is a summary chart of the AUC of the ROC curves in Figures C and D; F is a summary chart of the number of participants corresponding to the prediction of ICP by the PS model, with an optimal cut-off value of 0.18; G-H are the same as D-F, which are the result charts of the external validation cohort. Detailed implementation mode

[0056] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0057] In some processes described in the specification, claims and above-mentioned drawings of the present invention, a plurality of operations appear in a specific order. However, it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The operation numbers such as 101, 102, etc. are only used to distinguish different operations, and the numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions such as "first" and "second" in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, nor do they limit that "first" and "second" are of different types.

[0058] Embodiment

[0059] I. Patients

[0060] This retrospective study (NCT06366659) has been approved by the Medical Science Research Ethics Committee of Peking University Third Hospital (M2024049) and the Medical Ethics Committee of West China Second University Hospital, Sichuan University (2022YFC2704703 and 2024068). All participants were fully informed and understood the detailed information related to the study and provided written informed consent. Samples not used in routine clinical procedures could be used without obtaining informed consent from the subjects. Participants could choose to withdraw from the study at any time. We excluded subjects with skin lesions such as pruritus accompanied by eczema, pruritus caused by other liver-related diseases such as primary cholestasis, and pruritus lasting at least 4 - 6 weeks after childbirth. Plasma samples were collected from participants recruited from Peking University Third Hospital in the third trimester of pregnancy, and the pruritus intensity was recorded. Data from the discovery cohort and the retrospective multicenter cohort were collected from multiple medical centers in Sichuan, China, during the period from 2019 to 2023, and the plasma samples were leftovers from routine clinical operations.

[0061] II. Experimental Materials

[0062] 1) Animals: All wild-type (WT) animals were purchased from Charles River Laboratories (Beijing, China). Male and female rats (6 - 8 weeks old, 200 - 300 g) were housed (3 - 5 rats per cage) under a standard 12-hour light / 12-hour dark cycle. Behavioral experiments were conducted during the light cycle. All animal experiments were approved by the Animal Care and Use Committee of the School of Life Sciences, Peking University.

[0063] 2) Stable cell lines: The stable cell lines used in this study were constructed as described in the method of H. S. Yu et al. published in eLife in 2019 (MRGPRX4 is a bile acid receptor for human cholestatic itch). Briefly, the DNA encoding human MRGPRXI, MRGPRX2, MRGPRX3, and MRGPRX4 was subcloned into the PiggyBac transposon vector and co-transfected with the highly active PiggyBac transposon into HEK293T cells using polyethyleneimine (PEI). Positive cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 1 μg / ml puromycin, 100 μg / ml penicillin, and 100 μg / ml streptomycin under a humidified atmosphere at 37°C and 5% CO 2 2.

[0064] III. Experimental Methods

[0065] 1) FLIPR assay: Twenty-four hours before measurement, HEK293T cells stably expressing human MRGPRX were seeded in 96-well plates at a density of 50,000 cells per well. The next day, the medium was removed, and the cells were loaded with Fluo-8 AM (AAT Bio) for 1 hour and then washed with Hanks' balanced salt solution containing 5 mM HEPES (pH 7.4) (referred to as HHBS). Before adding the drug, the baseline signal of Fluo-8 was recorded as F 0 . Then the test compound was added to the wells at different concentrations, and the Fluo-8 signal was immediately measured using the FLIPR TETRA system (Molecular Devices). The peak signal was defined as F, and the activation response was calculated by (F - F 0 ) / F 0 .

[0066] 2) TGFα shedding assay: An AP-TGF-α stable cell line expressing hX4 was used. The isolated cell pellet was separated by centrifugation (190 g, 5 min), resuspended in 10 ml of PBS, and incubated at room temperature for 10 min. Then the cells were recentrifuged and resuspended in 4 ml of HHBS, and the recovered cells were seeded in 96-well plates (90 μl / well) and incubated at 37°C and 5% CO 2 for 30 min. After 30 min, 10 μl of 10× compound stock solution was added to each well. Then the culture plates were incubated for 1 hour, and then the alkaline phosphatase activity in the conditioned medium and the cells was measured (using the substrate).

