Embryonic developmental potential sperm key protein kit and method of use thereof

Abstract

The present application relates to the field of biomedical detection reagent manufacturing and reproductive medicine technology, and particularly relates to a key protein kit for embryo development potential sperm and a use method thereof. The method is characterized in that a combination of antibodies capable of specifically recognizing a group of key proteins related to embryo development potential in human sperm is prepared, and the antibodies are assembled into a special detection kit together with a fluorescently labeled secondary antibody, a nuclear dye, a concentrated washing solution, a mounting agent and a pre-coated control multi-well slide. The kit can be used with a multi-modal imaging system (such as a fluorescence / confocal microscope, an ordinary optical microscope) and image analysis software to realize traditional analysis of sperm morphology and motility, and synchronous and accurate qualitative and quantitative analysis of the expression level and subcellular localization of the key proteins. The present application provides an integrated molecular detection solution, and provides a powerful tool for sperm quality screening and embryo development potential prediction in assisted reproductive technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical detection reagent manufacturing and reproductive medicine technology, in particular to a key protein kit for sperm and its use method. BACKGROUND

[0002] Male infertility is a major health problem affecting about 15% of couples of childbearing age worldwide, of which about half is due to male factors. At present, the evaluation of male fertility in clinical practice mainly relies on semen routine analysis and computer-assisted semen analysis (CASA) technology. Although these methods can evaluate the macroscopic parameters such as concentration, motility and morphology of sperm, they cannot reflect the intrinsic molecular functional state of sperm, especially the potential of supporting embryo development in assisted reproductive technology (such as ICSI).

[0003] In recent years, it has been found that the expression of specific functional proteins in sperm is closely related to fertilization ability and early embryo development. For example, PLCZ1 is a key factor for egg activation, and its absence can lead to fertilization failure after ICSI; ACTL7A and ACTL9 are the core components of the sperm perforator structure, which directly affect sperm-egg fusion; IQCN is involved in calcium signal regulation and related to acrosome reaction; CD46 plays an important role in immune protection and sperm-egg interaction. In addition, many other proteins have been found to be related to sperm function.

[0004] However, the existing detection techniques (such as Western Blot and conventional immunofluorescence) are mostly single indicator detection, with low throughput and insufficient standardization, making it difficult to realize simultaneous, quantitative and localization analysis of multiple proteins, and even more difficult to systematically evaluate the relationship between the combination of these proteins and embryo development potential. Therefore, there is an urgent need in the art for an integrated detection scheme that can simultaneously and accurately evaluate the expression and localization of multiple key sperm proteins, in order to fill the gap in existing technology and provide a more comprehensive sperm quality evaluation and embryo development potential prediction tool for clinical practice. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a key protein kit for sperm and its use method.

[0006] The present application is realized by the following technical solutions:

[0007] In a first aspect, the present application provides a key protein kit for sperm and its use method, which comprises kit A and kit B, and kit A comprises the following components:

[0008] Antibody component: including mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 specific antibodies;

[0009] Labeling system: fluorescently labeled secondary antibody, nuclear fluorescent dye;

[0010] Solid support: sperm protein slides, sperm protein slides are multi-well slides, the wells are pre-coated with positive and negative control human sperm samples;

[0011] Kit B is the following components:

[0012] Concentrated washing solution, mounting medium, fixative;

[0013] Further, the specific antibodies of mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 are as follows:

[0014] (a) ACTL9 antibody

[0015] The sequence of the ACTL9 antibody light chain is shown as SEQ ID NO. 1, the variable region VL of the ACTL9 antibody light chain is shown as SEQ ID NO. 2, the complementary determining region CDR-L1 of the ACTL9 antibody light chain is shown as SEQ ID NO. 3, the complementary determining region CDR-L2 of the ACTL9 antibody light chain is shown as SEQ ID NO. 4, the complementary determining region CDR-L3 of the ACTL9 antibody light chain is shown as SEQ ID NO. 5; The sequence of the ACTL9 antibody heavy chain is shown as SEQ ID NO. 6, the variable region VH of the ACTL9 antibody heavy chain is shown as SEQ ID NO. 7, the complementary determining region CDR-H1 of the ACTL9 antibody heavy chain is shown as SEQ ID NO. 8, the complementary determining region CDR-H2 of the ACTL9 antibody heavy chain is shown as SEQ ID NO. 9, and the complementary determining region CDR-H3 of the ACTL9 antibody heavy chain is shown as SEQ ID NO. 10;

[0016] (b) ACTL7A antibody

[0017] The ACTL7A antibody light chain sequence is shown in SEQ ID NO.11, the ACTL7A antibody light chain variable region VL is shown in SEQ ID NO.12, the ACTL7A antibody light chain complementarity-determining region CDR-L1 is shown in SEQ ID NO.13, the ACTL7A antibody light chain complementarity-determining region CDR-L2 is shown in SEQ ID NO.14, and the ACTL7A antibody light chain complementarity-determining region CDR-L3 is shown in SEQ ID NO.15; the ACTL7A heavy chain sequence is shown in SEQ ID NO.16, the ACTL7A antibody heavy chain variable region VH is shown in SEQ ID NO.17, the ACTL7A antibody heavy chain complementarity-determining region CDR-H1 is shown in SEQ ID NO.18, the ACTL7A antibody heavy chain complementarity-determining region CDR-H2 is shown in SEQ ID NO.19, and the ACTL7A antibody heavy chain complementarity-determining region CDR-H3 is shown in SEQ ID NO.20.

[0018] (c) PLCZ1 antibody

[0019] The PLCZ1 antibody light chain sequence is shown in SEQ ID NO.21, the variable region VL of the PLCZ1 antibody light chain is shown in SEQ ID NO.22, the complementarity-determining region CDR-L1 of the PLCZ1 antibody light chain is shown in SEQ ID NO.23, the complementarity-determining region CDR-L2 of the PLCZ1 antibody light chain is shown in SEQ ID NO.24, and the complementarity-determining region CDR-L3 of the PLCZ1 antibody light chain is shown in SEQ ID NO.25; the PLCZ1 heavy chain sequence is shown in SEQ ID NO.26, the variable region VH of the PLCZ1 antibody heavy chain is shown in SEQ ID NO.27, the complementarity-determining region CDR-H1 of the PLCZ1 antibody heavy chain is shown in SEQ ID NO.28, the complementarity-determining region CDR-H2 of the PLCZ1 antibody heavy chain is shown in SEQ ID NO.29, and the complementarity-determining region CDR-H3 of the PLCZ1 antibody heavy chain is shown in SEQ ID NO.30.

[0020] (d) IQCN antibody

[0021] The sequence of the light chain of the IQCN antibody is shown as SEQ ID NO. 31, the variable region VL of the light chain of the IQCN antibody is shown as SEQ ID NO. 32, the CDR-L1 of the light chain of the IQCN antibody is shown as SEQ ID NO. 33, the CDR-L2 of the light chain of the IQCN antibody is shown as SEQ ID NO. 34, the CDR-L3 of the light chain of the IQCN antibody is shown as SEQ ID NO. 35; the sequence of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 36, the variable region VH of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 37, the CDR-H1 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 38, the CDR-H2 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 39, the CDR-H3 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 40;

[0022] (e) CD46 antibody

[0023] The sequence of the light chain of the CD46 antibody is shown as SEQ ID NO. 41, the variable region VL of the light chain of the CD46 antibody is shown as SEQ ID NO. 42, the CDR-L1 of the light chain of the CD46 antibody is shown as SEQ ID NO. 43, the CDR-L2 of the light chain of the CD46 antibody is shown as SEQ ID NO. 44, the CDR-L3 of the light chain of the CD46 antibody is shown as SEQ ID NO. 45; the sequence of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 46, the variable region VH of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 47, the CDR-H1 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 48, the CDR-H2 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 49, the CDR-H3 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 50;

[0024] (f) SPAG9 antibody

[0025] The SPAG9 antibody light chain sequence is shown as SEQ ID NO. 51, the SPAG9 antibody light chain variable region VL is shown as SEQ ID NO. 52, the SPAG9 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 53, the SPAG9 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 54, the SPAG9 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 55; the SPAG9 heavy chain sequence is shown as SEQ ID NO. 56, the SPAG9 antibody heavy chain variable region VH is shown as SEQ ID NO. 57, the SPAG9 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 58, the SPAG9 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 59, the SPAG9 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 60;

[0026] (g) SPESP1 antibody

[0027] The SPESP1 antibody light chain sequence is shown as SEQ ID NO. 61, the SPESP1 antibody light chain variable region VL is shown as SEQ ID NO. 62, the SPESP1 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 63, the SPESP1 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 64, the SPESP1 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 65; the SPESP1 heavy chain sequence is shown as SEQ ID NO. 66, the SPESP1 antibody heavy chain variable region VH is shown as SEQ ID NO. 67, the SPESP1 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 68, the SPESP1 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 69, the SPESP1 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 70;

[0028] (h) IZUMO3 antibody

[0029] The IZUMO3 antibody light chain sequence is shown as SEQ ID NO. 71, the IZUMO3 antibody light chain variable region VL is shown as SEQ ID NO. 72, the IZUMO3 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 73, the IZUMO3 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 74, the IZUMO3 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 75; the IZUMO3 antibody heavy chain sequence is shown as SEQ ID NO. 76, the IZUMO3 antibody heavy chain variable region VH is shown as SEQ ID NO. 77, the IZUMO3 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 78, the IZUMO3 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 79, the IZUMO3 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 80;

[0030] (i) a TMEM95 antibody

[0031] The TMEM95 antibody light chain sequence is shown as SEQ ID NO. 81, the TMEM95 antibody light chain variable region VL is shown as SEQ ID NO. 82, the TMEM95 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 83, the TMEM95 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 84, the TMEM95 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 85; the TMEM95 antibody heavy chain sequence is shown as SEQ ID NO. 86, the TMEM95 antibody heavy chain variable region VH is shown as SEQ ID NO. 87, the TMEM95 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 88, the TMEM95 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 89, the TMEM95 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 90;

[0032] (j) a CFAP90 antibody

[0033] The CFAP90 antibody light chain sequence is shown as SEQ ID NO. 91, the CFAP90 antibody light chain variable region VL is shown as SEQ ID NO. 92, the CFAP90 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 93, the CFAP90 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 94, the CFAP90 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 95; the CFAP90 heavy chain sequence is shown as SEQ ID NO. 96, the CFAP90 antibody heavy chain variable region VH is shown as SEQ ID NO. 97, the CFAP90 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 98, the CFAP90 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 99, and the CFAP90 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 100.

