A biomarker for spermatogenesis disorders and its application

Abstract

This invention provides a biomarker for spermatogenesis disorders and its application, wherein the biomarker is the MNDA molecule. This application characterizes the functional abnormalities or spermatogenesis disorders of Sertoli cells by detecting the expression level of MNDA molecules on cells in testicular tissue sections, and can therefore be used for the clinical diagnosis of non-obstructive azoospermia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomarker for spermatogenesis disorders and its application. Background Technology

[0002] Azoospermia refers to the absence of sperm in semen, and is mainly divided into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). OA patients have normal spermatogenesis and development, and fertility can be restored after removing the obstructive factors. NOA, on the other hand, is caused by testicular factors, characterized by abnormal testicular abnormalities leading to the absence or obstruction of sperm production. Currently, NOA accounts for up to 60% of azoospermia cases, making it one of the leading causes of male infertility. Its etiology is complex, and there is currently no effective clinical treatment.

[0003] NOA patients primarily rely on testicular sperm retrieval for reproduction, a procedure that is technically challenging and has a low success rate. Therefore, accurately assessing the spermatogenesis status of NOA patients is a prerequisite for conducting appropriate sperm retrieval surgery. Consequently, pathological examination of testicular aspiration samples from NOA patients is particularly important, serving as crucial reference material for physicians to assess the development of germ cells. Currently, pathological diagnosis of testicular aspiration samples mainly relies on a rough visual observation of the cellular structure within the seminiferous tubules using HE staining. This method provides relatively few indicators and cannot accurately determine the specific characteristics of spermatogenesis blockage within the seminiferous tubules.

[0004] Sertoli cells, also known as testicular supporting cells, are the only known gonadal cells that coexist with germ cells in the seminiferous tubules, playing a crucial role in regulating germ cell growth and development and triggering spermatogenesis. During the maturation of sex organs from puberty to adulthood, Sertoli cells also undergo a differentiation process from a pre-pubescent immature state to a mature state (a complex transition involving cell proliferation, morphological changes, protein expression, tight junction tissue, and blood-testis barrier penetration) to the mature adult state (Mature-Sertoli). The differentiation and maturation of Sertoli cells during puberty are closely related to normal spermatogenesis, while abnormal maturation can lead to a series of symptoms of testicular hypoplasia. Studies have shown that Sertoli cells in NOA patients (NOA-Sertoli) are in an abnormal immature state (Liangyu Zhao, et al. 2020, Single-cell analysis of developing and azoospermia human testicles reveals central role of Sertoli cells). This suggests that along with the spermatogenesis disorders in NOA patients, their Sertoli cells also exhibit physiological abnormalities. However, currently, there are no detection technologies or diagnostic indicators for the abnormal physiological state of Sertoli cells in NOA patients.

[0005] Therefore, it is still necessary to develop new clinical testing technologies or pathological indicators to more comprehensively and accurately reflect the pathological type of spermatogenesis disorders in patients, thereby providing more assistance for subsequent implementation of reasonable clinical diagnosis and treatment measures. Summary of the Invention

[0006] To overcome the above-mentioned defects, this invention provides a novel pathological marker for azoospermia, MNDA, based on Sertoli cells: the abnormal state of Sertoli cell function is characterized by detecting the expression level of MNDA on cells in testicular tissue sections.

[0007] In a first aspect, the present invention provides the use of a biomarker in the preparation of products for the diagnosis or prognostic assessment of spermatogenesis disorders, said biomarker including the myeloid cell nuclear differentiation antigen MNDA molecule, said MNDA molecule serving as a target for diagnosis or prognostic assessment.

[0008] Preferably, the MNDA molecule includes the MNDA protein, gene, and / or mRNA.

[0009] Preferably, compared to healthy samples, the biomarker is upregulated in samples with spermatogenesis disorders.

[0010] Preferably, the immunohistochemical results of the biomarkers in the test sample are positive compared to healthy samples.

[0011] Preferably, the biomarker is highly expressed in the test sample compared to a healthy sample.

[0012] Preferably, the biomarker sample is derived from testicular tissue; more preferably, the sample is derived from Sertoli cells of the testicular tissue.

[0013] Preferably, the MNDA molecule is specifically expressed in Sertoli cells.

[0014] Preferably, the MNDA molecule is expressed at low levels in spermatogenic cells. More preferably, the spermatogenic cells include spermatogonia, premeiotic spermatocytes, meiotic spermatocytes, haploid spermatocytes, and deformed spermatocytes, etc.

[0015] Preferably, the biomarkers also include other specific biomarkers of spermatogenic cells at various stages and / or Sertoli cells, such as melanoma-associated antigen A4 (MAGEA4) for spermatogonia, testis-expressed protein 19 (TEX19) for premeiotic spermatocytes, spermatogenesis-associated protein 8 (SPATA8) for meiotic spermatocytes, sperm acrosome-associated protein 4 (SPACA4) for haploid spermatocytes, transition protein 1 (TNP1) for amorphous spermatocytes, and SRY-box transcription factor 9 (SOX9) for Sertoli cells.

