Sperm DNA fragment index detection reference and preparation method and application thereof

Abstract

The invention relates to a sperm DNA fragment index detection reference and a preparation method and application thereof. The reference product comprises sperm fixed cells, sperm oxidized cells and sperm supernate; wherein the sperm fixing cells are sperm cells subjected to fixing treatment, and the sperm oxidation cells are sperm cells subjected to oxidation treatment. Reference products with different DFI values can be flexibly prepared, detection parameters and maps are almost consistent with those of clinical samples, various detection requirements are met, and the accuracy and flexibility of detection are remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a sperm DNA fragmentation index (DFI) detection reference substance and a preparation method and application thereof. Background Art

[0002] More than 15% of couples worldwide suffer from infertility problems, of which about 50% of cases are caused by male factors. Multiple in vitro and in vivo studies have shown that the integrity of sperm DNA plays a vital role in key reproductive processes such as fertilization, early embryonic development, implantation and pregnancy. Compared with the concentration, motility, morphology and total amount of sperm, the detection of sperm DNA fragmentation (SDF) also has a higher diagnostic value in disease diagnosis and prognosis. Sperm DNA fragmentation index (DFI) is an important indicator to measure sperm DNA integrity and is of great significance for evaluating male fertility.

[0003] At present, a variety of methods for detecting sperm DNA fragmentation (SDF) have been developed, including sperm chromatin structure analysis (SCSA), sperm chromatin diffusion assay (SCD) and comet assay, etc. Among them, SCSA has been widely used in clinical and scientific research due to its high degree of standardization, simple operation, strong stability and high detection efficiency.

[0004] However, the current testing of clinical semen samples generally lacks scientific and effective reference standards, and there are large differences between the testing products and analytical methods of different manufacturers. In addition, the existing methods cannot effectively evaluate the abnormalities that may occur during the collection, transportation and storage of semen samples, as well as the testing process. Therefore, the development of a sperm DNA fragmentation index (DFI) test reference is crucial to improving the accuracy and reliability of sperm DFI testing.

[0005] At present, the quality control products that can be used for DFI testing include sperm DFI quality control products prepared using simulated sperm cells and real sperm sample reference products prepared by aliquoting. These methods have the following problems:

[0006] 1. Sperm DFI quality control products based on simulated sperm cells:

[0007] This control is usually made of chicken red blood cells or microspheres that simulate sperm cells and contains two main components:

[0008] Chicken red blood cells: To simulate sperm with DNA damage, these cells have normal cells with intact DNA and cells with DNA damage mixed in a pre-set ratio.

[0009] Internal reference material: composed of green and red microspheres with stable fluorescence intensity, used to monitor the key operating steps and quality of sperm DNA reagents.

[0010] Although this quality control product can simulate the damage of sperm DNA to a certain extent, it has the following problems:

[0011] Simulated sperm cell differences: The differences in particle size and morphology between sperm DFI quality control products and human sperm cells cannot accurately reflect the detection status of sperm cells, affecting the accuracy of the test.

[0012] Inconsistent test parameters: The test parameters and spectra of sperm DFI quality control products are inconsistent with those of real sperm cells, which cannot effectively control abnormalities that may occur during the test process, such as sample liquefaction state, interference, sample contamination, and interference between different experiments;

[0013] Difficulty in gating quality control: Due to inconsistencies in detection parameters and spectra, it is difficult for sperm DFI quality control products to reasonably analyze the fluorescence intensity of sperm cells, and quality control of the gated area cannot be achieved.

[0014] 2. Select a single semen sample with a specific DFI value and prepare a reference sample by aliquoting:

[0015] The specific steps are as follows: Select a semen sample that shows about 15% DFI. Dilute the sample to a working concentration of 1–2×10^6 cells / mL using cold TNE buffer. Aliquot 300μL of the dilution into 0.5mL snap-cap vials. Store at -70°C or in liquid nitrogen.

[0016] This method uses real semen samples as reference, but has the following problems:

[0017] Large individual differences: Due to the differences between individual semen samples, it is difficult to meet the fixed value requirements, affecting the consistency of quality control samples;

[0018] Limited quality control range: Real sperm samples generally use the test value as the standard value, which makes it difficult to effectively control the sperm DFI test range (e.g. DFI 1% to 99%);

[0019] Sample quantity and variability: The actual number of sperm samples from a single individual cannot be guaranteed for a long time, and samples from different batches have large intra-batch and inter-batch variability. Summary of the invention

[0020] Based on this, the present application provides a sperm DNA fragmentation index (DFI) detection reference material and its preparation method and application, which can flexibly prepare reference materials with different DFI values. The detection parameters and spectra are almost consistent with clinical samples, meeting various detection needs and significantly improving the accuracy and flexibility of detection.