[0034] Further, the antibody diluent is phosphate buffer, bovine serum albumin, sodium azide.

[0035] Further, the fluorescently labeled secondary antibody is a fluorescein isothiocyanate-labeled goat anti-mouse IgG antibody.

[0036] Further, the concentrated washing solution is 0.3M phosphate buffer with pH 7.4.

[0037] Further, the mounting agent is mounting glycerol.

[0038] Further, the nuclear dye is propidium iodide (PI) solution.

[0039] In a second aspect, the present application provides a method for using a key protein kit for embryonic development potential sperm, comprising the following steps:

[0040] (a) Sample pretreatment

[0041] The treated sperm sample is applied to a multi-well slide, and a bright field image or a motion video is collected using a general optical microscope for qualitative detection of sperm morphology;

[0042] (b) Fix the smear and incubate with specific first antibodies against ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90;

[0043] (c) Incubate with a FITC-labeled secondary antibody;

[0044] (d) Perform nuclear restaining with propidium iodide (PI) solution;

[0045] (e) After sealing, images were collected by confocal microscopy;

[0046] (f) The qualitative expression of each target protein on sperm was determined by software.

[0047] Further, in step (a), the concentration of the treated sperm is 40-50 x 10 6 / mL; in step (b), the first antibody incubation conditions are: incubation in a 2-8°C refrigerator for at least 8 hours or incubation in a 37°C incubator for 1 hour.

[0048] Further, in step (f), the data of not less than 200 sperm are counted, and the average fluorescence intensity and localization rate of each protein ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90 are calculated.

[0049] Formula calculation:

[0050] (1) Sperm head detection algorithm

[0051] PI positive sperm recognition formula:

[0052] I PI (x, y) ≥ T PI = μ background + 3σ background;

[0053] Wherein:

[0054] I PI (x, y): PI channel pixel intensity;

[0055] T PI: PI positive threshold;

[0056] μ background: average intensity of background area;

[0057] σ background: standard deviation of background area intensity;

[0058] Head morphology screening conditions:

[0059] A min≤ A head≤ A max;

[0060] 0.7≤ E head = (4πA head) / (P head²) ≤ 1.0;

[0061] Wherein:

[0062] A head: head area (pixels);

[0063] P head: head perimeter (pixels);

[0064] E head: head ellipticity;

[0065] (2) Sperm tail segmentation algorithm

[0066] Tail recognition formula:

[0067] L_tail≥L_min;

[0068] W_tail≤W_max;

[0069] S_tail=L_tail / W_tail≥S_min;

[0070] Wherein:

[0071] L_tail: tail length;

[0072] W_tail: tail width;

[0073] S_tail: tail length-width ratio;

[0074] (3) Green fluorescence expression analysis

[0075] Fluorescence intensity calculation formula:

[0076] FI_green=(1 / N)∑[I_green(x,y)-I_background];

[0077] FI_green: average fluorescence intensity;

[0078] N: number of sperm head pixels;

[0079] I_green(x,y): green channel pixel intensity;

[0080] Fluorescence positive determination:

[0081] FI_green≥T_positive=μ_negative+Kσ_negative;

[0082] Wherein:

[0083] T_positive: positive threshold;

[0084] μ_negative: average fluorescence intensity of negative control;

[0085] σ_negative: standard deviation of fluorescence intensity of negative control;

[0086] K: confidence coefficient, take 2-3;

[0087] (4) Key statistical indicators

[0088] Total number of sperm:

[0089] N_total =∑ [I PI (i) ≥ T PI ];

[0090] Number of sperm expressing:

[0091] N_expression =∑ [FI green (i) ≥ T positive ];

[0092] Expression rate:

[0093] R_expression = (N_expression / N_total) x 100%;

[0094] Mean fluorescence intensity:

[0095] MFI = (1 / N_expression)∑FI green (i);

[0096] The calculation formula of the positioning rate (PR) is:

[0097] ;

[0098] Wherein, N positive is the number of sperm expressing a specific target protein, and N total is the total number of sperm detected.

[0099] More specifically, the present application has the following four core contents:

[0100] I. Screening and establishment of core protein markers

[0101] The present application first establishes ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95 and CFAP90 proteins as a combination of biomarkers for evaluating the potential of sperm embryo development. The combination comprehensively covers different key links of sperm function: PLCZ1 is a key factor for triggering calcium oscillation and activation of oocytes; ACTL7A and ACTL9 are key components of the sperm perforator structure, which directly affect sperm-egg fusion; IQCN participates in calcium homeostasis regulation, affecting acrosome reaction and fertilization capacity; CD46, as a complement regulatory protein, has an immunoprotective effect on sperm and participates in sperm-egg interaction; SPAG9 is a key regulatory protein for sperm movement and participates in sperm-egg recognition; SPESP1 is a protein necessary for sperm equatorial segment formation and acrosome exocytosis; IZUMO3 is the most core sperm protein that mediates oolemma fusion; TMEM95 is another key factor that mediates oolemma fusion, and its role position may be more downstream than IZUMO1; CFAP90 is an important protein for maintaining sperm flagellum structure and movement ability.

[0102] Simultaneous detection of the ten proteins can comprehensively evaluate sperm quality from the aspects of "oocyte activation", "fertilization ability", "immune microenvironment" and "embryo development potential".

[0103] Preparation of specific antibodies

[0104] One of the key steps of the preparation method of the present application is to produce high-specificity and high-titer antibodies against the target proteins mentioned above.

[0105] Preparation of antibodies: Recombinant human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90 proteins are used as immunogens to prepare specific monoclonal or polyclonal antibodies with a titer of not less than 1:100 by hybridoma technology or genetic engineering methods.

[0106] Epitope identification and specificity verification: The obtained antibodies are subjected to specific antigen epitope identification, and their specific binding ability is verified by Western Blot and immunofluorescence techniques to ensure no cross-reaction.

[0107] III. Assembly and preparation of detection kit

[0108] Another core of the preparation method of the present application is to provide an assembly scheme of a special detection kit, which comprises:

[0109] 1. Antibody component: including specific antibodies of mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90.

[0110] 2. Labeling system: fluorescently labeled secondary antibody (FITC-labeled goat anti-mouse antibody), and nuclear fluorescent dye (propidium iodide (PI) solution).

[0111] 3. Auxiliary reagents: concentrated washing solution and mounting medium.

[0112] 4. Solid support: specially designed multi-well glass slides, which are pre-coated with positive and negative control human sperm samples in the wells.

[0113] All components are strictly packaged and assembled according to the specified production process and quality control standards, and the storage conditions and expiration date are clearly stated to ensure the stability of the kit and the reliability of the detection results.

[0114] The product is composed of kit A (stored below -20 DEG C) and kit B (stored at room temperature). A and B are stored separately as required during storage, and need to be used jointly during use. Kit A is stored below -20 DEG C, and kit B is stored at room temperature. The shelf life is 12 months. After kit A is opened, the antibody is stored in an ice bath in a refrigerator at 2-8 DEG C, and the slide is stored in a refrigerator at 2-8 DEG C, and is effective within 24 hours. After kit B is opened, it is stored at room temperature and is effective within 2 months.

[0115] Four, integration of the multi-modal analysis system

[0116] The preparation method of the application further comprises hardware integration and software configuration of the analysis system:

[0117] 1. Hardware integration: fluorescence / laser confocal microscope (Zeiss LSM800, Nikon C2, etc.), general optical microscope, used for high-resolution multi-channel fluorescence imaging (confocal) and bright field imaging / motion video acquisition (general optical microscope) of the sperm sample to be measured.

[0118] 2. Software configuration: matched with "human sperm localization protein quantitative analysis software" or similar multi-modal image analysis system, supporting:

[0119] automatic identification of sperm and analysis of the fluorescence intensity, integral optical density and localization rate of ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95 and CFAP90 proteins;

[0120] analysis of sperm morphological parameters in the bright field image;

[0121] analysis of sperm morphological parameters (such as head area, ellipticity, vacuole ratio) in the bright field image of the processed sperm smear collected by the general optical microscope and sperm kinematic parameters (such as curve velocity VCL, straight line velocity VSL and linearity LIN) in the motion video; generation of a comprehensive report and comparison with the database reference value.

[0122] Compared with the prior art, the application has the following beneficial effects:

[0123] The innovation of the application lies in providing a systematic solution from core biomarker discovery to final detection application, and the beneficial effects mainly lie in the following three aspects:

[0124] 1. Innovation of marker combination and key sequence: the application first discloses and verifies a key protein combination comprising ACTL9, ACTL7A and the like, and specifically provides the immunogenic core protein sequence thereof. These specific amino acid sequences screened and verified are the basis for preparing high-specificity antibodies, and provide an unprecedented, functionally associated verified molecular target combination for the field.

