MiRNA markers for diagnosis of polycystic ovary syndrome and application thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a miRNA marker for diagnosing polycystic ovary syndrome and application thereof. The miRNA marker comprises one or a combination of hsa-miR-185-5p and hsa-miR-4433b-3p. The application first uses a plurality of extracellular vesicle (EVs) miRNAs such as hsa-miR-185-5p for diagnosing polycystic ovary syndrome patients, and it is found through verification in real clinical samples collected in the application that the plurality of extracellular vesicle (EVs) miRNAs such as hsa-miR-185-5p have good diagnostic efficiency for diagnosing polycystic ovary syndrome, and have high accuracy, sensitivity and specificity, and can be used for effective diagnosis of polycystic ovary syndrome. The application provides a brand-new thought and strategy for research and development of polycystic ovary syndrome related diagnostic products, and has a wide application prospect and important transformation significance in the technical field of polycystic ovary syndrome diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a miRNA biomarker for the diagnosis of polycystic ovary syndrome and its application. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women of reproductive age, characterized by chronic anovulation and hyperandrogenemia, with an unclear etiology. Currently, the diagnosis of PCOS is complex, requiring a combined analysis of clinical manifestations, imaging features, and laboratory hormone tests. However, due to the high clinical heterogeneity of PCOS, it easily overlaps with the normal population and is confused with various other endocrine disorders, leading to controversy and inconsistencies in diagnostic criteria. Therefore, clinical practice still needs to identify and characterize PCOS-specific serological molecular markers.

[0003] Extracellular vesicles (EVs) are a type of vesicle with a diameter of approximately 30–1000 nm, secreted or released by cells, and possessing a lipid membrane structure. Studies have shown that EVs are widely present in almost all body fluids and can transport proteins, lipids, messenger RNA (mRNA), microRNA (miRNA), and other substances, mediating intercellular communication and information exchange. They are closely related to the occurrence and development of various diseases and are ideal indicators for liquid biopsy.

[0004] Minimal RNAs (miRNAs) are a class of single-stranded, non-coding small RNA molecules, approximately 22 nucleotides in length. They can bind complementary to the 3' untranslated region (UTR) of downstream target gene mRNAs, regulating target gene expression at the post-transcriptional level and playing important regulatory roles in gene expression, cell cycle, and organismal developmental timing. Evidence shows that abnormal miRNA expression is closely related to various diseases, including cardiovascular disease, diabetes, and cancer, participating in disease occurrence and development by regulating the expression of multiple genes. Furthermore, peripheral blood extracellular vectors (EVs) from patients with different diseases exhibit specific miRNA profiles, representing a class of biological markers and therapeutic targets with significant clinical application potential.

[0005] Numerous studies have confirmed that the expression profile of circulating extracellular vesicle (EVs) miRNAs in PCOS patients differs significantly from that in controls, demonstrating their potential as novel indicators for PCOS detection, disease monitoring, and prognostic assessment. Exploring the significantly and stably altered EVs miRNAs in the peripheral blood of PCOS patients compared to controls could open up new avenues and methods for the detection, monitoring, and prognostic assessment of complex diseases like PCOS, holding significant application prospects and importance. Summary of the Invention

[0006] To address the shortcomings of existing hematological markers and detection methods for PCOS, this invention aims to provide a combination of extracellular vesicle (EV) microRNAs (miRNAs) for detecting PCOS and their applications. The detection method based on this biomarker can provide non-invasive, rapid, and accurate auxiliary diagnostic detection of PCOS, and has significant application value in PCOS screening and diagnostic research.

[0007] Therefore, the present invention provides the following technical solution.

[0008] The first aspect of the present invention provides a miRNA biomarker for diagnosing polycystic ovary syndrome, said miRNA biomarker comprising one or a combination of two of hsa-miR-185-5p and hsa-miR-4433b-3p.

[0009] In a preferred embodiment of the present invention, the miRNA marker further includes one or a combination of two of hsa-miR-199a-5p and hsa-miR-194-5p.

[0010] The sequence information of the four miRNAs mentioned above is shown in the table below: In this invention, verification in real clinical samples collected by this invention revealed that, as mentioned above, miRNA biomarkers have good diagnostic efficacy for polycystic ovary syndrome. The diagnostic efficacy is verified by receiver operating characteristic (ROC) curves. The area under the curve (AUC) is the area under the ROC curve, which is well 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.

[0011] 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., samples from patients with polycystic ovary syndrome and normal or 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 herein). Typically, the aforementioned characteristic data are sorted in ascending order across the entire population (e.g., patient group and 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).

[0012] Receiver operating characteristic (ROC) curves can be generated for other single calculations or for single characteristics to provide a single sum value. For example, two or more characteristics can be mathematically combined (e.g., addition, subtraction, multiplication, etc.), and this single sum value can be represented by a ROC curve. Additionally, it is possible to plot combinations of multiple characteristics from which a single calculation value can be derived using ROC curves. These combinations of characteristics can constitute a test. The aforementioned ROC curve is a graph representing the true positive rate (sensitivity) of a test relative to the false positive rate (specificity) of the test.

[0013] The sensitivity and specificity of biomarkers in diagnosing polycystic ovary syndrome (PCOS) can be evaluated by plotting the ROC curve and calculating the area under the curve (AUC). The criteria are as follows: AUC < 0.5 indicates no diagnostic significance; AUC = 0.5-0.7 indicates low diagnostic accuracy; AUC = 0.7-0.9 indicates moderate diagnostic accuracy; and AUC > 0.9 indicates high diagnostic accuracy. In other words, in the ROC curve, the AUC value represents the area covered by the ROC curve; the larger the AUC value, the higher the accuracy of the model's prediction.

