Azoospermia-related biomarker and use thereof

Abstract

This application relates to a biomarker associated with azoospermia and its application, specifically providing a biomarker for predicting sperm retrieval outcomes in azoospermia patients. The biomarker includes SOX9, DAZL, and SYCP3 molecules. The biomarker in this application was validated using immunofluorescence co-localization technology, demonstrating high specificity and sensitivity to target cells, with intuitive and reliable results. Compared to traditional staining methods, this application directly labels target cells with specific signals, significantly improving the objectivity, accuracy, and reproducibility of diagnosis. For the first time, it clearly links molecular pathological subtyping with clinical treatment and prognosis, providing patients with precise fertility solutions and achieving a closed loop from diagnosis to treatment.

Description

Technical Field

[0001] This application relates to the field of medical diagnostic technology, specifically to a biomarker related to azoospermia and its application. Background Technology

[0002] The pathological classification of azoospermia is mainly divided into: hypospermatogenesis, spermatogenesis arrest, and Sertoli cell-only syndrome.

[0003] The deleted-in-azoospermia-like (DAZL) gene in azoospermia is an important member of the DAZ gene family and a highly conserved gene. Located on an autosome, the DAZL gene encodes an RNA-binding protein that can bind to other mRNAs and is an essential regulatory factor for human germ cell formation. DAZL protein is expressed in spermatogonia and spermatocytes of patients with normal spermatogenesis, insufficient spermatogenesis, and maturation arrest, but is not expressed in the testes of patients with Sertoli cell-only syndrome. Detection of DAZL protein expression in testicular tissue allows for the identification and localization of spermatogonia within the seminiferous tubules.

[0004] Synaptonemal complex protein 3 (SYCP3) is a component of the synaptonemal complex, formed between homologous chromosomes during prophase of meiosis. This protein plays a role in centromere pairing during meiosis in male germ cells. SYCP3 itself forms a highly elongated helical tetramer structure, with its amino-terminal rod-like structure binding to the DNA double strand. Simultaneously, SYCP3 self-assembles into a network structure, using these two structures to firmly link two homologous chromosomes together. SYCP3 is essential for meiosis and is a crucial factor for successful homologous chromosome pairing; its absence leads to meiotic arrest, and even if a few cells do not arrest, they will ultimately only form aneuploid gametes.

[0005] Sertoli cells are the only somatic cells in the seminiferous tubules of the testis. They directly regulate the proliferation and differentiation of germ cells, maintain the typical seminiferous tubule structure, and induce germ cells to ensure sperm production in men. SRY-box transcription factor 9 (SOX9), as a transcription factor, is crucial for cell differentiation during embryogenesis of Sertoli cells in the testis. Since SOX9 expression is limited to Sertoli cells, it can serve as a specific marker for Sertoli cells in testis. By detecting SOX9 protein expression in testis, the identification and localization of Sertoli cells within the seminiferous tubules can be achieved.

[0006] The clinical diagnosis of azoospermia primarily relies on hematoxylin and eosin (HE) staining of testicular biopsy tissue for morphological observation. However, HE staining can only assess cell morphology and arrangement. When the cell arrangement within the seminiferous tubules is disordered or focal lesions are present, the accuracy and reproducibility of the diagnosis are severely challenged, easily leading to misdiagnosis. Furthermore, traditional methods struggle to precisely locate and differentiate supporting cells, spermatogenic cells, and spermatocytes, failing to provide reliable cytological evidence for subsequent personalized treatments (such as microsurgical sperm retrieval and in vitro spermatogenesis). Therefore, there is an urgent clinical need for a precise diagnostic tool capable of specifically identifying key cell types within the testes, objectively quantifying their state, and correlating them with clinical treatment outcomes. Summary of the Invention

[0007] To overcome the above-mentioned deficiencies, this application provides a method based on supporting cell nuclear marker SOX9, spermatogenic cytoplasmic marker DAZL, and spermatocyte nuclear marker SYCP3 as biomarkers: by detecting the expression of biomarkers, the pathological diagnosis and clinical treatment guidance of testicular tissue in patients with azoospermia can be determined.

[0008] A first aspect of this application provides the use of a biomarker detection reagent in the preparation of products for the typing, detection, and / or prediction of sperm collection efficacy in azoospermia, wherein the biomarkers include SOX9, DAZL, and SYCP3 molecules.

[0009] Preferably, the molecules include proteins, genes, and / or mRNA.

[0010] Preferably, the detection reagents include reagents for detecting SOX9, DAZL, and SYCP3 molecules.

[0011] Preferably, the detection reagent includes any detection reagent for SOX9, DAZL and SYCP3 molecules, such as antibodies against SOX9 protein, antibodies against DAZL protein and antibodies against SYCP3 protein, or substrates for SOX9, DAZL and SYCP3 proteins, primers or probes for detecting SOX9, DAZL and SYCP3 genes and / or mRNA.

[0012] Preferably, the substrate includes, but is not limited to, fluorescent markers or enzyme markers.