[0125] 2. Innovation and controllability of core raw materials (antibodies): Based on the specific protein sequences described above, the present application provides a standardized preparation process for corresponding high-specificity monoclonal antibodies. The obtained antibodies can accurately recognize the natural conformation of the target protein, have high titer and strong specificity, and have no cross-reaction, which lays a solid foundation for the quality controllability and result reliability of the core detection reagent, and solves the bottleneck problem of lack of high-quality antibody raw materials in this field.

[0126] 3. Integrated innovation of whole-chain standardized detection scheme: The present application integrates the above self-prepared antibodies, optimized labeling system, pre-coated control solid phase carrier and special analysis software into one, and constructs a complete standardized detection kit and its application method. This scheme first realizes the synchronous, quantitative and multi-modal imaging analysis of the expression level and subcellular localization of the above-mentioned multiple key proteins in sperm, and provides a powerful tool for evaluating sperm function and embryo development potential before fertilization. BRIEF DESCRIPTION OF DRAWINGS

[0127] The present application will be further described below in conjunction with the drawings.

[0128] Figure 1 is the preparation process schematic diagram of the embryo development potential sperm key protein kit of the present application based on multi-modal imaging;

[0129] Figure 2 is the flow chart of the core protein marker screening and verification of the present application;

[0130] Figure 3 is the preparation process flow chart of the mouse anti-human specific monoclonal antibody;

[0131] Figure 4 is the composition and assembly schematic diagram of the detection kit of the present application;

[0132] Figure 5 is the picture processing schematic diagram of the image analysis software in the analysis system. DETAILED DESCRIPTION

[0133] The present application will be described in detail below through specific embodiments.

[0134] Example 1: Screening and verification preparation of core protein markers

[0135] This embodiment details the screening and verification preparation process of the core protein markers ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95 and CFAP90 in the present application. The flow chart of the screening and verification of the core protein markers of the present application is shown in Figure 2 .

[0136] First, bioinformatics and literature mining screening were performed. By searching PubMed, GEO, proteomics database, with "sperm functional proteome", "oocyte activation", "acrosome reaction", "sperm-egg fusion", "embryo development" as keywords, the candidate proteins specifically expressed in human sperm and with clear function were screened. By using GO and KEGG pathway enrichment analysis, the molecules closely related to "calcium ion signal regulation", "acrosome reaction", "sperm-egg recognition and fusion", "zygotic genome activation" and other reproductive events were focused on, and PLCZ1, ACTL7A, ACTL9, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 were preliminarily locked.

[0137] Next, clinical sample cohort validation was performed. Ethically approved and informed consented clinical samples were collected, including normal fertile males and males diagnosed with low fertilization rate, low cleavage rate or embryo developmental arrest after intracytoplasmic sperm injection (ICSI). After total protein extraction, semi-quantitative analysis was performed by Western Blot. The results confirmed that the expression levels of PLCZ1 and ACTL9 proteins in the ICSI failure or embryo developmental failure group were significantly down-regulated compared with the fertile group (p<0.01), and the expression level of PLCZ1 was significantly positively correlated with the fertilization rate and high-quality embryo rate. IQCN and CD46 also showed abnormal expression patterns in some immunological infertility or fertilization failure samples. The expression of SPAG9 in weak teratospermia and some patients with unexplained fertilization failure was significantly reduced (p<0.05), and its expression level was positively correlated with the percentage of sperm forward movement and acrosome reaction rate. Abnormal localization or expression loss of SPESP1 protein at the equatorial segment was highly related to acrosome reaction dysfunction and complete fertilization failure. In the case group with complete fertilization failure, the expression level of SPESP1 was more than 50% lower than that in the fertile group (p<0.01). The protein level of IZUMO3 in patients with sperm-egg binding disorders showed significant heterogeneity, and IZUMO3 protein was completely undetectable in some samples, suggesting its important role in mediating sperm-egg interaction. The down-regulation of TMEM95 expression was closely related to the specific phenotype of sperm attachment to the egg membrane but unable to complete fusion during ICSI. The normal cleavage rate of the TMEM95 deficiency group was significantly lower than that of the control group (p<0.01). The protein expression level of CFAP90 was generally low in severe oligospermia group, and was significantly negatively correlated with the sperm flagellum abnormality index (p<0.001), further confirming its basic role in maintaining sperm flagellum structure and movement function.

[0138] Table 1. Results of clinical sample cohort validation

[0139] .

[0140] Finally, functional assays were performed to confirm the results, as shown in Table 2. Sperm-specific protein inhibitors or antibodies were used to block the activities of PLCZ1 or IQCN in healthy donor sperm. Computer-assisted sperm analysis (CASA) and flow cytometry confirmed that the inhibition treatment had no significant effect on sperm motility and morphology. Subsequent mouse ICSI experiments showed that the fertilization rate was significantly lower in the PLCZ1 activity inhibition group than in the control group (p < 0.001); although the fertilization rate did not significantly decrease in the IQCN inhibition group, the early embryonic development arrest rate significantly increased (p < 0.01). The function of SPAG9 was inhibited by its specific antibodies. CASA analysis showed that the treatment did not significantly affect the basic movement parameters of sperm. However, in the in vitro fertilization (IVF) model, the ability of SPAG9-inhibited sperm to penetrate the zona pellucida significantly decreased (p < 0.01), but the fertilization rate returned to normal after the sperm were injected into the egg by ICSI technology. This indicates that SPAG9 is mainly involved in the early stage of sperm-egg recognition, rather than in the post-fertilization egg activation event. After the activity of SPESP1 was inhibited, flow cytometry detection found that the sperm acrosome reaction rate significantly decreased (p < 0.001). After ICSI experiments, although the sperm nuclear depolymerization was normal, the pronucleus formation rate was significantly lower than that of the control group (p < 0.05), indicating that even if the natural fertilization step is bypassed by microinjection, the defect of SPESP1 still damages the ability of sperm to activate the egg, suggesting its core role in preparing for fertilization ability. Antibodies against IZUMO3 and TMEM95 were used for co-inhibition experiments. The results showed that inhibiting these two proteins had no direct effect on sperm motility and acrosome reaction. However, in the sperm-egg co-culture system, the fusion rate of the treated group was almost zero. Further ICSI experiments confirmed that the absence of these two proteins did not affect the development of embryos after fertilization, clearly positioning their function in the key step of sperm-egg plasma membrane fusion. Disturbing the function of CFAP90 presented a different phenotype. After treatment with its inhibitor, CASA system immediately captured a significant decrease in sperm flagellar swing and movement speed (p < 0.001). However, after screening normal morphological sperm from these motility-impaired sperm for ICSI, there was no difference in fertilization rate and early embryonic development rate compared with the control group. This directly proves that the core function of CFAP90 is to maintain sperm motility, and it has less effect on the intrinsic potential of fertilization and embryonic development.

[0141] Table 2. Results of functional assay verification

[0142] .

[0143] In summary, through the loss-of-function experiments of the system, we accurately analyzed the specific role of each key protein in the fertilization chain: PLCZ1 as the core indicator of predicting fertilization ability, and IQCN closely related to the early embryonic development potential. ACTL7A / ACTL9 and CD46 are key synergistic markers of fertilization and immune microenvironment, respectively. SPAG9 acts on the zona pellucida penetration, SPESP1 is the preparer of acrosome reaction and oocyte activation ability, IZUMO3 and TMEM95 are the "terminal effectors" of membrane fusion, and CFAP90 is the guarantee of sperm motility. These findings are confirmed by clinical cohort data, which together construct a complete molecular path diagram from sperm motility, recognition, activation to final fusion, providing a solid theoretical basis and potential target for the precise diagnosis and intervention of male infertility.

[0144] Example 2 Preparation of specific monoclonal antibody

[0145] This example details the preparation process of rabbit-derived monoclonal antibodies specific to human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90. The preparation process flow chart of the mouse anti-human specific monoclonal antibody of the present application is shown in Figure 3 .

[0146] 1. Design and preparation of immunogen

[0147] The preparation method of the immunogen is consistent with the "screening and verification preparation of core protein markers" process described in Example 1. That is, the target immunogenic protein is synthesized and coupled with the KLH carrier protein as the immunogen.

[0148] 2. Animal immunization

[0149] Taking the preparation of anti-ACTL9 monoclonal antibody as an example:

[0150] Experimental animals: healthy, 12-16 week old New Zealand white rabbits were selected.

[0151] Immunization procedure:

[0152] Primary immunization: 200 μg of KLH-ACTL9 protein conjugate was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously in multiple points on the back of the rabbit.

[0153] Boosting immunization: boosting immunization was performed every 3 weeks, with the same dose as the primary immunization, but using Freund's incomplete adjuvant for emulsification. A total of 3-4 times of boosting immunization was performed.

[0154] Titer monitoring: On the 10th day after each boost, blood was collected from the central artery of the rabbit ear, and serum was separated. The synthetic ACTL9 protein was used as the coating antigen, and the serum antibody titer was detected by indirect ELISA. When the serum titer reached more than 1:100,000 and was stable, it was considered to be immune successful.

[0155] 3. Peripheral blood B lymphocyte separation and single B cell sorting

[0156] On the 7th day after the last boost, the peripheral blood of the immunized rabbit was collected, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium.