[0014] A second aspect of the invention provides the use of a reagent for detecting the expression level of the miRNA markers as described above in the preparation of products for diagnosing polycystic ovary syndrome.

[0015] In a preferred embodiment of the present invention, the reagent is an antibody for the miRNA marker, primers for PCR, reagents for mass spectrometry analysis, or reagents for chromatographic analysis.

[0016] In a preferred embodiment of the present invention, the product includes a chip, test strip, formulation, reagent, kit, or high-throughput screening platform.

[0017] A third aspect of the invention provides a kit for diagnosing polycystic ovary syndrome, comprising reagents for detecting miRNA markers as described above.

[0018] In a preferred embodiment of the present invention, the detection is a quantitative detection of the expression level of the miRNA biomarker in the subject sample.

[0019] In a preferred embodiment of the present invention, the reagent is an antibody for the miRNA marker, primers for PCR, reagents for mass spectrometry analysis, or reagents for chromatographic analysis.

[0020] In a preferred embodiment of the present invention, the kit is a PCR kit.

[0021] PCR technology can be used to detect transcribed RNA of biomarkers. The RNA or fragments to be detected are amplified by PCR, and then quantitatively analyzed using methods such as gel electrophoresis or real-time quantitative PCR.

[0022] The provided kit for polycystic ovary syndrome includes primers designed based on miRNA biomarkers. Using these primers, the corresponding miRNA biomarkers can be rapidly detected without the need for nucleic acid extraction. This method is simple, fast, reduces contamination, avoids loss, lowers costs, and has a low technical barrier. It also saves sample, requiring no more than 1 μL of sample to detect a single miRNA. Furthermore, the results can be further analyzed for greater reliability and wide applicability.

[0023] For example, considering detection efficiency and convenience, the PCR kit includes a reverse transcription reaction system and a qRT-PCR reaction system.

[0024] In a preferred embodiment of the present invention, the PCR kit includes a primer set for detecting the miRNA marker.

[0025] In a preferred embodiment of the present invention, the primer set includes: specific reverse transcription primers for amplifying each miRNA marker and specific primers based on fluorescent dye PCR reaction.

[0026] In a preferred embodiment of the present invention, the nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO. 5-6.

[0027] In a preferred embodiment of the present invention, the nucleotide sequence of the specific primer is shown in SEQ ID NO. 7-9.

[0028] In some embodiments, the test sample includes samples obtained from cell, tissue, or body fluid collections from any subject. Specifically, the sample includes, but is not limited to: tissue or cell samples that may be derived from fresh, frozen, and / or preserved solid tissue of an organ or tissue sample, or from a biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid. Tissue samples may be primary or in vitro cultured cells or cell lines. Optionally, tissue or cell samples may be obtained from diseased tissue / organ. Tissue samples may contain compounds naturally mixed with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or similar compounds. In a specific embodiment of the invention, the test sample is a serum / plasma sample derived from a subject.

[0029] A fourth aspect of the invention provides the use of the miRNA marker as described above in the preparation of a formulation for the diagnosis of polycystic ovary syndrome.

[0030] For example, biomarkers for polycystic ovary syndrome (PCOS) can be used to study disease mechanisms, pathophysiological processes, and potential therapeutic targets. By analyzing changes in these biomarkers throughout the disease progression, we can gain a deeper understanding of the disease's development and metastasis mechanisms, providing a more comprehensive understanding for its prevention and treatment.

[0031] Biomarkers also play a crucial role in drug development. They serve as indicators for drug efficacy evaluation, helping researchers assess the safety and effectiveness of new drugs. By monitoring the impact of drugs on biomarkers, potential drug toxicity and adverse reactions can be detected early, guiding the adjustment and optimization of drug dosage.

[0032] Biomarkers can also be used in individual health management and preventive medicine. By regularly monitoring specific biomarkers, changes in an individual's health status can be detected in a timely manner, the risk of disease can be predicted, and corresponding health management measures can be taken, such as adjusting lifestyle, dietary habits, or drug treatment, to maintain health and slow disease progression.

[0033] Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these biomarkers, the extent to which different lifestyles affect health can be assessed, and individuals can be guided to take appropriate lifestyle interventions, such as weight loss, smoking cessation, and increased physical activity, thereby improving health status and reducing disease risk.

[0034] A fourth aspect of the present invention provides a method for in vitro screening of candidate drugs for the treatment of polycystic ovary syndrome, the method comprising the following steps: Treat in vitro culture systems expressing or containing miRNA markers as described above with the substances to be screened. The expression levels of the miRNA biomarkers in the in vitro culture system were detected. When the substance to be screened inhibits the expression level of the miRNA marker, the substance to be screened is a candidate drug for the treatment of polycystic ovary syndrome.

[0035] The reliability and accuracy of this method depend on the sensitivity and specificity of the selected detection method, as well as the determination of the reference range or threshold. Therefore, when implementing this method, it is necessary to strictly control experimental conditions and combine clinical experience and data analysis for the interpretation and diagnosis of results. Furthermore, the detection results of any single biomarker should be carefully evaluated and interpreted to avoid uncertainty in diagnostic results due to the potential for misinterpretation by a single biomarker.