[0013] Preferably, the detection reagents include reagents used in immunofluorescence and / or immunohistochemical staining.

[0014] More preferably, the detection reagent can be prepared into a kit and / or chip, the kit and / or chip containing any of the detection reagents described above.

[0015] Preferably, the biomarker sample is derived from the testis.

[0016] Preferably, the azoospermia includes obstructive azoospermia (OA) or non-obstructive azoospermia (NOA).

[0017] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0018] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0019] For type I azoospermia, the treatment options are medication, sperm collection, or in vitro spermization (IVS), with medication or sperm collection being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0020] Furthermore, the typing, detection, and / or sperm collection effect prediction includes: detecting whether biomarkers are expressed in the sample to be tested.

[0021] Preferably, the typing, detection, and / or sperm collection effect prediction includes the amount of biomarker expression in the detection sample.

[0022] Preferably, the typing, detection, and / or sperm collection effect prediction also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0023] A second aspect of this application provides a product comprising a detection reagent for azoospermia-related biomarkers, the product being used for the typing, detection, and / or prediction of sperm retrieval outcomes in azoospermia patients, the biomarkers including SOX9, DAZL, and SYCP3 molecules.

[0024] Preferably, the molecules include proteins, genes, and / or mRNA.

[0025] Preferably, the biomarker sample is derived from the testis.

[0026] Preferably, the detection reagents include reagents for detecting SOX9, DAZL, and SYCP3 molecules.

[0027] Preferably, the detection reagent includes any detection reagent for SOX9, DAZL and SYCP3 molecules, such as antibodies against SOX9 protein, antibodies against DAZL protein and antibodies against SYCP3 protein, or substrates for SOX9, DAZL and SYCP3 proteins, primers or probes for detecting SOX9, DAZL and SYCP3 genes and / or mRNA.

[0028] Preferably, the substrate includes, but is not limited to, fluorescent markers or enzyme markers.

[0029] Preferably, the detection reagents include reagents used in immunofluorescence and / or immunohistochemical staining.

[0030] Preferably, the azoospermia includes obstructive azoospermia or non-obstructive azoospermia.

[0031] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0032] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0033] For type I azoospermia, the treatment options are medication, sperm collection, or in vitro spermization (IVS), with medication or sperm collection being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0034] More preferably, the product includes a kit and / or a chip, the kit and / or chip containing any of the above-mentioned detection reagents.

[0035] Furthermore, the typing, detection, and / or sperm collection effect prediction includes: detecting whether biomarkers are expressed in the sample to be tested.

[0036] Preferably, the typing, detection, and / or sperm collection effect prediction includes the amount of biomarker expression in the detection sample.

[0037] Preferably, the typing, detection, and / or sperm collection effect prediction also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0038] A third aspect of this application provides a biomarker associated with azoospermia, said biomarker including SOX9, DAZL and SYCP3 molecules.

[0039] Preferably, the molecules include proteins, genes, and / or mRNA.

[0040] Preferably, the biomarker sample is derived from the testis.

[0041] Preferably, the azoospermia includes obstructive azoospermia or non-obstructive azoospermia.

[0042] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0043] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0044] For type I azoospermia, the treatment options are medication, sperm retrieval, or in vitro spermatogenesis and spermiogenesis (IVS), with medication or sperm retrieval being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0045] A fourth aspect of this application provides the application of a biomarker in the preparation of products for the typing, detection, and / or prediction of sperm collection efficacy in azoospermia, wherein the biomarker includes SOX9, DAZL, and SYCP3 molecules, and the SOX9, DAZL, and SYCP3 molecules serve as targets for the typing, detection, and / or prediction of sperm collection efficacy in the products.

[0046] Preferably, the molecules include proteins, genes, and / or mRNA.

[0047] Preferably, the product includes detection reagents, which include reagents for detecting SOX9, DAZL, and SYCP3 molecules.

[0048] Preferably, the detection reagent includes any detection reagent for SOX9, DAZL and SYCP3 molecules, such as antibodies against SOX9 protein, antibodies against DAZL protein and antibodies against SYCP3 protein, or substrates for SOX9, DAZL and SYCP3 proteins, primers or probes for detecting SOX9, DAZL and SYCP3 genes and / or mRNA.

[0049] Preferably, the substrate includes, but is not limited to, fluorescent markers or enzyme markers.

[0050] Preferably, the detection reagents include reagents used in immunofluorescence and / or immunohistochemical staining.

[0051] Preferably, the biomarker sample is derived from the testis.

[0052] Preferably, the azoospermia includes obstructive azoospermia or non-obstructive azoospermia.

[0053] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0054] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0055] For type I azoospermia, the treatment options are medication, sperm collection, or in vitro spermization (IVS), with medication or sperm collection being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0056] More preferably, the detection reagent can be prepared into a kit and / or chip, the kit and / or chip containing any of the detection reagents described above.

[0057] Furthermore, the typing, detection, and / or sperm collection effect prediction includes: detecting whether biomarkers are expressed in the sample to be tested.

