A detection system and method for evaluating sperm motility function in asthenozoospermia

Abstract

This invention relates to the fields of biomedical diagnostic reagent manufacturing and reproductive medicine technology, and particularly to a detection system and method for assessing sperm motility in patients with asthenospermia. This invention establishes a complete, accurate, and efficient molecular-level sperm quality evaluation system by integrating specific fluorescent labeling, high-resolution confocal imaging, and automated computer analysis software. This system not only overcomes the limitations of existing technologies but also further expands its functional scope by integrating an optical microscopy analysis module. It possesses good clinical applicability and scalability, providing a powerful tool for the etiological diagnosis, efficacy evaluation, and scientific research of male infertility, and has significant clinical application value and market prospects.

Description

Technical Field

[0001] This invention relates to the fields of biomedical testing reagent manufacturing and reproductive medicine technology, and in particular to a testing system and method for assessing sperm motility function in patients with asthenospermia. Background Technology

[0002] Asthenospermia is a major cause of male infertility, and its diagnosis has long relied on phenotypic indicators such as sperm motility. However, patients with the same phenotype of asthenospermia may have drastically different underlying molecular defects, which may stem from abnormal flagellar pulsation, insufficient energy production, impaired acrosome function, or genetic factors. Traditional semen analysis (such as sperm motility, viability, and morphology) and automated detection systems (such as computer-aided sperm analysis, CASA) can only reflect macroscopic phenotypic results of sperm motility and cannot elucidate the molecular mechanisms behind "low motility."

[0003] While techniques such as immunofluorescence can detect specific proteins, they are cumbersome to operate and provide limited information on a single target. For example, a single test can only verify the status of 1-2 proteins, while the molecular etiology of asthenospermia often involves synergistic abnormalities of multiple pathways and targets; single-target detection cannot construct a complete etiological map. This invention aims to solve this problem by providing an integrated system that can simultaneously and automatically acquire sperm motility performance and the underlying multidimensional molecular functional status. By detecting five proteins covering key sperm functional modules (HE12-acrosomal, PP1R7-flagellate motility, KT3K-genetic association, TMCO3-energy, UAP1-fertilization recognition), combined with motility analysis, it provides clinicians with in-depth diagnostic information far exceeding that of traditional methods. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a detection system and method for assessing sperm motility in patients with asthenospermia. This system integrates specific immunofluorescence labeling and high-resolution imaging analysis software, enabling automated and quantitative analysis of the expression levels and cellular localization of various sperm functional proteins. It can also simultaneously or independently perform morphological and kinematic analyses of sperm, thereby achieving the diagnostic purpose of asthenospermia.

[0005] In a first aspect, the present invention provides a detection system for assessing sperm motility function in patients with asthenospermia, including a sperm protein fluorescent labeling kit, an image acquisition system, and a sperm protein quality analysis platform.

[0006] This kit employs a two-component design (Kit A and Kit B), each catering to different storage conditions and application requirements, ensuring reagent stability and detection repeatability. All components of the kit have strictly defined storage conditions and expiration dates. Once opened, they must be stored according to the specified conditions and used within the defined time frame to maximize detection performance.

[0007] Kit A (stored below -20°C) contains the following components:

[0008] Antibody components: include specific antibodies against rabbit anti-human HE12, PP1R7, KT3K, TMCO3, and UAP1; each antibody has an immunofluorescence titer of 1:100 (concentration 0.05 mg / mL), and is dissolved in antibody dilution buffer containing phosphate-buffered saline (PBS), bovine serum albumin (BSA), and sodium azide to effectively maintain antibody activity and specificity;

[0009] Labeling system: fluorescently labeled secondary antibody (FITC-labeled goat anti-rabbit IgG secondary antibody, titer 1:100, 0.01 mg / mL) and nuclear fluorescent dye (0.01 mg / mL) for multiplex fluorescent labeling;

[0010] Solid support: sperm protein slides. The sperm protein slides are multi-well (12-well) slides, with each well pre-coated with positive and negative control human sperm samples. Each well of the 12-well multi-well slide undergoes strict quality control and is equipped with batch-specific reference range values ​​to ensure the comparability and accuracy of each batch of tests.

[0011] Kit B (stored at room temperature) contains the following components:

[0012] 30× concentrated washing solution (pH 7.4, 0.3 M PBS);

[0013] Mounting glycerol (glycerol dissolved in pH 7.4, 0.01 M PBS) is used for sample washing and mounting to ensure the clarity and stability of the fluorescence signal;

[0014] Fixative: Methanol.

[0015] The preferred specific antibody sequences for rabbits against human HE12, PP1R7, KT3K, TMCO3, and UAP1 are as follows:

[0016] (a) HE12 antibody

[0017] The HE12 antibody light chain sequence is shown in SEQ ID NO.1; the HE12 antibody light chain variable region VL is shown in SEQ ID NO.2; the HE12 antibody light chain complementarity-determining region CDR-L1 is shown in SEQ ID NO.3; the HE12 antibody light chain complementarity-determining region CDR-L2 is shown in SEQ ID NO.4; and the HE12 antibody light chain complementarity-determining region CDR-L3 is shown in SEQ ID NO.5. The HE12 antibody heavy chain sequence is shown in SEQ ID NO.6; the HE12 antibody heavy chain variable region VH is shown in SEQ ID NO.7; the HE12 antibody heavy chain complementarity-determining region CDR-H1 is shown in SEQ ID NO.8; the HE12 antibody heavy chain complementarity-determining region CDR-H2 is shown in SEQ ID NO.9; and the HE12 antibody heavy chain complementarity-determining region CDR-H3 is shown in SEQ ID NO.10.

[0018] (b) PP1R7 antibody

[0019] The PP1R7 antibody light chain sequence is shown in SEQ ID NO.11, the PP1R7 antibody light chain variable region VL is shown in SEQ ID NO.12, the PP1R7 antibody light chain complementarity-determining region CDR-L1 is shown in SEQ ID NO.13, the PP1R7 antibody light chain complementarity-determining region CDR-L2 is shown in SEQ ID NO.14, and the PP1R7 antibody light chain complementarity-determining region CDR-L3 is shown in SEQ ID NO.15; the PP1R7 heavy chain sequence is shown in SEQ ID NO.16, the PP1R7 antibody heavy chain variable region VH is shown in SEQ ID NO.17, the PP1R7 antibody heavy chain complementarity-determining region CDR-H1 is shown in SEQ ID NO.18, the PP1R7 antibody heavy chain complementarity-determining region CDR-H2 is shown in SEQ ID NO.19, and the PP1R7 antibody heavy chain complementarity-determining region CDR-H3 is shown in SEQ ID NO.20.

[0020] (c) KT3K antibody

[0021] The KT3K antibody light chain sequence is shown in SEQ ID NO.21; the variable region VL of the KT3K antibody light chain is shown in SEQ ID NO.22; the complementarity-determining region CDR-L1 of the KT3K antibody light chain is shown in SEQ ID NO.23; the complementarity-determining region CDR-L2 of the KT3K antibody light chain is shown in SEQ ID NO.24; and the complementarity-determining region CDR-L3 of the KT3K antibody light chain is shown in SEQ ID NO.25. The KT3K heavy chain sequence is shown in SEQ ID NO.26; the variable region VH of the KT3K antibody heavy chain is shown in SEQ ID NO.27; the complementarity-determining region CDR-H1 of the KT3K antibody heavy chain is shown in SEQ ID NO.28; the complementarity-determining region CDR-H2 of the KT3K antibody heavy chain is shown in SEQ ID NO.29; and the complementarity-determining region CDR-H3 of the KT3K antibody heavy chain is shown in SEQ ID NO.30.

