Application of biomarker in preparation of product for predicting risk of recurrent abortion

Abstract

The invention discloses application of a biomarker in preparation of a product for predicting the risk of recurrent abortion, and creatively finds that CSN2, PLIN1 and / or CHRDL2 proteins can be used as an effective biomarker for diagnosing the risk of recurrent abortion due to unknown reasons or predicting the risk of recurrent abortion due to unknown reasons for the first time. The biomarker has excellent diagnostic efficiency for diagnosing the unexplained recurrent abortion or predicting the risk of the unexplained recurrent abortion, is high in accuracy, sensitivity and specificity, provides a brand new thought and strategy for research and development of diagnostic products related to the unexplained recurrent abortion, and has wide application prospects. The method has a wide application prospect and important conversion significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of biomarkers in the preparation of products for predicting the risk of recurrent miscarriage. Background Technology

[0002] Unexplained recurrent pregnancy loss (URPL) is the most challenging subtype of recurrent pregnancy loss (RPL), accounting for approximately 30%-50% of all RPL cases. While routine clinical examinations, including chromosome analysis, uterine anatomy, endocrine testing, and immunological studies, are used to investigate the cause, many patients remain in a state of uncertainty due to a lack of clear pathogenic evidence. This leads to reliance on empirical interventions (such as luteal support and low-molecular-weight heparin anticoagulation), increasing the waste of medical resources and potentially impacting pregnancy outcomes due to misdirected interventions. Developing biomarkers for URPL can uncover potential pathogenic mechanisms at the molecular level (such as immune imbalance, vascular endothelial dysfunction, and embryo-maternal interface dysregulation), providing quantifiable diagnostic evidence for unexplained causes, filling gaps in existing screening systems, and shifting clinical diagnosis from empirical judgment to precise localization.

[0003] Currently, treatment for URPL lacks clear etiological indicators, leading to the prevalence of broad-spectrum interventions in clinical practice. Some patients may experience repeated treatment failures due to a lack of targeted treatment for the core pathogenic factors, while others may suffer unnecessary drug side effects (such as the infection risk from immunosuppressants and the bleeding risk from anticoagulants). The development of diagnostic biomarkers can serve as a treatment navigation tool—by detecting biomarker levels, clinicians can accurately determine the specific molecular cause of a patient's URPL (e.g., whether it is caused by abnormalities in specific cytokines or coagulation disorders), thereby developing individualized treatment plans (e.g., targeted regulation of the immune microenvironment, precise improvement of endometrial receptivity). Simultaneously, biomarkers can also serve as dynamic monitoring indicators of treatment effectiveness, allowing for real-time assessment of intervention efficacy, timely adjustments to the treatment plan, avoiding ineffective treatment that prolongs the patient's fertility period, ultimately increasing live birth rates and reducing the dual physiological and psychological harm caused by recurrent miscarriages.

[0004] Recurrent miscarriages not only cause irreversible damage to women's reproductive health (such as thinning of the endometrium and increased risk of intrauterine adhesions), but also easily trigger psychological problems such as anxiety and depression. Because the cause of urinary tract infection (URPL) is unknown, the psychological stress on patients is even more significant. The development of diagnostic biomarkers can shift the treatment of URPL from post-hoc intervention to pre-hoc prevention—by detecting biomarker levels during the preconception stage or early pregnancy, high-risk groups can be identified in advance (such as patients with abnormal biomarkers indicating a high risk of embryo implantation failure), and targeted preventive measures can be taken (such as optimizing the uterine microenvironment in advance and intervening in potential immune abnormalities), reducing the probability of miscarriage from the source. Furthermore, risk stratification based on biomarkers can enable precise allocation of medical resources, concentrating key intervention resources on high-risk groups while providing scientific pregnancy guidance to low-risk patients, reducing the burden of over-medicalization, and building a more efficient URPL prevention and treatment system. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the application of biomarkers in the preparation of products for predicting the risk of recurrent miscarriage.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: A first aspect of the invention provides the use of reagents for detecting the expression levels of biomarkers CSN2, PLIN1 and / or CHRDL2 in samples in the preparation of products for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

[0007] In this invention, CSN2 comprises the CSN2 gene and the CSN2 protein. The CSN2 gene is transcribed and translated into the CSN2 protein product in the research subjects (subjects). The CSN2 gene has a Gene ID of 1447, and its full name is casein beta [Homo sapiens (human)]. Detailed information about the gene can be obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) based on the aforementioned Gene ID.

[0008] In this invention, PLIN1 comprises the PLIN1 gene and the PLIN1 protein. The PLIN1 gene is transcribed and translated into the PLIN1 protein product in the research subject (subject). The PLIN1 gene has a Gene ID of 5346, and its full name is perilipin 1 [Homo sapiens (human)]. Detailed information about the gene can be obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) based on the aforementioned Gene ID.