[0058] Preferably, the typing, detection, and / or sperm collection effect prediction includes the amount of biomarker expression in the detection sample.

[0059] Preferably, the typing, detection, and / or sperm collection effect prediction also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0060] A fifth aspect of this application provides a method for detecting azoospermia-related biomarkers, the method comprising using the product as described in the second aspect to detect the biomarkers.

[0061] A sixth aspect of this application provides a method for predicting the sperm collection effect in patients with azoospermia, the method comprising using the aforementioned product to detect the aforementioned biomarkers in the sample.

[0062] The products described in this application include all products related to azoospermia, and preferably, the products include, but are not limited to, reagent kits, diagnostic chips, etc.

[0063] A seventh aspect of this application provides a method for diagnosing and classifying azoospermia, the method comprising the following steps: a. Obtain testicular tissue samples from the subject; b. Perform immunofluorescence and / or immunohistochemical staining of the samples for biomarkers; c. Based on the staining results, determine the expression status and cellular localization of biomarkers to achieve specific identification and quantification of supporting cells, spermatogenic cells, and spermatocytes.

[0064] Preferably, the biomarker is as described in the third aspect.

[0065] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0066] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0067] For type I azoospermia, the treatment options are medication, sperm collection, or in vitro spermization (IVS), with medication or sperm collection being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0068] The eighth aspect of this application provides a treatment guideline for patients with azoospermia, the method comprising detecting the biomarkers described in the third aspect.

[0069] Preferably, the azoospermia is classified into the following types based on the expression of biomarkers: Type I, expression of SOX9, DAZL and SYCP3: Sertoli cells, spermatogenic cells and spermatocytes undergoing meiosis are present in the seminiferous tubules; Type II, SOX9 and DAZL expressed, SYCP3 not expressed: Sertoli cells and spermatogenic cells present, but spermatocytes absent; Type III, SOX9 expression, no DAZL and SYCP3 expression: only Sertoli cells are present, no spermatogenic cells or spermatocytes, which meets the diagnosis of "Sertoli cell only syndrome"; Type IV, no expression of SOX9, DAZL and SYCP3: seminiferous tubules show hyalinization and fibrosis, with no seminiferous epithelium.

[0070] Preferably, the spermatogenic cells include spermatogonial stem cells, spermatogonia, spermatocytes, and sperm cells.

[0071] For type I azoospermia, the treatment options are medication, sperm collection, or in vitro spermization (IVS), with medication or sperm collection being the preferred treatment. Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval, with IVS being the preferred treatment. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

[0072] The method described in this application can be for therapeutic or non-therapeutic purposes. This method only assesses which treatment strategies can be used to treat azoospermia-related conditions; that is, treatment effectiveness is not guaranteed. The evaluation of treatment strategies is merely a possibility.

[0073] The application described in this application may be for therapeutic or non-therapeutic purposes. This application is only for screening which products can be used for the classification, detection or prediction of sperm retrieval effects in azoospermia patients. That is, the diagnostic and / or therapeutic effects are not certain, but only a possibility.

[0074] The term “diagnosis” in this application means determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression or possible future progression of a disease.

[0075] The term "treatment" as used in this application refers to slowing, interrupting, preventing, controlling, stopping, reducing, mitigating, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0076] The term "effective amount" as used in this application refers to the amount or dose of the drug of this application that provides the desired treatment or prevention after being administered to an individual or organ in one or more doses.

[0077] The terms “comprising” or “including” in this application are open-ended descriptions that include the specified components or steps described, as well as other specified components or steps that do not materially affect them.

[0078] The term "and / or" as used in this application includes all combinations of items connected by the term, and should be regarded as each combination having been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0079] The beneficial technical effects of this application are as follows: Compared with the immunohistochemical detection method based on SOX9, DAZL, and SYCP3 specific antibodies described in this application, the traditional hematoxylin-eosin staining method has the following main drawbacks in the pathological diagnosis of azoospermia, which highlight the necessity and significant advancement of the technical solution in this application: 1. Lacks molecular specificity, making it impossible to accurately pinpoint cell types. HE staining: This method relies solely on the morphology, size, location, and staining intensity of the cell nucleus (blue-purple) and cytoplasm (pink). In testicular tissue, distinguishing between Sertoli cells, spermatogonia, and spermatocytes highly depends on their classic, regularly arranged morphological characteristics.

[0080] Disadvantages: When cells are disordered, development is arrested, or disease occurs, cell morphology may become atypical or similar, making it impossible to reliably distinguish between supporting cells and immature spermatogenic cells, spermatogenic cells at different stages, or even interstitial cells. In contrast, IHC can clearly "mark" target cells using specific antibodies (such as anti-SOX9), unaffected by morphological abnormalities.

[0081] 2. Diagnosis relies heavily on subjective experience, resulting in poor consistency and reproducibility. HE staining: Interpretation relies entirely on the pathologist's visual observation and experience, making it a subjective qualitative analysis. Significant inter-observer variability: Different pathologists may reach different conclusions from the same HE slide, especially in complex cases. Observer-specific variability: Even the same pathologist may have biases when interpreting the same slide at different times. High training costs: Long-term specialized training is required to master the fine morphological interpretation of testicular seminiferous epithelium. In contrast, the positive signal (brown) of IHC is objective and conspicuous, significantly lowering the interpretation threshold and improving consistency in interpretation between different observers and at different time points.