[0021] The technical solutions include the following:

[0022] Sperm DNA fragmentation index detection reference substances, the reference substances comprising sperm fixed cells, sperm oxidized cells and semen supernatant;

[0023] Wherein, the fixed sperm cells are sperm cells that have been fixed, and the oxidized sperm cells are sperm cells that have been oxidized.

[0024] In one embodiment, the sperm oxidized cells account for 0% to 100% of the total cell number of the reference sample.

[0025] In one embodiment, the DFI value of the reference product is about 1% to 99%.

[0026] In one embodiment, the oxidized sperm cells are a mixture of sperm cells treated with oxidation for different time periods.

[0027] In one embodiment, the cell concentration in the reference sample is 1×10^6 to 1×10^8 cells / mL.

[0028] The preparation method of the reference material for sperm DNA fragmentation index detection comprises the following steps:

[0029] The sperm fixed cells, sperm oxidized cells and semen supernatant are mixed to prepare a sperm DNA fragmentation index detection reference product.

[0030] In one embodiment, the semen supernatant is prepared after centrifuging a semen sample containing sperm with a size of 3 to 5 μm.

[0031] Optionally, the pellet after centrifugation is a sperm cell pellet.

[0032] Optionally, the semen supernatant after centrifugation also includes the steps of re-centrifugation and filtration.

[0033] In one embodiment, a fixative is added to the sperm cell pellet to perform a fixation reaction to prepare the fixed sperm cells.

[0034] Optionally, the conditions for the fixation reaction include a temperature of room temperature or 2 to 8° C. and a time of 12 to 48 hours.

[0035] In one embodiment, the fixing solution comprises a fixing agent.

[0036] Optionally, the fixative comprises one or more of formaldehyde and paraformaldehyde.

[0037] Optionally, the fixative comprises, by volume fraction, 1.5% to 4% formaldehyde, 1% to 10% glycerol and PBS buffer.

[0038] Optionally, the fixative comprises, by volume fraction, 1.5% to 4% paraformaldehyde, 1% to 10% glycerol and PBS buffer.

[0039] In one embodiment, an oxidizing solution is added to the sperm cell precipitate, and an oxidation reaction is performed to prepare the sperm oxidized cells.

[0040] Optionally, the oxidation reaction conditions include an oxidation temperature of room temperature or 35-39° C., and an oxidation time of 0.5-4 hours.

[0041] In one embodiment, the sperm oxidized cells are a mixture of sperm oxidized cells oxidized at different times.

[0042] Optionally, sperm oxidized cells prepared with an oxidation time of 0.5 to 1.5 hours, sperm oxidized cells prepared with an oxidation time of 1.5 to 2.5 hours, and sperm oxidized cells prepared with an oxidation time of 2.5 to 4 hours are mixed at a cell number ratio of 1:(0.8-1.2):(0.8-1.2) to prepare a mixture of sperm oxidized cells oxidized at different times.

[0043] In one embodiment, the oxidizing liquid includes an oxidant.

[0044] Optionally, the oxidizing agent comprises hydrogen peroxide.

[0045] Optionally, the oxidizing liquid comprises 3% to 30% hydrogen peroxide by mass.

[0046] Optionally, the oxidizing solution includes 3% to 30% hydrogen peroxide and PBS buffer solution by mass fraction.

[0047] The sperm DNA fragmentation index detection reference product is used in the following applications:

[0048] (1) Application in establishing standardized analytical methods;

[0049] (2) Application in the gating area of ​​the quality control fragmentation index;

[0050] (3) Application in monitoring abnormal conditions that occur during the testing of real semen samples;

[0051] (4) Application in evaluating the status of test samples, optionally, evaluating abnormal status of test samples during collection, transportation and / or storage;

[0052] (5) Application in studying DFI and high DFI in sperm;

[0053] (6) Application in the study of other aspects of sperm DNA damage.

[0054] A performance test product for a sperm DNA fragmentation index detection kit, the product comprising the sperm DNA fragmentation index detection reference product.

[0055] Compared with the traditional technology, this application has the following beneficial effects:

[0056] This application proposes a sperm DNA fragmentation index (DFI) detection reference material and a preparation method thereof, which mainly includes the following technical effects:

[0057] 1. Adjustable DFI value range:

[0058] It is possible to flexibly prepare reference samples with different DFI values. The detection parameters and spectra are almost consistent with those of clinical samples, meeting various detection needs and significantly improving the accuracy and flexibility of detection.