[0157] Using fluorescence-labeled recombinant target protein (such as ACTL9) as a probe, single B lymphocytes with antigen specificity were sorted by fluorescence-activated cell sorting (FACS) technology.

[0158] 4. Single B cell antibody gene cloning and expression

[0159] The sorted single antigen-specific B cells were lysed, and the variable region genes of rabbit antibody light and heavy chains were amplified by reverse transcription-polymerase chain reaction (RT-PCR).

[0160] The amplified antibody variable region genes were cloned into expression vectors containing human antibody constant regions, respectively, to construct full humanized rabbit IgG expression vectors.

[0161] The heavy chain and light chain expression vectors were co-transfected into HEK293F mammalian expression cells for transient expression.

[0162] The cell supernatant was collected, and rabbit monoclonal antibodies were obtained by Protein A affinity chromatography purification.

[0163] 5. Antibody characterization and functional verification

[0164] (1) Antibody titer and affinity quantitative analysis

[0165] The titer of the purified antibody was determined by indirect ELISA. Each recombinant target protein (such as ACTL9) was coated on an enzyme-labeled plate, and the antibody was gradiently diluted for detection. The results showed that the ELISA titers of all ten antibodies were ≥ 1:128,000, indicating that they had extremely high binding activity.

[0166] The affinity of the antibody to the corresponding antigen was determined by surface plasmon resonance (SPR) technology. The antibody was immobilized on the chip, and the antigen solution was flowed through to analyze the binding and dissociation curves. The affinity constant (KD) of the ten antibodies was determined as shown in Table 3, and the KD values of all antibodies reached the nanomolar (nM) level, showing excellent affinity.

[0167] (2) Antibody specificity verification

[0168] Western Blot analysis: The detection was performed using human sperm protein extract. Each antibody recognized a single, clear band at the expected molecular weight, and was consistent with the theoretical value. No cross-reaction with other non-target proteins proved the high specificity of the antibody.

[0169] Immunofluorescence localization verification: Staining of normal human sperm smears. Each antibody showed a clear subcellular localization pattern consistent with literature reports and expectations, with high signal-to-noise ratio. For example: Anti-ACTL9 and anti-ACTL7A antibody signals were concentrated in the equatorial plate region of sperm; Anti-PLCZ1 antibody signals were mainly located in the post-acrosomal region and equatorial segment of sperm; Anti-IZUMO3 and anti-TMEM95 antibodies showed distribution in the whole membrane structure of sperm head; Anti-CFAP90 antibody signals showed continuous linear distribution along the main axis of sperm flagellum. These precise localization results further confirmed the antibody's ability to specifically recognize its native conformation antigen.

[0170] (3) Functional verification

[0171] To prove that the prepared antibodies not only can bind, but also can target the key epitope of function, functional inhibition experiments were performed:

[0172] Anti-PLCZ1 antibody: In the mouse ICSI model, the antibody was injected into the oocyte cytoplasm, and compared with the control group (injected with irrelevant IgG), the fertilization rate was significantly reduced from 85% to 20% (p<0.001), proving that it effectively blocked the oocyte activation function of PLCZ1.

[0173] Anti-IZUMO3 and anti-TMEM95 antibody mixture: In the sperm-egg co-culture system, after adding the antibody, the sperm-egg fusion rate was reduced from 75% to less than 5% (p<0.001), confirming the key role of the two in membrane fusion and the effective blocking of the antibody.

[0174] Anti-SPESP1 antibody: After pretreatment of sperm, flow cytometry detection showed that the acrosome reaction rate induced by calcium ion carrier was reduced from 60% to 18% (p<0.001).

[0175] These functional data strongly prove that the antibodies prepared by the present application successfully target the functionally active region of the protein, and have value for functional detection and research.

[0176] Table 3 Antibody quality control and performance parameter summary

[0177] .

[0178] Note: IZUMO3 and TMEM95 antibodies are used in combination in sperm-egg fusion inhibition experiments, with an inhibition rate of >90%. Functional inhibition rate refers to the percentage of activity reduction in the antibody treatment group compared with the control group in a specific functional experiment (such as ICSI fertilization inhibition, acrosome reaction inhibition, fusion inhibition).

[0179] The following are the sequences of the protein-protein:

[0180] (1) ACTL9 immunogen: Synthesize the protein sequence shown below (SEQ ID NO. 101):

[0181] MDASRPKSSESQSSLEAPRPGPNPSPNVVNKPLQRDSPGMVADRLPPKTGAVVIDMGTGTCKVGFAGQASPTYTVATILGCQPKKPATSGQSGLQTFIGEAARVLPELTLVQPLRSGIVVDWDAAELIWRHLLEHDLRVATHDHPLLFSDPPFSPATNREKLVEVAFESLRSPAMYVASQSVLSVYAHGRVSGLVVDTGHGVTYTVPVFQGYNLLHATERLDLAGNNLTAFLAEMLLQAGLPLGQQDLDLVENIKHHYCYVASDFQKEQARPEQEYKRTLKLPDGRTVTLGKELFQCPELLFNPPEVPGLSPVGLSTMAKQSLRKLSLEMRADLAQNVLLCGGSSLFTGFEGRFRAELLRALPAETHVVVAAQPTRNFSVWIGGSILASLRAFQSCWVLREQYEEQGPYIVYRKCY;

[0182] (2) ACTL7A immunogen: Synthesize the protein sequence shown below (SEQ ID NO. 102):

[0183] MWAPPAAIMGDGPTKKVGNQAPLQTQALQTASLRDGPAKRAVWVRHTSSEPQEPTESKAAKERPKQEVTKAVVVDLGTGYCKCGFAGLPRPTHKISTTVGKPYMETAKTGDNRKETFVGQELNNTNVHLKLVNPLRHGIIVDWDTVQDIWEYLFRQEMKIAPEEHAVLVSDPPLSpHTNREKYAEMLFEAFNTPAMHIAYQSRLSMYSYGRTSGLVVEVGHGVSYVVPIYEGYPLPSITGRLDYAGSDLTAYLLGLLNSAGNEFTQDQMGIVEDIKKKCCFVALDPIEEKKVPLSEHTIRYVLPDGKEIQLCQERFLCSEMFFKPSLIKSMQLGLHTQTVSCLNKCDIALKRDLMGNILLCGGSTMLSGFPNRLQKELSSMCPNDTPQVNVLPERDSAVWTGGSILASLQGFQPLWVHRFEYEEHGPFFLYRRCF;

[0184] (3) PLCZ1 immunogen: the protein sequence shown below (SEQ ID NO. 103) was synthesized:

[0185] 306-325: CETHERKGSDKRGDNQDKETG-NH2 (this short peptide is a specific epitope selected by bioinformatics analysis, used to stimulate high-specificity antibodies);

[0186] (4) IQCN immunogen: the protein sequence shown below (SEQ ID NO. 104) was synthesized:

[0187] 295-310: CETPLSRRYDQAVTRPS-NH2 (this short peptide is a specific epitope selected by bioinformatics analysis, used to stimulate high-specificity antibodies);

[0188] (5) CD46 immunogen: the protein sequence shown below (SEQ ID NO. 105) was synthesized:

[0189] MEPPGRRECPFPSWRFPGLLLAAMVLLLYSFSDACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDVWVIAVIVIAIDIFKGGRRKGKQMVELNMPLTRLNQPLQQSREAE;

[0190] (6) SPAG9, the immunogenic protein sequence (SEQ ID NO. 106) is as follows:

[0191]

[0192] (7) SPESP1, the immunogenic protein sequence (SEQ ID NO. 107) of which is as follows:

[0193] MKPLVLLVALLLWPSSVPAYPSITVTPDEEQNLNHYIQVLENLVRSVPSGEPGREKKSNSPKHVYSIASKGSKFKELVTHGDASTENDVLTNPISEETTTFPTGGFTPEIGKKKHTESTPFWSIKPNNVSIVLHAEEPYIENEEPEPEPEPAAKQTEAPRMLPVVTESSTSPYVTSYKSPVTTLDKSTGIGISTESEDVPQLSGETAIEKPEEFGKHPESWNNDDILKKILDINSQVQQALLSDTSNPAYREDIEASKDHLKRSLALAAAAEHKLKTMYKSQLLPVGRTSNKIDDIETVINMLCNSRSKLYEYLDIKCVPPEMREKAATVFNTLKNMCRSRRVTALLKVY;

[0194] (8) IZUMO3, the immunogenic protein sequence (SEQ ID NO. 108) of which is as follows:

[0195] MGDLWLFLLLPLSAFHGVKGCLECDPKFIEDVGSLLGNLIPSEVPGRTQLLERQIKEMIHLSFKVSHSDKRLRVLAVQQVVKLRTWLKNEFYKLGNETWKGVFIYQGKLLDVCQNLESKLKELLKNFSEIACSEDCIVVEGPILDCWTCLRMTNRCFKGEYCGDEDPRKAENREIALFLILLATAVILGSAVLLFHFCIFHRRKMKAIRRSLKEYVEKKLEELMGKIDEKEEKDFRLRK;

[0196] (9) TMEM95, the immunogenic protein sequence (SEQ ID NO. 109) of which is as follows:

[0197] MWRLALGGVFLAAAQACVFCRLPAHDLSGRLARLCSQMEARQKECGASPDFSAFALDEVSMNKVTEKTHRVLRVMEIKEAVSSLPSYWSWLRKTKLPEYTREALCPPACRGSTTLYNCSTCKGTEVSCWPRKRCFPGSQDLWEAKILLLSIFGAFLLLGVLSLLVESHHLQAKSGL;

[0198] (10) CFAP90, the immunogenic protein sequence (SEQ ID NO. 110) is as follows:

[0199] MEDDEEETTASTLRGKPRPPPVSAQSAFSYIPPRRLDPKEHSYYYRPARTGIISLYDCIFKRRLDYDQKLHRDDREHAKSLGLHVNEEEQERPVGVLTSSVYGKRINQPIEPLNRDFGRANHVQADFYRKNDIPSLKEPGFGHIAPS.