[0016] Preferably, the MNDA molecule is upregulated in non-obstructive azoospermia.

[0017] Furthermore, the diagnostic or prognostic assessment includes: detecting biomarkers in the test sample; and comparing the expression levels of biomarkers in the test sample and healthy samples.

[0018] Preferably, the detected biomarker is the amount of expression of the biomarker in the sample, and the amount of expression can be displayed by the fluorescence intensity of immunohistochemistry.

[0019] Preferably, if the expression of the biomarkers is upregulated in the test sample compared to a healthy sample, it can be diagnosed as a spermatogenesis disorder, or the expected treatment effect is poor;

[0020] Preferably, a high expression of the biomarker in the test sample compared to a healthy sample can be diagnosed as a spermatogenesis disorder.

[0021] Preferably, the prognosis of spermatogenesis disorders can be assessed. For example, high expression of the biomarker in the test sample indicates a low probability of obtaining sperm in the sperm retrieval procedure, while no expression or low expression of the biomarker in the test sample indicates a high probability of obtaining sperm in the sperm retrieval procedure.

[0022] Preferably, the spermatogenesis disorder includes azoospermia; more preferably, the azoospermia is non-obstructive azoospermia; and more preferably, the non-obstructive azoospermia includes, but is not limited to, spermatocyte blockade type, spermatogonial / spermatomatous blockade type, and Sertoli cell-only syndrome type of azoospermia.

[0023] Preferably, the spermatogenesis disorder includes moderate or severe spermatogenesis disorder, such as severe spermatogenesis disorder.

[0024] Preferably, the diagnostic or prognostic assessment also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0025] A second aspect of the present invention provides the use of a biomarker detection reagent in the preparation of products for the diagnosis or prognostic assessment of spermatogenesis disorders, wherein the biomarker includes the MNDA molecule.

[0026] Preferably, the biomarker sample is derived from testicular tissue; more preferably, the sample is derived from Sertoli cells of the testicular tissue.

[0027] Preferably, the MNDA molecule is specifically expressed in Sertoli cells.

[0028] Preferably, the MNDA molecule is expressed at low levels in spermatogenic cells. More preferably, the spermatogenic cells include spermatogonia, premeiotic spermatocytes, meiotic spermatocytes, haploid spermatocytes, and deformed spermatocytes, etc.

[0029] Preferably, the biomarkers also include other specific biomarkers of spermatogenic cells at various stages and / or Sertoli cells, such as spermatogonial specific biomarker MAGEA4, premeiotic spermatocyte specific biomarker TEX19, meiotic spermatocyte specific biomarker SPATA8, haploid spermatocyte specific biomarker SPACA4, amorphous spermatocyte specific biomarker TNP1, and Sertoli cell specific biomarker SOX9.

[0030] Preferably, the spermatogenesis disorder includes azoospermia; more preferably, the azoospermia is non-obstructive azoospermia; and more preferably, the non-obstructive azoospermia includes, but is not limited to, spermatocyte blockade type, spermatogonial / spermatomatous blockade type, and Sertoli cell-only syndrome type of azoospermia.

[0031] Preferably, the spermatogenesis disorder includes moderate or severe spermatogenesis disorder, such as severe spermatogenesis disorder.

[0032] Preferably, the detection reagent is used to detect the expression level of a biomarker in a sample, and the expression level can be indicated by the fluorescence intensity of immunohistochemistry.

[0033] Preferably, the MNDA molecule is upregulated in non-obstructive azoospermia.

[0034] Furthermore, the diagnostic or prognostic assessment includes: detecting biomarkers in the test sample; and comparing the expression levels of biomarkers in the test sample and healthy samples.

[0035] Preferably, when the expression of the biomarkers is upregulated in the test sample compared to a healthy sample, it can be diagnosed as a spermatogenesis disorder, or the expected treatment effect is poor;

[0036] Preferably, high expression of the biomarkers in the test sample compared to a healthy sample can be diagnosed as spermatogenesis disorder, or...

[0037] Preferably, the prognosis of spermatogenesis disorders can be assessed. For example, high expression of the biomarker in the test sample indicates a low probability of obtaining sperm in the sperm retrieval procedure, while no expression or low expression of the biomarker in the test sample indicates a high probability of obtaining sperm in the sperm retrieval procedure.

[0038] Preferably, the diagnostic or prognostic assessment also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0039] Preferably, the MNDA molecule includes the MNDA protein, gene, and / or mRNA.

[0040] Preferably, the detection reagent includes reagents for detecting the molecular level.

[0041] Depending on the specific implementation requirements, the detection reagent can be any reagent in the prior art, as long as it can detect the molecular level.