[0036] The implementation of this method requires starting with experimental design and sample collection. During the design phase of a study or clinical trial, the drug, treatment regimen, and evaluation indicators to be assessed must be clearly defined. Simultaneously, the selection criteria for subjects need to be determined, and appropriate biological samples, such as serum, plasma, tissue, or cell samples, need to be collected for subsequent experimental analysis.

[0037] For the chosen drug or treatment regimen, any one or more of the miRNA biomarkers described above can be used as biomarkers to evaluate its therapeutic effect. The selection of these biomarkers can be determined based on previous studies or clinical experience, or inferred from the drug's mechanism of action and expected effects.

[0038] Once the biological sample is obtained, appropriate techniques and reagents can be used to detect the level of the selected biomarker. These detection methods can be immunological techniques, such as ELISA and immunofluorescence analysis, molecular biological techniques, such as PCR and in situ hybridization, or mass spectrometry. These methods allow for the accurate quantification of biomarker levels in the subject's biological sample.

[0039] Next, the medication needs to be administered to the subject, and treatment will proceed according to the predetermined protocol. During treatment, the subject's biological samples will be monitored regularly, and the levels of the selected biomarkers will be repeatedly tested. This allows for timely monitoring of the drug's effectiveness and whether the intended treatment goals have been achieved.

[0040] Finally, the therapeutic effect of the drug can be evaluated and determined based on the obtained test results. If the level of the selected biomarker changes significantly and shows a clear difference compared to before treatment, it can be inferred that the drug may have the potential to treat polycystic ovary syndrome. Conversely, if the biomarker level does not change significantly or does not achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

[0041] A fifth aspect of the present invention provides a risk prediction system for polycystic ovary syndrome, the risk prediction system including an assessment unit; The assessment unit predicts the risk of a subject having polycystic ovary syndrome based on the expression levels of miRNA markers as described above. Preferably, the assessment unit uses the following method to determine the risk of polycystic ovary syndrome (PCOS): when the expression level of the miRNA marker in the subject's sample is significantly higher than that in the normal human sample, the subject is judged to have a high risk of PCOS.

[0042] In a preferred embodiment of the present invention, the risk prediction system further includes a detection unit for detecting the expression level of the miRNA biomarker as described above.

[0043] In a preferred embodiment of the present invention, the risk prediction system further includes a display unit for displaying the conclusions reached by the evaluation unit.

[0044] It should be understood that the terms "system" and "unit" used herein are a method of 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.

[0045] Those skilled in the art will recognize that this invention can be implemented as an apparatus, method, or computer program product. Therefore, this disclosure can be specifically implemented in the following forms: it can be entirely hardware, entirely software (including firmware, resident software, microcode, etc.), or a combination of hardware and software, generally referred to herein as a "unit" or "system".

[0046] In this invention, when a biomarker is used to indicate an abnormal process, disease, or other condition in an individual, or as a marker of an abnormal process, disease, or other condition, the biomarker is typically described as overexpressed or underexpressed when compared to the expression level or value of a biomarker that indicates a normal process, absence of disease, or other condition in the individual. The term may also refer to the value or level of a biomarker in a biological sample that is greater than the value or level (or range of values ​​or levels) of a biomarker detectable at different stages of a particular disease.

[0047] Furthermore, compared to the "normal" expression level or value of a biomarker that indicates normal progression or absence of disease or other conditions in an individual, overexpressed or underexpressed biomarkers can also be referred to as "differentially expressed" or having "differential levels" or "differential values." Therefore, "differential expression" of a biomarker can also be referred to as a variation of the "normal" expression level of the biomarker.

[0048] The terms “differential biomarker expression” and “differential expression” are used interchangeably to refer to a biomarker whose expression is activated at a higher or lower level in subjects with a specific disease, relative to its expression in normal subjects, or relative to its expression in patients who respond differently to a particular treatment or have different prognoses. The term also includes biomarkers whose expression is activated at higher or lower levels at different stages of the same disease. It should also be understood that differentially expressed biomarkers can be activated or inhibited at the nucleic acid or protein level, or can undergo alternative splicing to produce different polypeptide products. This difference can be demonstrated by a variety of alterations, including at the mRNA level, microRNA level, antisense transcript level, or protein surface expression, secretion, or other partitioning of the polypeptide. Differential biomarker expression can include a comparison of expression between two or more genes or their gene products; or a comparison of the ratio of expression between two or more genes or their gene products; or even a comparison of two differently processed products of the same gene that differ between normal and diseased subjects; or that differ at different stages of the same disease. Differential expression includes, for example, quantitative and qualitative differences in transient expression patterns or cellular expression patterns of markers between normal and diseased cells or between cells experiencing different disease events or stages.

[0049] "Increased expression" or "upregulation" means that the content shows an increase of 10% or more compared to the control, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more.

[0050] The fifth aspect of the present invention provides the use of a reagent for detecting the expression level of the biomarker in a sample in the preparation of a system or device for diagnosing polycystic ovary syndrome.

[0051] Furthermore, the biomarker is the biomarker as described above.

[0052] The present invention also provides a method for diagnosing polycystic ovary syndrome, the method comprising the following steps: (1) Collect samples from the subjects; (2) Detect the expression levels of the miRNA biomarkers in samples from subjects; (3) Diagnose whether the subject has polycystic ovary syndrome based on the expression level of the miRNA markers obtained by detection.