[0022] (d) TMCO3 antibody

[0023] The TMCO3 antibody light chain sequence is shown in SEQ ID NO.31, the TMCO3 antibody light chain variable region VL is shown in SEQ ID NO.32, the TMCO3 antibody light chain complementarity-determining region CDR-L1 is shown in SEQ ID NO.33, the TMCO3 antibody light chain complementarity-determining region CDR-L2 is shown in SEQ ID NO.34, and the TMCO3 antibody light chain complementarity-determining region CDR-L3 is shown in SEQ ID NO.35; the TMCO3 heavy chain sequence is shown in SEQ ID NO.36, the TMCO3 antibody heavy chain variable region VH is shown in SEQ ID NO.37, the TMCO3 antibody heavy chain complementarity-determining region CDR-H1 is shown in SEQ ID NO.38, the TMCO3 antibody heavy chain complementarity-determining region CDR-H2 is shown in SEQ ID NO.39, and the TMCO3 antibody heavy chain complementarity-determining region CDR-H3 is shown in SEQ ID NO.40.

[0024] (e) UAP1 antibody

[0025] The UAP1 antibody light chain sequence is shown in SEQ ID NO.41, the UAP1 antibody light chain variable region VL is shown in SEQ ID NO.42, the UAP1 antibody light chain complementarity-determining region CDR-L1 is shown in SEQ ID NO.43, the UAP1 antibody light chain complementarity-determining region CDR-L2 is shown in SEQ ID NO.44, and the UAP1 antibody light chain complementarity-determining region CDR-L3 is shown in SEQ ID NO.45; the UAP1 heavy chain sequence is shown in SEQ ID NO.46, the UAP1 antibody heavy chain variable region VH is shown in SEQ ID NO.47, the UAP1 antibody heavy chain complementarity-determining region CDR-H1 is shown in SEQ ID NO.48, the UAP1 antibody heavy chain complementarity-determining region CDR-H2 is shown in SEQ ID NO.49, and the UAP1 antibody heavy chain complementarity-determining region CDR-H3 is shown in SEQ ID NO.50.

[0026] The core of the image acquisition system is a fluorescence microscope. A high-resolution model such as the Nikon C2 or Zeiss LSM800 is recommended. This system features a multi-laser excitation device (488 nm for FITC excitation, 561 nm for PI excitation), a high-sensitivity photomultiplier tube (PMT) detector, and precise Z-axis tomography, supporting multi-channel synchronous or sequential image acquisition. The system can simultaneously acquire high-quality two-dimensional or three-dimensional images of the bright-field channel (sperm morphology), FITC channel (target protein signal), and PI channel (nuclear signal). Optical tomography effectively avoids fluorescence cross-contamination, providing raw image data for subsequent quantitative analysis.

[0027] In addition, the system can be equipped with a conventional optical microscope module to acquire high-resolution bright-field images or motion videos of sperm for morphological and kinematic analysis.

[0028] Image acquisition must follow standardized operating procedures (SOPs): For confocal imaging, this includes power-on warm-up, inverted slide placement, focus calibration (initial Z-axis position 3800 μm), PFS (Perfect Focus System) activation to ensure focus stability during continuous shooting, and multi-field system acquisition (≥200 sperm per sample). Acquired images are saved in multi-channel formats such as ND2 and can be split into single-channel or composite images, exported to standard formats such as TIFF for software analysis. For ordinary optical microscope imaging, the corresponding bright-field focusing and video acquisition procedures must be followed.

[0029] The sperm protein quality analysis platform is an integrated computer workstation system developed using Microsoft Visual Studio 2008 C++, running on a Windows 7 / 8.1 64-bit operating system. Access is authorized via an encryption dongle to ensure data security. The sperm protein quality analysis platform adopts a modular design and includes the following core functional modules:

[0030] The medical record management module is patient-centric, comprehensively recording patients' basic information, clinical data, and test indicators. It supports the creation, modification, querying, and deletion of medical records. All information is structured and stored in an SQL Server 2008 database for easy subsequent statistical analysis and data traceability.

[0031] The protein analysis module is the core analysis unit, supporting the import of multi-channel fluorescence images (bright field, FITC, PI). It integrates multi-channel information from the same field of view through image registration and synthesis algorithms; it automatically identifies and segments sperm cells based on the strong nuclear signal of the PI channel; for each sperm cell, it calculates the average fluorescence intensity (MFI) and fluorescently positive area of ​​specific cellular regions such as the head, neck, and tail in the FITC channel; and it integrates data from multiple fields of view (≥200 sperm cells) to statistically analyze parameters such as the average fluorescence intensity and localization rate of each target protein. The protein analysis module provides a manual correction interface, allowing users to verify and adjust the automatic identification results, such as adding / deleting sperm identification boxes, correcting the matching relationship between fluorescence signals and cells, and adjusting segmentation sensitivity to ensure analytical accuracy.

[0032] Morphology and Motility Analysis Module: This module supports importing brightfield images or video streams from ordinary optical microscopes. For static images, it automatically identifies sperm outlines and analyzes morphological abnormalities in the head, neck, and tail. For motion videos, it automatically tracks sperm movement trajectories and calculates key motion parameters such as the proportion of forward-moving sperm, curve velocity (VCL), linear velocity (VSL), and average path velocity (VAP).

[0033] Data Management Module: Enables comprehensive management of detection data (including protein quantification data and morphological motion data), supporting multi-condition queries, detailed data viewing, single-item / comprehensive report generation and printing, and data export. It utilizes an ADO interface to interact with the database, ensuring efficient and stable data reading and writing.

[0034] The system management module provides user permission management (account creation, authorization, and password modification for ordinary users and administrators), hospital information settings, customized medical record loading methods (by sample number, name, or medical record number), and automatic data backup and recovery functions to ensure system flexibility and security.

[0035] Data backup supports both instant and automatic modes, and can be set to back up daily, weekly or monthly to effectively address the risk of data loss.

[0036] The sperm protein quality analysis platform is a customized computer analysis system specifically designed for reagent kits. It automatically performs multi-channel fluorescence image registration, target recognition based on sperm nuclear signals, and quantitative calculation of fluorescence intensity and localization rate according to predefined cell localization regions of target proteins through built-in algorithms. The analysis results are compared with built-in clinical reference intervals and a diagnostic report is generated.

[0037] The built-in algorithm is as follows:

[0038] (1) Sperm head detection algorithm

[0039] PI positive sperm identification formula:

[0040] I_PI(x,y)≥T_PI=μ_background+3σ_background;

[0041] in:

[0042] I_PI(x,y): Pixel intensity of the PI channel;

[0043] T_PI: PI positive threshold;

[0044] μ_background: Average intensity of the background area;

[0045] σ_background: Standard deviation of background area intensity;

[0046] Head shape screening criteria:

[0047] A_min≤A_head≤A_max;

[0048] 0.7≤E_head=(4πA_head) / (P_head²)≤1.0;

[0049] in:

[0050] A_head: Head area (pixels);

[0051] P_head: Head perimeter (pixels);

[0052] E_head: Head ellipticity;

[0053] (2) Sperm tail segmentation algorithm

[0054] Tail recognition formula:

[0055] L_tail ≥ L_min;

[0056] W_tail≤W_max;

[0057] S_tail=L_tail / W_tail≥S_min;

[0058] in:

[0059] L_tail: Tail length;

[0060] W_tail: Tail width;

[0061] S_tail: Tail length-to-width ratio;

[0062] (3) Green fluorescence expression analysis

[0063] Fluorescence intensity calculation formula:

[0064] FI_green=(1 / N)∑[I_green(x,y)-I_background];

[0065] FI_green: Average fluorescence intensity;

[0066] N: Number of pixels in the sperm head;

[0067] I_green(x,y): Pixel intensity of the green channel;

[0068] Determination of positive fluorescence:

[0069] FI_green≥T_positive=μ_negative+Kσ_negative;

[0070] in:

[0071] T_positive: Positive threshold;

[0072] μ_negative: Average fluorescence intensity of the negative control;

[0073] σ_negative: Standard deviation of fluorescence intensity in the negative control;

[0074] K: Confidence coefficient;

[0075] (4) Key statistical indicators

[0076] Total sperm count:

[0077] N_total=∑[I_PI(i)≥T_PI];

[0078] Expressing sperm count:

[0079] N_expression=∑[FI_green(i)≥T_positive];

[0080] Positioning rate:

[0081] R_expression=(N_expression / N_total)×100%;

[0082] Average fluorescence intensity:

[0083] MFI=(1 / N_expression)∑FI_green(i).