[0200] The following is the sequence for preparing the antibody:

[0201] (a) ACTL9 antibody sequence (SEQ ID NO. 1-SEQ ID NO. 10):

[0202] Light chain:

[0203] DIQMTQSPSSLSASVGDRVTITCSASQDISNYFNWYQQKPGKAPKVLIYFTGTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYATFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0204] Light chain variable region (VL):

[0205] DIQMTQSPSSLSASVGDRVTITCSASQDISNYFNWYQQKPGKAPKVLIYFTGTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYATFPWTFGQGTKVEIK;

[0206] Complementarity determining regions (CDRs):

[0207] CDR-L1: SASQDISNYFN;

[0208] CDR-L2: FTGTLHS;

[0209] CDR-L3: QQYATFPWT;

[0210] Heavy chain:

[0211] EVQLVESGGGLVQPGGSLRLSCAASGYTFTQYGMNWVRQAPGKGLEWVGWINTYTTDPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYpHWYGSTHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0212] Heavy chain variable region (VH):

[0213] EVQLVESGGGLVQPGGSLRLSCAASGYTFTQYGMNWVRQAPGKGLEWVGWINTYTTDPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYpHWYGSTHWYFDVWGQGTLVTVSS;

[0214] Complementarity determining regions (CDRs):

[0215] CDR-H1: GYTFTQYGMN;

[0216] CDR-H2: WINTYTTDPTYAADFKRR;

[0217] CDR-H3: YpHWYGSTHWYFDV.

[0218] (b) ACTL7A antibody sequences (SEQ ID NO. 11-SEQ ID NO. 20):

[0219] Heavy chain:

[0220] AIQLTQSPSSLSASVGDRVTITCRASQGIGSAFVWYQQKPGKAPKLLIYEASSFESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNEYITFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0221] Light chain variable region (VL):

[0222] AIQLTQSPSSLSASVGDRVTITCRASQGIGSAFVWYQQKPGKAPKLLIYEASSFESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNEYITFGPGTKVDIK;

[0223] Complementarity determining regions (CDRs):

[0224] CDR-L1: ASQGIGSAFV;

[0225] CDR-L2: EASSFES;

[0226] CDR-L3: QQFNEYIT;

[0227] Heavy chain:

[0228] QVQLVQSGAEVKKPGASVKVSCKASGYTFTQKYMHWVRQAPGQGLEWMGIINWLSGSTSNAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDILEAFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0229] Heavy chain variable region (VH):

[0230] QVQLVQSGAEVKKPGASVKVSCKASGYTFTQKYMHWVRQAPGQGLEWMGIINWLSGSTSNAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDILEAFDFWGQGTMVTVSS

[0231] Complementarity determining regions (CDRs):

[0232] CDR-H1: GYTFTQKYMH

[0233] CDR-H2: GIINWLSGSTSNAQKFQGR

[0234] CDR-H3: DILEAFDF

[0235] (c) PLCZ1 antibody sequences (SEQ ID NO. 21-SEQ ID NO. 30):

[0236] Light chain:

[0237] EIVLTQSPATLSLSPGERATLSCRASQGIAAYLAWYQQKPGQAPRLLIYDATQKATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSEYYLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0238] Heavy chain variable region (VH):

[0239] EIVLTQSPATLSLSPGERATLSCRASQGIAAYLAWYQQKPGQAPRLLIYDATQKATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSEYYLTFGQGTNLEIK;

[0240] Complementarity determining regions (CDRs):

[0241] CDR-H1 : GFTFSDYWMD

[0242] CDR-H2: INPYNGNTKYNEKFKG

[0243] CDR-H3: RNYGS GYT YADSVKG

[0244] Heavy chain:

[0245] QVQLQQWGAGLLKPSETLSLTCAVYGGSLGEPYWNWIRQPPGKGLEWIGEIQKKSSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSEPDWNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0246] Heavy chain variable region (VH):

[0247] QVQLQQWGAGLLKPSETLSLTCAVYGGSLGEPYWNWIRQPPGKGLEWIGEIQKKSSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSEPDWNWFDPWGQGTLVTVSS

[0248] Complementarity determining regions (CDRs):

[0249] CDR-H1 : AVYGGSLGEPYW;

[0250] CDR-H2: GEIQKKSSTNSNPSLKSR;

[0251] CDR-H3: AFGYSEPDWNWFDP.

[0252] (d) IQCN antibody sequences (SEQ ID NO. 31-SEQ ID NO. 40):

[0253] Light chain:

[0254] DIQMTQSPSSLSASVGDRVTITCRASQDVGALGAWYQQKPGKAPKLLIYSASVIWTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYIWKYATFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0255] Light chain variable region (VL):

[0256] DIQMTQSPSSLSASVGDRVTITCRASQDVGALGAWYQQKPGKAPKLLIYSASVIWTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYIWKYATFGQGTKVEIK;

[0257] Complementarity determining regions (CDRs):

[0258] CDR-L1 : RASQDVGALGA;

[0259] CDR-L2: SASVIWT;

[0260] CDR-L3: QQYIWKYAT;

[0261] Heavy chain:

[0262] EVQLVESGGGLVQPGGSLRLSCAASGFTFSEAYFHWVRQAPGKGLEWVAWISPYGGAGWWADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRKYWSGFDYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] Heavy chain variable region (VH):

[0264] EVQLVESGGGLVQPGGSLRLSCAASGFTFSEAYFHWVRQAPGKGLEWVAWISPYGGAGWWADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRKYWSGFDYWGQGTLVTVS

[0265] Complementarity determining regions (CDRs):

[0266] CDR-H1: GFTFSEAYFH;

[0267] CDR-H2: WISPYGGAGWWADSVKG;

[0268] CDR-H3: RRKYWSGFDY.

[0269] (e) CD46 antibody sequences (SEQ ID NO. 41-SEQ ID NO. 50):

[0270] Light chain:

[0271] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLWASQQKNYLAWYQQKPGQSPKLLIYWAGAHDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQWWGPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0272] Light chain variable region (VL):

[0273] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLWASQQKNYLAWYQQKPGQSPKLLIYWAGAHDSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQWWGPRTFGGGTKLEIK;

[0274] Complementarity determining regions (CDRs):

[0275] CDR-L1 : KSSQSLLWASQQKNYLA;

[0276] CDR-L2: WAGAHDS;

[0277] CDR-L3: QQWWGPRT;

[0278] Heavy chain:

[0279] QVQLQQSGPEVVKPGASVKMSCKASGYTFERTYIHWVRQKPGQGLDWIGYINPYQESYDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKEQWSTGAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0280] Heavy chain variable region (VH):

[0281] QVQLQQSGPEVVKPGASVKMSCKASGYTFERTYIHWVRQKPGQGLDWIGYINPYQESYDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKEQWSTGAWFAYWGQGTLVTVSS

[0282] Complementarity determining regions (CDRs):

[0283] CDR-H1: GYTFERTYIH;

[0284] CDR-H2: YINPYQESYDYDEKFKG;

[0285] CDR-H3: AREKEQWSTGAWFAY.

[0286] (f) SPAG9 antibody sequences (SEQ ID NO. 51-SEQ ID NO. 60):

[0287] Light chain:

[0288] DIQMTQSPSSLSASVGDRVTITCRASQDIGQPFNWYQQKPGKAPKLLIYYTGKFHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHAFYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0289] Heavy chain variable region (VH):

[0290] DIQMTQSPSSLSASVGDRVTITCRASQDIGQPFNWYQQKPGKAPKLLIYYTGKFHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHAFYYTFGQGTKVEIK;

[0291] Complementarity determining regions (CDRs):

[0292] CDR-L1 : RASQDIGQPFN;

[0293] CDR-L2: YTGKFHS;

[0294] CDR-L3: QQGHAFYYT;

[0295] Heavy chain:

[0296] EVQLVQSGAEVKKPGASVKVSCKASGYTFTEGPLSWVRQAPGQGLEWIGDMYPDNGDGGWQQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAYHPWFSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0297] Heavy chain variable region (VH):

[0298] EVQLVQSGAEVKKPGASVKVSCKASGYTFTEGPLSWVRQAPGQGLEWIGDMYPDNGDGGWQQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAYHPWFSVWGQGTLVTVSS

[0299] Complementarity determining regions (CDRs):

[0300] CDR-H1: GYTFTEGPLS;

[0301] CDR-H2: DMYPDNGDGGWQQKFQGR;

[0302] CDR-H3: VLAYHPWFSV.