[0067] 3) Protein-ligand docking: Use the web version of CB-Dock2 ( https: / / cadd.labshare.cn / cb- dock2 / php / index.php ) to perform cavity search and automatic blind docking of ligands PM5 and PM3 with the hX4 protein.

[0068] 4) Isolation, electroporation, and culture of rat dorsal root ganglion neurons

[0069] Adult rat DRG tissues were collected, cut into pieces smaller than 1 mm, and then digested at 37°C in a solution containing 5 mg / ml dispase and 1 mg / ml collagenase for 1 h. After mechanical grinding and centrifugation, the cells were washed with 15% (w / v) BSA solution and resuspended in DMEM / F12 supplemented with 10% FBS. Then, according to the manufacturer's protocol, DRG neurons were electroporated using the P3 Primary Cell 4D Nucleofector X Kit L (Lonza). Then the cells were plated on glass coverslips pre-coated with poly-D-lysine and laminin and cultured for another 72 hours to allow transgene expression.

[0070] 5) Confocal Ca 2+ Imaging

[0071] In the Ca 2+ imaging experiment, cells were loaded with 5 μM Fluo-8 AM (AAT Bio) for 1 hour at 37˚C and then rinsed to remove the dye. Various compounds were perfused onto the cells in the chamber using a custom-built 6-channel perfusion valve control system, and real-time fluorescence images were recorded using a Nikon A1 confocal microscope.

[0072] 6) Construction of hX4 humanized rats

[0073] To construct hX4 humanized rats, we used CRISPR / Cas9 gene editing technology to modify the rat MrgA gene. The nucleotide sequence of 28 amino acids before the stop codon of exon 2 was replaced with the hX4-3*Flag-P2A-iCre-WPRE-polyA sequence, and the CAG-loxP-Frt-Stop-loxP-Frt-hX4-3*Flag-iP2A-mScarlet-WPRE-polyA construct was inserted into the ROSA26 locus using CRISPR / Cas9. Fl offspring were screened by tail biopsy to confirm the presence of gene modification. Finally, humanized rats expressing hX4 were established by breeding MrgA::hX4-Cre rats with Rosa26::loxP-STOP-loxP-hX4-3*flag mScarlet (Rosa26::LSL-hX4) rats.

[0074] 7) Animal behavior studies

[0075] Behavioral tests were conducted and analyzed by an experimenter blinded to the genotype and the compounds used. Before the experiment, animals were placed in the experimental chamber for 1 hour per day for 5 consecutive days. On the 6th day, the animals were first allowed to acclimate to the experimental chamber, and the basal scratching or wiping behavior was recorded for 60 min using a camera. Then, the test compound (dissolved in 50 μl of a vehicle consisting of 91% saline and 9% Tween-80) was injected intradermally into the nape or cheek of the animal, and the scratching or wiping behavior was recorded again for 60 min. For antagonist pretreatment, 1-55 was injected intradermally into the nape, and the test compound was injected at the same site 20 min later. Scratching or wiping was defined as the continuous movement of the ipsilateral hind or forepaw towards the injection site. Scratching or wiping behavior was quantified by counting the number of episodes during the 60-min observation period before and after injection.

[0076] 8) Human pruritus test

[0077] This study (NCT06364969) was approved by the Human and Animal Protection Committee of the Department of Psychology, Peking University (2021-06-02) and conducted in accordance with the description in the aforementioned article by H. S. Yu et al. Volunteers were students and teachers at Peking University, regardless of gender. All subjects received the experimental protocol and provided written informed consent. All compounds were administered subcutaneously using the INJEX 30 needle-free injection system (INJEX Pharma GmbH, Berlin, Germany).

[0078] First, each test compound was dissolved in physiological saline containing 8% Tween-80 (Sigma-Aldrich) to the final concentration. The injection site was cleaned with alcohol swabbing. 25 μl of each solution was intradermally injected on the volar surface of both arms. Itching was defined as the desire to scratch during the experiment, and the subjects were instructed to rate the perceived intensity of itching caused by the given stimulus using a generalized labeling scale during a 30-minute observation period. After these 30 minutes, the area of erythema around the injection site was measured.