[0084] Secondly, the present invention provides a method for using a detection system for assessing sperm motility function in patients with asthenospermia, comprising the following steps:

[0085] (a) The processed semen sample was spread on a porous glass slide, and bright-field images or motion videos were acquired using a conventional optical microscope to qualitatively detect sperm morphology.

[0086] (b) Fix the smear obtained in step (a) and incubate it with the rabbit-derived primary antibodies HE12, PP1R7, KT3K, TMCO3, and UAP1 that are specific to the target protein; wash and dry after incubation.

[0087] (c) Incubate the washed and dried smear from step (b) with the FITC-labeled second antibody; wash and dry after incubation;

[0088] (d) Counterstain the smears washed and dried in step (c) with propidium iodide (PI) solution; then wash and dry, and then mount.

[0089] (e) Acquire images of the smear after mounting in step (d) using a fluorescence microscope;

[0090] (f) Use software to determine the qualitative expression of each target protein in sperm.

[0091] The comprehensive diagnostic and etiological reference basis for asthenospermia in this invention includes:

[0092] Standards for sperm motility parameters: percentage of progressively motile sperm <32% and / or total sperm motility <40%;

[0093] Protein expression suggests an underlying cause (abnormal motor parameters):

[0094] If the PP1R7 localization rate is <75% and / or the fluorescence intensity is significantly reduced, it suggests a disorder in flagellar motility regulation.

[0095] If the fluorescence intensity of TMCO3 is <6500 au, it suggests that mitochondrial energy metabolism may be insufficient;

[0096] If the HE12 localization rate is <70%, it suggests that there may be underlying acrosome functional defects, which may affect the overall fertilization potential assessment.

[0097] If multiple target sites are abnormal at the same time, it suggests impaired sperm pluripotency, and the cause may be more complex.

[0098] After acquiring multi-channel fluorescence images, software is used to automatically analyze the bright-field images or motility videos of sperm, quantitatively calculating motility parameters; calculating the average fluorescence intensity and fluorescence-positive area of ​​each sperm in the FITC channel; statistically analyzing data from at least 200 sperm to calculate the average fluorescence intensity and localization rate of the target protein; generating a quantitative analysis report and comparing it with a reference interval; the formula for calculating the localization rate is:

[0099] .

[0100] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0101] The detection system and method for assessing sperm motility in asthenospermia provided by this invention establishes a complete, accurate, and efficient molecular-level sperm quality evaluation system by integrating specific fluorescent labeling, high-resolution confocal imaging, and automated computer analysis software. This system not only overcomes the limitations of existing technologies but also further expands its functional scope by integrating an optical microscopy analysis module. It possesses good clinical applicability and scalability, providing a powerful tool for the etiological diagnosis, efficacy evaluation, and scientific research of male infertility, and has significant clinical application value and market prospects. Attached Figure Description

[0102] The invention will now be further described with reference to the accompanying drawings.

[0103] Figure 1 This is a schematic diagram of the overall composition and workflow of the sperm protein quality analysis system and method for diagnosing asthenospermia as described in this invention.

[0104] Figure 2 This is a schematic diagram of a glass slide for the sperm protein fluorescent labeling kit described in this invention;

[0105] Figure 3 This is the case creation interface for the sperm protein quality analysis system;

[0106] Figure 4 This is the protein analysis interface of the sperm protein quality analysis system;

[0107] Figure 5 This is an example image of a sperm protein quality analysis report generated by the sperm protein quality analysis system described in this invention. Detailed Implementation

[0108] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. The following embodiments are merely illustrative and are not intended to limit the scope of protection of this invention.

[0109] The overall composition and workflow of the sperm protein quality analysis system and method for diagnosing asthenospermia described in this invention are illustrated in the following diagram. Figure 1 As shown.

[0110] Example 1: Screening of key sperm functional proteins and determination of antibody target sequences for etiological analysis of asthenospermia

[0111] The core of this invention lies in selecting five key proteins—HE12, PP1R7, KT3K, TMCO3, and UAP1—as biomarkers for analyzing the etiology of asthenospermia. The design of this protein combination aims to provide a comprehensive and complementary molecular assessment of sperm quality from multiple core dimensions, including acrosome function, motility regulation, energy metabolism, sperm-egg recognition, and genetic association, overcoming the limitations of single-indicator assessments. The screening and antibody development are based on the following:

[0112] Criteria for screening key proteins

[0113] By integrating and analyzing human sperm proteomics databases, infertility-related transcriptomics studies, and existing literature, this invention screens out protein targets that play a key role in sperm acrosome reaction, motility, energy metabolism, and DNA integrity maintenance, and whose abnormal expression is significantly associated with clinical infertility.

[0114] (1) PP1R7: directly regulates flagellar movement. Its expression or localization defects are one of the direct molecular causes of decreased sperm motility (VCL / VSL) and increased non-progressive motility.

[0115] (2) TMCO3: a mitochondrial functional protein. Insufficient expression of TMCO3 leads to reduced ATP production, which is a common cause of low sperm motility and poor sperm endurance.

[0116] (3) HE12: Acrosome functional protein. Patients with asthenospermia may have acrosome dysfunction. Detecting HE12 helps to assess the subsequent potential of sperm to complete fertilization even if the sperm successfully reaches the egg.

[0117] (4) KT3K: a genetically associated protein. Its abnormal expression may indicate that there are genetic factors behind asthenospermia, providing clues as to whether further gene testing is needed.

[0118] (5) UAP1: sperm-egg recognition protein, which assesses the ability of sperm to combine with egg. If asthenospermia is accompanied by UAP1 abnormality, it suggests that there may be a double obstacle in the fertilization process.

[0119] The combination of these five proteins enables a comprehensive assessment of asthenospermia, from "motor execution" to "energy supply," then to "final fertilization capacity" and "potential genetic background."