[0042] Preferably, the detection reagent includes reagent components used in chromatography, capillary electrophoresis, liquid chromatography-mass spectrometry, biosensor methods, immunoassays, or nucleic acid detection methods. The detection reagent refers to the key reagent components used in the corresponding detection method.

[0043] More preferably, the chromatographic method includes ion exchange chromatography, liquid chromatography, gas chromatography, etc.

[0044] Preferably, the detection reagent includes any detection reagent for the molecule, such as antibodies or substrates for detecting proteins, primers or probes for detecting genes and / or mRNA.

[0045] Preferably, the detection reagents include reagent kits for detecting biomarkers, diagnostic chips, etc.

[0046] A third aspect of the present invention provides a product comprising a detection reagent for biomarkers related to spermatogenesis disorders, the product being used for diagnosis or prognostic assessment of spermatogenesis disorders, wherein the biomarkers are as described in the first aspect.

[0047] Preferably, the detection reagent includes reagents for detecting the molecular level.

[0048] Depending on the specific implementation requirements, the detection reagent can be any reagent in the prior art, as long as it can detect the molecular level.

[0049] Preferably, the detection reagent includes reagent components used in chromatography, capillary electrophoresis, liquid chromatography-mass spectrometry, biosensor methods, immunoassays, or nucleic acid detection methods. The detection reagent refers to the key reagent components used in the corresponding detection method.

[0050] More preferably, the chromatographic method includes ion exchange chromatography, liquid chromatography, gas chromatography, etc.

[0051] Preferably, the detection reagent includes any detection reagent for the molecule, such as antibodies or substrates for detecting proteins, primers or probes for detecting genes and / or mRNA.

[0052] Preferably, the spermatogenesis disorder includes azoospermia; more preferably, the azoospermia is non-obstructive azoospermia; and more preferably, the non-obstructive azoospermia includes, but is not limited to, spermatocyte blockade type, spermatogonial / spermatomatous blockade type, and Sertoli cell-only syndrome type of azoospermia.

[0053] Preferably, the spermatogenesis disorder includes moderate or severe spermatogenesis disorder, such as severe spermatogenesis disorder.

[0054] Preferably, the detection reagent is used to detect the expression level of a biomarker in a sample, and the expression level can be indicated by the fluorescence intensity of immunohistochemistry.

[0055] Furthermore, the diagnostic or prognostic assessment includes: detecting biomarkers in the test sample; and comparing the expression levels of biomarkers in the test sample and healthy samples.

[0056] Preferably, when the expression of the biomarker is upregulated in the test sample compared to a healthy sample, it can be diagnosed as a spermatogenesis disorder; or the expected treatment effect is poor.

[0057] Preferably, high expression of the biomarkers in the test sample compared to a healthy sample can be diagnosed as spermatogenesis disorder, or...

[0058] Preferably, the prognosis of spermatogenesis disorders can be assessed. For example, high expression of the biomarker in the test sample indicates a low probability of obtaining sperm in the sperm retrieval procedure, while no expression or low expression of the biomarker in the test sample indicates a high probability of obtaining sperm in the sperm retrieval procedure.

[0059] Preferably, the product includes a kit or diagnostic chip for detecting biomarkers.

[0060] Preferably, the diagnostic or prognostic assessment also includes diagnosis based on a combination of clinical symptoms and / or other test results.

[0061] Furthermore, the samples are derived from any animal, including human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. Mammals include non-human primates, mice, rats, guinea pigs, goats, sheep, dogs, rabbits, pigs, cats, and cattle. Non-mammals include chickens, amphibians, or reptiles.

[0062] Preferably, the sample is derived from humans.

[0063] A fourth aspect of the present invention provides a method for detecting biomarkers of spermatogenesis disorders, the method comprising using a product as described in the third aspect to detect the biomarkers.

[0064] A fifth aspect of the present invention provides a method for diagnosing spermatogenesis disorders, the method comprising using the above-described product to detect the above-described biomarkers in a sample.

[0065] The products described in this invention include all products related to the diagnosis of spermatogenesis disorders. Preferably, the products include, but are not limited to, antibodies, substrates, primers, probes, kits, diagnostic chips, etc., for detecting biomarkers.

[0066] The terms “comprising” or “including” in this invention are open-ended descriptions that include the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0067] In this invention, "severe spermatogenesis disorder" refers to a clinical pathological report or microscopic examination indicating the absence of sperm in the seminiferous tubules; characterized by the presence of only Sertoli cells in the seminiferous tubules, with virtually no spermatogenic cells or sperm. "Moderate spermatogenesis disorder" refers to a clinical pathological report or microscopic examination indicating the absence of sperm in the seminiferous tubules; characterized by the presence of spermatogenic cells but no sperm. "Mild spermatogenesis disorder" refers to a clinical pathological report or microscopic examination indicating the presence of sperm in the seminiferous tubules; characterized by the presence of spermatogenic cells and sperm, with a relatively complete spermatogenic process.