[0079] For the antihistamine pretreatment experiment, approximately 1.5 g of topical antihistamine cream (doxepin hydrochloride cream, Chongqing Huabang Pharmaceutical Co., Ltd.) or placebo (cold cream, Avene) was applied 2 hours before injection; all unabsorbed cream was wiped off with alcohol swabbing. PM5S and histamine were prepared and injected as described above. Then the subjects were instructed to rate the itching sensation as described above.

[0080] 9) Blood sample preparation

[0081] Whole blood specimens from ICP patients and healthy pregnant women were placed in anticoagulant tubes. Plasma was separated from the blood by centrifugation at 12000 g for 10 min at 4°C and stored at -80°C. For protein precipitation, 100 μl of each plasma sample was mixed with 425 μl of methanol and then vortexed for 10 minutes. After centrifugation at 14000 g for 10 minutes, the supernatant was transferred to a new tube, stored at -80°C, and then subjected to HPLC-MS / MS analysis.

[0082] 10) HPLC-MS / MS analysis

[0083] HPLC-MS / MS analysis was performed using an Agilent UPLC 1290-MS / MS 6495 system and an API 5000 tandem mass spectrometer. 5 μl of each sample was injected into the system. A sulfonated progesterone was separated using an EC-Cl8 column (Agilent Poroschell 120, 50×4.6 mm, 2.7 μm particle size, Agilent Technologies). The mobile phase was LC-MS grade water and methanol, with 0.01% formic acid and 10 mM ammonium acetate added. The gradient was 70% methanol for 1.2 min; changed to 75% methanol within 6.8 min; changed to 100% methanol within 0.2 min, maintained at 100% methanol for 2.2 min, restored to 70% methanol within 0.25 min, and then maintained at 70% methanol for 2.35 min for re-equilibration. The flow rate was maintained at 0.3 ml / min, and the HPLC column was maintained at 20 °C.

[0084] For MS analysis, the negative electrospray ionization mode was used, with an ionization voltage of 5500 V, and multiple reaction monitoring was combined.

[0085] 11) Standard compounds and calibration curves

[0086] Stock solutions of all standard compounds were prepared using 100% methanol at a concentration of 1 mg / ml. Working solutions containing different concentrations were prepared by diluting the stock solutions to the following final concentrations using charcoal-stripped blank human plasma: concentrations of PM4S (Steraloids Inc.), PM5S (Toronto Research Chemicals), PM6S (Steraloids Inc.), and PM7S (Steraloids Inc.) were 0.64, 1.28, 2.57, 5.13, 10.27, 20.53, 41.07, 82.14, 164.27, 657.09, and 2628.38 ng / ml; concentration of Preg17olS (Steraloids Inc.) was 2.5, 5.0, 10.1, 20.1, 40.3, 80.6, 161.1, 644.6, 2578.4, and 10313.5 ng / ml (Steraloids Inc.); concentrations of PM3S and 3β5α-diolS (Steraloids Inc.) were 0.61, 1.22, 2.44, 4.89, 9.78, 19.56, 39.12, 78.24, 156.47, 625.89, and 2503.56 ng / ml; concentration of PregS (Toronto Research Chemicals) was 0.64, 1.28, 2.55, 5.11, 10.21, 20.42, 40.84, 81.69, 163.38, 653.52, and 2614.06 ng / ml. Calibration curves were obtained by plotting the MS response of the standard solutions against the concentration. All solutions were stored at -80°C.

[0087] 12) Generation and evaluation of prediction models

[0088] The R package caret (version 6.0 - 94) was used to generate a model for diagnosing ICP. A diagnostic model for ICP was established using the plasma sulfated progesterone level in the third trimester of pregnancy. The data was carefully examined and incomplete values were removed. To avoid multicollinearity, we used the "findCorrelation" function in R to exclude highly correlated variables, with a cut-off threshold of 0.8. To avoid multicollinearity, we used a generalized linear model (GLM) with a binomial family ("1" for positive and "0" for negative) to indicate the presence or absence of ICP. The model was trained using the R package caret and a ten-fold cross-validation procedure was used to ensure reusability and reliability. Specifically, in each iteration of cross-validation, the data was divided into 10 groups, 9 groups were used to train the GLM model, and the 10th group was used as a test set to evaluate the performance of the model. The model was evaluated using metrics such as ROC, AUC, accuracy (calculated using the formula [(TP + TN) / (P + N)]), specificity (true negative rate calculated using the formula TN / (TN + FP)), and sensitivity (true positive rate calculated using the formula [TP / (TP + FN)]) to assess the performance of patients, where T represents true, F represents false, P represents positive, and N represents negative. The ROC curve of a single sulfated progesterone biomarker was calculated and implemented using the ROC module in IBM SPSS Statistics (version 27).