[0120] The sequences of the five proteins are as follows:

[0121] HE12, the immunogenic protein sequence (SEQ ID NO.51) is shown below:

[0122] MTRWSSYLLGWTTFLLYSYESSGGMHEECVFPFTYKGSVYFTCTHIHSLSPWCATRAVYNGQWKYCQSEDYPRCIFPFIYRGKAYNSCISQGSFLGSLWCSVTSVFDEKQQW KFCETNEYGGNSLRKPCIFPSIYRNNVVSDCMEDESNKLWCPTTENMDKDGKWSFCADTRISALVPGFPCHFPFNYKNKNYFNCTNEGSKENLVWCATSYNYDQDHTWVYC;

[0123] The immunogenic protein sequence (SEQ ID NO.52) of PP1R7 is shown below:

[0124] MAAERGAGQQQSQEMMEVDRRVESEESGDEEGKKHSSGIVADLSEQSLKDGEERGEEDPEEEHELPVDMETINLDRDAEDVDLNHYRIGKIEGFEVLKKVKTLCLRQNLIKCIENLEELQSLRELDLYDNQIKKIENLEALTELEILDISFNLLRNIEGVDKLTRLKKLFLVNNKISKIE NLSNLHQLQMLELGSNRIRAIENIDTLTNLESLFLGKNKITKLQNLDALTNLTVLSMQSNRLTKIEGLQNLVNLRELYLSHNGIEVIEGLENNNKLTMLDIASNRIKKIENISHLTELQEFWMNDNLLESWSDLDELKGARSLETVYLERNPLQKDPQYRRKVMLALPSVRQIDATFVRF;

[0125] The immunogenic protein sequence of KT3K (SEQ ID NO.53) is shown below:

[0126] MEELLRRELGCSSVRATGHSGGGCISQGRSYDTDQGRVFVKVNPKAEARRMFEGEMASLTAILKTNTVKVPKPIKVLDAPGGGSVLVMEHMDMRHLSSHAAKLGAQLADLHLDNKKLGEMRLKEAGTVGRGGGQEERPFVARFGFDVVTCCGYLPQVNDWQEDWVVFYARQRIQPQMDMVEKESGDREALQLWSALQLKIPDLFRDLEIIPALLHGDLWGGNVAEDSSGPVIFDPASFYGHSEYELAIAGMFGGFSSSFYSAYHGKIPKAPGFEKRLQLYQLFHYLNHWNHFGSGYRGSSLNIMRNLVK;

[0127] TMCO3, whose immunogenic protein sequence (SEQ ID NO.54) is shown below:

[0128] MKVLGRSFFWVLFPVLPWAVQAVEHEEVAQRVIKLHRGRGVAAMQSRQWVRDSCRKLSGLLRQKNAVLNKLKTAIGAVEKDVGLSDEEKLFQVHTFEIFQKELNESENSVFQAVYGLQRALQGDYKDVVNMKESSRQRLEALREAAIKEETEYMELLAAEKHQVEALKNMQHQNQSLSMLDEILEDVRKAADRLEEEIEEHAFDDNKSVKGVNFEAVLRVEEEEANSKQNITKREVEDDLGLSMLIDSQNNQYILTKPRDSTIPRADHHFIKDIVTIGMLSLPCGWLCTAIGLPTMFGYIICGVLLGPSGLNSIKSIVQVETLGEFGVFFTLFLVGLEFSPEKLRKVWKISLQGPCYMTLLMIAFGLLWGHLLRIKPTQSVFISTCLSLSSTPLVSRFLMGSARGDKEGDIDYSTVLLGMLVTQDVQLGLFMAVMPTLIQAGASASSSIVVEVLRILVLIGQILFSLAAVFLLCLVIKKYLIGPYYRKLHMESKGNKEILILGISAFIFLMLTVTELLDVSMELGCFLAGALVSSQGPVVTEEIATSIEPIRDFLAIVFFASIGLHVFPTFVAYELTVLVFLTLSVVVMKFLLAALVLSLILPRSSQYIKWIVSAGLAQVSEFSFVLGSRARRAGVISREVYLLILSVTTLSLLLAPVLWRAAITRCVPRPERRSSL;

[0129] UAP1, whose immunogenic protein sequence (SEQ ID NO. 55) is as follows:

[0130] .

[0131] Determination of antibody sequence

[0132] For the five target proteins mentioned above, this embodiment used the following strategy to determine and obtain the sequences of their specific antibodies:

[0133] 1. Immunogen Design: Complete amino acid sequences of various proteins were obtained from authoritative protein databases such as UniProt. Their antigenicity, hydrophilicity, and surface accessibility were analyzed using bioinformatics tools, and highly immunogenic fragments were ultimately selected as immunogens.

[0134] 2. Antibody Development: New Zealand white rabbits were immunized with synthetic immunogenic peptides, and cell fusion and screening were performed using standard hybridoma technology. Positive clones were initially screened using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

[0135] 3. Sequence Determination and Selection: For the selected high-affinity and high-specificity monoclonal hybridoma cell lines, the variable region sequences of their antibody heavy and light chains were obtained through cDNA cloning and sequencing. The finally selected antibody sequences had their complementarity-determining regions (CDRs) validated, demonstrating that they could efficiently and specifically bind to specific epitopes of the target protein and showed no cross-reactivity with other human proteins.

[0136] Conclusion: Through the above-mentioned systematic bioinformatics analysis and experimental verification, this embodiment successfully identified five key proteins that are highly correlated with sperm quality: HE12, PP1R7, KT3K, TMCO3, and UAP1. Corresponding high-performance rabbit monoclonal antibodies with well-defined sequences were also obtained, laying the molecular foundation for the subsequent development of kits and analyzers.

[0137] The antibody sequence is as follows:

[0138] (a) HE12 antibody sequence (SEQ ID NO.1-SEQ ID NO.10):

[0139] Light chain:

[0140] QIVSTQSPAIMSASPGEKVTMTCSASSSKTPLQWYQQKPGTSPKRWIYDTTHIGSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQHTTWTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0141] Light chain variable region:

[0142] QIVSTQSPAIMSASPGEKVTMTCSASSSKTPLQWYQQKPGTSPKRWIYDTTHIGSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQHTTWTFGGGTKLEIK;

[0143] Complementary Determinant Regions (CDRs):

[0144] CDR-L1: SASSSKTPL;

[0145] CDR-L2: DTTHIG;

[0146] CDR-L3: HQHTTWTF;

[0147] Heavy chain:

[0148] QLQQSGTVLARPGASVKMSCKASGYSFTHPYLHWIKQRPGQGLEWIGAIYPGNSESTWNQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSREWAPYFDFWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0149] Heavy chain variable region:

[0150] QLQQSGTVLARPGASVKMSCKASGYSFTHPYLHWIKQRPGQGLEWIGAIYPGNSESTWNQKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSREWAPYFDFWGQGTTLTVSS;

[0151] Complementary determining regions (CDRs):

[0152] CDR-H1: GYSFTHPYLH;

[0153] CDR-H2: AIYPGNSEST;

[0154] CDR-H3: REWAPYFDF;

[0155] (b)PP1R7 antibody sequence (SEQ ID NO.11 - SEQ ID NO.20):

[0156] Light chain:

[0157] EIVLTQSPDFQSVTPKEKVTITCRASQSIATTIHWYQQKPDQSPKLLIKYATNTLSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQTTTIPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0158] Light chain variable region:

[0159] EIVLTQSPDFQSVTPKEKVTITCRASQSIATTIHWYQQKPDQSPKLLIKYATNTLSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQTTTIPFTFGPGTKVDIK;

[0160] Complementary determining regions (CDRs):

[0161] CDR-L1: RASQSIATTIH

[0162] CDR-L2: ATNTLS

[0163] CDR-L3: HQTTTIPFT

[0164] Heavy chain:

[0165] QVQLVESGGGVVQPGRSLRLSCAASGFTFSLWAVNWVRQAPGKGLEWVAIIWYDGDQNWWADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLKSAYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0166] Heavy chain variable region:

[0167] QVQLVESGGGVVQPGRSLRLSCAASGFTFSLWAVNWVRQAPGKGLEWVAIIWYDGDQNWWADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLKSAYFDYWGQGTLVTVSS;

[0168] Complementary determining regions (CDRs):

[0169] CDR-H1: GFTFSLWAVN;

[0170] CDR-H2: IIWYDGDQNWWADSVKG;

[0171] CDR-H3: ARDLKSAYFDY;

[0172] (c) KT3K antibody sequence (SEQ ID NO.21 - SEQ ID NO.30):

[0173] Light chain:

[0174] DIQMTQSPSSLSASVGDRVTITCKASQNYEHWLNWYQQKPGKAPKLLIYNTQQIETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHLTKYRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0175] Light chain variable region:

[0176] DIQMTQSPSSLSASVGDRVTITCKASQNYEHWLNWYQQKPGKAPKLLIYNTQQIETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHLTKYRTFGQGTKVEIK;

[0177] Complementary determining regions (CDRs):

[0178] CDR-L1: KASQNYEHWLN;

[0179] CDR-L2: NTQQIET;

[0180] CDR-L3: LQHLTKYRT;

[0181] Heavy chain:

[0182] QVQLQESGPGLVRPSQTLSLTCTVSGFTFTELWLNWVRQPPGRGLEWIGFIRDKAKAWSSEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGKSGGPFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0183] Heavy chain variable region:

[0184] QVQLQESGPGLVRPSQTLSLTCTVSGFTFTELWLNWVRQPPGRGLEWIGFIRDKAKAWSSEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGKSGGPFDYWGQGSLVTVSS;

[0185] Complementary determining regions (CDRs):

[0186] CDR-H1: GFTFTELWLN;

[0187] CDR-H2: FIRDKAKAWSSEYNPSVKG;

[0188] CDR-H3: AREGKSGGPFDY;

[0189] (d) TMCO3 antibody sequence (SEQ ID NO.31 - SEQ ID NO.40):

[0190] Light chain:

[0191] DIQMTQSPSSLSASVGDRVTITCKASQDVSLFTAWYQQKPGKVPKLLIYWAGAKRTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYGGWYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0192] Light chain variable region:

[0193] DIQMTQSPSSLSASVGDRVTITCKASQDVSLFTAWYQQKPGKVPKLLIYWAGAKRTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYGGWYYTFGQGTKVEIK;

[0194] Complementary determining regions (CDRs):

[0195] CDR-L1: KASQDVSLFTA;

[0196] CDR-L2: WAGAKRT;

[0197] CDR-L3: QQYGGWYYT;

[0198] Heavy chain:

[0199] EVQLVESGGGLVQPGGSLRLSCAASGFDFGKWPMSWVRQAPGKGLEWIGEINPDSSAFQWAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARYESQYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0200] Heavy chain variable region:

[0201] EVQLVESGGGLVQPGGSLRLSCAASGFDFGKWPMSWVRQAPGKGLEWIGEINPDSSAFQWAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARYESQYWYFDVWGQGTLVTVSS;

[0202] Complementary determining regions (CDRs):

[0203] CDR-H1: GFDFGKWPMS;

[0204] CDR-H2: EINPDSSAFQWAPSLKD;

[0205] CDR-H3: ARYESQYWYFDV;

[0206] (e) UAP1 antibody sequence (SEQ ID NO.41 - SEQ ID NO.50):

[0207] Light chain:

[0208] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0209] Light chain variable region:

[0210] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKVEIK;

[0211] Complementary determining regions (CDRs): <000043�>CDR-L1: RASQGIRNDLG;

[0213] CDR-L2: AASSLQS;

[0214] [[ID=X]]CDR-L3: QQYNSYPLT;

[0215] Heavy chain:

[0216] It should be noted that there is a small error in the original text where "CDR-L3" is mislabeled as "CDR-LX" in the translation. It should be corrected to "CDR-L3" in the English translation. The corrected translation is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0209] Light chain variable region:

[0210] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKVEIK; <0OO0436> Complementary determining regions (CDRs):

[0212] CDR-L1: RASQGIRNDLG;

[0213] CDR-L2: AASSLQS;

[0214] CDR-L3: QQYNSYPLT;

[0215] Heavy chain:

[0216] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDRGYYGSSYWYFDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0217] Heavy chain variable region:

[0218] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDRGYYGSSYWYFDVWGQGTTVTVSS;

[0219] Complementary Determinant Regions (CDRs):

[0220] CDR-H1: GYSFTGYTMN;

[0221] CDR-H2: GIIPIFGTANYAQKFQG;

[0222] CDR-H3: DRGYYGSSYWYFDV.

[0223] Example 2: Preparation and Validation of Key Protein Antibodies

[0224] To support the core components of the reagent kit of this invention, specific rabbit monoclonal antibodies against five key sperm functional proteins, namely HE12, PP1R7, KT3K, TMCO3, and UAP1, were successfully prepared, and the performance of the system was verified.

[0225] 1. Successful preparation and basic characteristics of antibodies

[0226] Five hybridoma cell lines capable of stably secreting target antibodies were successfully obtained using recombinant protein immunoassay hybridoma technology. After antibody purification, the purity of all five antibodies was greater than 95% as analyzed by SDS-PAGE and high-performance liquid chromatography (HPLC). As shown in Table 1, all five antibodies exhibited high titers as determined by indirect ELISA, producing strong positive signals even at dilutions exceeding 1:100,000, ensuring detection sensitivity at low working concentrations (0.05 mg / mL). More importantly, in Western blotting validation of clinical samples with asthenospermia, all five proteins showed varying degrees of abnormal expression patterns. PP1R7 and TMCO3 showed the highest abnormality rates (68% and 60%, respectively), which highly coincides with the core pathophysiological aspects of asthenospermia—flagellate motility dysfunction and energy metabolism disorders—strongly suggesting that these two can serve as key targets for assessing the molecular etiology of asthenospermia. Abnormalities in HE12, UAP1, and KT3K reveal acrosome dysfunction, decreased fertilization potential, and potential genetic risks often associated with asthenospermia, demonstrating the comprehensive advantages of this multi-target detection protocol in fully assessing sperm function and infertility risk.

[0227] Table 1. Basic characteristics and potency analysis of key antibodies

[0228] .

[0229] 2. Validation of antibody affinity and functional localization

[0230] Surface plasmon resonance (SPR) analysis showed that the equilibrium dissociation constants (KD) of the five antibodies to their corresponding antigens were all in the nanomolar range (10⁻⁶). -9 M) to Anamore (10 -10 The antibodies exhibited excellent high affinity at the M) level, which is key to achieving high-contrast, low-background fluorescence detection. The functionality of the antibodies was ultimately verified through immunofluorescence experiments. As shown in Table 2, using the kit procedure provided by this invention, all antibodies produced bright and precisely localized specific fluorescence signals on normal human sperm samples, with a high signal-to-noise ratio. Furthermore, the localization consistency among different sperm exceeded 95.7%, fully meeting the requirements for accurate quantitative analysis.

[0231] Table 2 Immunofluorescence localization and performance analysis

[0232] .

[0233] The localization rate and mean fluorescence intensity (MFI) of all five proteins in the asthenospermia group were significantly lower than those in the normal group (p<0.001), confirming the widespread functional molecular defects in asthenospermia sperm from both spatial distribution and abundance perspectives. The expression of PP1R7 and TMCO3 was most significantly decreased in the asthenospermia group, with their localization rate and mean MFI values ​​being significantly lower than normal levels. This corroborates the Western Blot results in Table 1, further confirming that abnormal flagellar motility regulation (PP1R7) and insufficient mitochondrial energy production (TMCO3) are among the most important molecular causes of asthenospermia. The expression of HE12 (acrosomal region) and UAP1 (fertilization recognition) was also significantly reduced, suggesting that a considerable proportion of asthenospermia patients (approximately 35%-30%) may also face functional disorders in the fertilization process, which has important guiding significance for prognosis and the selection of assisted reproductive technologies. The extremely high localization concordance (>95%) in the normal group demonstrates the high accuracy and reproducibility of the antibody and detection methods in this system, ensuring the reliability of clinical test results.