[0059] 2. Simple raw material acquisition and preparation method:

[0060] The raw materials are easy to obtain and the preparation process is simple, which is suitable for routine laboratory operations, reduces operational complexity and time cost, and improves experimental efficiency.

[0061] 3. Establish standardized analytical methods:

[0062] Through the use of reference materials, standardized analytical methods are established to identify sperm with DNA damage (DFI part) and sperm with excessive histone abnormalities (HDS part), improve the consistency and reliability of detection, and standardize laboratory operations.

[0063] 4. Evaluate the status of the test samples:

[0064] Use reference materials to evaluate abnormal conditions during semen sample collection, transportation and storage, identify and resolve abnormal conditions during the testing process, and ensure sample quality.

[0065] 5. Study on DFI and high DFI in sperm:

[0066] Prepare medium-DFI and high-DFI reference materials to conduct in-depth research on sperm DNA damage and provide data support for clinical diagnosis and treatment.

[0067] 6. Other studies on sperm DNA damage:

[0068] Prepare reference materials that match the DNA damage of real semen samples, study other aspects of sperm DNA damage, and improve the overall assessment of sperm quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0070] Figure 1 The process flow chart for the preparation of reference products.

[0071] Figure 2 is the DFI value of sperm fixed cells (the lower limit of the DFI value of the reference product).

[0072] Figure 3 is the DFI value of sperm oxidized cells (the upper limit of the DFI value of the reference product).

[0073] Figure 4 To prepare a DFI reference product with a cell concentration of 50×10^6 cells / mL, Figure A is a DFI value 40% reference product (DFI value 39.33%); Figure B is a DFI value 25% reference product (DFI value 25.88%).

[0074] Figure 5 For the medium DFI 40% reference and the high DFI 40% reference. Figure A is the medium DFI reference, with a total DFI value of 40.14%, a medium DFI value of 37.46%, and a high DFI value of 2.66%; Figure B is the high DFI reference, with a total DFI value of 41.81%, a medium DFI value of 1.92%, and a high DFI value of 41.81%.

[0075] Figure 6 Gated sperm DFI classification plot for SCSA data.

[0076] Figure 7 To prepare the cell concentration of 5×10^6 cells / mL, the DFI value was 40% (41.84%) and 25% (24.33%) DFI reference.

[0077] Figure 8 The DFI value was 0.78% for sperm fixed cells prepared with 2% paraformaldehyde and 10% glycerol.

[0078] Fig. 9 The DFI value was 2.15% for sperm fixed cells prepared with 1.5% formaldehyde and 10% glycerol.

[0079] Fig.10 The DFI value was 0.78% for sperm fixed cells prepared with 4% formaldehyde and 1% glycerol.

[0080] Fig.11 The results of sperm oxidation cell test using 30% hydrogen peroxide preparation showed a DFI value of 99.79%.

[0081] Fig.12 The sperm oxidation cell test results were prepared by oxidation at room temperature for 2 hours, and the DFI value was 96.45%.

[0082] Fig.13 DFI references, 40% references (40.74%) and 25% references (25.59%) were prepared for 2 h oxidation at room temperature.

[0083] Fig.14 To prepare DFI reference products with DFI values ​​of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% respectively.

[0084] Fig.15 In order to prepare a DFI reference that fits a real semen sample, the cell concentration was 50×10^6 cells / mL and the DFI value was 30% of the reference.

[0085] Fig.16 These are the results of clinical semen sample testing. Figure A shows a DFI value of 8.06% (DFI ≤ 15% is normal); Figure B shows a DFI value of 23.30% (15% < DFI < 30% is average); Figure C shows a DFI value of 45.75% (DFI ≥ 30% is considered poor integrity).

[0086] Fig.17 R1 is a sperm cell with normal morphology;

[0087] Fig.18 is the main sperm population P1;

[0088] Fig.19 Set up gate areas for the DFI section and the HDS section;

[0089] Fig. 20 It is a blank template image;

[0090] Figure 21A-Figure 21D To detect abnormal conditions; there are bubbles in the flow cytometer fluid path ( Fig.21A ), A1 is the instrument with bubbles, A2 is the normal condition after equipment maintenance; Inter-experimental interference ( Fig. 21B ), B1 other experimental residue examples, B2 normal conditions after maintenance; clinical samples with high cellular debris ( Fig. 21C ), C1 and C2 are high fragmentation cases detected in clinical samples; R1 and P1 < 50% in their spectra can be judged as high cell apoptosis or incomplete liquefaction, see high fragmentation sample-1 and high fragmentation sample-2; clinical samples with abnormal complete DNA ( Fig.21D); D1 is moderate abnormal impact, and D2 is severe abnormal impact.