[0089] 13) Statistical analysis

[0090] Statistical analysis was performed using OriginPro 2020. Inter-group data was analyzed using Student's t-test, one-way ANOVA, or Mann-Whitney U test, and the differences were statistically significant (p < 0.05). Unless otherwise stated, all summary data is presented as mean + SEM.

[0091] IV. Experimental results

[0092] 1) High-throughput detection showed that sulfated progesterone highly specifically activates the pruritus receptor hX4

[0093] Considering the increased intensity of ICP-related pruritus during pregnancy and the possible role of the pruritus receptor hX4 in this symptom, the inventors attempted to identify an endogenous metabolite that changes in parallel with the intensity of pruritus and activates hX4, i.e., a potential ICP-related pruritogen. First, through a literature survey, 28 endogenous metabolites were identified that increase during pregnancy and decrease postpartum ( Figure 1A) of which. More than 40% of the metabolites are steroids. Therefore, to increase the likelihood of identifying hits, we incorporated the commercial steroid library of Steraloids Inc. (https: / / www.steraloids.com / ) and obtained a total of 1490 potential candidate compounds to screen as possible pruritogens ( Figure 1 A) of which. Then, we performed high-throughput screening using a fluorescence imaging reader (FLIPR) calcium assay to determine whether any of these candidates could activate hX4 in a stable HEK293T cell line expressing hX4. The known hX4 ligand deoxycholic acid (DCA) was used as a positive control. After screening 1490 compounds, the inventors identified 22 metabolites that strongly activated hX4, with each metabolite inducing a calcium response at least 0.85-fold higher than that induced by DCA ( Figure 1 B) of which. These metabolites can be classified into five categories based on their structures, namely sulfated progesterone (i.e., sulfated progestin derivatives), bile acid derivatives, androgen derivatives, estrogen derivatives, and "others" ( Figure 1 B) of which. Progesterone is an essential hormone for pregnancy to term, and its levels gradually increase during pregnancy and then decrease at parturition. Given the similarity between this time course and the progression of ICP-related pruritus, the present invention focused on nine sulfated progesterones by further examining their ability to activate hX4 and induce ICP-related pruritus ( Figure 1 B) of which.

[0094] These nine sulfated progesterones can be further classified into three categories based on their non-sulfated precursors: pregnanolone derivatives, pregnanediol derivatives, and pregnenolone derivatives ( Figure 1 B) of which. To quantify the ability of compounds to activate hX4, we developed an α-index. The higher the α-index, the more effective the activation of hX4, and DCA was defined as having an α-index of 1.0. Among the nine sulfated progesterones tested, PM4S and PM3S were the most effective hX4 activators, while PregS was the least activator. Among these pruritogens, the levels of PM5S, PM3S, and PM3DiS were significantly elevated in ICP patients. Given the higher endogenous levels of PM5S and PM3S in ICP, the possible role of sulfated progesterones in ICP-related pruritus was further investigated using these two compounds.