[0234] The data in Tables 1 and 2 together demonstrate that the five proteins targeted by this invention—HE12, PP1R7, KT3K, TMCO3, and UAP1—not only possess excellent high specificity, high affinity, and precise localization capabilities at the technical level, but have also been clinically proven to be closely related to various molecular causes of asthenospermia. This provides a solid experimental data foundation and theoretical basis for constructing a diagnostic, classification, and etiological analysis system for asthenospermia based on multi-target protein expression.

[0235] 3. Batch consistency

[0236] To ensure the stability and reliability of the reagent kit, this invention conducted batch-to-batch consistency assessments on three independently produced antibody batches. The coefficient of variation (CV) of fluorescence intensity in the immunofluorescence assay results was less than 10%, demonstrating the stability of the antibody production process and the controllability of product quality.

[0237] Conclusion: In summary, this invention not only successfully prepared five high-performance rabbit-derived monoclonal antibodies, but also demonstrated their high titer, high specificity, high affinity, and precise localization ability through systematic experimental data. These core reagents provide a solid foundation for the reliable operation of the sperm protein quality analyzer of this invention.

[0238] Example 3: Sperm protein fluorescent labeling kit

[0239] The reagent kit provided by this invention consists of the following components:

[0240] Table 3. Components of the reagent kit

[0241] ;

[0242] .

[0243] Storage conditions and shelf life

[0244] 1. Kit A should be stored below -20°C, and Kit B should be stored at room temperature. Shelf life is 12 months.

[0245] 2. After opening, reagent kit A should be stored with the antibody at 2-8°C and the slides at 2-8°C. It is effective for 24 hours. After opening, reagent kit B should be stored at room temperature. It is effective for 2 months.

[0246] Applicable instruments

[0247] The core image acquisition equipment is a fluorescence microscope. Recommended models are the Zeiss LSM800 and Nikon C2; other models require technical support and calibration from the manufacturer before use. The system can also be equipped with a standard optical microscope module for morphological and kinematic analysis. The case creation interface of the sperm protein quality analysis system is shown below. Figure 3 As shown, the protein analysis interface of the sperm protein quality analysis system is as follows: Figure 4 As shown.

[0248] Sample processing and storage

[0249] 1. Fresh semen samples should be used immediately after liquefaction, or stored in a refrigerator at 2-8℃ for up to 12 hours before use.

[0250] 2. Immunofluorescence assays cannot be performed on the same day. Place the methanol-fixed sperm smears into a slide box containing a desiccant, seal the box in a self-sealing bag, and store at -20°C or below for no more than one year. Use within the expiration date of the kit.

[0251] Sample requirements: Fresh semen.

[0252] Interpretation of test results

[0253] 1. The magnitude of deviation from the normal reference range (marked with ↑ or ↓) indicates the sperm quality level.

[0254] 2. Improper sperm washing, concentration adjustment, and smear preparation during semen sample processing can all affect the acquisition of fluorescence microscope images.

[0255] 3. Invalid results: If the results of the control wells do not meet the required range, contact the manufacturer's technical personnel to check for problems with the reagent kit and equipment system, and then retest.

[0256] Factors affecting human sperm protein quality analyzer

[0257] Table 4 Factors affecting human sperm protein quality analyzer

[0258] ;

[0259] .

[0260] Example 4: Validation of the reagent kit's analytical performance

[0261] To evaluate the reliability and effectiveness of the sperm protein fluorescent labeling kit described in this invention in the diagnosis of asthenospermia, in addition to routine performance verification, this invention also conducted specific analysis and verification on a clinical sample cohort of asthenospermia patients. An example image of the sperm protein quality analysis report generated by the sperm protein quality analysis system of this invention is shown below. Figure 5 As shown.

[0262] 1. Precision

[0263] Using the same batch of reagent kits, one normal sperm sample and one sample diagnosed with asthenospermia were repeatedly tested (n=20). The results are as follows:

[0264] (1) Normal samples: The intra-batch precision CV% of the five target proteins is 4.2%-8.5%.

[0265] (2) Asthenospermia samples: The intra-assay precision coefficients (CVs) for the five target proteins ranged from 5.8% to 9.7%. Although the asthenospermia samples themselves exhibited high heterogeneity, the CVs remained below 10%, indicating that the kit still demonstrated good reproducibility when detecting abnormal samples. Inter-assay precision (three batches) assessment showed that the coefficients of variation for the detection results of typical abnormal proteins in asthenospermia (such as PP1R7 and TMCO3) were all less than 12%, meeting the requirements for clinical testing.

[0266] 2. Correlation between accuracy and asthenospermia samples

[0267] The results of this kit (immunofluorescence assay) and Western blotting (WB) were compared on 30 paired samples (15 normal, 15 asthenospermia). The overall correlation coefficient r = 0.96. Further analysis showed:

[0268] In asthenospermia samples, the results of the two methods were highly consistent for PP1R7 and TMCO3 proteins, which were significantly downregulated (r=0.98).

[0269] This kit can more sensitively detect "partially defective" samples where the WB bands are lighter but not completely absent, demonstrating its advantage in quantifying the gradient expression of proteins related to asthenospermia.

[0270] 3. Clinical Validity: Focusing on Asthenospermia

[0271] A prospective study was conducted, including 150 semen samples (100 with asthenospermia and 50 with normal sperm). While using this kit to detect five target proteins, WHO standard semen analysis parameters (focusing on the proportion of progressively motile sperm) were recorded.

[0272] Diagnostic consistency: The gold standard for asthenospermia is a progressive motility rate of <32%. The results of this kit (based on the core indicators of PP1R7 and TMCO3) showed a concordance rate of 94.7%.

[0273] Etiological Implications: Among 100 patients with asthenospermia, 68 cases were identified as "motor core disorder type" (significantly abnormal PP1R7 / TMCO3) based on the five-target protein expression profile. Subsequent clinical examinations (such as flagellar ultrastructure analysis and mitochondrial function assessment) revealed clear corresponding abnormalities in 85.3% of these cases. This demonstrates that the protein detection results of this kit can effectively reveal the specific molecular causes behind asthenospermia.

[0274] Predictive value: A 3-month follow-up of 50 patients with asthenospermia who received targeted treatment revealed that patients who were diagnosed as "motor core disorder type" before treatment and whose PP1R7 / TMCO3 expression was significantly improved after treatment had a significantly higher increase in the proportion of forward motility sperm than patients with other subtypes or no improvement (p<0.01).

[0275] Conclusion: Supplementary data show that the kit of the present invention not only has excellent analytical performance, but also demonstrates high diagnostic accuracy, accurate etiological classification ability and potential predictive value for treatment response in the clinical detection of asthenospermia, fully verifying its clinical effectiveness in serving the accurate diagnosis of asthenospermia.

[0276] Example 5: Method for performing asthenospermia-related analysis using the kit and system described in Example 3

[0277] I. The reagent kit operation steps are as follows:

[0278] (a) Provide a human semen sample;

[0279] (b) The sample was washed with 1× washing buffer (prepared by diluting 30× concentrated washing buffer with deionized water at a ratio of 1:29), centrifuged (1500 rpm, 5 min), and resuspended. The washing was repeated twice to adjust the sperm concentration to 40-50×10⁻⁶. 6 / mL;

[0280] (c) Take 1 μL of the treated sperm sample and spread it on a porous glass slide (see schematic diagram of the slide for the sperm protein fluorescent labeling kit). Figure 2Immediately acquire bright-field images or motion videos (unfixed state) of sperm in the designated wells (as shown);

[0281] (d) The analysis software automatically analyzes the sperm morphology and motility parameters and generates a morphological and kinematic analysis report;

[0282] (e) The sperm sample was then fixed: dried at 37°C for 30 minutes, fixed with methanol for 5-10 minutes, dried at room temperature, and then incubated with the specific rabbit-derived first antibody for ≥8 hours to form an antigen-antibody complex.