[0082] 3. It is not sensitive to early or focal lesions and is easily missed. HE staining: It can only be detected when there are obvious changes in cell morphology (such as vacuolization, shedding, or obvious disordered arrangement).

[0083] Disadvantages: HE staining is prone to missing early lesions or focal lesions where molecular abnormalities have been observed but morphological changes are not yet obvious. For example, spermatogenesis may arrest in the early stages of meiosis (SYCP3 expression may already be abnormal), but cell morphology may not show significant abnormalities under a light microscope. IHC, on the other hand, can directly reveal functional abnormalities through the absence of marker expression (such as SYCP3-) before morphological changes occur.

[0084] 4. Inability to perform semi-quantitative or quantitative analysis. HE staining: It can usually only provide qualitative descriptions (such as "present" or "absent") or rough grading (such as "slight reduction"), and it is difficult to perform precise cell counting.

[0085] Disadvantages: It cannot accurately quantify key pathological parameters such as the number of supporting cells and the ratio of spermatogenic cells to supporting cells. However, IHC combined with image analysis software can count and analyze the density of positive cells, providing semi-quantitative and even quantitative data, making the diagnosis more precise and providing more accurate indicators for prognostic assessment.

[0086] 5. Lack of direct molecular association with downstream treatment decisions HE staining: Diagnostic conclusions (such as "Serpentine cell-only syndrome" or "low spermatogenesis") are based on a comprehensive morphological assessment, and their correlation with specific treatment options (especially sperm retrieval success rate) is empirical and indirect.

[0087] Disadvantages: It cannot provide accurate typing based on specific molecular marker expression combinations as described in this application, making it difficult to directly and powerfully predict the success probability of different treatment methods (such as sperm retrieval and IVS), thus limiting its guiding value.

[0088] Traditional HE staining, as a fundamental morphological technique, suffers from significant drawbacks in the diagnosis of azoospermia due to its "non-specificity" and "subjectivity." It's like navigating a complex, unlabeled map using topographical features, easily becoming lost in a chaotic landscape. In contrast, the IHC technology employed in this application adds specific "molecular tags" to key landmarks (Stemonae cells, Spermatogenic cells, and Spermatocytes) on the map, making localization direct, objective, and accurate. This not only solves the diagnostic challenges of HE staining in difficult cases but also closely links diagnostic results with individualized treatment pathways, achieving a paradigm shift from morphological description to functional guidance.

[0089] In summary, this application includes the following advantages, contributions to the prior art, or beneficial technical effects: 1. High specificity and high sensitivity: Immunofluorescence co-localization techniques (such as co-localization of SOX9 nuclear staining and DAZL cytoplasmic staining, and co-localization of SOX9 nuclear staining and SYCP3 nuclear staining) have confirmed that the selected markers are highly specific to the target cell types, and the detection sensitivity meets the requirements of pathological detection, with intuitive and reliable results.

[0090] 2. Improved Diagnostic Accuracy and Operability: Immunohistochemical staining results showed that the diagnostic results of this combination were consistent with those of immunofluorescence. Compared with traditional HE staining, this application directly "locks on" target cells through specific brown signals (IHC) or fluorescence signals, significantly reducing the difficulty and workload of morphological interpretation caused by cell arrangement disorder or focal lesions. It does not rely on the observer's experience and greatly improves the objectivity, accuracy, and reproducibility of diagnosis.

[0091] 3. Direct Guidance for Clinical Treatment: This application is the first to explicitly link specific molecular pathological subtypes with clinical treatment pathways and prognoses (sperm retrieval success rate, IVS applicability). It provides clinicians with quantitative decision-making support, helping to avoid unnecessary surgical trauma and enabling patients to choose the most suitable and economical fertility solutions, thus achieving a precision medicine closed loop from diagnosis to treatment. Attached Figure Description

[0092] The embodiments of this application will be described in detail below with reference to the accompanying drawings: Figure 1 HE image under 10x magnification, showing the morphology of various cells in the seminiferous tubules.

[0093] Figure 2 HE image under 20x magnification, showing the morphology of various cells in the seminiferous tubules.

[0094] Figure 3A Immunofluorescence co-staining pattern, showing SOX9 (red, nucleus), located in the nucleus of supporting cells.

[0095] Figure 3B Immunofluorescence co-staining pattern showing DAZL (green, cytoplasm), located in the cytoplasm of spermatogenic cells.

[0096] Figure 3C Immunofluorescence co-staining image, showing Hurst staining (blue, nucleus), locating the nuclei of all cells.

[0097] Figure 3D Immunofluorescence co-staining image, a composite image, showing the co-localization of SOX9 and DAZL in the seminiferous tubules.