[0303] (g) SPESP1 antibody sequences (SEQ ID NO. 61-SEQ ID NO. 70):

[0304] Light chain:

[0305] DIQMTQSPSSLSASVGDRVTITCLASEDIWEQFAWYQQKPGKAPKLLIYYTGGIQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQEDGWPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0306] Light chain variable region (VL):

[0307] DIQMTQSPSSLSASVGDRVTITCLASEDIWEQFAWYQQKPGKAPKLLIYYTGGIQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQEDGWPWTFGGGTKVEIK;

[0308] Complementarity determining regions (CDRs):

[0309] CDR-L1: LASEDIWEQFA;

[0310] CDR-L2: YYTGGIQD;

[0311] CDR-L3: LQEDGWPWT;

[0312] Heavy chain:

[0313] EVTLKESGPALVKPTQTLTLTCTFSGFSLTTWSLSVGWIRQPPGKALEWLANIWWEEEHYYNPSLKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIGPIHWYAAPYRYFDFWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;-

[0314] Heavy chain variable region (VH):

[0315] EVTLKESGPALVKPTQTLTLTCTFSGFSLTTWSLSVGWIRQPPGKALEWLANIWWEEEHYYNPSLKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIGPIHWYAAPYRYFDFWGQGTMVTVSS;

[0316] Complementarity determining regions (CDRs):

[0317] CDR-H1: FSGFSLTTWSLS;

[0318] CDR-H2: NIWWEEEHYYNPSLKN;

[0319] CDR-H3: RIGPIHWYAAPYRYFDF.

[0320] (h) IZUMO3 antibody sequences (SEQ ID NO. 71-SEQ ID NO. 80):

[0321] Light chain:

[0322] DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSTQNHNYLAWYQQKPGQAPKLLIYFTGAKESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQRWEAPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0323] Heavy chain variable region (VH):

[0324] DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSTQNHNYLAWYQQKPGQAPKLLIYFTGAKESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQRWEAPYTFGGGTKLEIK;

[0325] Complementarity determining regions (CDRs):

[0326] CDR-H1: GYWMN

[0327] CDR-H2: VINPGDGDTNYNGKFKG

[0328] CDR-H3: WYQQKPGQAPKLLIY

[0329] Heavy chain:

[0330] QVQLVQSGAEVVKPGASVKVSCKASGYSFASWSMNWVRQAPGQNLEWIGLINPYNASAGWNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEWHPSGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0331] Heavy chain variable region (VH):

[0332] QVQLVQSGAEVVKPGASVKVSCKASGYSFASWSMNWVRQAPGQNLEWIGLINPYNASAGWNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEWHPSGDYFDYWGQGTTLTVSS

[0333] Complementarity determining regions (CDRs):

[0334] CDR-H1: GYSFASWSMN;

[0335] CDR-H2: LINPYNASAGWNQKFQG;

[0336] CDR-H3: SEWHPSGDYFDY.

[0337] (i) TMEM95 antibody sequences (SEQ ID NO. 81-SEQ ID NO. 90):

[0338] Light chain:

[0339] DIQMTQSPSSLSASVGDRVTITCKASQDVGATFAWYQQKPGKAPKLLIYSAGWKPTGVPS RFSGSGSGTDFTLTISSLQPEDFAVYYCQQHWLAYLTFGQGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0340] Heavy chain:

[0341] DIQMTQSPSSLSASVGDRVTITCKASQDVGATFAWYQQKPGKAPKLLIYSAGWKPTGVPS RFSGSGSGTDFTLTISSLQPEDFAVYYCQQHWLAYLTFGQGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0342] Complementarity determining regions (CDRs):

[0343] CDR-H1 : GFTFSDYWMD

[0344] CDR-H2: INPYNGDTKYNEKFKG

[0345] CDR-H3: RNYGS GYT YADSVKG

[0346] Heavy chain:

[0347] QVQLVQSGAEVKKPGASVKVSCKASGYTFTATSFQWVRQAPGQGLEWMGWINTHSGLWRWAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGLSGGYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0348] Heavy chain variable region (VH):

[0349] QVQLVQSGAEVKKPGASVKVSCKASGYTFTATSFQWVRQAPGQGLEWMGWINTHSGLWRWAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGLSGGYWYFDVWGQGTLVTVSS

[0350] Complementarity determining regions (CDRs):

[0351] CDR-H1: GYTFTATSFQ

[0352] CDR-H2: WINTHSGLWRWAEDFKGR

[0353] CDR-H3: SGLSGGYWYFDV

[0354] (j) CFAP90 antibody sequences (SEQ ID NO. 91-SEQ ID NO. 100):

[0355] Light chain:

[0356] DIQMTQSPSSLSASVGDRVTITCGASENIWATINWYQQKPGKAPKLLIYGTSQIADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNFIQSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0357] Light chain variable region (VL):

[0358] DIQMTQSPSSLSASVGDRVTITCGASENIWATINWYQQKPGKAPKLLIYGTSQIADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNFIQSPLTFGQGTKVEIK;

[0359] Complementarity determining regions (CDRs):

[0360] CDR-L1 : GASENIWATIN;

[0361] CDR-L2: GTSQIAD;

[0362] CDR-L3: QNFIQSPLT;

[0363] Heavy chain:

[0364] QVQLVQSGAEVKKPGASVKVSCKASGYIFSQWYLQWVRQAPGQGLEWMGEILPGSGTSDWTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYLLATSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0365] Heavy chain variable region (VH):

[0366] QVQLVQSGAEVKKPGASVKVSCKASGYIFSQWYLQWVRQAPGQGLEWMGEILPGSGTSDWTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYLLATSPNWYFDVWGQGTLVTVSS;

[0367] Complementary determining regions (CDRs):

[0368] CDR-H1: GYIFSQWYLQ.

[0369] CDR-H2: EILPGSGTSDWTENFKDR.

[0370] CDR-H3: RYLLATSPNWYFDV.

[0371] In summary, the present application has successfully completed the large-scale preparation of specific rabbit monoclonal antibodies for ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90, ten key proteins of human sperm. All antibodies have passed strict quality control (see Table 3), with high titer, high affinity, high specificity, and clear functional inhibition activity. The successful preparation of this batch of high-quality antibodies is the core guarantee for the precise and reliable detection of the kit, and also provides key tool reagents for related basic research and clinical diagnosis in the field of reproductive medicine.

[0372] Example 3: Assembly and preparation of the detection kit

[0373] This example details the preparation, quality control and final assembly process of each component of the detection kit. The preparation flowchart of the present application based on multi-modal imaging of sperm key protein for embryo development potential is shown in Figure 1 The composition and assembly of the detection kit are shown in Figure 4 .

[0374] Antibody working solution preparation and dispensing: After purification, the mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90 monoclonal antibodies were diluted to their respective optimal working concentrations with 1% BSA in PBS antibody diluent. Under the clean bench, 150 μL (corresponding to 5 tests per tube) was dispensed into 0.5 mL brown light-protective centrifuge tubes, and labeled.

[0375] 2. Labeling reagents and dye dispensing: FITC-labeled goat anti-mouse IgG secondary antibody and propidium iodide (PI) nuclear dye solution were diluted to working concentration with special diluent, and 750 μL per tube was dispensed;

[0376] The working concentration of FITC secondary antibody was obtained by diluting the stock solution (0.01 mg / ml) by 1:100;

[0377] The working concentration of PI dye solution is 0.01 mg / ml;

[0378] The "special diluent" used to dilute the FITC-labeled goat anti-mouse IgG secondary antibody and propidium iodide (PI) nuclear dye solution should have the following standard formula:

[0379] Phosphate buffer solution (PBS) containing bovine serum albumin (BSA) as a stabilizer and sodium azide as a preservative. Preparation method of phosphate buffer solution: take 1L 1x PBS buffer solution (pH 7.4), add 10g BSA, slowly stir until completely dissolved; add 0.2g sodium azide, mix well; filter sterilization with 0.22μm filter membrane; store at 2-8℃ after sub-packaging.

[0380] 3. Preparation of control slides:

[0381] Sperm sample preparation: Collect the semen samples of strictly screened fertile volunteers (positive control) and patients diagnosed with severe oligoasthenoteratozoospermia or azoospermia (as negative control), wash them thoroughly with PBS, fix them with pre-cooled methanol, and adjust the concentration to about 50x106 / mL.

[0382] Spotting and fixing: Use a non-contact spotting instrument to accurately spot 1μL of positive control sample and negative control sample into the designated hole positions (such as A1, B1 holes) of 12-hole slides. After drying at room temperature, seal and store in a desiccator environment away from light.

[0383] Quality control and calibration: Randomly select 3 slides from each batch, process them using the standardized immunofluorescence staining process, and analyze at least 200 sperm using the image analysis software of the system to calculate the average fluorescence intensity (MFI) and localization rate of each target protein (ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90), determine the reference value range of the batch, and print it on the product label.

[0384] 4. Sub-packaging of auxiliary reagents: 30x PBS concentrated washing solution is sub-packaged at 50mL per bottle; mounting glycerol (glycerol dissolved in pH 7.4, 0.01 M PBS) is sub-packaged at 3mL per bottle.

[0385] 5. Assembly and packaging: Put all components into the foam tray of the customized mold in the specification of 5 servings per box, including: ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 ten kinds of primary antibody each 1, FITC secondary antibody 1, propidium iodide (PI) liquid 1, 30x washing solution 1 bottle, mounting glycerol 1 (the preparation method of mounting glycerol is: measure 90 mL of glycerol into a beaker, add 10 mL of 0.01M PBS (pH 7.4), mix thoroughly until uniform and transparent, filter impurities with 0.22 μm filter membrane, and store in the dark), 1 pre-set control slide, 5 coverslips. Put the tray into an aluminum foil bag and heat seal, and finally paste the label printed with product batch number, expiration date, storage condition (-20℃, avoid light).

[0386] Table 4 Composition of the kit

[0387] ;

[0388] ;

[0389] ;

[0390] .

[0391] Example 4

[0392] This example evaluates the accuracy and precision of the kit, as shown in the following table:

[0393] Table 5 Accuracy and precision evaluation of the kit

[0394]

[0395] .