[0091] Fig. 22 The results of sperm oxidation cell test using an acidic solution containing hydrochloric acid at pH 1.2 show a DFI value of 94.75%. Impurity residues similar to oxidation abnormalities are present at the position marked by the red circle.

[0092] Figure 23A-23C The results of three batches of sperm fixed cell oxidation tests are shown in Figures A1, B1, and C1, and the results of three batches of sperm fixed cell oxidation tests are shown in Figures A2, B2, and C2, respectively.

[0093] Fig.24 These are the detection diagrams of Comparative Example 3, Example 1 and clinical semen samples; wherein, Figures E1 to E2 are the quality control products (DFI value 40%) prepared using chicken red blood cells in Comparative Example 3; Figures E3 to E5 are the reference products prepared in Example 1, wherein E3 is a visible light forward scatter and side scatter diagram, which is used to determine the size and morphology of cells, and gate R1 is set to divide cell populations; E4 and E5 are fluorescence diagrams (FITC is a green fluorescence diagram, and Percp is a red fluorescence diagram), and the data in Figure E4 comes from the R1 gated data in the preceding Figure E3, and E5 is the final data analysis diagram, and the data comes from the data in the gate P1 of Figure E4; Figures E6 to E8 are the detection diagrams of clinical semen samples. DETAILED DESCRIPTION

[0094] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0096] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0097] In this article, the explanations of the English abbreviations involved are as follows:

[0098] DNA (deoxyribonucleic acid): deoxyribonucleic acid;

[0099] DFI (DNA fragmentation index): DNA fragmentation index;

[0100] HDS (high DNA stainability) high DNA stainability.

[0101] An embodiment of the present application provides a sperm DNA fragmentation index detection reference product, which includes sperm fixed cells, sperm oxidized cells and semen supernatant; wherein the sperm fixed cells are sperm cells that have been fixed, and the sperm oxidized cells are sperm cells that have been oxidized.

[0102] The DFI value range in the reference product can be adjusted by adjusting the proportion of sperm oxidized cells in the reference product. The detection parameters and spectra of the reference product in this application are almost consistent with those of clinical semen samples, meeting various detection needs, and providing a comprehensive and accurate reference product solution by simulating the DNA damage characteristics of real sperm.

[0103] In a specific example, the sperm oxidized cells account for 0% to 100% of the total sperm cells in the reference sample. When the proportion is 0%, the sperm cells in the reference sample are composed of sperm fixed cells, and the DFI value of the sperm fixed cell detection is the lower limit of the DFI value of the reference sample; when the proportion is 100%, the sperm cells in the reference sample are composed of sperm oxidized cells, and the DFI value of the sperm oxidized cells detection is the upper limit of the DFI value of the reference sample.

[0104] In a specific example, the DFI value of the reference product is about 1% to 99%.

[0105] In a specific example, the DFI value of the reference product can be selected as but not limited to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 99%, or a range between any two of the above values.

[0106] In a specific example, the sperm cell concentration in the reference is 1×10^6 to 1×10^8 cells / mL. In a specific example, the sperm cell concentration in the reference may be selected as but not limited to 1×10^6 cells / mL, 2×10^6 cells / mL, 5×10^6 cells / mL, 8×10^6 cells / mL, 1×10^7 cells / mL, 2×10^7 cells / mL, 5×10^7 cells / mL, 8×10^7 cells / mL, 1×10^8 cells / mL, or a range between any two of the above values. In a specific example, 5×10^6 to 5×10^7 cells / mL. Those skilled in the art can adjust the concentration range, and the concentration range or its lower limit is adjusted according to the standard of "Chinese Expert Consensus on the Diagnosis and Treatment of Oligospermia", "Extreme oligospermia: 0 < sperm concentration < 1 × 10^6 / mL, sperm concentration < 5 × 10^6 / mL is severe oligospermia, and the normal human sperm cell concentration is (20-250) × 10^6". For example, the sperm cell concentration in the reference product is 5 × 10^6 cells / mL, and the reference product can be used for clinical sample quality control of severe oligospermia.

[0107] In a specific example, based on the total cell count of the reference sample, the percentage of oxidized sperm cells can be selected as but not limited to 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, or a range between any two of the above values.

[0108] In a specific example, the detection reference products of medium DFI and high DFI are prepared by adjusting the oxidation treatment time of sperm oxidized cells.

[0109] In a specific example, the sperm oxidized cells are a mixture of sperm oxidized cells with different oxidation times.