[0009] In this invention, CHRDL2 comprises the CHRDL2 gene and the CHRDL2 protein. The CHRDL2 gene is transcribed and translated into the CHRDL2 protein product in the research subjects (subjects). The CHRDL2 gene has a Gene ID of 25884, short for chordin-like 2 [Homo sapiens (human)], and its detailed information can be obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) based on the aforementioned Gene ID.

[0010] In this invention, the biomarker refers to a molecular indicator with specific biological characteristics, biochemical features, or other properties, which can be used to determine the presence or absence of a specific disease or condition and / or the severity of a specific disease or condition. In a specific embodiment of this invention, the biomarker is CSN2, PLIN1, and / or CHRDL2.

[0011] Furthermore, the reagent is: Reagents for detecting the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in samples; Reagents for detecting the DNA levels of CSN2, PLIN1, and / or CHRDL2 in a sample; or A reagent for detecting the RNA levels of CSN2, PLIN1, and / or CHRDL2 in a sample.

[0012] Furthermore, the reagents used to detect the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample are antibodies, aptamers, agglutinants, and / or small molecule compounds that specifically bind to proteins encoded by CSN2, PLIN1, and / or CHRDL2.

[0013] Furthermore, the reagents used to detect the DNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample are reagents for detecting the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, and / or DNA hydroxymethylation level of CSN2, PLIN1, and / or CHRDL2 in the sample. Optionally, the reagent used to detect the RNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample is a reagent used to detect the expression levels of mRNA, lncRNA, and / or miRNA of CSN2, PLIN1, and / or CHRDL2 in the sample.

[0014] In some implementations, the reagents used to detect the DNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample include reagents that detect the levels using sequencing technology.

[0015] In some embodiments, the reagents used to detect the RNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample include primers that specifically amplify CSN2, PLIN1, and / or CHRDL2 and / or probes that specifically recognize CSN2, PLIN1, and / or CHRDL2.

[0016] In some embodiments, the antibody refers to a specific immunoglobulin against an antigen. The antibody in this invention refers to an antibody that specifically binds to the CSN2, PLIN1, and / or CHRDL2 proteins described in this invention, and can be manufactured according to conventional methods in the art. The antibody may take the form of a polyclonal or monoclonal antibody, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing antigen binding, provided that the antibody exhibits the desired biological binding activity.

[0017] In a specific embodiment of the present invention, the reagent used to detect the expression levels of biomarkers CSN2, PLIN1 and / or CHRDL2 in the sample is an antibody against CSN2, an antibody against PLIN1 and / or an antibody against CHRDL2.

[0018] In a specific embodiment of the present invention, the anti-CSN2 antibody, the anti-PLIN1 antibody, and the anti-CHRDL2 antibody are respectively derived from the following kits: CSN2 enzyme immunoassay kit (human casein β (CSN2) ELISA quantitative detection kit, catalog number YJ624879, specification 96T / 48T, used for quantitative detection of human CSN2 in cell culture supernatant, serum, and plasma, purchased from Shanghai Yuanju Biotechnology Center); PLIN1 enzyme immunoassay kit (human perilipid droplet 1 (PLIN1) ELISA quantitative detection kit, catalog number YJ523686, specification 96T / 48T, used for quantitative detection of human PLIN1 in cell culture supernatant, serum, and plasma, purchased from Shanghai Yuanju Biotechnology Center); and CHRDL2 enzyme immunoassay kit (human tendinoid protein 2). (CHRDL2) ELISA quantitative detection kit, catalog number YJ387457, specification 96T / 48T, is used for quantitative detection of human CHRDL2 in cell culture supernatant, serum, and plasma, purchased from Shanghai Yuanju Biotechnology Center.

[0019] In some implementations, the primers refer to 7-50 nucleic acid sequences capable of forming base pairs complementary to the template strand and serving as a starting point for template strand replication. Primers are typically synthesized, but naturally occurring nucleic acids can also be used. The primer sequence does not necessarily need to be identical to the template sequence, as long as it is sufficiently complementary to hybridize with the template.

[0020] In some embodiments, the probe refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to hundreds of bases in length, which can specifically bind to mRNA and can determine the presence of a specific mRNA through labeling. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0021] In some implementations, the reagents used to detect the DNA expression levels of the biomarkers by the sequencing technology refer to any reagents capable of detecting the DNA expression levels, DNA methylation levels, DNA acetylation levels, DNA phosphorylation levels, and / or DNA hydroxymethylation levels of CSN2, PLIN1, and / or CHRDL2 in a sample. Exemplarily, the sequencing technologies include, but are not limited to: Sanger sequencing technology, Illumina sequencing technology, Ion Torrent sequencing technology, PacBio sequencing technology, and Oxford Nanopore long-read sequencing technology.

[0022] Furthermore, the samples are serum samples, plasma samples, blood samples, tissue samples, exosome samples, and / or cell samples.