[0098] Figure 4A Immunofluorescence co-staining image, showing SYCP3 (red, nucleus), located in the spermatocyte nucleus.

[0099] Figure 4B Immunofluorescence co-staining pattern showing DAZL (green, cytoplasm), located in the cytoplasm of spermatogenic cells.

[0100] Figure 4C Immunofluorescence co-staining image, showing Hurst staining (blue, nucleus), locating the nuclei of all cells.

[0101] Figure 4D Immunofluorescence co-staining image, a composite image, showing the co-localization of SYCP3 and DAZL in the seminiferous tubules.

[0102] Figure 5 HE staining results of sample 101 under 100x magnification: J score is 2, showing only Sertoli cells and no spermatogenic cells.

[0103] Figure 6 HE staining results of sample 101 under a 400x microscope.

[0104] Figure 7 : This indicates that SYCP3 is located in the spermatocyte nucleus and appears only in local tissue areas.

[0105] Figure 8: This shows that DAZL is located in the cytoplasm of spermatogenic cells, consistent with the location of SYCP3, and appears only in local tissues. Detailed Implementation

[0106] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application. Example 1: Biomarker Specificity Validation

[0107] Testicular biopsy tissue from patients clinically diagnosed with azoospermia was subjected to multicolor immunofluorescence staining. Primary antibodies included anti-SOX9, anti-DAZL, and anti-SYCP3, while secondary antibodies were labeled with different fluorophores.

[0108] 1 Fixed tissue Testicular tissue was obtained from patients with azoospermia (tissue acquisition methods: testicular incision, testicular puncture, testicular aspiration, etc.) and fixed with 10% neutral formalin fixative for 24 hours.

[0109] 2 embeddings The fixed tissue was dehydrated and embedded in paraffin to prepare tissue blocks.

[0110] 3 slices The paraffin blocks were prepared into tissue sections, and DAZL protein was detected using immunofluorescence.

[0111] 4. Baked Sheets Place the slides on a staining rack and bake them in a 60°C oven for 1 hour.

[0112] 5. Dewaxing and Rehydration Place the staining racks into the corresponding staining tanks in the following order for dewaxing and rehydration of the paraffin sections. Xylene 1 10min Xylene 2 10min Anhydrous ethanol 1 5 min Anhydrous ethanol 2.5 min; 90% ethanol for 5 minutes 80% ethanol for 5 minutes 70% ethanol for 5 minutes 50% ethanol for 5 minutes Purified water 5 min Purified water 5 min Purified water 5 min 6 Antigen Repair Place 500ml of antigen retrieval solution into an antigen retrieval container (stainless steel medical alcohol container) and heat it directly to boiling on an induction cooker. Then place the antigen retrieval container in the boiling pot and continue heating for approximately 10 minutes. Next, place the staining rack (containing tissue sections) into the antigen retrieval container, cover, and continue heating for 20 minutes (antigen retrieval time). After this, remove the antigen retrieval container and allow it to cool naturally. Once cooled to room temperature, place the staining rack in a staining jar containing TBST solution and gently agitate and wash it three times on a shaker for 5 minutes each time.

[0113] Antigen retrieval buffer composition: Citric acid 0.36g 3g sodium citrate 1L of distilled water pH 6.0 Preparation of TBST solution: Take TBS buffer (ready-to-use dry powder), dissolve it in 2000 mL of deionized water, add 1 mL of LTween-20, and mix well.

[0114] 7 Transparency After drying the tissue sections, draw circles around the tissue sections with an immunohistochemical pen, making two sections (note the size, each section should hold 20 μL). Place the tissue sections flat in a humidified chamber (with a small amount of water added). Then, add 20 μL of 1% (v / v) Triton X-100 solution (prepared with 1×PBS) to each circle of the tissue section. After permeation at room temperature for 10 min, place the tissue sections on a staining rack and then place them in a staining jar containing TBST solution. Gently shake and wash the sections three times on a shaker for 5 min each time.

[0115] Preparation of 1% (v / v) Triton X-100 solution: 100 μL Triton X-100 + 9.9 mL 1×PBS 8 closed After drying the tissue sections, place them flat in a humidified chamber. Add 20 μL of blocking solution to each circle of the tissue section and incubate at room temperature for 1 hour.

[0116] Blocking buffer preparation: 30 g / L BSA + 0.1% Proclean 300 (V / V) + 1×PBS (0.3g BSA+10μL proclean300+10mL 1×PBS) 9. Primary antibody incubation Primary antibody dilution: Antibodies were diluted using blocking buffer: SYCP3 rabbit multi-antibody (1:2000), SOX9 rabbit monoclonal antibody (1:4000), DAZL mouse monoclonal antibody (1:2000). After gently wiping off the blocking solution, place the tissue section flat in a humidified chamber, add 20 μL of primary antibody working solution to the circle of the tissue section, and incubate overnight at 4°C.

[0117] 10 Second Antibody Incubation Secondary antibody dilution: The secondary antibodies goat anti-rabbit - Alexa Fluor™ 555 (catalog number A-21428) and goat anti-mouse - Alexa Fluor™ 488 (catalog number A0428) were diluted 1:500 using blocking buffer.