[0110] One embodiment of the present application also provides a method for preparing a reference substance for detecting sperm DNA fragmentation index, comprising the following steps:

[0111] The sperm fixed cells, sperm oxidized cells and semen supernatant are mixed to prepare a sperm DNA fragmentation index detection reference product.

[0112] The raw materials of sperm cells used in the preparation of reference products in this application are very easy to obtain. The remaining samples from routine semen tests or sperm DFI tests in clinical laboratories can be used as raw materials for the preparation of reference products.

[0113] In a specific example, the sperm cell concentration in the reference product is 1×10^6 to 1×10^8 per mL. Optionally, the sperm oxidized cells account for 0% to 100% of the total sperm cell count of the reference product. Optionally, the DFI value of the reference product is 1% to 99%.

[0114] In a specific example, the method includes the following steps: mixing the sperm fixed cells with the semen supernatant to obtain a mixture A;

[0115] After mixing the sperm oxidized cells with the semen supernatant, a mixture B is obtained; and

[0116] Mixture A and mixture B were mixed to prepare the reference sample.

[0117] In a specific example, the DFI value of the reference product is 1% to 99%.

[0118] In a specific example, in mixture A, the concentration of sperm fixed cells is 1×10^6 to 1×10^8 cells / mL. In a specific example, in mixture A, the concentration of sperm fixed cells can be selected as but not limited to 1×10^6 cells / mL, 2×10^6 cells / mL, 5×10^6 cells / mL, 8×10^6 cells / mL, 1×10^7 cells / mL, 2×10^7 cells / mL, 5×10^7 cells / mL, 8×10^7 cells / mL, 1×10^8 cells / mL, or a range between any two of the above values.

[0119] In a specific example, in mixture B, the concentration of sperm oxidized cells is 1×10^6 to 1×10^8 cells / mL. In a specific example, in mixture B, the concentration of sperm oxidized cells may be selected as but not limited to 1×10^6 cells / mL, 2×10^6 cells / mL, 5×10^6 cells / mL, 8×10^6 cells / mL, 1×10^7 cells / mL, 2×10^7 cells / mL, 5×10^7 cells / mL, 8×10^7 cells / mL, 1×10^8 cells / mL, or a range between any two of the above values.

[0120] In a specific example, a semen sample containing sperm with a size of about 3 to 5 μm is centrifuged to prepare a semen supernatant.

[0121] The size of sperm in the semen sample should be similar to that of human sperm, i.e. about 3-5 μm. Human semen can be selected.

[0122] In one specific example, the pellet after centrifugation is a sperm cell pellet.

[0123] In a specific example, the semen supernatant after centrifugation further includes the steps of centrifugation and filtration again.

[0124] The purpose of centrifugation and filtration is to remove incompletely liquefied viscous substances and large particles in clinical semen samples to prevent clogging of the detection channel of the flow cytometer.

[0125] Specifically, the semen supernatant is prepared by centrifuging a semen sample containing sperm of 3 to 5 μm in size, transferring the semen supernatant into a centrifuge tube, centrifuging at 10,000 to 12,000 g for 10 minutes, and filtering 2 to 3 times using a 0.22 μm filter membrane.

[0126] In a specific example, a fixative solution is added to the sperm cell pellet, and a fixation reaction is performed to prepare the sperm fixed cells.

[0127] In a specific example, the conditions of the fixation reaction include a temperature of room temperature or 2 to 8° C. and a time of 12 to 48 hours.

[0128] In a specific example, the temperature of the fixation reaction can be selected as but not limited to room temperature, or 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or a range between any two of the above values.

[0129] In a specific example, the fixed reaction time can be selected as 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, or a range between any two of the above values.

[0130] In one specific example, the fixing solution includes a fixing agent.

[0131] In one specific example, the fixative includes formaldehyde and / or paraformaldehyde.

[0132] Optionally, the fixative is formaldehyde, which is a typical cross-linking fixative that allows protein side chains to form strong covalent bonds with each other, thereby preventing enzymatic degradation and microbial erosion from damaging the structure and chemical composition of the specimen.

[0133] In one specific example, the fixative solution includes formaldehyde, glycerol, and PBS buffer.

[0134] In a specific example, the volume fraction of formaldehyde in the fixing liquid is 1.5% to 4%, which may be selected from but not limited to 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or a range between any two of the above values.

[0135] In a specific example, the volume fraction of glycerol in the fixing liquid is 1% to 10%. In a specific example, the volume fraction of glycerol in the fixing liquid is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values.

[0136] In a specific example, the fixative solution includes, by volume, 1.5% to 4% paraformaldehyde, 1% to 10% glycerol, and PBS buffer.