[0023] Furthermore, the product is a test kit, a test chip, or a test strip; Optionally, the detection kit is a detection kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker; Optionally, the detection kit detects the biomarker via an antigen-antibody reaction; Optionally, the detection kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit; Optionally, the detection kit includes reagents for detecting the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample.

[0024] In some embodiments, the sample refers to a composition obtained from or derived from the target subject, which contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics.

[0025] In some embodiments, the sample may be a tissue sample, blood, or other fluid sample of biological origin, such as a biopsy tissue sample or tissue culture or cells derived therefrom, obtained from a subject (the subject may be a human or a non-human mammal, preferably a human). The source of the tissue sample may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma. The term "sample" includes biological samples that have been processed in any way after their acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. In this invention, there are no particular limitations on the sample, and applications related to the detection of CSN2, PLIN1, and / or CHRDL2 in any sample for the purpose of diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage fall within the scope of this invention.

[0026] In some embodiments, the samples described in this invention include, but are not limited to: serum, tissue, blood, tissue-derived cells, blood-derived cells, plasma, lymph, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, synovial fluid, pleural effusion, peritoneal effusion, serous cavity effusion, lymph, cerebrospinal fluid, uterine fluid, digestive juices, bile, organs, and any combination thereof.

[0027] In this invention, verification in real clinical samples collected by this invention revealed that the biomarkers CSN2, PLIN1, and / or CHRDL2 have good diagnostic efficacy for unexplained recurrent miscarriage. The diagnostic efficacy is verified by receiver operating characteristic (ROC) curves. The area under the curve (AUC) is the area under the ROC curve known to those skilled in the art. Measuring the area under the curve (AUC) helps to compare the accuracy of classifiers across the overall data range.

[0028] A classifier with a larger area under the curve (AUC) has a greater ability to accurately classify unknowns between two groups of interest (e.g., disease samples, healthy control samples). Receiver operating characteristic (ROC) curves have performance in distinguishing between the two groups for graphical representation of specific characteristics (e.g., any items of biomarkers and / or additional biomedical information described in this invention). Typically, the aforementioned characteristic data are sorted in ascending order across the entire population (e.g., patient group and healthy control group) based on a single characteristic value. Then, for each value of the aforementioned characteristic, the true positive rate and false positive rate are calculated for the data. The true positive rate is determined by dividing the number of cases above the value for its characteristic by the total number of cases. The false positive rate is determined by dividing the number of control groups above the value for its characteristic by the total number of control groups. Although this definition refers to the case where the characteristic is higher in the patient group than in the control group, it also applies to the case where the characteristic is lower in the patient group than in the control group (in which case the number of samples below the value for the aforementioned characteristic can be calculated).

[0029] A second aspect of the invention provides a product for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

[0030] Furthermore, the product comprises the reagents described in the first aspect of the present invention for detecting the expression levels of biomarkers CSN2, PLIN1, and / or CHRDL2 in a sample.

[0031] Furthermore, the product is a test kit, a test chip, or a test strip; Optionally, the detection kit is a detection kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker; Optionally, the detection kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescence immunoassay kit.

[0032] In some implementations, the ELISA test kit is used to diagnose or predict unexplained recurrent miscarriage (URPL), targeting the biomarkers CSN2, PLIN1, and / or CHRDL2, and is designed using a double-antibody sandwich method.

[0033] In some implementations, the ELISA kit comprises an enzyme-labeled plate pre-coated with capture antibodies targeting the target biomarker, a biotin-labeled detection antibody, streptavidin-conjugated horseradish peroxidase (SA-HRP), a substrate chromogenic solution (such as TMB), a stop solution, and accompanying diluents and washing solutions. During detection, a pre-treated sample (such as serum or plasma) is added to the wells of the ELISA kit, allowing the target biomarker in the sample to bind to the capture antibody. After washing to remove unbound components, a biotinylated detection antibody is added to form a "capture antibody-biomarker-detection antibody" complex. The enzyme labeling is then introduced through the specific binding of SA-HRP to biotin. Finally, after substrate chromogenic development (blue) and stop solution termination (turning to yellow), the absorbance is measured at 450 nm using an ELISA reader. The expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample are calculated using a standard curve, providing a quantitative basis for the diagnosis or risk prediction of URPL. Its advantages include ease of operation, low cost, high specificity, and suitability for batch sample testing, meeting the needs of routine clinical testing scenarios.

[0034] In some implementations, the fluorescent immunoassay kit uses CSN2, PLIN1, and / or CHRDL2 as core detection markers to achieve quantitative detection of target proteins based on fluorescent immunoassay technology, which is suitable for the diagnosis and risk prediction of URPL.