[0068] Current clinical diagnosis of NOA primarily relies on HE staining from testicular biopsy, judging the severity of spermatogenesis disorders based on visual observation and experience. Diagnostic indicators are singular and lack quantifiable metrics. This invention, through in-depth analysis of scRNA-seq data from NOA patients, integrates various clinical pathological types of NOA spermatogenesis disorders, focusing on the abnormal physiological state of Sertoli cells. It screens and identifies MNDA as specifically highly expressed in Sertoli cells of NOA patients, and its expression intensity can characterize the abnormal physiological state of Sertoli cells within the seminiferous tubules. Furthermore, because MNDA expression levels in germ cells are low, its development and utilization can avoid ethical issues related to germ cells, making it more practically feasible and controllable.

[0069] MNDA is a novel pathological biomarker for azoospermia in Sertoli cells. By detecting the expression intensity of MNDA, the abnormal physiological state of Sertoli cells can be characterized, thus providing a new and specific quantitative indicator for the clinical diagnosis of NOA. Attached Figure Description

[0070] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0071] Figure 1 ① t-SNE cell clustering diagram of scRNA-seq sequencing analysis results of testicular tissue from NOA patient No. 1, showing the different cell types identified. Among them, 0, 1, 2, 3, and 7 are spermatogonia, 4 and 5 are premeiotic spermatocytes, 10 is meiotic spermatocytes, 6 is Leydig interstitial cells, 8 is Sertoli cells, 9 is macrophages, and 11 is mast cells.

[0072] Figure 2 ① A scatter plot combining scRNA-seq sequencing results from testicular tissue of NOA patient No. 1, showing the expression of MAGEA4, TEX19, SPATA8, SPACA4, TNP1, and SOX9 genes in different cell populations. The redder the color, the higher the gene expression level.

[0073] Figure 3 : t-SNE cell clustering diagram of scRNA-seq sequencing analysis results of testicular tissue from NOA patient No. 2, showing the identified cell types, where 0 represents mast cells, 1 and 9 represent epithelial cells, 2 represents Sertoli cells, 3 represents endothelial cells, 4 represents macrophages, 5 represents granulocytes, 6 represents peritubular myoid cells, 7 represents Leydig interstitial cells, 8 represents T cells, and 10 represents lymphangiocytes.

[0074] Figure 4 Bubble graph of scRNA-seq sequencing results from testicular tissue of NOA patient No. 2, showing representative genes of different cell types. The larger the bubble diameter and the darker the blue, the higher the expression level of the gene in the cell type.

[0075] Figure 5 : 2. A scatter plot of combined scRNA-seq sequencing results from testicular tissue of NOA patient No. 2, showing the expression of MAGEA4, TEX19, SPATA8, SPACA4 and TNP1 in different cell types. The higher the red intensity, the higher the gene expression level.

[0076] Figure 6 t-SNE cell clustering diagram of scRNA-seq sequencing analysis results of SRR6860519, where,

[0077] Cell cluster 0 represents haploid spermatocytes, cell clusters 1 and 3 represent amorphous spermatocytes, cell cluster 2 represents Leydig interstitial cells, cell clusters 4 and 6 represent spermatogonia, cell cluster 5 represents premeiotic spermatocytes, cell cluster 7 represents meiotic spermatocytes, cell cluster 8 represents apoptotic cells, cell cluster 9 represents Sertoli cells, cell cluster 10 represents endothelial cells, and cell cluster 11 represents macrophages.

[0078] Figure 7 The combined scatter plot of scRNA-seq sequencing analysis results of SRR6860519 shows the expression status in NS individual cell types. The higher the red intensity, the higher the gene expression level.

[0079] Figure 8t-SNE cell clustering diagram of scRNA-seq sequencing analysis results of SRR6860520, where cell cluster 0 represents Leydig mesenchymal cells, cell cluster 1 represents apoptotic cells, cell clusters 2 and 7 represent deformed spermatocytes, cell cluster 3 represents endothelial cells, cell cluster 4 represents peritubular myoid cells, cell clusters 5 and 12 represent spermatogonia, cell cluster 6 represents premeiotic spermatocytes, cell cluster 8 represents haploid spermatocytes, cell cluster 9 represents macrophages, cell cluster 10 represents meiotic spermatocytes, cell cluster 11 represents Sertoli cells, and cell cluster 13 represents smooth muscle cells.

[0080] Figure 9 The combined scatter plot of scRNA-seq sequencing analysis results of SRR6860520 shows the expression status in NS individual cell types. The higher the red intensity, the higher the gene expression level.