[0283] (f) After gently rinsing with 1× washing solution, immerse in three washing tanks containing 1× washing solution for 5 minutes each, rinse with deionized water and dry, and then incubate with FITC-labeled secondary antibody (10 μL per well) at 37°C for 30 min in the dark.

[0284] (g) After washing and drying, the samples were counterstained with propidium iodide (PI) nuclear dye (10 μL per well);

[0285] (h) After adding one drop of mounting glycerin to each well and covering it with a coverslip, use a fluorescence microscope (40x objective lens, 488nm and 561nm laser channels) to acquire FITC and PI signal images. Ensure that the system acquires at least 200 valid images of sperm per sample.

[0286] (i) Based on the acquired multi-channel fluorescence images, the dedicated analysis software automatically performs image registration and synthesis, identifies and segments sperm based on PI channel signals, calculates the average fluorescence intensity (MFI) and localization rate of each target protein in specific cell regions of sperm, generates a quantitative analysis report, and compares the results with the built-in reference interval.

[0287] Based on the above process, this system was applied to over 300 clinical samples (including 200 cases of asthenospermia), accumulating key operational experience and data related to asthenospermia detection:

[0288] Sample processing and motility analysis: For asthenospermia samples, due to their low motility, the observation time should be appropriately extended to 3-5 seconds when collecting motility videos in step (c), and the ambient temperature should be strictly controlled at 37°C to obtain statistically significant motility parameters. Data analysis revealed that the average VCL in the asthenospermia group was 20.3±7.1 μm / s, significantly lower than the 38.5±8.9 μm / s in the normal group (p<0.001), and the proportion of progressively motile sperm was significantly positively correlated with the protein expression level of PP1R7 / TMCO3 (r=0.72, p<0.001).

[0289] Immunofluorescence staining: During the immunofluorescence staining process in steps (e)-(g), it was found that asthenospermia samples occasionally had a lot of background (such as cell debris and non-sperm cells), requiring strict adherence to the washing procedure. After applying the standard procedure, 98.5% of asthenospermia samples could obtain fluorescence images that met the requirements for quantitative analysis.

[0290] Image Analysis and Report Interpretation: The software analysis in step (i) is crucial for asthenospermia samples. The system successfully identified the characteristic "protein expression heterogeneity" in asthenospermia samples, that is, the presence of sperm with normal expression, weakened expression, and absent expression in the same sample.

[0291] Example 6: Clinical Application and Criteria for Asthenospermia

[0292] Based on the kit and analysis system described in this invention, the fluorescence intensity and localization rate of five key target proteins (HE12, PP1R7, KT3K, TMCO3, and UAP1) in sperm samples were quantitatively analyzed. The results were interpreted according to the following evaluation criteria, and molecular subtyping of asthenospermia was performed in combination with protein expression patterns, providing a basis for clinical diagnosis and treatment.

[0293] I. Criteria for Evaluating the Quality of Key Sperm Functional Proteins

[0294] The table below shows the reference ranges and clinical significance of the localization rate and mean fluorescence intensity (MFI) of each target protein. The expression level of each protein is divided into three levels: "Excellent", "Good", and "Needs Improvement".

[0295] Table 5. Criteria for Evaluating the Protein Quality of Key Sperm Functions

[0296] .

[0297] Based on the abnormal combination patterns of five target proteins, the system classifies asthenospermia into the following four molecular subtypes and provides corresponding clinical recommendations:

[0298] Table 6. Molecular subtyping and clinical significance of asthenospermia based on five-target protein expression profiles.

[0299] .

[0300] III. Comprehensive Evaluation and Clinical Application Guidelines

[0301] Sub-evaluation: The "localization rate" and "average fluorescence intensity" of each protein were analyzed independently, and the expression level was determined with reference to Table 5.

[0302] Comprehensive classification: Based on the abnormal patterns of each protein, asthenospermia is classified into molecular types according to Table 6, and a comprehensive diagnostic report is generated.

[0303] Clinical suggestion:

[0304] 1. If PP1R7 / TMCO3 is abnormal, it indicates motor dysfunction, and targeted vitality improvement treatment can be carried out.

[0305] 2. If HE12 / UAP1 is abnormal, it indicates a defect in the fertilization process, and ICSI should be given priority in assisted reproduction.

[0306] 3. If multiple target points are abnormal, it suggests severe functional impairment, and the prognosis needs to be carefully assessed.

[0307] 4. Threshold Explanation: The thresholds listed in the table are based on clinical research data. The actual diagnostic thresholds should be based on the product instructions and calibrated with each batch of the kit.

Claims

1. A test system for assessing sperm motility function in asthenozoospermia, characterized in that, The kit comprises a sperm protein fluorescence labeling kit, an image acquisition system, and a sperm protein quality analysis platform. The antibody components include specific antibodies of rabbit anti-human HE12, PP1R7, KT3K, TMCO3, and UAP1. The specific antibodies of rabbit anti-human HE12, PP1R7, KT3K, TMCO3, and UAP1 have the following specific sequences: (a) HE12 antibody The HE12 antibody light chain sequence is shown in SEQ ID NO. 1, the HE12 antibody light chain variable region VL is shown in SEQ ID NO. 2, the HE12 antibody light chain complementarity determining region CDR-L1 is shown in SEQ ID NO. 3, the HE12 antibody light chain complementarity determining region CDR-L2 is shown in SEQ ID NO. 4, the HE12 antibody light chain complementarity determining region CDR-L3 is shown in SEQ ID NO. 5; the HE12 antibody heavy chain sequence is shown in SEQ ID NO. 6, the HE12 antibody heavy chain variable region VH is shown in SEQ ID NO. 7, the HE12 antibody heavy chain complementarity determining region CDR-H1 is shown in SEQ ID NO. 8, the HE12 antibody heavy chain complementarity determining region CDR-H2 is shown in SEQ ID NO. 9, and the HE12 antibody heavy chain complementarity determining region CDR-H3 is shown in SEQ ID NO. 10; (b) PP1R7 antibody The PP1R7 antibody light chain sequence is shown in SEQ ID NO. 11, the PP1R7 antibody light chain variable region VL is shown in SEQ ID NO. 12, the PP1R7 antibody light chain complementarity determining region CDR-L1 is shown in SEQ ID NO. 13, the PP1R7 antibody light chain complementarity determining region CDR-L2 is shown in SEQ ID NO. 14, the PP1R7 antibody light chain complementarity determining region CDR-L3 is shown in SEQ ID NO. 15; the PP1R7 heavy chain sequence is shown in SEQ ID NO. 16, the PP1R7 antibody heavy chain variable region VH is shown in SEQ ID NO. 17, the PP1R7 antibody heavy chain complementarity determining region CDR-H1 is shown in SEQ ID NO. 18, the PP1R7 antibody heavy chain complementarity determining region CDR-H2 is shown in SEQ ID NO. 19, and the PP1R7 antibody heavy chain complementarity determining region CDR-H3 is shown in SEQ ID NO. 20; (c) KT3K antibody The KT3K antibody light chain sequence is shown in SEQ ID NO. 21, the KT3K antibody light chain variable region VL is shown in SEQ ID NO. 22, the KT3K antibody light chain complementarity determining region CDR-L1 is shown in SEQ ID NO. 23, the KT3K antibody light chain complementarity determining region CDR-L2 is shown in SEQ ID NO. 24, the KT3K antibody light chain complementarity determining region CDR-L3 is shown in SEQ ID NO. 25; the KT3K heavy chain sequence is shown in SEQ ID NO. 26, the KT3K antibody heavy chain variable region VH is shown in SEQ ID NO. 27, the KT3K antibody heavy chain complementarity determining region CDR-H1 is shown in SEQ ID NO. 28, the KT3K antibody heavy chain complementarity determining region CDR-H2 is shown in SEQ ID NO. 29, and the KT3K antibody heavy chain complementarity determining region CDR-H3 is shown in SEQ ID NO.