[0053] The term "subject" includes both human and non-human subjects, including birds and non-human mammals such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, and cattle); and non-domesticated animals such as large felines. The term "subject" applies regardless of the stage of an organism's life cycle. Therefore, depending on the organism (i.e., whether the organism is a mammal or a bird, whether domesticated or wild), the term "subject" applies to organisms in the womb or in an egg.

[0054] The Evs miRNA and its combination described in this invention can be applied to the detection of PCOS patients, for example, to supplement new detection indicators for PCOS patients, and for screening, auxiliary screening, detection or auxiliary detection, diagnosis or auxiliary diagnosis of polycystic ovary syndrome.

[0055] This invention is the first to apply multiple extracellular vesicle (EV) miRNAs, such as hsa-miR-185-5p, to the diagnosis of polycystic ovary syndrome (PCOS). Validation using real clinical samples collected during this invention revealed that these EV miRNAs exhibit good diagnostic efficacy for PCOS, with high accuracy, sensitivity, and specificity, making them effective for the diagnosis of PCOS. This invention provides a novel approach and strategy for the research and development of diagnostic products related to PCOS, and has broad application prospects and significant translational value in the field of PCOS diagnosis.

[0056] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0057] Figure 1 A flowchart of the screening process for miRNA biomarkers for the diagnosis of polycystic ovary syndrome (PCOS) of the present invention is shown.

[0058] Figure 2 The results of qRT-PCR detection of changes in four serum EVs miRNAs in patients in the PCOS rescreening group are shown.

[0059] Figure 3 The results of qRT-PCR detection of changes in four serum EVs miRNAs in PCOS validation group patients are shown.

[0060] Figure 4 The ROC curves for the diagnosis of PCOS patients show specific changes in serum EVs miRNA.

[0061] Figure 5 The ROC curves of the combined effects of hsa-miR-185-5p and hsa-miR-4433b-3p are shown to differentiate between PCOS patients and healthy controls. Detailed Implementation

[0062] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0063] Studies have confirmed that EVs miRNAs are closely related to the occurrence and development of various diseases, making them ideal liquid biopsy markers. EVs are widely present in almost all body fluids, and their expression profiles and levels change significantly under different cell origins and physiological or pathological conditions. Detecting changes in specific EVs miRNA levels can reflect the physiological and pathological state of cells, making them the most promising liquid biopsy molecular markers for clinical translational applications in disease diagnosis, efficacy monitoring, and prognostic assessment. They exhibit good specificity and sensitivity for early disease diagnosis and disease prediction, and demonstrate good stability and reliable results. Furthermore, numerous studies have shown that EVs miRNAs can serve not only as novel biomarkers for the potential diagnosis, prognosis, or efficacy evaluation of molecular endocrine diseases such as diabetes, obesity, and metabolic syndrome, but also participate in pathological processes such as abnormal hormone secretion, insulin resistance, and the secretion of adipose-derived inflammatory factors. EVs carrying overexpression of specific protein molecules or miRNAs can effectively participate in the development of endocrine diseases such as diabetes. EVs carrying specific miRNA inhibitors can increase the sensitivity of pancreatic islet cells or adipocytes to hormones, suggesting that EV miRNAs are not only potential novel molecular markers for endocrine diseases but also key factors in disease development and progression, and hold promise as drugs with natural biological activity for precision treatment. The specimen used in this invention is peripheral blood from the subject; the sample is readily available, clinically feasible, and non-invasive to the subject.

[0064] Therefore, this invention analyzes the expression profiles of miRNAs in serum EVs of PCOS patients and healthy controls. It was found that compared to healthy controls, the expression of 87 miRNAs was increased and the expression of 126 miRNAs was decreased in serum EVs of PCOS patients. Further, based on the miRNA expression level (copy number > 50 in serum EVs of healthy controls and PCOS patients) and the fold change (increase > 2-fold in serum EVs of PCOS patients), four miRNAs were initially screened out: miR-185-5p, miR-199a-5p, miR-194-5p, and miR-4433b-3p. These miRNAs are deemed suitable for clinical application as molecular markers for PCOS patients in diagnostic reagents or kits.

[0065] Further experimental studies revealed that among the four EVs miRNAs, miR-185-5p and miR-4433b-3p were significantly elevated in serum EVs of PCOS patients (P < 0.05), consistent with sequencing results. ROC curve analysis showed that the area under the curve (AUC) for serum EVs miR-185-5p in distinguishing between PCOS patients and healthy controls was 0.780 (95% CI: 0.675–0.884), with a sensitivity of 68.8% and a specificity of 80.6% at a cutoff value of 33.21. The AUC for serum EVs miR-4433-3p in distinguishing between PCOS patients and healthy controls was 0.927 (95% CI: 0.872–0.981), with a sensitivity of 83.3% and a specificity of 100% at a cutoff value of 24.27. The area under the curve (AUC) for the combined use of miR-185-5p and miR-4433b-3p to distinguish between PCOS patients and healthy controls was 0.914 (95% CI: 0.852–0.976). At a cutoff value of 60.87, the sensitivity was 83.3% and the specificity was 94.4%.

[0066] ROC curve results indicate that EVs miR-185 and miR-4433b-3p can be used alone or in combination as molecular markers for PCOS screening or auxiliary screening, detection or auxiliary detection, diagnosis or auxiliary diagnosis, providing multiple new application directions for the diagnosis and treatment of PCOS.

[0067] The following detailed description uses specific embodiments. Unless otherwise specified, all other materials, reagents, etc., used in the following embodiments of this application are commercially available.