[0095] 2) Sulfation is required for progesterone metabolites to activate hX4

[0096] This sulfated group is required for the above nine pruritogens to activate hX4 because their direct non-sulfated precursors cannot activate hX4 ( Figure 1in C and D). To determine why the sulfonic acid group is crucial for hX4 activation, we attempted to dock sulfonated progesterone with the reported hX4 structure using CB-Dock2. The docking results showed that PM5S and PM3S were located in the orthosteric pocket of hX4, and specific residues in the pocket interacted with these sulfonated progesterones. Some of these residues had molecular interactions with hX4 agonists, and mutations of most of these residues significantly reduced or eliminated the activation of hX4 by PM5S and PM3S. Moreover, the hX4 binding pocket formed a positively charged environment, indicating that the negatively charged sulfonic acid group might increase the interaction between the metabolite and the receptor. In addition, the potency of some drugs modified to contain negatively charged phosphate groups to activate hX4 was higher than that of the unmodified part. Based on the structural model, we predicted that the sulfonic acid group in sulfonated progesterone interacted with the positively charged arginine residue at position 82 (R82 in the hX4 binding pocket), and the same residue was reported to have a strong charge interaction with the phosphate group in the agonist. To further investigate this interaction, arginine was replaced with negatively charged aspartic acid at R82, which greatly reduced the potency of PM5S and PM3S to activate hX4. Therefore, based on the study of the structure-function relationship, it can be concluded that sulfonated progesterone activates the hX4 receptor by binding in the orthosteric binding pocket, and the sulfonamide group in the metabolite is necessary for this interaction. In addition, sulfonated progesterone selectively activates the hX4 receptor and cannot activate hMRGPRX1, hMRGPRX2, or hMRGPRX3 (other members of the human MRGPRX family) even at a high concentration of 100 μM ( Figure 1 in E).

[0097] 3) Sulfonated progesterone activates DRG neurons through hX4

[0098] Considering that the itching sensation is mainly conducted by DRG neurons, it was next investigated whether sulfonated progesterone could activate DRG neurons through hX4. hX4 is a primate-specific receptor, and no homologous gene has been found in rodents; therefore, for Ca 2+ imaging in vitro, we heterologously expressed hX4 in cultured rat DRG neurons by electroporation ( Figure 2 in A). We found that PM5S significantly increased the intracellular Ca 2+ in a dose-dependent manner in hX4-expressing (hX4 rat DRG neurons) cells, but had no effect on the intracellular Ca - in untransfected (hX4 2+ ) neurons ( Figure 2 in B). Similar results were obtained for PM3S ( Figure 2in C). Whether pretreatment of DRG neurons with hX4 antagonists can inhibit P5S-induced DRG neuron activation. The hX4 antagonist compound "1-55" (developed by Escient Pharmaceuticals company) can dose-dependently block PM5S from activating hX4. In a time-course experiment, we found that applying 10 μM PM5S to hX4 + DRG neurons induced a strong Ca 2+ signal, which was abolished in the presence of 1-55 ( Figure 2 in D). In addition, the non-sulfonated precursors of PM5S and PM3S failed to activate hX4 + DRG neurons, which is consistent with the concept that sulfonamide groups are required for hX4 activation.

[0099] 4) Sulfonated progesterone induces pruritic behavior by activating hX4

[0100] It was demonstrated that sulfonated progesterone activated DRG neurons expressing hX4, and then the neck-injection model was used to detect whether these metabolites could induce hX4-dependent pruritus in vivo ( Figure 2 in E). Statistical results confirmed that in wild-type (WT) rats, sulfonated progesterone could not induce pruritic behavior ( Figure 2 in H); as a positive control, 5-HT (5-hydroxytryptamine, a potent pruritogen) caused a strong pruritic response in WT rats. In contrast, PM3S was reported to induce scratching behavior in mice by activating TGR5. Therefore, rats expressing hX4 (i.e., hX4 humanized) provide a valuable tool for studying the role of sulfonated progesterone in pruritus.

[0101] hX4 humanized rats were generated by crossing MrgA::hX4-Cre rats with Rosa26::LSL-hX4 rats ( Figure 2 in E). The results showed that injecting PM5S or PM3S into the nape of hX4 humanized rats induced a strong sense of pruritus (quantified by the number of scratching times) in a dose-dependent manner ( Figure 2 in F), but had no effect on WT rats ( Figure 2 in H). In addition, the non-sulfonated precursor of PM5S could not activate hX4 in vitro and failed to induce pruritus in hX4 humanized rats; similarly, PregS, which had the lowest α index among all nine metabolites, also failed to cause a significant scratching response in humanized rats, which was consistent with its relatively low potency in activating hX4. As an additional control, in the cheek-injection model, we measured the pain and pruritus levels of rats by quantifying cheek wiping and scratching respectively. PM5S had no effect, while injecting capsaicin induced a strong pain response in WT rats. Figure 4in E). Then we found that when PM5S was injected into the cheek of hX4 humanized rats, it induced a strong scratching response but no pain response.