[0396] Instructions:

[0397] In actual detection, the total number of sperm should be ensured to be ≥200 to improve statistical accuracy.

[0398] The classification of sperm number and MFI should be based on comparison with the positive control. The reference value of the positive control is provided on the control slide of the kit, which may be slightly different for each batch of product.

[0399] The comprehensive classification combines the expression rate and MFI: if both are "excellent", the comprehensive evaluation is "excellent"; if one is "good" and the other is "excellent", the comprehensive evaluation is "good"; if either indicator is "poor", the comprehensive evaluation is "poor".

[0400] Kit

[0401] Stability:

[0402] Precision: Excellent, the batch and batch precision CV% mainly between 5%-12%, far better than the acceptable standard of 20%, indicating that the detection results are stable and reliable.

[0403] Accuracy: Excellent, highly correlated with the gold standard WB method (r=0.94), and the prediction accuracy of clinical outcomes is more than 91%.

[0404] Example 5: Analysis system integration and detection application

[0405] This embodiment details the hardware integration, software configuration of the multi-modal analysis system, and the complete process of using it for detection.

[0406] 1. Hardware system integration: fluorescence / laser confocal microscope, general optical microscope, used for fluorescence image, bright field image and motion video acquisition.

[0407] 2. Software configuration: matched with "human sperm localization protein quantitative analysis software" or similar multi-modal image analysis system, supporting:

[0408] Automatic identification of sperm and analysis of fluorescence intensity, integrated optical density, localization rate of ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 proteins; analysis of sperm morphology parameters in bright field images; analysis of sperm morphology parameters (such as head area, ellipticity, vacuole ratio) in bright field images of processed sperm smears collected by general optical microscope and sperm kinematics parameters (such as curve velocity VCL, straight line velocity VSL, linearity LIN) in motion video; generate comprehensive report and compare with database reference value.

[0409] 3. System detection application: the picture processing schematic diagram of the image analysis software in the analysis system is shown in Figure 5 .

[0410] (1) Sample pretreatment and smear preparation

[0411] Take the liquefied fresh semen sample, add 6 mL of 1x washing solution (diluted from 30x concentrated washing solution with deionized water at a ratio of 1:29), and mix gently. Place the mixed solution in a horizontal centrifuge, centrifuge at 1500 rpm for 5 minutes, discard the supernatant. Repeat washing twice: each time add 6 mL of 1x washing solution to resuspend the sperm precipitate, centrifuge again to discard the supernatant. Finally, resuspend the sperm with an appropriate amount of 1x washing solution, adjust the sperm concentration to 40-50x10 6 / mL (ensuring 40-50 sperm are visible in 40x field). Take 1 μL of adjusted semen sample and evenly spread in the blank wells of the multi-well slide (5 samples per slide, 2 wells per sample). Place the smear in a 37°C constant temperature dry incubator for 30 minutes for complete drying.

[0412] (2) Common optical microscope detection: Morphology and kinematics analysis

[0413] Directly use the dried smear for common optical microscope observation. Take 5 μL of physiological saline or special sperm diluent and cover on the smear well, cover with a cover glass to avoid air bubbles. Use 40x objective lens to collect bright field images and motion video (recommended to record for not less than 30 seconds). Import the images and video into "human sperm localization protein quantification analysis software" for the following analysis:

[0414] (a) Morphology parameters: automatically identify sperm head, calculate head area, ellipticity, vacuole ratio, etc.

[0415] (b) Kinematics parameters: track sperm motion trajectory, quantitatively calculate curve velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), forwardness (STR), wobble (WOB), amplitude of lateral head excursion (ALH) and beat cross frequency (BCF);

[0416] (c) Generate preliminary morphology and kinematics analysis report.

[0417] (3) Sample fixation and immunofluorescence staining

[0418] (a) After completing the bright field detection, immerse the smear in methanol for 5-10 minutes, take it out and dry naturally at room temperature for 5-10 minutes, and then perform immunofluorescence staining;

[0419] (b) Primary antibody incubation: according to the protein to be tested (ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90), add 10 μL of corresponding primary antibody working solution (including A, B control wells) in each well. Place the slide in a light-proof wet box and incubate in a 2-8°C refrigerator for at least 8 hours (or in a 37°C incubator for 1 hour);

[0420] (c) Washing: take out the slide, gently rinse it with 1x washing solution, then immerse it in 3 cylinders of fresh 1x washing solution, each for 5 minutes (can be gently shaken), and finally immerse it in deionized water for 30 seconds, and dry;

[0421] (d) Secondary antibody incubation: Add 10 μL of FITC-labeled goat anti-mouse IgG secondary antibody to each well, and incubate at 37°C for 30 minutes in a dark humidified chamber;

[0422] (e) Re-washing: Wash and dry as above;

[0423] (f) Nuclei counterstaining: Add 10 μL of propidium iodide (PI) solution to each well, and incubate at room temperature for 15 minutes in the dark;

[0424] (g) Final washing: Wash and dry as above;

[0425] (h) Mounting: Add one drop of mounting glycerol to each well, and cover with a coverslip to avoid air bubbles.

[0426] (4) Fluorescence / laser confocal microscopy detection

[0427] Using a fluorescence / laser confocal microscope (such as Zeiss LSM800 or Nikon C2), collect multi-channel fluorescence images under 40x objective: FITC channel (Ex / Em: 488nm) for detecting target proteins, and PI channel (Ex / Em: 561nm) for nuclei localization. Collect at least 200 images of sperm per sample. First, check if the fluorescence signals of A, B control wells are within the batch-specific reference range, and confirm the system is correct before collecting whole-slide images.

[0428] (5) Image analysis and report generation

[0429] Import fluorescence images into analysis software, and perform the following processing:

[0430] (a) Automatically identify the head region of sperm, and calculate the mean fluorescence intensity (MFI), integrated optical density (IOD), and localization rate (PR) of ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90;

[0431] (b) Combine previous brightfield analysis results to generate a comprehensive report, including morphology, kinematics, and protein expression data;

[0432] (c) Compare results with reference intervals in the database, and output the final evaluation report;

[0433] Generate a quantitative analysis report, and compare with reference intervals, where the localization rate is calculated as:

[0434] .

[0435] N positive is the number of sperm expressing the specific protein in the correct position, and N total is the total number of sperm detected.

[0436] Note: All wash steps require fresh 1x wash solution, which cannot be reused. Strictly avoid light during operation to prevent fluorescence quenching.

[0437] 4. Formula calculation:

[0438] (1) Sperm head detection algorithm

[0439] PI positive sperm recognition formula:

[0440] I PI (x, y) ≥ T PI = μ background + 3σ background;

[0441] Where:

[0442] I PI (x, y): PI channel pixel intensity;

[0443] T PI: PI positive threshold;

[0444] μ background: background area average intensity;

[0445] σ background: background area intensity standard deviation;

[0446] Head morphology screening conditions:

[0447] A min≤ A head≤ A max;

[0448] 0.7≤ E head = (4πA head) / (P head²)≤ 1.0;

[0449] Where:

[0450] A head: head area (pixels);

[0451] P head: head perimeter (pixels);

[0452] E head: head ellipticity.

[0453] (2) Sperm tail segmentation algorithm

[0454] Tail recognition formula:

[0455] L tail≥ L min;

[0456] W tail≤ W max;

[0457] S tail = L tail / W tail≥ S min;

[0458] Where:

[0459] L_tail: tail length;

[0460] W_tail: tail width;

[0461] S_tail: tail aspect ratio.

[0462] (3) Green fluorescence expression analysis

[0463] Fluorescence intensity calculation formula:

[0464] FI_green = (1 / N)∑[I_green(x,y) - I_background];

[0465] FI_green: average fluorescence intensity;

[0466] N: number of sperm head pixels;

[0467] I_green(x,y): green channel pixel intensity;

[0468] Fluorescence positive determination:

[0469] FI_green ≥ T_positive = μ_negative + Kσ_negative;

[0470] Where:

[0471] T_positive: positive threshold;

[0472] μ_negative: average fluorescence intensity of negative control;

[0473] σ_negative: standard deviation of fluorescence intensity of negative control;

[0474] K: confidence coefficient (usually 2-3).

[0475] (4) Key statistical indicators

[0476] Total number of sperm:

[0477] N_total = ∑[I_PI(i) ≥ T_PI];

[0478] Number of expressing sperm:

[0479] N_expression = ∑[FI_green(i) ≥ T_positive];

[0480] Expression rate:

[0481] R_expression = (N_expression / N_total) × 100%;

[0482] Mean fluorescence intensity:

[0483] MFI = (1 / N expression)∑FI green(i).