[0081] Figure 10 The t-SNE plot shows the integrated clustering results of scRNA-seq data of Sertoli cells from NOA patients and normal adult Sertoli cells. The blue cell cluster represents Mature-Sertoli cells, and the red cell cluster represents NOA-Sertoli.

[0082] Figure 11 The t-SNE split diagram shows the data source composition structure in the NOA patient Sertoli cell population (NOA-Sertoli cell population) and the adult normal Sertoli cell population (Mature-Sertoli cell population). Among them, the SRR series represents the encoding of scRNA-seq sequencing data of testicular tissue from NS adult individuals in the GEO database, and NOA-① and NOA-② represent the scRNA-seq sequencing data of testicular tissue from NOA patients No. ① and No. ② in this application.

[0083] Figure 12 The violin plot illustrates the differential expression of MNDA in scRNA-seq data of NOA patient Sertoli cells (NOA-Sertoli cells) and normal adult Sertoli cells (Mature-Sertoli cells).

[0084] Figure 13 The expression of MNDA in spermatogenic cells at all levels in normally spermatogenic samples. The t-SNE clustering of scRNA-seq data of spermatogenic cells at all levels in normally spermatogenic adult individuals shows the types of spermatogenic cells at each level. Among them, cluster 0 and 1 are spermatogonia, cluster 2-4 are spermatocytes, and cluster 5-8 are spermatids.

[0085] Figure 14The expression level of the MNDA gene in spermatogenic cells at each stage is shown in the figure, with the specific percentage of positively expressed cells for the MNDA gene at each stage marked in the figure.

[0086] Figure 15 Fluorescent histochemical staining of testicular pathological sections from patients with severe clinical spermatogenesis disorders. Blue fluorescence represents DAPI staining for cell nuclei, and green fluorescence represents SOX9 protein staining. The cell types represented by the fluorescent signals are marked on the figure with dashed arrows.

[0087] Figure 16 Fluorescent histochemical staining of testicular pathological sections from patients with moderate spermatogenesis disorders in clinical practice. Blue fluorescence represents DAPI staining for cell nuclei, and green fluorescence represents SOX9 protein staining. The cell types represented by the fluorescent signals are marked on the figure with dashed arrows.

[0088] Figure 17 Fluorescent histochemical staining of testicular pathological sections from patients with mild clinical spermatogenesis disorders. Blue fluorescence represents DAPI staining for cell nuclei, and green fluorescence represents SOX9 protein staining. The cell types represented by the fluorescent signals are marked on the figure with dashed arrows.

[0089] Figure 18 The image shows the immunofluorescence expression of MNDA in the testicular tissue of patients with mild azoospermia. Blue signal represents DAPI staining of cell nuclei, green signal represents SOX9, and red signal represents MNDA.

[0090] Figure 19 The image shows the immunofluorescence expression of MNDA in the testicular tissue of patients with moderate azoospermia. Blue signal represents DAPI staining of cell nuclei, green signal represents SOX9, and red signal represents MNDA.

[0091] Figure 20 The image shows the immunofluorescence expression of MNDA in the testicular tissue of patients with severe azoospermia. Blue signal represents DAPI staining of cell nuclei, green signal represents SOX9, and red signal represents MNDA.

[0092] Figure 21 Statistical results of MNDA fluorescence signal intensity in three groups of clinical pathological sections of testicular tissue. Each point represents a patient sample. MNDA signal intensity is the average signal intensity of red fluorescence within the physiological structure of seminiferous tubules. *: p < 0.05; **: p < 0.001; t-test.

[0093] Figure 22Immunohistochemical expression of MNDA in testicular tissue of patients with mild azoospermia (a clinical type of mild spermatogenesis disorder). Brown indicates positive MNDA signal.

[0094] Figure 23 Immunohistochemical expression of MNDA in testicular tissue of patients with moderate azoospermia (a clinically predisposed to moderate spermatogenesis disorder). Brown indicates a positive signal for MNDA.

[0095] Figure 24 Immunohistochemical expression of MNDA in testicular tissue of patients with severe azoospermia (a clinical type of spermatogenesis disorder). Brown indicates positive MNDA signal.

[0096] Figure 25 Statistical results of MNDA signal intensity in three groups of clinical pathological sections of testicular tissue. Each point represents a patient sample. MNDA signal intensity is the average brown signal intensity within the physiological structure of the seminiferous tubules. *: p < 0.05; t-test.