[0068] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0069] The following embodiments involve and mention: 1. Main Instruments High-speed refrigerated centrifuge (Model 5418, Eppendorf, Germany); Ultra-low temperature freezer (DW-86L628, Haier, China); Medical low-temperature freezer (Model DW-25L282, Haier, China); Protein electrophoresis apparatus (1658001, Bio-Rad, USA); PCR amplification instrument (Axygen® Maxygene II, Axygen); Real-time quantitative PCR instrument (LightCycler480 II, Roche); Microplate reader (Multiscan GO, Thermo Fisher Scientific, USA).

[0070] 2. Main reagents EV extraction kit (ExoQuick exosome precipitation solution, SBI, USA); Trizol reagent (Thermo Fisher Scientific, USA); isopropanol (Shanghai Chemical Reagent Co., Ltd.); anhydrous ethanol (Shanghai Chemical Reagent Co., Ltd.); miRNA probe (Thermo Fisher Scientific, USA); PCR reagents including dNTPs, AMV reverse transcriptase; rTaq enzyme, etc. (Takara, Dalian).

[0071] Example 1: Preliminary screening of biomarkers 1. Collection of clinical samples (1) Sample source The clinical cases were derived from PCOS patients who visited the Reproductive Medicine Department of Jiangsu Provincial Hospital of Traditional Chinese Medicine. The baseline for inclusion was that the patients had not used any hormone drugs and / or traditional Chinese medicine treatment or had stopped taking medication for more than two months.

[0072] (2) Diagnostic criteria The diagnostic criteria for PCOS are the Rotterdam criteria proposed by the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine (ESHRE / ASRM): ① infrequent ovulation or anovulation; ② clinical or biochemical hyperandrogenism; ③ ultrasound imaging showing an ovarian volume >10 mL, or ≥12 follicles with a diameter of 2–9 mm. If two of the above three criteria are met and other diseases that cause elevated androgens are excluded.

[0073] (3) Inclusion criteria Patient inclusion criteria were: ① meeting the above-mentioned Rotterdam diagnostic criteria for PCOS; ② being aged 25 to 40 years and married; ③ being willing to participate in this study and signing an informed consent form; and ④ not having used any hormone drugs or drugs affecting lipid metabolism in the past two months.

[0074] (4) Exclusion criteria Exclusion criteria for patients include: ① Hyperandrogenemia due to other causes (such as hyperprolactinemia and thyroid disease, congenital adrenal hyperplasia, Cushing's syndrome, androgen-secreting tumors, 21-hydroxylase deficiency atypical adrenal hyperplasia, 17-hydroxyprogesterone < 3 nmol / L, use of exogenous androgens, etc.). ② Severe diseases, such as kidney disease (creatinine clearance < 60 mL / min), liver dysfunction, autoimmune diseases, or cancer. ③ Received drug treatment in the past 3 months (cortisol, other hypoglycemic treatments such as insulin, acarbose, antidepressants, hormonal contraceptives, hormonal ovulation-inducing drugs, or other drugs); or took medroxyprogesterone acetate or similar drugs within the past 6 months. ④ Patients with mental illness, such as unstable periodic mood disorders, bipolar disorder, or suicidal ideation. ⑤ Pregnant or lactating women. ⑥ Those with language barriers or inability to understand the test content.

[0075] Based on the above criteria, this application has collected a sample set for biomarker screening, which consists of the following: Twenty PCOS patients were included: PCOS patients who visited the Department of Reproductive Medicine at Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022 and had not received any hormonal drugs and / or traditional Chinese medicine treatment or had stopped medication for more than two months. Clinical data and laboratory test results of patients and controls were collected.

[0076] Twenty healthy controls were included: age- and sex-matched healthy controls who underwent health checkups at Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022; healthy controls excluded individuals with PCOS, endocrine disorders, tumors, diabetes, neurological disorders, and kidney diseases.

[0077] 2. Sample Collection and Processing (1) Sample collection Collect 3-5 mL of venous blood from PCOS patients and healthy controls after fasting for more than 12 hours using vacuum biochemical blood collection tubes. After thorough mixing, centrifuge at 1500 g for 10 min at room temperature to separate serum and blood cells. Centrifuge the obtained serum sample again at 12000 g for 5 min at 4 ℃ to further remove any possible residual blood cells. Carefully transfer the serum to clean, enzyme-free Eppendorf tubes and store at -80 ℃ for subsequent studies.

[0078] (2) Isolation and identification of serum EVs 2.1 Experimental Methods Using an EV extraction kit (SBI Pharmaceuticals, USA), serum EVs were isolated from 20 PCOS patients and 20 healthy controls according to the kit's instructions. The EVs isolated from PCOS patients and controls were mixed separately, and the particle size distribution of the isolated EVs was detected using Nanosight, followed by EV RNA extraction. The specific procedures are as follows: ①Separation of serum EVs: The serum, frozen at -80℃, was brought to room temperature and allowed to thaw completely. The entire serum was centrifuged at 2000g for 30 min at room temperature. After centrifugation, the serum was placed on ice. Using a 200 μL pipette tip, 150 μL of the supernatant was carefully aspirated into a new enzyme-free EP tube (avoiding floating lipids and impurities). 1 / 5 volume (30 μL) of extraction reagent (Reagent name: ExoQuick exosome precipitation solution, manufacturer: System Biosciences (SBI), catalog number: EXOQ5A-1) was added, and the tube was immediately vortexed thoroughly until a cloudy appearance was achieved. The tube was first incubated upright at 4℃ for 30 min, then centrifuged at 10000g for 10 min at room temperature. All supernatant was discarded, and the precipitate was retained as serum EVs. The precipitate at the bottom of the tube was resuspended appropriately and PBS was added for particle size analysis.