[0102] To verify whether the pruritic response induced by sulfated progesterone is indeed mediated by the hX4 receptor, we pretreated hX4 humanized rats with the hX4 antagonist 1-55 (or vehicle), and then determined the ability of PM5S and PM3S to induce pruritus. Compared with vehicle-treated rats, sulfated progesterone failed to cause pruritus in "1-55"-treated rats ( Figure 2 in G). Thus, pharmacologically inhibiting hX4 activation with an antagonist can prevent sulfated progesterone-induced pruritus, suggesting a potentially viable strategy for preventing ICP-related pruritus.

[0103] 5) Sulfated progesterone induces pruritus in humans

[0104] Next, we tested whether sulfated progesterone can act as a pruritogen and induce pruritus when injected subcutaneously in human subjects ( Figure 3 in A). We found that a single injection of PM5S rapidly induced a pruritic response, peaking within 10 minutes and then gradually declining within 30 minutes ( Figure 3 in B-D), similar to pruritus caused by human bile acids. In addition. PM5S induced pruritus in both female and male subjects, with similar potency in terms of time course or degree of pruritic response ( Figure 3 in E-G).

[0105] To test whether the typical pruritogen histamine is involved in sulfated progesterone-induced pruritus, we examined the allergic reactions of human subjects ( Figure 3 in A). We found that, unlike histamine, which causes a strong allergic reaction as shown by the "red and swollen" area at the injection site, PM5S did not trigger an allergic reaction ( Figure 3 in H). In addition, pretreatment with antihistamines failed to prevent PM5S-induced pruritus ( Figure 3 in I-J). These results indicate that sulfated progesterone-induced pruritus is independent of the histaminergic system. To further examine in detail the role of the hX4 pathway in sulfated progesterone-induced pruritus, we tested two metabolites with weak (PregS) or absent (3β5α-diolS) hX4 activation effects. Neither of these two metabolites caused significant pruritus in human subjects, similar to the results we obtained in hX4 humanized rats, and these findings suggest that sulfated progesterone most likely induces rapid pruritic sensations in humans by activating hX4.

[0106] 6) Sulfated progesterone levels are correlated with the intensity of ICP pruritus

[0107] Clinically, we examined whether sulfated progesterone levels were associated with the presence and / or intensity of ICP-related pruritus. We collected plasma samples from ICP patients and healthy pregnant women at each trimester and postpartum, and measured the intensity of pruritus in the third trimester of pregnancy ( Figure 4 A in Figure 4 ). We separated and quantified various sulfated progesterones in plasma samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) ( Figure 4 B in Figure 4 ). Since it was difficult to completely separate chiral isomers, such as PM4S, PM5S, and PM7S isomers, we calculated the total plasma concentration of PM4S, PM5S, and PM7S isomers, hereinafter referred to as "PMS" ( Figure 4 B in Figure 4 ). Similarly, "PM3S+" represents the sum of two chiral isomers, SPM3s and 3β5α-diolS. We also measured the plasma concentrations of PregS and Pregl7olS; we were unable to measure PM3DiS (full name: 5β-pregnan-3α, 20α-diol-3, 20-disulfate) because there was no standard available on the market. We found that in ICP patients, except for PregS, almost all measured endogenous sulfated progesterone levels gradually increased during pregnancy, peaked in the third trimester, and then decreased after delivery ( 2+ B in Figure 4 ), which was consistent with the time course of ICP-related pruritus intensity. Importantly, in women without ICP-related pruritus (

[0108] 7) Diagnosis of ICP by sulfated progesterone levels

[0109] Figure 4 B in Figure 4 ), the levels of sulfated progesterone showed minimal or slight changes throughout pregnancy. Next, we examined whether a combination of sulfated progesterone at pathophysiological relevant levels was sufficient to activate hX4. Based on our quantification results ( Figure 4 B in 2+ ), we prepared sulfated progesterone mixtures similar to the plasma sulfated progesterone levels of healthy pregnant women in the third trimester ("healthy mixture") or ICP patients ("ICP mixture"), respectively. We found that the ICP mixture, but not the healthy mixture, induced a significant Ca 2+ signal in HEK293T cells expressing hX4. Notably, in the third trimester of pregnancy, we found a strong correlation between sulfated progesterone levels and the intensity of pruritus reported by ICP patients, and PM3S+ showed the highest positive correlation among the five sulfated progesterones ( Figure 4 C in