[0097] Figure 26 ROC curves of MNDA positive signals at different fluorescence thresholds (1500, 1000, 700) showed AUC values ​​of 0.97, 0.95, and 0.90, respectively. Detailed Implementation

[0098] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0099] Example 1: Screening of MNDA, a pathological marker gene in Sertoli cells

[0100] 1.1 Single-cell RNA sequencing (scRNA-seq) and data analysis of testicular tissue from NOA patients

[0101] With ethical and patient consent, fresh testicular tissue samples from two NOA patients were obtained from a collaborating hospital and labeled NOA-① and NOA-②. A digestion solution was prepared using 1 mg / mL collagenase IA (Sigma), 1 mg / mL collagenase IV, 1 mg / mL DNase I, 0.5% BSA, 50% HBSS solution (containing calcium and magnesium), and 50% DMEM / F12K medium to digest the tissues into single cells. Subsequently, 10×Genomics library construction was performed using the Chromium single-cell 3' library and Gel Bead Kit V3 (Illumina) to prepare scRNA-seq single-cell transcriptome libraries. Paired-end sequencing was performed using an X-Ten sequencer to obtain raw sequencing data.

[0102] The raw scRNA-seq sequencing data were processed using software packages such as UMI-tools, STAR, FeatureCounts, and Samtools.

[0103] (1) The Cell Barcode sequence (16bp) and UMI sequence (12bp) in the R1 sequencing reads were extracted using the whitelist function of UMI-tools;

[0104] (2) Use the extract function of UMI-tools to add the Cell Barcode and UMI sequence to the R2 end sequencing reads;

[0105] (3) The processed R2 end sequencing data was compared with the human genome GRCh38 using STAR to obtain the BAM file;

[0106] (4) Use the featureCounts function to annotate the BAM file;

[0107] (5) Use Samtools' sort and index functions to extract and sort the BAM files;

[0108] (6) Use the count function of UMI-tools to obtain the cell-gene expression matrix.

[0109] For testicular tissue with normal spermatogenic function, scRNA-seq sequencing results can identify the complete spermatogenic process, including spermatogonia (MAGEA4+) → premeiotic spermatocytes (TEX19+) → meiotic spermatocytes (SPATA8+) → haploid spermatocytes (SPACA4+) → deformed spermatocytes (TNP1+). Cluster analysis of the cell-gene expression matrix from single-cell sequencing of testicular tissue from NOA patients ① and ② was performed using Seurat software to obtain different cell groups. The marker genes for each cell group were obtained using the FindALLMarkers function (note in parentheses above, P-value < 0.05 & Log2Foldchang > 0.5), and cell types were then annotated based on the marker genes.

[0110] The results showed that for patient ① NOA ( Figures 1-2 ),

[0111] Figure 1 The t-SNE cell clustering diagram shows the identified cell types, including spermatogonia (0, 1, 2, 3, 7) at different stages, premeiotic spermatocytes (4, 5), meiotic spermatocytes (10), and Sertoli cells (8).

[0112] Figure 2 The scatter plot shows the expression of biomarkers in spermatogenic cells at different stages and in Sertoli cells across different cell types. Spermatogonia (MAGEA4+), premeiotic spermatocytes (TEX19+), and a small number of meiotic spermatocytes (SPATA8+) were detected in the testicular tissue; haploid spermatocytes (SPACA4-) and amorphous spermatocytes (TNP1-) were not detected. Figure 1 The test results were consistent. It is evident that spermatogenesis in NOA patient #1 was arrested during the prophase of meiosis. Furthermore, Sertoli cells (SOX9+) were also identified.

[0113] For NOA patient number ② ( Figures 3-5 ),

[0114] Figure 3 The t-SNE cell clustering diagram shows the identified cell types, including Leydig mesenchymal cells (7), peritubular myoid cells (6), macrophages (4), epithelial cells (1, 9), endothelial cells (3), mast cells (0), supporting cells (2), T cells (8), and granulocytes (5). The sample contained no germ cells.

[0115] Figure 4The bubble diagram shows representative genes from different cell types. The larger the bubble diameter and the deeper the blue color, the higher the expression level of the gene in the cell type. Nine cell types were mainly detected in the testicular tissue: mast cells (TPSB2+), epithelial cells (TNFAIP6+), Sertoli cells (SOX9+), endothelial cells (STC1+), macrophages (ID1+), granulocytes (S100A9+), peritubular myoid cells (MFAP4+), Leydig interstitial cells (ABCA8+), and T cells (KLRB1+).

[0116] Figure 5 The composite scatter plot shows the expression of spermatogenic cells at different stages in different cell types. No spermatogenic cells (MAGEA4- / TEX19- / SPATA8- / SPACA4- / TNP1-) were observed.

[0117] Therefore, it can be seen that patient number ② presents with Sertoli cell-only syndrome, where only Sertoli cells are present in the seminiferous tubules and no spermatogenic cells are present.

[0118] Meanwhile, Sertoli cells were successfully detected in the scRNA-seq data of both NOA patients ① and ②.

[0119] 1.2 Acquisition and analysis of scRNA-seq sequencing data from testicular tissue of adult individuals with normal spermatogenesis (NS)

[0120] Data from relevant published literature were collected, and scRNA-seq sequencing data from testicular tissues of 12 adult male NS individuals were performed using the 10×Genomics sequencing method. The raw sequencing data (paired-end FastQ sequencing files) were downloaded.