[0118] After placing the tissue sections on a staining rack, place them in a staining jar containing TBST solution and gently agitate and wash them three times for 5 minutes each time on a shaker. After drying the tissue sections, place them flat in a humidified chamber and add 20 μL of secondary antibody solution (prepared blocking solution) to the circle of the tissue section. Incubate at room temperature in the dark for 1 hour.

[0119] 11 Hoechst staining Hearst solution: Dilute Hearst dye 1:5000 using TBST.

[0120] After placing the tissue sections on a staining rack, place them in a staining jar containing TBST solution and gently agitate and wash them three times on a shaker for 5 minutes each time. After drying the tissue sections, place them flat in a humidified chamber and add 20 μL of Hurst's solution (prepared with TBST) to each circle of the tissue section. Incubate at room temperature in the dark for 10 minutes.

[0121] 12 Producers After placing the tissue sections on a staining rack, place them in a staining jar containing TBST solution and gently agitate and wash them three times on a shaker for 5 minutes each time. After drying the tissue sections, lay them flat in a dry box, add one drop of anti-quenching agent to the circle of the tissue section, and then cover with a coverslip to complete the slide preparation.

[0122] Note: This step must be completed in a dark environment.

[0123] 13 Microscopic photographs Immunofluorescence staining results of tissue sections were captured using a fluorescence microscope.

[0124] HE staining results are as follows Figures 1-2 Immunofluorescence staining results are as follows Figures 3A-3D , Figures 4A-4DAs shown, SOX9 specifically localizes to the nucleus of Sertoli cells, DAZL specifically localizes to the cytoplasm of spermatogenic cells such as spermatogonia and spermatocytes, and SYCP3 specifically localizes to the nucleus of spermatocytes. Co-staining images clearly show that SOX9+ nuclei and DAZL+ cytoplasm are present in Sertoli cells and spermatogenic cells, respectively, and are thus distinguished. DAZL+ cytoplasm and SYCP3+ nuclei are present in the cytoplasm and nucleus of spermatocytes, respectively, and are thus distinguished. These results confirm that the methodological specificity and sensitivity of the three antibodies meet the requirements for co-staining and co-localization.

[0125] Example 2: Study on the Accuracy of Clinical Diagnosis For patients with azoospermia, diagnostic testicular / epididymal sperm retrieval may be considered for diagnostic and treatment purposes. Open incision or percutaneous testicular aspiration is commonly used, followed by histopathological examination with hematoxylin and eosin (HE) staining. The Johnsen score is recommended for testicular biopsy pathology results. The Johnsen score is a method for assessing testicular spermatogenesis through testicular biopsy, using a 10-point scale to quantify the health status of spermatogenesis. This scoring system is based on the quantity and morphology of various germ cells in testicular tissue, providing physicians with intuitive and accurate diagnostic information. A higher score indicates better testicular spermatogenesis; conversely, a lower score suggests impaired spermatogenesis.

[0126] Table 1. Testicular biopsy pathology results. Recommended use of the Johnsen scoring method.

[0127] Immunohistochemistry utilizes the principle of specific binding between antigens and antibodies in immunology. It detects tissue cell antigens by detecting the color of enzymes (horseradish peroxidase, HRP) labeled on antibodies and chromogenic substances through a chemical reaction, allowing for their localization and characterization. After dewaxing, hydration, and retrieval, tissue sections are incubated with SYCP3 antibody reagent, which binds to the SYCP3 antigen on the tissue. Then, enzyme-labeled secondary antibody is added, binding to the primary antibody molecule. Finally, DAB chromogenic solution is added; the horseradish peroxidase on the secondary antibody polymer catalyzes the decomposition of H2O2 in the DAB chromogenic solution, causing DAB precipitation and resulting in a brownish-yellow staining at the antigen sites in the tissue sections. After counterstaining and mounting the samples, the staining of SYCP3 protein on the tissue sections is observed and analyzed using an optical microscope. The principles of DAZL and SOX9 antibody reagents are the same.

[0128] 1. Experimental reagents: Anhydrous ethanol, xylene, dewaxing solution, washing solution, immunohistochemical antigen retrieval buffer, immunochromatographic reagents (including peroxidase blocking agent, primary antibody post-reagent, polymer, DAB solution, hematoxylin), SYCP3 antibody, SOX9 antibody, DAZL antibody, glycine, BSA, ProClean300, 1×PBS.

[0129] 2. Experimental apparatus Forced drying oven, fume hood, BOND-III fully automated immunohistochemistry and in situ hybridization staining platform, fluorescence microscope.

[0130] 3. Primary antibody reagent: Antibody working solution preparation: 0.5% glycine, 1% BSA, ProClean300, 1×PBS, SYCP3 rabbit multi-antibody (1:2000), SOX9 rabbit monoclonal antibody (1:4000), DAZL mouse monoclonal antibody (1:2000).