Claims

1. An embryonic developmental potential sperm key protein kit characterized in that, Kit A and Kit B, Kit A is the following components: Antibody components: including mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 specific antibodies; Labeling system: fluorescently labeled secondary antibody, nuclear fluorescent dye; Solid support: sperm protein slide, sperm protein slide is a multi-well glass slide, the wells are pre-coated with positive and negative control human sperm samples; Kit B is the following components: Concentrated washing solution, mounting medium, fixative; The specific sequences of mouse anti-human ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, CFAP90 specific antibodies are as follows: (a) ACTL9 antibody The ACTL9 antibody light chain sequence is shown as SEQ ID NO. 1, the ACTL9 antibody light chain variable region VL is shown as SEQ ID NO. 2, the ACTL9 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 3, the ACTL9 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 4, the ACTL9 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 5; The ACTL9 antibody heavy chain sequence is shown as SEQ ID NO. 6, the ACTL9 antibody heavy chain variable region VH is shown as SEQ ID NO. 7, the ACTL9 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 8, the ACTL9 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 9, the ACTL9 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 10; (b) ACTL7A antibody The ACTL7A antibody light chain sequence is shown as SEQ ID NO. 11, the ACTL7A antibody light chain variable region VL is shown as SEQ ID NO. 12, the ACTL7A antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 13, the ACTL7A antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 14, the ACTL7A antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 15; The ACTL7A heavy chain sequence is shown as SEQ ID NO. 16, the ACTL7A antibody heavy chain variable region VH is shown as SEQ ID NO. 17, the ACTL7A antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 18, the ACTL7A antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 19, the ACTL7A antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 20; (c) PLCZ1 antibody The ACTL9 antibody light chain sequence is shown as SEQ ID NO. 1, the ACTL9 antibody light chain variable region VL is shown as SEQ ID NO. 2, the ACTL9 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 3, the ACTL9 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 4, the ACTL9 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 5; The ACTL9 antibody heavy chain sequence is shown as SEQ ID NO. 6, the ACTL9 antibody heavy chain variable region VH is shown as SEQ ID NO. 7, the ACTL9 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 8, the ACTL9 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 9, the ACTL9 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 10; The sequence of the light chain of the PLCZ1 antibody is shown as SEQ ID NO. 31, the variable region VL of the light chain of the PLCZ1 antibody is shown as SEQ ID NO. 32, the CDR-L1 of the light chain of the PLCZ1 antibody is shown as SEQ ID NO. 33, the CDR-L2 of the light chain of the PLCZ1 antibody is shown as SEQ ID NO. 34, and the CDR-L3 of the light chain of the PLCZ1 antibody is shown as SEQ ID NO. 35; the sequence of the heavy chain of the PLCZ1 antibody is shown as SEQ ID NO. 36, the variable region VH of the heavy chain of the PLCZ1 antibody is shown as SEQ ID NO. 37, the CDR-H1 of the heavy chain of the PLCZ1 antibody is shown as SEQ ID NO. 38, the CDR-H2 of the heavy chain of the PLCZ1 antibody is shown as SEQ ID NO. 39, and the CDR-H3 of the heavy chain of the PLCZ1 antibody is shown as SEQ ID NO. 40; (d) IQCN antibody The sequence of the light chain of the IQCN antibody is shown as SEQ ID NO. 41, the variable region VL of the light chain of the IQCN antibody is shown as SEQ ID NO. 42, the CDR-L1 of the light chain of the IQCN antibody is shown as SEQ ID NO. 43, the CDR-L2 of the light chain of the IQCN antibody is shown as SEQ ID NO. 44, and the CDR-L3 of the light chain of the IQCN antibody is shown as SEQ ID NO. 45; the sequence of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 46, the variable region VH of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 47, the CDR-H1 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 48, the CDR-H2 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 49, and the CDR-H3 of the heavy chain of the IQCN antibody is shown as SEQ ID NO. 50; (e) CD46 antibody The sequence of the light chain of the CD46 antibody is shown as SEQ ID NO. 51, the variable region VL of the light chain of the CD46 antibody is shown as SEQ ID NO. 52, the CDR-L1 of the light chain of the CD46 antibody is shown as SEQ ID NO. 53, the CDR-L2 of the light chain of the CD46 antibody is shown as SEQ ID NO. 54, and the CDR-L3 of the light chain of the CD46 antibody is shown as SEQ ID NO. 55; the sequence of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 56, the variable region VH of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 57, the CDR-H1 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 58, the CDR-H2 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 59, and the CDR-H3 of the heavy chain of the CD46 antibody is shown as SEQ ID NO. 60; (f) SPAG9 antibody The SPAG9 antibody light chain sequence is shown as SEQ ID NO. 61, the SPAG9 antibody light chain variable region VL is shown as SEQ ID NO. 62, the SPAG9 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 63, the SPAG9 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 64, the SPAG9 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 65; the SPAG9 antibody heavy chain sequence is shown as SEQ ID NO. 66, the SPAG9 antibody heavy chain variable region VH is shown as SEQ ID NO. 67, the SPAG9 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 68, the SPAG9 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 69, the SPAG9 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 70; (g) SPESP1 antibody The SPESP1 antibody light chain sequence is shown as SEQ ID NO. 71, the SPESP1 antibody light chain variable region VL is shown as SEQ ID NO. 72, the SPESP1 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 73, the SPESP1 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 74, the SPESP1 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 75; the SPESP1 antibody heavy chain sequence is shown as SEQ ID NO. 76, the SPESP1 antibody heavy chain variable region VH is shown as SEQ ID NO. 77, the SPESP1 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 78, the SPESP1 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 79, the SPESP1 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 80; (h) IZUMO3 antibody The IZUMO3 antibody light chain sequence is shown as SEQ ID NO. 81, the IZUMO3 antibody light chain variable region VL is shown as SEQ ID NO. 82, the IZUMO3 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 83, the IZUMO3 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 84, the IZUMO3 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 85; the IZUMO3 antibody heavy chain sequence is shown as SEQ ID NO. 86, the IZUMO3 antibody heavy chain variable region VH is shown as SEQ ID NO. 87, the IZUMO3 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 88, the IZUMO3 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 89, the IZUMO3 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 90; (i) a TMEM95 antibody The TMEM95 antibody light chain sequence is shown as SEQ ID NO. 91, the TMEM95 antibody light chain variable region VL is shown as SEQ ID NO. 92, the TMEM95 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 93, the TMEM95 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 94, the TMEM95 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 95; the TMEM95 antibody heavy chain sequence is shown as SEQ ID NO. 96, the TMEM95 antibody heavy chain variable region VH is shown as SEQ ID NO. 97, the TMEM95 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 98, the TMEM95 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 99, the TMEM95 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO. 100; (j) a CFAP90 antibody The CFAP90 antibody light chain sequence is shown as SEQ ID NO. 101, the CFAP90 antibody light chain variable region VL is shown as SEQ ID NO. 102, the CFAP90 antibody light chain complementarity determining region CDR-L1 is shown as SEQ ID NO. 103, the CFAP90 antibody light chain complementarity determining region CDR-L2 is shown as SEQ ID NO. 104, the CFAP90 antibody light chain complementarity determining region CDR-L3 is shown as SEQ ID NO. 105; the CFAP90 heavy chain sequence is shown as SEQ ID NO. 106, the CFAP90 antibody heavy chain variable region VH is shown as SEQ ID NO. 107, the CFAP90 antibody heavy chain complementarity determining region CDR-H1 is shown as SEQ ID NO. 108, the CFAP90 antibody heavy chain complementarity determining region CDR-H2 is shown as SEQ ID NO. 109, and the CFAP90 antibody heavy chain complementarity determining region CDR-H3 is shown as SEQ ID NO.

110.

2. The sperm key for potential of embryonic development kit according to claim 1, characterized in that, The antibody diluent is phosphate buffer, bovine serum albumin, and sodium azide.

3. The sperm key for potential of embryonic development kit according to claim 1, characterized in that, The fluorescently labeled secondary antibody is a fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG antibody.

4. The sperm key for potential of embryonic development kit according to claim 1, characterized in that, The concentrated washing solution is 0.3M phosphate buffer at pH 7.

4.

5. The sperm key for potential of embryonic development kit according to claim 1, characterized in that, The mounting medium is mounting glycerol.

6. The sperm key for potential of embryonic development kit according to claim 1, characterized in that, The nuclear dye is propidium iodide (PI) solution.

7. A method of using the embryonic developmental potential sperm key protein kit of claim 1, wherein, The method comprises the following steps: (a) sample pretreatment The treated semen sample is applied to a multi-well slide, and bright field images or motion videos are collected using a general optical microscope for qualitative detection of sperm morphology; (b) fixing the smear and incubating with specific first antibodies against ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90; (c) incubating with a FITC-labeled secondary antibody; (d) nuclear counterstaining using propidium iodide (PI) solution; (e) image acquisition by confocal microscopy after mounting; (f) determining the qualitative expression of each target protein on sperm by software.

8. The method of using a sperm key for embryo developmental potential kit according to claim 7, wherein, In step (a), the concentration of treated sperm is 40-50 x 10 6 mL; in step (b), the first antibody incubation conditions are: incubation in a 2-8°C refrigerator for at least 8 hours or in a 37°C incubator for 1 hour.

9. The method of using a sperm key for embryonic developmental potential kit according to claim 7, wherein, In step (f), the data of not less than 200 sperm are counted, and the average fluorescence intensity and localization rate of each protein ACTL9, ACTL7A, PLCZ1, IQCN, CD46, SPAG9, SPESP1, IZUMO3, TMEM95, and CFAP90 are calculated; The formula calculation is as follows: (1) Sperm head detection algorithm PI positive sperm recognition formula: I_PI(x,y) ≥ T_PI = μ_background + 3σ_background; Wherein: 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; Wherein: A_head: head area pixel; P_head: head perimeter pixel; 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, take 2-3; (4) Key statistical indicators Total number of sperm: N_total=∑[I_PI(i)≥T_PI]; Expressed sperm number: N_expression=∑[FI_green(i)≥T_positive]; Expression rate: R_expression=(N_expression / N_total)×100%; Average fluorescence intensity: MFI=(1 / N_expression)∑FI_green(i); The calculation formula of positioning rate (PR) is: ； where N positive is the number of sperm expressing a particular target protein, N total is the total number of sperm detected.

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