[0108] 7) Diagnosis of ICP by sulfated progesterone levels

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[0108] 7) Diagnosis of ICP by sulfated progesterone levels <000027Currently, the diagnostic criteria for ICP are insufficient and lack specificity, especially considering the confounding effects of other factors such as other skin diseases, liver diseases, and dietary changes. Therefore, next, we investigated whether sulfated progesterone levels could be a reliable diagnostic biomarker for ICP. Since women usually develop ICP in the late pregnancy, we collected plasma samples from 38 ICP patients and 68 women with uncomplicated term pregnancies ("healthy controls") in the late stage as the discovery cohort ( Figure 5 A in Figure 5 ). We found a significant difference in the individual sulfated progesterone levels between the ICP group and the healthy control group ( Figure 5 B). In addition, PCA and t-SNE analyses were able to separate ICP patients and healthy control individuals into two groups, and then the receiver operating characteristic (ROC) curves for each sulfated progesterone were calculated to distinguish ICP patients from healthy controls. The area under the curve (AUC) of PM3S+ was the highest, at 0.94 ( Figure 5 C and E), showing improvement compared with TBA (AUC - 0.92), the most commonly used biomarker for diagnosing ICP. Overall, all five sulfated progesterones were able to diagnose ICP ( Figure 5 C and E). To improve the diagnostic algorithm, we examined whether the combination of five sulfated progesterones could improve the diagnostic performance. Therefore, we generated a combined model (here called the "PS model") using a generalized linear model (GLM) with ten-fold cross-validation ( Figure 5 A and D) and found that the combined model had robust performance, with an AUC of 0.97, an accuracy of 0.93, a sensitivity of 0.89, and a specificity of 0.95 (using the optimal cut-off value of 0.18), which was a great improvement over individual sulfated progesterones and classical biomarkers used for diagnosing ICP ( Figure 5 E and F). Overall, sulfated progesterone levels can accurately identify ICP patients in the late pregnancy.

[0110] To validate the diagnostic performance of sulfated progesterone, we collected external cohort data from 37 ICP patients and 38 healthy control individuals in an independent medical center ( Figure 5 A). We again found that the endogenous levels of various sulfated progesterones in the ICP group were significantly higher than those in the healthy control group. In addition, similar to the results we obtained in the discovery cohort, we found that a single type of sulfated progesterone was able to distinguish ICP patients from healthy control individuals. Importantly, applying the PS model in this independent validation cohort showed high diagnostic performance, with an AUC of 0.95 ( Figure 5 G and H). Based on these results, we believe that sulfated progesterone levels can be a reliable biomarker for diagnosing ICP.

[0111] It should be understood that the systems, devices and methods described in the present invention can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division. In actual implementation, there may be other division methods. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the couplings or direct couplings or communication connections shown or discussed with each other can be indirect couplings or communication connections through some interfaces, devices or units, and can be in electrical, mechanical or other forms. The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they can be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0112] In addition, the functional units in the various embodiments of the present invention can be integrated in a processing unit, or each unit can exist physically alone, or two or more units can be integrated in one unit. The above integrated units can be implemented in the form of hardware or in the form of software functional units.

[0113] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A use of a sulfonated progesterone metabolite in the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that: The sulfonated progesterone metabolites include one or more of Preg17olS, PM6S, PM3S+, PregS, and PMS; Preferably, the sulfonated progesterone metabolite is a combination of Preg17olS, PM6S, PM3S+, PregS, and PMS; Preferably, the PMS comprises a combination of three isomers: PM4S, PM5S and PM7S; Preferably, the PM3S+ comprises a combination of two isomers of PM3S and 3β5α-diolS.