[0079] ② Particle size analysis to identify EV diameter and concentration: EV samples can only be loaded after the instrument performance test using standard products is qualified; EV samples are serially diluted and injected into the sample cell to ensure that the number of sample particles displayed on the software interface is between 50 and 400; 1 to 2 mL of the diluted EV suspension is injected into the sample cell, and the particle size and concentration information of EVs are detected using the NanoFCM instrument (FlowNanoAnalyzer).

[0080] 2.2 Results of serum EV separation and particle size analysis Particle size analysis showed that the diameter of serum EVs from PCOS patients and controls was between 30 and 200 nm, with concentrations of 1.7E + 10 Particles / mL for Control and 7.5E + 10 Particles / mL for PCOS, respectively. There was no order of magnitude difference in the size and concentration of serum EVs between PCOS patients and healthy controls, indicating that serum EVs were successfully separated.

[0081] (3) Extraction of serum EVs miRNA Add 1 mL of Trizol to an EP tube containing EVs, vortex to mix, and let stand for 5 min. Add 200 μL of Trizol Pal, and immediately vortex vigorously to mix. After thorough mixing, the sample will be a light pink color. Let stand at room temperature for 10 min, and layering will be visible. Then, place the sample in a high-speed refrigerated centrifuge and centrifuge at 4°C and 12000 g for 20 min. After centrifugation, the sample will be clearly divided into three layers: an aqueous layer, a protein layer, and an organic layer. Transfer 500 μL of the upper aqueous phase to a new 1.5 mL EP tube, add an equal volume of isopropanol and 1 μL of nucleic acid precipitate glycogen, invert to mix thoroughly, and let stand overnight at -20°C. Place the overnight sample in a high-speed refrigerated centrifuge and centrifuge at 4°C and 12000 g for 20 min. After centrifugation, discard the supernatant and retain the precipitate at the bottom of the EP tube. Add 1 mL of freshly prepared 75% ethanol solution to the tube, invert until the precipitate floats, then centrifuge the sample again at 4℃ and 12000 g for 20 min. Discard the supernatant and retain the precipitate at the bottom of the EP tube. Invert the EP tube onto clean absorbent paper and allow it to air dry. Once fully dry, add 22 μL of DEPC water, vortex to mix, and ensure the precipitate at the bottom of the tube is completely dissolved. Proceed to the next experiment or store in a -80℃ ultra-low temperature freezer for long-term storage.

[0082] Following the above method, serum EVs miRNAs were extracted from PCOS patients and healthy controls, and their concentrations were measured and high-throughput small RNA sequencing was performed to determine the miRNA expression profiles in serum EVs. Based on the number of miRNA reads and the fold change between the patient group and the control group, miRNAs with significantly different and upregulated expression levels in serum EVs of the patient group and the control group were preliminarily screened. The results are shown in Table 1.

[0083] Table 1. miRNAs with significantly different and upregulated expression levels in serum EVs of PCOS patients and healthy controls. As shown in Table 1, compared with healthy controls, the expression of 87 miRNAs was increased and the expression of 126 miRNAs was decreased in the serum EVs of PCOS patients.

[0084] Based on the above small RNA sequencing results, and according to the miRNA expression level (copy number >50 in serum EVs of healthy controls and PCOS patients) and fold change (>2-fold increase in serum EVs of PCOS patients), four miRNAs were initially screened out: miR-185-5p, miR-199a-5p, miR-194-5p, and miR-4433b-3p. The results are shown in Table 2. The sequence information of the four significantly upregulated EVs miRNAs initially screened out is shown in Table 3.

[0085] Table 2. Four miRNAs with significantly upregulated copy numbers (greater than 50) in serum EVs of PCOS patients compared to healthy controls. Table 3. Four significantly upregulated EVs miRNA sequences Example 2: Rescreening of biomarkers 1. Secondary screening of the sample set A sample set for marker rescreening was collected according to the standard of Example 1. The marker rescreening sample set was a separate sample set different from the sample set mentioned above, and its specific composition was as follows: Fourteen PCOS patients were included: PCOS patients who visited the Reproductive Medicine Department of Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022 and had not received any hormone drugs and / or traditional Chinese medicine treatment or had stopped medication for more than two months. Clinical data and laboratory test results of patients and controls were collected.

[0086] Fourteen healthy controls were included: age- and sex-matched healthy controls who underwent health checkups at Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022; healthy controls excluded individuals with PCOS, endocrine disorders, tumors, diabetes, neurological disorders, and kidney diseases.

[0087] 2. Detection of serum EVs miRNA levels The four target miRNAs screened in Example 1 were reverse transcribed into cDNA using the stem-loop reverse transcription method based on TaqMan hydrolysis probes. The specific system of the reverse transcription PCR reaction is shown in Table 4.

[0088] Table 4. Reverse transcription PCR system based on TaqMan hydrolysis probes Note: The specific reverse transcription primers for miRNA markers, the specific primers for quantitative fluorescence reactions, and the probes for quantitative fluorescence reactions were all purchased from Thermo Fisher Scientific Inc.'s miRNA detection kit based on TaqMan hydrolysis probes.