30. The sequence of the light chain of the KT3K antibody is shown as SEQ ID NO. 21, the variable region VL of the light chain of the KT3K antibody is shown as SEQ ID NO. 22, the CDR-L1 of the light chain of the KT3K antibody is shown as SEQ ID NO. 23, the CDR-L2 of the light chain of the KT3K antibody is shown as SEQ ID NO. 24, and the CDR-L3 of the light chain of the KT3K antibody is shown as SEQ ID NO. 25; the sequence of the heavy chain of the KT3K antibody is shown as SEQ ID NO. 26, the variable region VH of the heavy chain of the KT3K antibody is shown as SEQ ID NO. 27, the CDR-H1 of the heavy chain of the KT3K antibody is shown as SEQ ID NO. 28, the CDR-H2 of the heavy chain of the KT3K antibody is shown as SEQ ID NO. 29, and the CDR-H3 of the heavy chain of the KT3K antibody is shown as SEQ ID NO. 30; (d) TMCO3 antibody The sequence of the light chain of the TMCO3 antibody is shown as SEQ ID NO. 31, the variable region VL of the light chain of the TMCO3 antibody is shown as SEQ ID NO. 32, the CDR-L1 of the light chain of the TMCO3 antibody is shown as SEQ ID NO. 33, the CDR-L2 of the light chain of the TMCO3 antibody is shown as SEQ ID NO. 34, and the CDR-L3 of the light chain of the TMCO3 antibody is shown as SEQ ID NO. 35; the sequence of the heavy chain of the TMCO3 antibody is shown as SEQ ID NO. 36, the variable region VH of the heavy chain of the TMCO3 antibody is shown as SEQ ID NO. 37, the CDR-H1 of the heavy chain of the TMCO3 antibody is shown as SEQ ID NO. 38, the CDR-H2 of the heavy chain of the TMCO3 antibody is shown as SEQ ID NO. 39, and the CDR-H3 of the heavy chain of the TMCO3 antibody is shown as SEQ ID NO. 40; (e) UAP1 antibody The sequence of the light chain of the UAP1 antibody is shown as SEQ ID NO. 41, the variable region VL of the light chain of the UAP1 antibody is shown as SEQ ID NO. 42, the CDR-L1 of the light chain of the UAP1 antibody is shown as SEQ ID NO. 43, the CDR-L2 of the light chain of the UAP1 antibody is shown as SEQ ID NO. 44, and the CDR-L3 of the light chain of the UAP1 antibody is shown as SEQ ID NO. 45; the sequence of the heavy chain of the UAP1 antibody is shown as SEQ ID NO. 46, the variable region VH of the heavy chain of the UAP1 antibody is shown as SEQ ID NO. 47, the CDR-H1 of the heavy chain of the UAP1 antibody is shown as SEQ ID NO. 48, the CDR-H2 of the heavy chain of the UAP1 antibody is shown as SEQ ID NO. 49, and the CDR-H3 of the heavy chain of the UAP1 antibody is shown as SEQ ID NO. 50; Labeling system: fluorescently labeled secondary antibody, nuclear fluorescent dye; Solid support: sperm protein slide, which is a multi-well slide pre-coated with positive and negative control human sperm samples in the wells; Kit B is the following components: Concentrated washing solution, mounting medium, fixative; The image acquisition system includes a fluorescence microscope with a multi-laser excitation device, a photomultiplier tube detector, and a Z-axis layer scanning function. The sperm protein quality analysis platform includes a medical record management module, a protein analysis module, a morphology and motion analysis module, a data management module, and a system management module.

2. The system for evaluating sperm motility function in asthenospermia according to claim 1, wherein The antibody diluent is phosphate buffer, bovine serum albumin, and sodium azide.

3. The system for evaluating sperm motility function in asthenospermia according to claim 1, wherein The fluorescently labeled secondary antibody is fluorescein isothiocyanate-labeled goat anti-mouse IgG antibody.

4. The system for evaluating sperm motility function in asthenospermia according to claim 1, wherein The concentrated washing solution is 0.3M phosphate buffer at pH 7.

4.

5. The system for evaluating sperm motility function in asthenospermia according to claim 1, wherein The nuclear fluorescent dye is propidium iodide (PI) solution.

6. The system for evaluating sperm motility function in asthenospermia according to claim 1, wherein The built-in algorithms of the sperm protein quality analysis platform are as follows: (1) Sperm head detection algorithm PI positive sperm recognition formula: I_PI(x,y)≥T_PI=μ_background + 3σ_background; Where: I_PI(x,y): PI channel pixel intensity; T_PI: PI positive threshold; μ_background: background area average intensity; σ_background: background area intensity standard deviation; Head morphology screening conditions: A_min≤A_head≤A_max; 0.7≤E_head=(4πA_head) / (P_head²)≤1.0; Where: A_head: head area pixels; P_head: head perimeter pixels; E_head: head ellipticity; (2) Sperm tail segmentation algorithm Tail recognition formula: L_tail≥L_min; W_tail≤W_max; S_tail=L_tail / W_tail≥S_min; Where: L_tail: tail length; W_tail: tail width; S_tail: tail length-width ratio; (3) Green fluorescence expression analysis Fluorescence intensity calculation formula: FI_green=(1 / N)∑[I_green(x,y) - I_background]; FI_green: average fluorescence intensity; N: number of sperm head pixels; I_green(x,y): green channel pixel intensity; Fluorescence positive judgment: FI_green≥T_positive=μ_negative + Kσ_negative; Where: T_positive: positive threshold; μ_negative: negative control average fluorescence intensity; σ_negative: negative control fluorescence intensity standard deviation; K: confidence coefficient; (4) Key statistical indicators Total number of sperm: N_total=∑[I_PI(i)≥T_PI]; Number of expressing sperm: N_expression=∑[FI_green(i)≥T_positive]; Localization rate: R_expression= (N_expression / N_total)×100%; Mean fluorescence intensity: MFI= (1 / N_expression)∑FI_green(i).

7. A method of using a test system for assessing sperm motility function in asthenospermia, characterized in that, The method comprises the following steps: (a) spreading the processed semen sample on a multi-well slide, collecting bright field images or motion videos using an optical microscope, and qualitatively detecting sperm morphology; (b) fixing the smear obtained in step (a) and incubating it with specific rabbit-derived first antibodies HE12, PP1R7, KT3K, TMCO3, and UAP1 against target proteins; after incubation, washing and drying; (c) incubating the washed and dried smear in step (b) with a FITC-labeled second antibody; after incubation, washing and drying; (d) using propidium iodide (PI) solution to perform nuclear restaining on the washed and dried smear in step (c); then washing and drying, and then performing mounting; (e) collecting images of the mounted smear in step (d) through a fluorescence microscope; (f) judging the qualitative expression of each target protein on sperm through software.

8. The method of using a system for assessing sperm motility function in asthenospermia according to claim 7, wherein, In step (b), methanol is fixed at room temperature for 5 to 10 minutes, and the specific rabbit-derived first antibody is incubated for ≥8 h.

9. The method of using a system for assessing sperm motility function in asthenospermia according to claim 7, wherein, In step (e), after mounting, the fluorescence microscope is used to collect FITC and PI signal images at 40 times magnification under 488 nm and 561 nm channels, and no less than 200 sperm are collected for each sample.

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