[0137] When the fixative and glycerol in the fixative were within the above concentration range, the cell size (FSC-H) of the reference samples prepared at different concentrations was slightly different from that of clinical semen samples, which had little effect on the detection of DFI value.

[0138] In one specific example, an oxidizing solution is added to a sperm cell pellet, and an oxidation reaction is performed to prepare sperm oxidized cells.

[0139] In a specific example, the conditions of the oxidation reaction include an oxidation temperature of room temperature or 35 to 39° C., and an oxidation time of 0.5 to 4 hours.

[0140] In a specific example, the oxidation temperature may be selected as but not limited to room temperature, or 35° C., 36° C., 37° C., 38° C., 39° C., or a range between any two of the above values.

[0141] In the present application, normal temperature refers to 15°C to 30°C, which can be 15°C, 18°C, 20°C, 25°C, 28°C, 30°C, or a range between any two of the above values.

[0142] In a specific example, the oxidation time may be selected as but not limited to 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or a range between any two of the above values.

[0143] In a specific example, sperm oxidized cells are a mixture of sperm oxidized cells with different oxidation times, and a reference product that is basically consistent with the clinical semen sample is prepared. The detection spectrum of the reference product is basically consistent with the clinical semen sample in terms of the gate setting (DFI and HDS) of the detection analysis and the scattered particle distribution. It can be used for research on fitting clinical semen samples. Commercialized quality control products cannot achieve this effect. The present application has irreplaceable advantages and has more far-reaching applications for in-depth research on clinical samples.

[0144] In a specific example, sperm oxidized cells prepared with an oxidation time of 0.5 to 1.5 hours, sperm oxidized cells prepared with an oxidation time of 1.5 to 2.5 hours, and sperm oxidized cells prepared with an oxidation time of 2.5 to 4 hours are mixed at a cell number ratio of 1:(0.8-1.2):(0.8-1.2) to prepare a mixture of sperm oxidized cells with different oxidation times.

[0145] The method for preparing a reference product that fits clinical semen samples in the present application is to use an oxidizing liquid to react in a 37°C environment for 0.5 to 1.5 hours, 1.5 to 2.5 hours, and 2.5 to 4 hours, respectively, and after mixing according to the sperm oxidized cell concentration ratio, a reference product that is basically consistent with the detection spectrum of the clinical semen sample can be prepared.

[0146] In a specific example, sperm oxidized cells prepared with an oxidation time of 1.5 hours, sperm oxidized cells prepared with an oxidation time of 2.5 hours, and sperm oxidized cells prepared with an oxidation time of 4 hours are mixed to prepare a mixture of sperm oxidized cells with different oxidation times.

[0147] In one specific example, the oxidizing liquid includes an oxidizing agent.

[0148] In one specific example, the oxidizing agent includes hydrogen peroxide.

[0149] In a specific example, the oxidizing solution includes 3% to 30% hydrogen peroxide by mass. The main component of the oxidizing solution is low-concentration hydrogen peroxide, in order to fragment the cell DNA.

[0150] In a specific example, the mass fraction of hydrogen peroxide in the oxidizing liquid may be selected as but not limited to 3%, 5%, 7.5%, 9%, 10%, 15%, 20%, 25%, 30%, or a range between any two of the above values.

[0151] In a specific example, the oxidizing solution also includes a mixture of hydrogen peroxide and PBS buffer.

[0152] The raw materials for preparing the reference products in this application, i.e., the remaining clinical semen samples from conventional semen testing or sperm DFI testing, are living cells, and the reference products prepared after fixation and oxidation treatment are dead cells, which refer to cells without cell activity, non-apoptotic or necrotic cells. The positive rate of the results of the V-FITC apoptosis detection kit and PI staining test is more than 90%, which is not suitable for purposes other than sperm DFI testing.

[0153] One embodiment of the present application also provides the application of the sperm DNA fragmentation index detection reference product in the following:

[0154] (1) Application in establishing standardized analytical methods;

[0155] (2) Application in the gating area of ​​the quality control fragmentation index;

[0156] (3) Application in monitoring abnormal conditions that occur during the testing of real semen samples;

[0157] (4) Application in evaluating the status of test samples, optionally, evaluating the abnormal status of test samples during collection, transportation and / or storage;

[0158] (5) Application in studying DFI and high DFI in sperm;

[0159] (6) Application in studying other aspects of sperm DNA damage;

[0160] (7) Detection of whole semen components.

[0161] The reference product prepared using sperm cells in this application not only has a fixed value effect, but also has detection conditions and analysis methods consistent with clinical semen samples, and can also assist laboratories in establishing detection and analysis templates for clinical samples and monitoring abnormalities of clinical samples.