2. Use of a reagent for detecting the level of the sulfonated progesterone metabolite described in claim 1 in a sample in the preparation of a product for diagnosing intrahepatic cholestasis of pregnancy; Preferably, the product includes a kit, a chip, a test paper, a high-throughput sequencing, a system, equipment, or a device.

3. The use according to claim 2, wherein the reagent detects the level of metabolite markers in the sample by one or more of targeted or non-targeted nuclear magnetic resonance, chromatography, spectroscopy, mass spectrometry, and chromatography-mass spectrometry; Preferably, the reagent is for detecting the level of metabolite markers in the sample by chromatography-mass spectrometry; Preferably, the sample is selected from blood, serum, and plasma; Preferably, the sample is plasma; Preferably, the product further comprises reagents for processing the sample.

4. A product for diagnosing intrahepatic cholestasis of pregnancy, characterized in that: The product includes a reagent for detecting the level of the sulfonated progesterone metabolite of claim 1 in a sample.

5. Use of the sulfonated progesterone metabolite according to claim 1 in constructing a diagnostic model for intrahepatic cholestasis of pregnancy.

6. A method for constructing a diagnostic model for intrahepatic cholestasis of pregnancy, characterized in that: The steps of the method include obtaining data on the level of the sulfonated progesterone metabolite of claim 1 and clinical characteristic data in a sample, inputting the data into a machine learning algorithm to construct a diagnostic model; Preferably, the diagnostic model obtains the classification result by the following criteria: when the level of one or more of the sulfonated progesterone metabolites described in claim 1 is higher than the optimal cutoff value, a classification result is obtained that the subject suffers from intrahepatic cholestasis of pregnancy or is at risk of suffering from intrahepatic cholestasis of pregnancy; if the level of the sulfonated progesterone metabolite described in claim 1 is lower than the optimal cutoff value, a classification result that the subject does not suffer from intrahepatic cholestasis of pregnancy is obtained; Preferably, the clinical characteristic data include whether the subject suffers from intrahepatic cholestasis of pregnancy and the subject's pregnancy stage.

7. The method according to claim 6, characterized in that The machine learning algorithm includes an algorithm model developed using various development tools; Preferably, the development tools include TensorFlow, Scikit Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, Spell; Preferably, the algorithm model includes a generalized linear model, principal component analysis, logistic regression analysis, LASSO regression analysis, nearest neighbor analysis, support vector machine, neural network model, and random forest model.

8. A diagnostic system for intrahepatic cholestasis of pregnancy, characterized in that: The system comprises a data classification unit, which is used to substitute the sulfonated progesterone metabolite level data into the diagnostic model constructed according to the method described in any one of claims 6-7 to obtain a classification result of whether the sample suffers from intrahepatic cholestasis of pregnancy or whether there is a risk of suffering from intrahepatic cholestasis of pregnancy; Preferably, the system further comprises a data acquisition unit, the data acquisition unit being used to acquire the level data of the sulfonated progesterone metabolite according to claim 1 in the sample; Preferably, the system further comprises an output unit, and the output unit is used to output the classification result.

9. A diagnostic device for intrahepatic cholestasis of pregnancy, characterized in that: The diagnostic device includes a memory and a processor; The memory is used to store program instructions; The processor is used to execute program instructions, and when the program instructions are executed, it is used to perform the following operations: obtaining the sulfonated progesterone metabolite level data of claim 1 in the sample, inputting the sulfonated progesterone metabolite level data into a diagnostic model constructed based on the method described in any one of claims 6-7, and obtaining a classification result of whether the sample suffers from intrahepatic cholestasis of pregnancy or whether there is a risk of suffering from intrahepatic cholestasis of pregnancy; Preferably, the sample is selected from blood, serum, and plasma; Preferably, the sample is plasma.

10. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by the processor, the following method is implemented: obtaining the sulfonated progesterone metabolite level data of claim 1 in the sample, inputting the sulfonated progesterone metabolite level data into a diagnostic model constructed based on the method described in any one of claims 6-7, and obtaining a classification result of whether the sample suffers from intrahepatic cholestasis of pregnancy or whether there is a risk of suffering from intrahepatic cholestasis of pregnancy; Preferably, the sample is selected from blood, serum, and plasma; Preferably, the sample is plasma.

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