[0035] In some implementations, the main components of the fluorescent immunoassay kit include a reaction carrier coated with capture antibodies (such as microplates or magnetic beads), a detection antibody labeled with fluorescein (such as FITC or Cy3), fluorescent calibrators, washing buffer, and sample diluent. In the detection process, the target biomarker in the sample first binds to the capture antibody on the carrier. After washing, the fluorescently labeled detection antibody is added to form a double-antibody complex. After washing to remove the free fluorescent antibody, a fluorescence detector is used to excite the fluorescein to generate a fluorescence signal at a specific wavelength (e.g., FITC excitation wavelength 488 nm, emission wavelength 525 nm). The fluorescence intensity is positively correlated with the concentration of the target biomarker in the sample. The advantages of this kit are higher sensitivity than traditional ELISA, shorter detection time, better fluorescence signal stability, and lower background interference. It can more accurately detect low-concentration biomarkers, and is particularly suitable for detecting URPL clinical samples with low expression levels of CSN2, PLIN1, or CHRDL2, providing more sensitive technical support for early risk prediction.

[0036] In some implementations, the chemiluminescent immunoassay kit uses CSN2, PLIN1, and / or CHRDL2 as detection targets, and achieves quantitative detection by relying on the high sensitivity of chemiluminescent immunoassay technology.

[0037] In some implementations, the key components of the chemiluminescent immunoassay kit include magnetic beads (or microplates) coated with capture antibodies, acrid ester (or luminol-based) labeled detection antibodies, a luminescent substrate (such as an H2O2-NaOH system), calibrators, and a matching buffer. During detection, the target biomarker in the sample is first captured by the magnetic beads (or microplate), then combines with the chemiluminescent labeled detection antibody to form an immune complex. After washing, the luminescent substrate is added, and the biomarker reacts chemically with the substrate to release photons. The luminescence intensity (RLU) is recorded by a chemiluminescence detector, and the concentration of the target biomarker in the sample is calculated based on a standard curve established using the calibrators. This kit combines high sensitivity (detection limit down to the pg level) with a wide linear range, effectively distinguishing the expression differences of CSN2, PLIN1, and CHRDL2 between URPL patients and healthy individuals. Furthermore, the detection process is highly automated and the results are reproducible, reducing human error. It is suitable for the efficient and accurate detection needs of clinical laboratories, providing reliable quantitative data support for the early diagnosis and risk stratification of URPL.

[0038] In some implementations, the detection chip uses biomarkers CSN2, PLIN1, and / or CHRDL2 as core detection targets, achieving simultaneous detection of multiple biomarkers based on protein chip technology. The chip carrier is typically a glass slide, silicon wafer, or polymer membrane, with its surface chemically modified (e.g., activated by amino or carboxyl groups) to immobilize specific capture antibodies against the target biomarkers, forming an ordered array of detection dots. Positive controls, negative controls, and internal control dots are also included to ensure the reliability of the detection results. During detection, a pre-treated sample (e.g., diluted serum or plasma) is added to the chip reaction area, where the target biomarker specifically binds to the corresponding capture antibody. After incubation and washing to remove unbound components, a fluorescein- or enzyme-labeled detection antibody is added. The signal intensity of each dot is acquired using a signal amplification and recognition system (e.g., a fluorescence scanner or chemiluminescence imager), and a quantitative model established using standards is used to calculate the expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample.

[0039] In some implementations, the test strip targets CSN2, PLIN1, and / or CHRDL2 and is designed based on colloidal gold immunochromatography (or latex-enhanced immunochromatography), offering the advantages of speed, convenience, and the absence of complex instruments. The test strip structure mainly includes a sample pad, a conjugate pad, a reaction membrane, an absorbent pad, and a base plate. The conjugate pad is pre-coated with detection antibodies labeled with colloidal gold (or colored latex particles). The reaction membrane features a detection line (T line, immobilized with another capture antibody targeting the target biomarker) and a control line (C line, immobilized with a secondary antibody against the detection antibody). During testing, pretreated samples (such as serum or plasma) are dropped onto the sample pad. The sample diffuses towards the absorbent pad via capillary action, binding with labeled antibodies on the conjugate pad to form a "labeled antibody-marker" complex. When the complex reaches the T-line of the reaction membrane, it binds with capture antibodies to form a "labeled antibody-marker-capture antibody" sandwich structure. The aggregated labeler causes the T-line to develop color (e.g., red or purple). Unbound labeled antibodies continue to diffuse to the C-line, binding with secondary antibodies to develop color (serving as a quality control for detection validity). The expression level of the target marker in the sample can be quickly determined by the color intensity of the T-line (which can be judged visually or quantitatively using a portable reader).

[0040] A third aspect of the present invention provides a system or apparatus for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

[0041] Furthermore, the system or device includes a processor, an input module, and an output module; The input module is used to input the expression levels of CSN2, PLIN1, and / or CHRDL2 in the subject's sample; the processor is used to perform logical operations on the input information using bioinformatics methods, and the processor contains a computer-readable medium storing instructions, which, when executed by the processor, perform algorithms on the input CSN2, PLIN1, and / or CHRDL2 expression levels; the output module is used to output the result of whether the subject is a patient with unexplained recurrent miscarriage or the risk of having unexplained recurrent miscarriage.