[0089] As another preferred embodiment, the stem-loop reverse transcription PCR system based on fluorescent dyes can also be used to reverse transcribe the four target miRNAs screened in Example 1 into cDNA. The specific system composition is shown in Table 5.

[0090] Table 5. Reverse transcription PCR system based on fluorescent dyes The reaction conditions for reverse transcription were: 16℃ for 30 min, 42℃ for 30 min, 85℃ for 5 min, and 4℃ +∞.

[0091] The reverse transcription reaction was carried out according to the above reverse transcription system and conditions to obtain cDNA, which was used for subsequent PCR reactions.

[0092] The cDNA was then amplified using qRT-PCR based on TaqMan hydrolysis probes, and the specific system is shown in Table 6. Table 6. qRT-PCR reaction system based on TaqMan hydrolysis probe Note: The specific reverse transcription primers for miRNA markers, the specific primers for quantitative fluorescence reactions, and the probes for quantitative fluorescence reactions were all purchased from Thermo Fisher Scientific Inc.'s miRNA detection kit based on TaqMan hydrolysis probes.

[0093] As another preferred embodiment, cDNA can also be amplified using a fluorescent dye-based qRT-PCR reaction, and the specific system composition is shown in Table 7: Table 7 qRT-PCR reaction system based on fluorescent dye method The amplification reaction was performed using a real-time quantitative PCR instrument (LightCycler480 II, Roche). The reaction program was: 95℃ for 5 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles. All other settings were the system default values.

[0094] The reverse transcription primers and qPCR amplification forward primers and probes for the stem-loop method real-time fluorescence quantitative PCR kit based on TaqMan hydrolysis probes were all from TaqMan™ MicroRNA Assay, Thermo Fisher Scientific (formerly ABI, later acquired by Thermo Fisher Scientific). The forward and reverse primers and the unified reverse primer for the qRT-PCR reaction based on the fluorescent dye method were all designed and synthesized in-house. The remaining reagents were from Takara.

[0095] The above four miRNAs were detected using qRT-PCR in the secondary screening sample set, and the results are shown in [Figure number missing]. Figure 2 .like Figure 2 The results showed that hsa-miR-185-5p, hsa-miR-199a-5p, hsa-miR-194-5p and miR-4433b-3p were significantly elevated in serum EVs of PCOS patients (P < 0.05), consistent with the changes in sequencing results.

[0096] Example 3: Validation of the marker 1. Validation sample set A sample set for marker verification was collected according to the standard of Example 1. The marker verification sample set is a separate sample set that is different from the sample set mentioned above, and its specific composition is as follows: Forty-eight PCOS patients were included: PCOS patients who visited the Department of Reproductive Medicine at Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022 and had not received any hormonal drugs and / or traditional Chinese medicine treatment or had stopped medication for more than two months. Clinical data and laboratory test results of patients and controls were collected.

[0097] 36 healthy controls: These were age- and sex-matched healthy controls who underwent health checkups at Jiangsu Provincial Hospital of Traditional Chinese Medicine between June and December 2022. Patients with PCOS, endocrine disorders, tumors, diabetes, neurological disorders, and kidney diseases were excluded.

[0098] 2. Serum Evs miRNA detection The four miRNAs (hsa-miR-185-5p, hsa-miR-199a-5p, hsa-miR-194-5p, and hsa-miR-4433b-3p) screened in Example 3 were validated and differentially analyzed in external validation set serum samples using the method described in Example 2. The selected biomarkers were hsa-miR-185-5p and hsa-miR-4433b-3p. Differential expression analysis plots and receiver operating characteristic (ROC) curves were plotted; the results are shown below. Figure 3 , Figure 4 and Figure 5 .

[0099] like Figure 3 As shown, serum EVs hsa-miR-185-5p and hsa-miR-4433b-3p are significantly and stably elevated in PCOS patients.

[0100] like Figure 4 ROC curve analysis showed that the area under the curve (AUC) for serum EVs hsa-miR-185-5p in distinguishing between PCOS patients and healthy controls was 0.780 (95% CI: 0.675–0.884), with a sensitivity of 68.8% and a specificity of 80.6% at a cutoff value of 33.21. The AUC for serum EVs hsa-miR-4433b-3p in distinguishing between PCOS patients and healthy controls was 0.927 (95% CI: 0.872–0.981), with a sensitivity of 83.3% and a specificity of 100% at a cutoff value of 24.27. Figure 5 ROC curve analysis showed that the area under the curve (AUC) for serum EVs hsa-miR-185-5p and miR-4433b-3p in distinguishing PCOS patients from healthy controls was 0.914 (95% CI: 0.852–0.976). When the cutoff value was 60.87, the sensitivity was 83.3% and the specificity was 94.4%.

[0101] The blood samples used in this invention are easy to collect, clinically feasible, and non-invasive. Furthermore, EVs miRNAs exhibit good stability and are convenient to detect. Therefore, EVs hsa-miR-185-5p and hsa-miR-4433b-3p are worthy of promotion and clinical application as serological biomarkers for PCOS patients.

[0102] In summary, this invention initially identified four serum EVs miRNAs associated with PCOS, and based on these four EVs miRNA biomarkers, a PCOS detection kit was prepared. An auxiliary diagnostic method for PCOS based on EVs miRNAs was developed, and the method was validated. Results showed that among the four serum EVs miRNAs provided by this invention, EVs hsa-miR-185-5p and hsa-miR-4433b-3p were significantly elevated in the serum EVs of PCOS patients, consistent with sequencing results. Further validation showed that EVs hsa-miR-185-5p and hsa-miR-4433b-3p have good sensitivity and specificity for PCOS diagnosis, contributing to the screening, detection, and auxiliary diagnosis of PCOS, and possessing potential clinical application, translational, and promotional value.