[0131] 4. Experimental steps: According to the instructions for the staining machine and its general reagents, add the SOX9 antibody working solution, SYCP3 antibody working solution, DAZL antibody working solution, dewaxing solution, immunohistochemical antigen retrieval buffer, washing solution (1×), and immunochromatographic reagent to the corresponding containers on the staining machine, and place the reagent containers into the reagent rack.

[0132] Place the test sample slides and negative / positive control slides in a 60℃ oven and bake for 1 hour.

[0133] Set the staining program in the staining machine software according to the table below, and place the sample slide to be tested on the staining rack to start staining.

[0134] Table 2 Staining Procedure

[0135] Soak and dehydrate the slides in the following order: 75% ethanol, 95% ethanol, anhydrous ethanol, and xylene (twice). Rinse the slides with xylene, then add a certain amount of neutral resin, cover with a coverslip, and allow to air dry before storage or observation. Store the slides at room temperature away from light.

[0136] Testicular biopsy tissues were collected from 23 patients with azoospermia and subjected to HE staining (gold standard) and immunohistochemical staining for SOX9, DAZL, and SYCP3, as described in this application. Figures 5-8The results in Tables 3-7 show that in samples with basically normal seminiferous tubule structure, the diagnostic concordance rate between IHC and HE is high. However, in samples with disordered arrangement of spermatogenic cells or focal developmental arrest, HE staining is difficult to interpret, and the diagnostic consistency among physicians is low. In particular, for specimens positive for SOX9+, DAZL+, and SYCP3+, the HE staining scores vary greatly, ranging from a very low score of 2 (101 specimens) to a very high score of 9 (2 and 3 specimens). In contrast, IHC staining, through a specific brown signal, can clearly and consistently identify Sertoli cells (SOX9+), the presence or absence of spermatogenic cells (DAZL+ / -), and spermatocytes (SYCP3+ / -), providing a clear diagnostic result. This indicates that the method of the present invention has higher sensitivity than HE.

[0137] Table 3. Johnsen score and IHC staining results

[0138] Reference method – usually refers to the “gold standard” or the current standard method.

[0139] Candidate method – A new method or system to be validated or evaluated.

[0140] In qualitative scenarios, the most commonly used indicator of "consistency" is "agreement rate" or "positive / negative agreement rate".

[0141] A four-line table is equivalent to a 2x2 contingency table (four-cell table).

[0142] Table 4. Four-cell table

[0143] Table 5. Results of SYCP3 Consistency Indicators

[0144] Sensitivity: 100.00%, 95% confidence interval: 75.29% to 100.00%; Specificity: 90.00%, 95% confidence interval: 55.50% to 99.75%; Positive likelihood ratio: 10.00, 95% confidence interval: 1.56 to 64.20; Positive predictive value: 92.86%, 95% confidence interval: 66.94% to 98.82%; Negative predictive value: 100.00%, 95% confidence interval: 66.37% to 100.00%; Accuracy: 95.65%, 95% confidence interval: 78.05% to 99.89%.

[0145] Table 6. Results of DAZL Consistency Index

[0146] Sensitivity: 100.00%, 95% confidence interval: 75.29% to 100.00%; Specificity: 90.00%, 95% confidence interval: 55.50% to 99.75%; Positive likelihood ratio: 10.00, 95% confidence interval: 1.56 to 64.20; Positive predictive value: 92.86%, 95% confidence interval: 66.94% to 98.82%; Negative predictive value: 100.00%, 95% confidence interval: 66.37% to 100.00% Accuracy: 95.65%, 95% confidence interval: 78.05% to 99.89%.

[0147] Table 7. Results of SOX9 Consistency Indicators

[0148] Sensitivity: 100.00%, 95% confidence interval: 83.89% to 100.00%; Specificity: 100.00%, 95% confidence interval: 15.81% to 100.00%; Positive predictive value: 100.00%, 95% confidence interval: 83.89% to 100.00%; Negative predictive value: 100.00%, 95% confidence interval: 15.81% to 100.00%; Accuracy: 100.00%, 95% confidence interval: 85.18% to 100.00%.

[0149] Currently, the J score in clinical practice adopts the HE (Hematologic Analysis) results. The above results indicate that the HE and IHC results have a good concordance rate and high consistency, making them clinically interchangeable. Furthermore, this method avoids the reliance on operator experience inherent in the HE method; it is more objective and reproducible. Further analysis of sample 101, which showed discrepancies, demonstrated that the IHC combined with biomarkers is more sensitive and accurate than HE.

[0150] Further investigation was conducted on the differences in repeatability between different testing institutions and personnel using the DAZL, SYCP3, and SOX9 detection kit (which includes DAZL, SYCP3, and SOX9 antibodies) for IHC staining and HE staining.

[0151] Seven testicular tissue samples from different spermatogenic stages were selected. IHC staining and HE staining slides from DAZL, SYCP3, and SOX9 detection kits were interpreted at four different testing institutions. The results, shown in Tables 8-10, indicate that the interpretations of DAZL, SYCP3, and SOX9 IHC staining by the four pathologists at the four institutions were consistent. However, the interpretations of HE staining showed some differences. This suggests that the IHC staining results for DAZL, SYCP3, and SOX9 are highly consistent across different medical institutions and by different testing personnel, demonstrating good reproducibility.