[0121] Data SRR11509931 and SRR11509938 are from the literature Zhao L, Yao C, Xing X, et al. 2020, Single-cell analysis of developing and azoospermia human testicles reveal central role of Sertoli cells, corresponding to the GSE149512 database;

[0122] Data SRR15613719, SRR15613720, and SRR15613722 are from the literature Nie X, Munyoki SK, Sukhwani M, et al. 2022, Single-cell analysis of human testis aging and correlation with elevated body mass index, corresponding to the GSE182786 database;

[0123] Data SRR6860519, SRR6860520, SRR6860521, SRR6860522, SRR6860523, and SRR6860524 are from the literature Guo J, Grow EJ, Mlcochova H, et al. 2018, The adult human testistranscriptional cell atlas, corresponding to the GSE120508 database;

[0124] Data SRR12164934 comes from the literature Di Persio S, Tekath T, Siebert-Kuss LM, et al. 2021, Single-cell RNA-seq unravels alterations of the human spermatogonialstem cell compartment in patients with impaired spermatogenesis, corresponding to the GSE153947 database.

[0125] The raw scRNA-seq sequencing data from 12 NS individuals were processed using the same methods described in section 1.1 to obtain the cell-gene expression matrix corresponding to each dataset. Cluster analysis of the cell-gene expression matrices from the testicular tissue scRNA-seq of the 12 NS individuals was performed using Seurat software. The results showed that all 12 NS individuals identified spermatogenic cells at each stage of the complete spermatogenesis process: spermatogonia (MAGEA4+) → premeiotic spermatocytes (TEX19+) → meiotic spermatocytes (SPATA8+) → haploid spermatocytes (SPACA4+) → deformed spermatocytes (TNP1+). A certain number of normal Sertoli cells (SOX9+) and different types of somatic cells were also identified in all individuals. Figure 6-9 Two exemplary results are given.

[0126] 1.3 Screening for MNDA, a pathological marker gene in Sertoli cells of NOA patients

[0127] Based on the scRNA-seq analysis results of testicular tissues from NOA patients ① and ② and 12 NS individuals, the cell-gene expression matrices of Sertoli cell populations were extracted separately using Seurat software. These matrices were then integrated using Seurat's ensemble function for unified cluster analysis. The t-SNE clustering results showed that Sertoli cells from NOA patients and normal adult Sertoli cells each clustered into a separate cell group (…). Figures 10-11 Differentially expressed genes between Sertoli cells from NOA patients and normal adult Sertoli cells were obtained using the FindALLMarkers function (P < 0.05 & Log2Foldchang > 0.5).

[0128] Genes highly expressed in Sertoli cells of NOA patients were retrieved and reintroduced into the scRNA-seq data of spermatogenic cells. Using the Dotplot function in Seurat, the proportion of positive cells for each highly expressed gene in each level of spermatogenic cell group was statistically analyzed to detect the expression level of candidate genes in each level of spermatogenic cell group. Through analysis, the target gene MNDA was successfully screened, which has the potential to be a marker gene for Sertoli cells of NOA patients: (1) Compared with normal adult Sertoli cells, MNDA was differentially highly expressed in Sertoli cells of NOA patients (P value < 0.05 & Log2Foldchang > 0.5) Figure 12 (2) MNDA is expressed at low levels in spermatogenic cells at all stages (the proportion of positive cells is <10%), which ensures that the MNDA signal in the seminiferous tubules is specifically displayed on Sertoli cells. Figure 13-14 ).

[0129] Example 1: Based on the analysis of single-cell transcript sequencing data of human testis tissue, MNDA was identified as a differentially expressed gene between NOA patient Sertoli cells and normal adult Sertoli cells, and it was abnormally highly expressed on NOA-Sertoli cells.

[0130] Example 2: Validation of MNDA, a pathological marker gene in NOA-Sertoli cells

[0131] Testicular tissue was collected from patients with clinical azoospermia, fixed with 4% PFA, and embedded in paraffin to prepare 5mm pathological sections for later use. The testicular biopsy sections were subjected to immunofluorescence histochemical double staining for SOX9 (488nm green fluorescence) and MNDA (555nm red fluorescence), and DAPI staining for nuclear DNA (405nm blue fluorescence). Fluorescence imaging of the sections was performed using a Nikon AI-SIM laser confocal microscope to obtain the staining results.

[0132] Based on the pathological reports and microscopic examination results of testicular puncture tissue from clinical NOA patients, the types of spermatogenesis disorders in the seminiferous tubules of the samples were classified using SOX9 and DAPI fluorescence signals. The criteria for judgment were as follows: DAPI staining showed that the nuclei of mature sperm cells exhibited small and bright sharp signals due to the high degree of chromatin condensation (Sperm-DAPI+), which corroborated the clinical test results and determined the presence of mature sperm; SOX9 is a protein specifically expressed in the nuclei of Sertoli cells in testicular tissue. A positive signal was used to mark Sertoli cells, and a negative signal could be used to determine the presence of spermatogenic cells such as spermatogonia or spermatocytes.