[0103] The above results confirm that serum EVs hsa-miR-185-5p and hsa-miR-4433b-3p have high accuracy in the detection and auxiliary diagnosis of PCOS patients.

[0104] Example 4: PCR kit for the diagnosis of polycystic ovary syndrome This embodiment provides a detection kit for polycystic ovary syndrome (PCOS). The kit is designed to detect the levels of two miRNAs, hsa-miR-185-5p and hsa-miR-4433b-3p, in the serum of subjects, thereby achieving accurate diagnosis of PCOS.

[0105] The kit consists of: (1) Primers capable of reverse transcription of hsa-miR-185-5p or hsa-miR-4433b-3p; The reverse transcription primers include the commercially available qRT-PCR nucleotide sequences shown in Table 8, which are based on the stem-loop method using TaqMan hydrolysis probes or the method based on fluorescent dyes. Table 8 Reverse transcription primers (2) Primers that can perform PCR amplification of cDNA corresponding to hsa-miR-185-5p or hsa-miR-4433b-3p; The PCR amplification primers include the nucleotide sequences shown in Table 9. The reverse primers are uniform reverse primers, and the forward primers are miRNA-specific primers. Table 9 PCR Amplification Primers (3) The reagents used for qRT-PCR reverse transcription include: enzyme-free water, reverse transcriptase, dNTPs, reverse transcriptase buffer, miRNA reverse transcription primers, etc. The reagents used for qRT-PCR real-time quantitative detection include: enzyme-free water, hot-start DNA polymerase, and PCR buffer (containing Mg). 2+ Components include dNTPs, miRNA reverse primers, miRNA forward primers, TaqMan hydrolysis probes (or SYBR Green fluorescent dye).

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A miRNA biomarker for diagnosing polycystic ovary syndrome, characterized in that, The miRNA markers include one or a combination of two of hsa-miR-185-5p and hsa-miR-4433b-3p; The nucleotide sequence of hsa-miR-185-5p is shown in SEQ ID NO. 1, and the nucleotide sequence of hsa-miR-4433b-3p is shown in SEQ ID NO.

4.

2. The miRNA biomarker according to claim 1, characterized in that, The miRNA markers also include one or a combination of two of hsa-miR-199a-5p and hsa-miR-194-5p; The nucleotide sequence of hsa-miR-199a-5p is shown in SEQ ID NO. 2, and the nucleotide sequence of hsa-miR-194-5p is shown in SEQ ID NO.

3.

3. The use of the reagent for detecting the expression level of the miRNA biomarker as described in claim 1 or 2 in the preparation of products for diagnosing polycystic ovary syndrome; The reagents are antibodies for the miRNA markers, primers for PCR, reagents for mass spectrometry analysis, or reagents for chromatographic analysis. Preferably, the product includes a chip, test strip, formulation, reagent, kit, or high-throughput screening platform.

4. A kit for diagnosing polycystic ovary syndrome, characterized in that, Includes reagents for detecting the miRNA markers as described in claim 1 or 2; Preferably, the detection is a quantitative detection of the expression level of the miRNA marker in the subject's plasma or serum; The reagents are antibodies against the miRNA markers, primers for PCR, reagents for mass spectrometry, or reagents for chromatographic analysis.

5. The reagent kit according to claim 4, characterized in that, The kit is a PCR kit; Preferably, the PCR kit includes a primer set for detecting the miRNA marker.

6. The reagent kit according to claim 5, characterized in that, The primer set includes: specific reverse transcription primers for amplifying each miRNA marker and specific primers based on fluorescent dye PCR reactions; Preferably, the nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO. 5-6; Preferably, the nucleotide sequence of the specific primer is shown in SEQ ID NO. 7-9.

7. Use of the miRNA marker of claim 1 or 2 in the preparation of a formulation for the diagnosis of polycystic ovary syndrome.

8. A method for in vitro screening of candidate drugs for the treatment of polycystic ovary syndrome, characterized in that, The method includes the following steps: Treat the in vitro culture system expressing or containing the miRNA markers of claim 1 or 2 with the substance to be screened; Detect the expression level of the miRNA marker of claim 1 or 2 in the in vitro culture system; Wherein, when the substance to be screened inhibits the expression level of the miRNA marker of claim 1 or 2, the substance to be screened is a candidate drug for the treatment of polycystic ovary syndrome.

9. A risk prediction system for polycystic ovary syndrome, characterized in that, The risk prediction system includes an assessment unit; The evaluation unit predicts the risk of a subject having polycystic ovary syndrome by the expression level of the miRNA biomarker according to claim 1 or 2. Preferably, the assessment unit uses the following method to determine the risk of polycystic ovary syndrome: when the expression level of the miRNA marker described in claim 1 or 2 in the subject sample is significantly higher than the expression level of the miRNA marker in the normal human sample, the subject is judged to have a high risk of polycystic ovary syndrome.

10. The risk prediction system according to claim 9, characterized in that, The risk prediction system further includes a detection unit, which is used to detect the expression level of the miRNA biomarker according to claim 1 or 2; Preferably, the risk prediction system further includes a display unit for displaying the conclusions reached by the evaluation unit.

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