[0162] An embodiment of the present application also provides a performance test product for a sperm DNA fragmentation detection reagent, which includes the sperm DNA fragmentation index detection reference product.

[0163] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0164] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0165] Examples of recommended chemical reagents involved in the following examples are shown in Table 1.

[0166] Table 1

[0167] Reagent name Part Number Specification Manufacturer Formaldehyde (37%~40%) solution A501912-0500 500mL / bottle Shanghai Bioengineering Co., Ltd. 4% paraformaldehyde E672002-0100 100mL / bottle Shanghai Bioengineering Co., Ltd. glycerin G8190-100mL 100mL / bottle Beijing Solable Technology Co., Ltd. Phosphate buffered saline SH30256 500mL / bottle HyClone Hydrogen Peroxide 30% 10011208 500mL / bottle Shanghai Test 7.5% Hydrogen Peroxide 7722-84-1 500mL / bottle Yonghua Chemical Co., Ltd.

[0168] Example 1

[0169] A method for preparing a sperm DNA fragmentation index (DFI) detection reference substance. Figure 1 Schematic diagram of the process for preparing reference products.

[0170] 1. Preparation of Cell Pretreatment Solution

[0171] 1.1 Preparation of fixative solution:

[0172] 1) Using phosphate buffer as a solvent, prepare a mixed solution of 3.5% formaldehyde and 9% glycerol.

[0173] 2) After being thoroughly mixed, the solution was heated in a water bath at 65°C until it became clear to obtain a stationary solution.

[0174] 1.2 Preparation of oxidation solution

[0175] 7.5% hydrogen peroxide was used as the oxidizing solution.

[0176] 2. Preparation of reference materials

[0177] 2.1 Preparation of semen supernatant and cell pellet:

[0178] 1) Divide the semen samples of 3-5 μm in size into multiple 1.5 mL centrifuge tubes, 1 mL per tube, and centrifuge at 6000 g for 5 minutes.

[0179] 2) The supernatant and cell pellet after centrifugation are recovered separately and marked as semen supernatant and sperm cell pellet.

[0180] 3) The recovered semen supernatant was centrifuged at 12000 g for 10 minutes, and then filtered 2 to 3 times using a 0.22 μm syringe filter to obtain a relatively pure semen separation supernatant.

[0181] 2.2 Preparation of sperm fixed cells:

[0182] 1) Take a portion of sperm cell pellet and add 1 mL of fixative solution, and react at 2℃~8℃ for 24 hours.

[0183] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0184] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0185] 4) Repeat the above steps 2 to 3 times, add 500 μL of semen separation supernatant and mix well to prepare sperm fixed cells.

[0186] Note: The concentration of sperm fixed cells is basically the same as the concentration of sperm cells in semen samples when the volumes are equal, while the concentration of sperm fixed cells prepared in step 4) is approximately twice the concentration of sperm cells in semen samples.

[0187] 2.3 Preparation of sperm oxidized cells:

[0188] 1) Take a portion of sperm cell pellet and add 1 mL of oxidizing solution, and react at 37°C for 2 hours.

[0189] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0190] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0191] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant, mix well and prepare sperm oxidation cells.

[0192] Note: The concentration of sperm oxidized cells and the concentration of sperm cells in semen samples are basically the same when the volumes are equal, while the concentration of sperm oxidized cells prepared in step 4) is approximately twice the concentration of sperm cells in semen samples.

[0193] 2.4 Preparation of reference materials:

[0194] 1) Cell concentration value: The recommended cell concentration range is 1×10^6 to 1×10^8 cells / mL, for example, 50×10^6 cells / mL. Adjust the cell concentration to the required range by centrifugation or dilution with semen separation supernatant.

[0195] NOTE: The concentration of sperm fixed cells and sperm oxidized cells can be determined by absolute counting by flow cytometry.

[0196] 2) DFI value: The DFI value range is 1% to 99%, and the lower limit of the DFI value is the DFI value of sperm fixed cell detection ( Figure 2 ), the upper limit of DFI value is the DFI value of sperm oxidation cell detection ( Figure 3 ). Since the raw materials come from mixed semen samples with a sperm size of 3 to 5 μm, the DFI values ​​detected after fixation and oxidation will be slightly different, and it is difficult to reach the ultimate DFI values ​​of 0% and 100%.

[0197] For example, the DFI values ​​of the reference product are 25% and 40%. The concentrations of sperm fixed cells and sperm oxidized cells are adjusted to be the same, and then the sperm fixed cells and sperm oxidized cells are mixed in a volume ratio of 3:1 and 3:2, respectively, and the ratio is adjusted to the actual test value to prepare reference products with DFI values ​​of 25% and 40% ( Figure 4 ).