[0042] In this invention, the system or apparatus is a method for distinguishing different components, elements, parts, sections, or assemblies at different levels. However, if other terms can achieve the same purpose, they can be replaced by other expressions. Those skilled in the art will know that this invention can be implemented as a device, method, or computer program product. Therefore, the disclosure of this invention can be specifically implemented in the following forms: entirely hardware, entirely software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. Furthermore, in some specific embodiments, this invention can also be implemented as a computer program product in one or more computer-readable media containing computer-readable program code.

[0043] A fourth aspect of the invention provides the use of reagents for detecting the expression levels of biomarkers CSN2, PLIN1, and / or CHRDL2 in a sample in the preparation of a system or apparatus for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

[0044] Furthermore, the present invention also provides a method for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage, the method comprising the following steps: detecting the expression levels of CSN2, PLIN1 and / or CHRDL2 in a sample from a subject, and diagnosing whether the subject is a patient with unexplained recurrent miscarriage or has the risk of unexplained recurrent miscarriage based on the detected expression levels of CSN2, PLIN1 and / or CHRDL2.

[0045] In this invention, any method that uses the biomarkers CSN2, PLIN1, and / or CHRDL2 (or in combination with other indicators) to diagnose unexplained recurrent miscarriage or predict the risk of unexplained recurrent miscarriage will fall within the protection scope of this invention. It is not limited to the specific method of using the biomarkers CSN2, PLIN1, and / or CHRDL2 (or in combination with other indicators). As long as the purpose of diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage can be achieved or substantially achieved, it will fall within the protection scope of this invention.

[0046] In some implementations, the subjects include various animal groups, including both human individuals and non-human animals. The specific types of non-human animals can be divided into vertebrates and non-vertebrates: Vertebrates include mammals and non-mammals. Mammals include non-human primates (especially higher primates closely related to humans), livestock such as sheep, pigs, and cattle, pets such as dogs and cats, and rodents commonly used in experimental research (such as mice and rats), guinea pigs, rabbits, and other mammals such as goats; non-mammals include birds such as chickens, amphibians (such as frogs and toads), reptiles (such as lizards and snakes), etc. In a specific implementation of this invention, from a practical point of view, the subjects are preferably humans.

[0047] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention is the first to creatively discover that CSN2, PLIN1, and / or CHRDL2 proteins can serve as effective biomarkers for diagnosing or predicting the risk of unexplained recurrent miscarriage. Clinical validation through exploratory and validation cohorts has shown that these biomarkers have excellent diagnostic efficacy for diagnosing or predicting the risk of unexplained recurrent miscarriage, with high accuracy, sensitivity, and specificity. This invention provides a key tool for the early diagnosis and accurate assessment of unexplained recurrent miscarriage, and offers novel ideas and strategies for the research and development of diagnostic products related to unexplained recurrent miscarriage, demonstrating broad application prospects and significant translational value. Attached Figure Description

[0048] Figure 1 The volcano plot illustrates the analysis of two groups of differentially expressed proteins. Figure 2 In the exploration cohort, the heatmap shows 9 significantly upregulated proteins and 25 significantly downregulated proteins; Figure 3 ROC curves of differentially expressed proteins in the exploration cohort; Figure 4 : The differential expression results of three proteins, CSN2, PLIN1 and CHRDL2, in the validation cohort; Figure 5 ROC curves of three proteins, CSN2, PLIN1, and CHRDL2, in the validation queue. Detailed Implementation

[0049] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0050] Example 1: Screening and validation of diagnostic biomarkers for unexplained recurrent miscarriage (URPL) 1. Sample Source (1) Exploration queue This study included 40 healthy controls (HC) and 40 patients with unexplained recurrent miscarriage (URPL) (serum sample from healthy controls: serum sample from patients with unexplained recurrent miscarriage (URPL) = 40:40). Strict screening was performed according to the following inclusion and exclusion criteria: The patients with unexplained recurrent miscarriage (URPL) and healthy controls included in this study were all outpatients and inpatients of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, from December 2023 to December 2024.

[0051] 1) Inclusion criteria URPL patients: Married women aged 18-45 who have regular sexual activity and have not used contraception; Meeting the diagnostic criteria for recurrent miscarriage: two or more consecutive spontaneous abortions, with no clear cause found through clinical examination (such as chromosomal abnormalities, anatomical abnormalities, infectious factors, autoimmune diseases, etc.).

[0052] Healthy control group: Has at least one history of normal full-term pregnancy, with no adverse pregnancy outcomes such as miscarriage or stillbirth; Age and sex were matched to the URPL group (±3 years); During the same period, a health check-up was conducted to rule out reproductive system diseases and autoimmune diseases.