[0133] Based on the characteristics of SOX9 and DAPI signals and clinical pathological test results, the samples were divided into three basic pathological types of spermatogenesis disorders: severe spermatogenesis disorder, moderate spermatogenesis disorder, and mild spermatogenesis disorder. Figure 15-17 As shown.

[0134] ① Severe spermatogenesis disorder: Clinical pathology reports or microscopic examination results indicate the absence of sperm in the seminiferous tubules; the DAPI cell nuclear signals in the seminiferous tubules show no Sperm-DAPI signal and are almost entirely SOX9 positive signals (DAPI+ / SOX9+), with no or occasional SOX9 negative cell nuclei (DAPI+ / SOX9-), indicating that only Sertoli cells exist in the seminiferous tubules, with virtually no spermatogenic cells and sperm.

[0135] ② Moderate spermatogenesis disorder: Clinical pathology reports or microscopic examination results indicate the absence of sperm in the seminiferous tubules; the DAPI cell nuclear signals in the seminiferous tubules show no Sperm-DAPI signal, while there are both SOX9 positive signals (DAPI+ / SOX9+) and obvious SOX9 negative cell nuclear signals (DAPI+ / SOX9-), indicating the presence of spermatogenic cells in the seminiferous tubules, but no sperm.

[0136] ③ Mild spermatogenesis disorder: Clinical pathology reports or microscopic examination results indicate the presence of sperm in the seminiferous tubules; the DAPI cell nuclear signals in the seminiferous tubules show Sperm-DAPI signals, as well as both SOX9 positive signals (DAPI+ / SOX9+) and obvious SOX9 negative cell nuclear signals (DAPI+ / SOX9-), indicating the presence of Sertoli cells, spermatogenic cells and sperm in the seminiferous tubules, with a relatively complete spermatogenesis process.

[0137] Further statistical analysis was conducted on the histochemical fluorescence staining intensity of MNDA in seminiferous tubules of various sample types. Using ImageJ software, the fluorescence-stained images were opened, and the observation area was located within the seminiferous tubules based on physiological structure. The colorimetric channel was adjusted to green fluorescence, displaying the SOX9 green fluorescence signal, and Sertoli cells within the seminiferous tubules were located based on its positive expression location. Then, the colorimetric channel was adjusted to red fluorescence, displaying the red fluorescence signal of MNDA. A uniform fluorescence threshold was then set, and the MNDA fluorescence signal intensity within the seminiferous tubules of all samples was statistically analyzed using the same threshold. Finally, the statistical differences in MNDA fluorescence expression intensity values ​​among the samples in the three groups were compared.

[0138] like Figures 18-25 As shown, both immunofluorescence and immunohistochemical staining results indicated that MNDA signals were specifically localized within the nuclei of Sertoli cells. Statistical results showed that the MNDA expression level in the seminiferous tubules of the severe and moderate spermatogenesis disorder groups was significantly higher than that of the mild spermatogenesis disorder group. Given that Sertoli cells in the moderate to severe spermatogenesis disorder samples are in a relatively abnormal physiological state, while Sertoli cells in the mild spermatogenesis disorder samples are in a relatively normal physiological state, these results suggest that the expression level of MNDA can characterize the abnormal physiological state of Sertoli cells.

[0139] like Figure 26 The ROC curves of MNDA positive signals at different fluorescence thresholds (1500, 1000, 700) are shown. The AUC values ​​are 0.97, 0.95, and 0.90, respectively, indicating that MNDA performs well as a marker for pathological detection.

[0140] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0141] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of a biomarker detection reagent in the preparation of diagnostic products that differentiate between different pathological types of non-obstructive azoospermia, characterized in that, The biomarker is MNDA molecule, and the pathological types are severe spermatogenesis disorder, moderate spermatogenesis disorder, and mild spermatogenesis disorder.

2. The application according to claim 1, characterized in that, The biomarker samples were derived from testicular tissue.

3. The application according to claim 1, characterized in that, The biomarker MNDA molecule is specifically expressed in Sertoli cells.

4. The application according to any one of claims 1-3, characterized in that, The detection reagent is used to detect the expression level of biomarkers in a sample.

5. The application according to claim 4, characterized in that, The MNDA molecule was upregulated in severe and moderate spermatogenesis disorders.

6. The application according to claim 5, characterized in that, The MNDA molecule includes the MNDA protein and / or gene.

7. The application according to claim 6, characterized in that, The detection reagents for the MNDA molecule include antibodies for detecting proteins and primers or probes for detecting genes.

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