[0152] Table 8. Differences in the detected structures based on DAZL IHC staining and HE results.

[0153] Table 9. Differences in the detected structures between SYCP3 IHC staining and HE results

[0154] Table 10. Differences in the detected structures based on SOX9 IHC staining and HE results.

[0155] Example 3: A prospective study on the therapeutic guidance value A case-control study included 114 male infertility patients with azoospermia who visited a male infertility clinic between 2023 and 2025. Patients underwent microsurgical sperm retrieval, and tissue samples were sent to pathologists for histopathological diagnosis of azoospermia type. Immunohistochemical staining with SOX9, DAZL, and SYCP3, as described in this application, was performed for biopsy typing. The results of microsurgical sperm retrieval for each patient and the diagnostic results of this combination are summarized below: Of the 59 patients with type I (SOX9+, DAZL+, SYCP3+), sperm was successfully retrieved in 45 cases using microsurgical sperm retrieval, with a success rate of approximately 76.27%.

[0156] There were 0 patients with type II (SOX9+, DAZL+, SYCP3-).

[0157] Forty-six patients with type III (SOX9+, DAZL-, SYCP3-) were diagnosed with Sertoli cell-only syndrome. Only 11 of them successfully retrieved sperm via microsurgical sperm retrieval, a success rate of only about 23.9%.

[0158] Nine patients with type IV (SOX9-, DAZL-, SYCP3-) had hyalinization in their testicular tissue. Only one of them successfully retrieved sperm via microsurgical sperm extraction, resulting in an extremely low success rate of 11.11%.

[0159] Table 11: Four clinical subtyping models based on the combined detection results of this application and their corresponding treatment guidance pathways

[0160] This study demonstrates that the application can provide biomarkers to accurately determine the stage of spermatogenesis failure, thereby predicting the outcome of testicular sperm extraction with better diagnostic performance. It has significant predictive value for the success rate of microsurgical sperm retrieval and can effectively guide the selection of subsequent treatment pathways, avoiding ineffective medical treatment.

[0161] The scope of protection claimed in this application is not limited to the SOX9, DAZL, and SYCP3 proteins disclosed in the above specific embodiments, but also includes other biomarkers with the same or similar functions and equivalent substitution capabilities. The localization of these three targets on specific cells within tissues is applicable not only to fluorescence staining methodologies but also to immunohistochemical staining. The expression patterns of these three target proteins are highly suitable for combined use in clinical diagnostic testing techniques, as well as any modifications, applications, and products that utilize this principle for guiding the diagnosis and treatment of azoospermia.

[0162] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0163] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. Use of a biomarker detection reagent in the manufacture of a product for the typing, detection and / or prediction of the effect of sperm retrieval in azoospermia, characterized in that, The biomarkers mentioned include SOX9, DAZL, and SYCP3 molecules.

2. Use according to claim 1, characterized in that, The molecules mentioned include proteins, genes, and / or mRNA.

3. Use according to claim 1, characterized in that, The detection reagents include antibodies against SOX9 protein, DAZL protein, and SYCP3 protein, or substrates for SOX9, DAZL, and SYCP3 proteins, and primers or probes for detecting SOX9, DAZL, and SYCP3 genes and / or mRNA.

4. Use according to claim 1, characterized in that, The detection reagents include reagents used in immunofluorescence and / or immunohistochemical staining.

5. The use according to claim 1, characterized in that, The biomarker samples were derived from the testes.

6. Use according to any one of claims 1 to 5, characterized in that, The aforementioned azoospermia is classified into the following types based on the expression of biomarkers: Type I, SOX9, DAZL and SYCP3 expression; Type II, SOX9 and DAZL expressed, SYCP3 not expressed; Type III, SOX9 expressed, DAZL and SYCP3 not expressed; Type IV, no expression of SOX9, DAZL and SYCP3; For type I azoospermia, treatment options include medication, sperm retrieval, or in vitro spermization (IVS). Type II azoospermia can be treated with in vitro fertilization (IVS) or sperm retrieval. Type III and IV azoospermia patients do not undergo sperm retrieval surgery.

7. Use according to claim 6, characterized in that, The treatment options for type I azoospermia are medication or sperm collection. The aforementioned type II azoospermia patients opted for in vitro spermization (IVS).

8. A product characterized by, The product includes a reagent for detecting biomarkers associated with azoospermia, including SOX9, DAZL, and SYCP3 molecules.

9. The product of claim 8, wherein, The detection reagents include antibodies against SOX9 protein, DAZL protein, and SYCP3 protein, or substrates for SOX9, DAZL, and SYCP3 proteins, and primers or probes for detecting SOX9, DAZL, and SYCP3 genes and / or mRNA.

10. The product according to any one of claims 8-9, characterized in that, The detection reagents include reagents used in immunofluorescence and / or immunohistochemical staining.

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