[0053] 2) Exclusion criteria ① There is a clear cause for recurrent miscarriage, including but not limited to: Chromosomal abnormalities: Abnormal karyotypes in either the couple or in the miscarried embryo; Anatomical abnormalities: uterine malformations (such as septate uterus, bicornuate uterus), intrauterine adhesions, uterine fibroids (submucosal or intramural fibroids that affect the shape of the uterine cavity), cervical insufficiency, etc. Infectious factors: Reproductive tract infections (such as chlamydia, mycoplasma, toxoplasmosis, cytomegalovirus, etc.); Autoimmune diseases: antiphospholipid syndrome, systemic lupus erythematosus, and other definitively diagnosed autoimmune diseases.

[0054] ② The patient has serious dysfunction of vital organs such as the heart, liver, and kidneys, or diseases such as malignant tumors or mental illnesses that may affect the research results.

[0055] ③Having bad lifestyle habits such as alcoholism or drug abuse, or being exposed to toxic and harmful substances for a long time (such as radioactive substances, chemical toxins, etc.).

[0056] (2) Verification queue To further validate the diagnostic efficacy of the screened biomarkers, this invention further collected data from 40 healthy controls (HC) and 40 patients with unexplained recurrent miscarriage (URPL) (serum samples from healthy controls (HC): serum samples from patients with unexplained recurrent miscarriage (URPL) = 40:40). Strict screening was performed using the inclusion and exclusion criteria described above. Both patients and healthy controls were from the outpatient and inpatient populations of Beijing Obstetrics and Gynecology Hospital, affiliated with Capital Medical University.

[0057] 2. Experimental Methods Data Independent Acquisition (DIA) label-free quantitative proteomics analysis is a newly developed and widely used quantitative technique in differential proteomics. It divides the entire mass spectrometry scan range into several windows, sequentially fragmenting all ions within each window and acquiring all fragment ion information. The experimental procedure is as follows: First, determine the detection protocol and collect samples; after sample collection, perform preprocessing, including sample extraction and protein detection; after protein digestion with enzymes, perform DIA mass spectrometry detection using liquid chromatography-mass spectrometry (LC-MS / MS); finally, use software for qualitative and quantitative proteomic analysis. Specific experimental methods are as follows: (1) Sample preparation Serum samples were processed using a deep blood proteomics kit (PH035, Beijing PreciseHealth Biotechnology Co., Ltd). The steps were as follows: 50 μL of each sample was added to 150 μL of beads and incubated at 37°C with shaking at 1500 rpm for 1 h. The supernatant was then magnetically discarded. 200 μL of wash buffer (10 mM Tris (pH = 7.4), 150 mM KCl, 0.05% CHAPS) was added to each sample, and the mixture was shaken at room temperature for 5 min. The supernatant was then magnetically discarded, and the washing was repeated 3 times. 50 μL of 100 mM TEAB, 0.1 μg Lysis-C, and 0.5 μg Trypsin were added, and the samples were digested at 37°C for 2 h. After desalting and lyophilization, the samples were ready for analysis.

[0058] (2) Mass spectrometry detection The chromatographic mobile phase A consisted of 0.1% FA, and phase B consisted of 80% ACN and 0.1% FA. The lyophilized peptides were thoroughly dissolved in solution A (0.1% FA) and centrifuged at 15000 g for 15 min. Concentration was determined using a nanodrop one (Thermo Scientific, USA). 500 ng of each sample was added to an internal tube and placed in an autosampler. The samples were loaded onto a self-made analytical column (1.9 μm particle size, 75 μm capillary inner diameter, 20 cm length) using an EASY-nLC 1200 liquid chromatograph (Thermo Scientific, USA) at 400 bar pressure with 12 μL of solution A. Gradient elution was then performed. Liquid chromatography was performed at a flow rate of 300 nL / min under the following elution conditions: from 0 min to 42 min, solution B (acetonitrile containing 0.1% formic acid) increased linearly from 6% to 30%; from 42 min to 51 min, solution B increased linearly from 30% to 42%, and then reached 95% within 5 min and maintained it until 60 min. A Thermo Scientific Q Exactive HF mass spectrometer with a Nanospray Flex ion source was used, with the ion spray voltage set to 2.3 kV and the ion transfer tube temperature set to 320 °C.

[0059] The mass spectrometer used DIA mode for data acquisition, with a first-stage scan resolution of 120,000 m / s and a scan range of 398-900 m / s, and a maximum injection time of auto. The second-stage scan resolution was 30,000 m / s, with 32 scan windows and a collision energy of 27%.

[0060] (3) Search parameters DIA data were searched using Spectronaut 17 (Biognosys) software. Search parameters included: protein databases for the corresponding species, trypsin digestion, a maximum of two missed cleavage sites, fixed modification as Carbamidomethyl (C), and variable modifications as Oxidation (M) and Acetyl (N-terminal). The false discovery rate (FDR) for peptides and proteins was <1.0%, with at least one specific peptide identified for each protein. Other parameters were left at default. The database used was the human database downloaded from Uniprot.

[0061] (4) ELISA was used to detect the protein expression levels of CSN2, PLIN1 and CHRDL2. The following kits were used to detect human CSN2 in cell culture supernatant, serum, and plasma: a CSN2 ELISA kit (human casein β (CSN2) quantification kit, catalog number YJ624879, 96T / 48T, purchased from Shanghai Yuanju Biotechnology Center), a PLIN1 ELISA kit (human perilipoplasmin 1 (PLIN1) quantification kit, catalog number YJ523686, 96T / 48T, purchased from Shanghai Yuanju Biotechnology Center), and a CHRDL2 ELISA kit (human tendinoid protein 2 (CHRDL2) quantification kit, catalog number YJ387457, 96T / 48T, purchased from Shanghai Yuanju Biotechnology Center). Specific experimental methods were described in the instructions for each kit.

[0062] When using the CHRDL2 ELISA kit to detect CHRDL2, firstly, all reagents should be equilibrated at room temperature for 30 minutes. Prepare diluted wash buffer, standards at seven concentration gradients (0.157-10 ng / mL), 1× biotinylated antibody (biotinylated anti-CHRDL2 antibody) working solution, and 1× SA-HRP working solution. Simultaneously, process cell culture supernatant, serum, plasma, and other samples as required (e.g., centrifuge to remove particles, dilute appropriately to reduce matrix effects). During the experiment, prepare the required ELISA plate, add 100 μL of standard or pretreated sample to each well, incubate at 37°C in the dark for 1 hour, and wash 3 times with 1× wash buffer. Next, add 100 μL of biotinylated anti-CHRDL2 antibody working solution to each well, incubate at 37°C in the dark for 1 hour, and wash 4 times. Then add 100 μL of 1× SA-HRP working solution, incubate at 37°C in the dark for 30 minutes, and wash 4 times. Finally, add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well sequentially. Incubate μL of chromogenic solution B at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader within 5 minutes. Finally, calculate the corrected OD values ​​(minus the OD values ​​of blank wells) for the standard and sample replicates. Plot a four-parameter logistic function standard curve with the standard concentration on the x-axis and the corrected OD value on the y-axis. Calculate the concentration of CHRDL2 in the sample based on the standard curve (if the sample OD value exceeds the upper limit, it needs to be diluted and retested, then multiplied by the dilution factor).

[0063] The core reagent in the aforementioned PLIN1 enzyme immunoassay kit is a biotinylated anti-PLIN1 antibody. This antibody specifically binds to PLIN1, laying the foundation for subsequent SA-HRP binding and signal development, and is a key specific reagent for achieving quantitative detection of PLIN1. The specific detection method is the same as above.

[0064] The core reagent in the aforementioned CSN2 enzyme immunoassay kit is a biotinylated anti-CSN2 antibody. This antibody specifically binds to CSN2, laying the foundation for subsequent SA-HRP binding and signal development, and is a key specific reagent for achieving quantitative detection of CSN2. The specific detection method is the same as described above.

[0065] (5) Validation of the diagnostic efficacy of biomarkers CSN2, PLIN1 and CHRDL2 in the exploratory and validation cohorts For the biomarkers CSN2, PLIN1, and CHRDL2 that were screened in this embodiment and showed significant differential expression between healthy controls and patients with unexplained recurrent miscarriage, receiver operating characteristic (ROC) curves were plotted using the R package "pROC". The accuracy, sensitivity, specificity, and AUC value of these biomarkers in differentiating between patients with unexplained recurrent miscarriage and healthy controls were analyzed to determine their diagnostic efficacy in the aforementioned exploration and validation cohorts.

[0066] In assessing the diagnostic efficacy of biomarkers CSN2, PLIN1, or CHRDL2 in the aforementioned screening and validation sets, the relative expression levels of CSN2, PLIN1, or CHRDL2 were analyzed, and the level corresponding to the point with the largest Youden index was selected as its cutoff value. The resulting AUC value is the area under the ROC curve and the coordinate axis, ranging from 0.5 to 1. A higher AUC value indicates greater accuracy in diagnosing and differentiating patients with unexplained recurrent miscarriage from healthy controls; an AUC value close to 1.0 indicates high reliability and excellent predictive ability of the diagnostic results based on the biomarker.

[0067] 3. Experimental Results In the exploratory cohort, there was no significant difference in age between the control group and the group of patients with unexplained recurrent miscarriage, ensuring the comparability of subsequent analyses. In serum proteomics testing, we first used a standard t-test to screen for differentially expressed proteins (…). Figure 1 The significance thresholds were set at an adjusted P-value < 0.05 and |log2FC| > 0.585, with the Benjamini-Hochberg method used for false positive control. Results showed that 9 significantly upregulated proteins (including CHRDL2) and 25 significantly downregulated proteins (including CSN2 and PLIN1) were identified. Figure 2 ).

[0068] In the exploratory cohort, ROC curves were plotted for the differentially expressed proteins in the control group and the unexplained recurrent miscarriage group. The results are as follows: Figure 3 As shown, the results indicate that three differentially expressed proteins had an AUC greater than 0.8: CSN2 (AUC: 0.9206, sensitivity: 80%, specificity: 95%), PLIN1 (AUC: 0.8406, sensitivity: 70%, specificity: 87.5%), and CHRDL2 (AUC: 0.8094, sensitivity: 82.5%, specificity: 75%).

[0069] In the validation cohort, the three differentially expressed proteins CSN2, PLIN1, and CHRDL2 were validated using ELISA kits. The results are shown below. Figure 4 As shown, the results indicate that, consistent with previous test results, compared with the control group, the serum levels of CSN2 and PLIN1 in the unexplained recurrent miscarriage group were significantly lower, while the level of CHRDL2 was significantly higher.

[0070] In the validation cohort, ROC curves were plotted for the three differentially expressed proteins CSN2, PLIN1, and CHRDL2 in both the control group and the unexplained recurrent miscarriage group. The results are as follows: Figure 5 As shown, the results indicate that the differentially expressed proteins all have high diagnostic efficacy and high AUC values, namely CSN2 (AUC: 0.9116, sensitivity: 90%, specificity: 85%), PLIN1 (AUC: 0.8700, sensitivity: 77.5%, specificity: 87.5%), and CHRDL2 (AUC: 0.7241, sensitivity: 80%, specificity: 62.5%).

[0071] In summary, the results show that CSN2, PLIN1, and CHRDL2 were significantly differentially expressed between the control group and the unexplained recurrent miscarriage disease group in both the exploratory and validation cohorts composed of real clinical samples collected by this invention. CSN2 and PLIN1 were significantly downregulated, while CHRDL2 was significantly upregulated. All of these expression levels showed high accuracy (AUC), sensitivity, and specificity, and can be used for the accurate and effective diagnosis of unexplained recurrent miscarriage.

Claims

1. Application of reagents for detecting the expression levels of biomarkers CSN2, PLIN1 and / or CHRDL2 in samples in the preparation of products for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

2. The application according to claim 1, characterized in that, The reagent is: Reagents for detecting the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in samples; Reagents for detecting the DNA levels of CSN2, PLIN1, and / or CHRDL2 in a sample; or A reagent for detecting the RNA levels of CSN2, PLIN1, and / or CHRDL2 in a sample.

3. The application according to claim 2, characterized in that, The reagents used to detect the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample are antibodies, aptamers, agglutinants, and / or small molecule compounds that specifically bind to proteins encoded by CSN2, PLIN1, and / or CHRDL2.

4. The application according to claim 2, characterized in that, The reagents used to detect the DNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample are reagents for detecting the DNA expression level, DNA methylation level, DNA acetylation level, DNA phosphorylation level, and / or DNA hydroxymethylation level of CSN2, PLIN1, and / or CHRDL2 in the sample. Optionally, the reagent used to detect the RNA levels of CSN2, PLIN1, and / or CHRDL2 in the sample is a reagent used to detect the expression levels of mRNA, lncRNA, and / or miRNA of CSN2, PLIN1, and / or CHRDL2 in the sample.

5. The application according to claim 1, characterized in that, The samples are serum samples, plasma samples, blood samples, tissue samples, exosome samples, and / or cell samples.

6. The application according to claim 1, characterized in that, The product is a test kit, a test chip, or a test strip; Optionally, the detection kit is a detection kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker; Optionally, the detection kit detects the biomarker via an antigen-antibody reaction; Optionally, the detection kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit; Optionally, the detection kit includes reagents for detecting the protein expression levels of CSN2, PLIN1, and / or CHRDL2 in the sample.

7. A product for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage, characterized in that, The product comprises the reagent described in claim 1 for detecting the expression levels of biomarkers CSN2, PLIN1, and / or CHRDL2 in a sample.

8. The product according to claim 7, characterized in that, The product is a test kit, a test chip, or a test strip; Optionally, the detection kit is a detection kit for ELISA, protein / peptide chip detection, immunoblotting detection, microbead immunoassay, or microfluidic immunoassay of the biomarker; Optionally, the detection kit is an ELISA detection kit, a fluorescence immunoassay kit, or a chemiluminescence immunoassay kit.

9. A system or apparatus for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage, characterized in that, The system or device includes a processor, an input module, and an output module; The input module is used to input the expression levels of CSN2, PLIN1, and / or CHRDL2 in the subject's sample; the processor is used to perform logical operations on the input information using bioinformatics methods, and the processor contains a computer-readable medium storing instructions, which, when executed by the processor, perform algorithms on the input CSN2, PLIN1, and / or CHRDL2 expression levels; the output module is used to output the result of whether the subject is a patient with unexplained recurrent miscarriage or the risk of having unexplained recurrent miscarriage.

10. The use of reagents for detecting the expression levels of biomarkers CSN2, PLIN1 and / or CHRDL2 in samples in the preparation of systems or devices for diagnosing unexplained recurrent miscarriage or predicting the risk of unexplained recurrent miscarriage.

Images