[0198] Example 2

[0199] A method for preparing a reference substance for detecting DFI in sperm.

[0200] The steps of Reference Example 1 are different from those of Example 1 in that the sperm oxidized cells are prepared by reducing the oxidation reaction time and then mixed with the sperm fixed cells to prepare the DFI reference product.

[0201] The method for preparing sperm oxidized cells comprises:

[0202] 1) Take a portion of sperm cell pellet and add 1 mL of oxidizing solution, and react at 37°C for 1.5 hours.

[0203] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0204] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0205] 4) Repeat the above steps 2-3 times, add the supernatant of semen separation, mix well and prepare sperm oxidation cells.

[0206] 5) Adjust the cell concentration to 50×10^6 cells / mL by centrifugation or dilution with semen supernatant.

[0207] 6) The concentrations of sperm fixed cells and sperm oxidized cells were adjusted to be consistent, and then the sperm fixed cells and sperm oxidized cells were pre-mixed at a volume ratio of 3:2, and the ratio was adjusted according to the actual detection value to prepare a sperm DFI detection reference product with a DFI value of 40% ( Figure 5 A).

[0208] Example 3

[0209] A method for preparing a sperm high DFI detection reference product.

[0210] The steps of Reference Example 1 are different from those of Example 1 in that the sperm oxidized cells prepared by increasing the oxidation reaction time are then mixed with the sperm fixed cells to prepare the high DFI reference product.

[0211] The method for preparing sperm oxidized cells comprises:

[0212] 1) Take a portion of sperm cell pellet and add 1 mL of oxidation solution, and react at 37°C for 4 hours.

[0213] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0214] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0215] 4) Repeat the above steps 2-3 times, add the supernatant of semen separation, mix well and prepare sperm oxidation cells.

[0216] 5) Adjust the cell concentration to 50×10^6 cells / mL by centrifugation or dilution with semen supernatant.

[0217] 6) The concentrations of sperm fixed cells and sperm oxidized cells were adjusted to be consistent, and then the sperm fixed cells and sperm oxidized cells were pre-mixed in a volume ratio of 3:2, and the ratio was adjusted according to the actual test value to prepare a sperm high DFI test reference product with a DFI value of 40% ( Figure 5 B).

[0218] Note: "Medium DFI and high DFI" refer to the degree of DNA damage in sperm cells. "Medium DFI" is comparable to moderate damage, and "high DFI" is comparable to severe damage, not the size of the DFI value. The size of the DFI value refers to the proportion of sperm cells with damaged DNA to the total sperm cells, that is, sperm oxidized cells / (sperm fixed cells + sperm oxidized cells). About "Medium DFI" and "High DFI" ( Figure 6 )For details, please see the "Expert Consensus on Laboratory Testing and Clinical Application of Sperm Fragmentation".

[0219] Example 4

[0220] A method for preparing a sperm DFI detection reference product

[0221] The steps of Reference Example 1 are different from those of Example 1 in that the sperm cell concentration is adjusted to prepare a DFI reference product with a low cell concentration, for example, 5×10^6 cells / mL.

[0222] 1) Adjust the cell concentration to 5×10^6 cells / mL by centrifugation or dilution with semen supernatant.

[0223] 6) The concentrations of sperm fixed cells and sperm oxidized cells were adjusted to be consistent, and then the sperm fixed cells and sperm oxidized cells were pre-mixed at a ratio of 3:2 and 3:1, and the ratio was adjusted according to the actual test value to prepare a reference product with a DFI value of 40% ( Figure 7 ).

[0224] Example 5

[0225] A method for preparing fixed sperm cells.

[0226] The steps of Reference Example 1 were followed, except that 2% paraformaldehyde and 10% glycerol were used to prepare the fixative.

[0227] 1. Prepare the fixative solution: Use phosphate buffer as solvent to prepare a mixed solution of 2% paraformaldehyde and 10% glycerol.

[0228] 2. Prepare sperm fixed cells using the above prepared fixative according to the steps of Example 1:

[0229] 1) Take a portion of sperm cell pellet and add 1 mL of fixative solution, and react at 2℃~8℃ for 24 hours.

[0230] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0231] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0232] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant and mix well to prepare sperm fixed cells, the DFI value of which is 0.78% ( Figure 8 The fixed sperm cells prepared in this example and the fixed sperm cells prepared in Example 1 ( Figure 2 ), indicating that the fixative of this example can be used to prepare sperm fixed cells. The prepared sperm fixed cells are prepared according to the reference sample preparation method in Example 1, and the prepared reference sample is consistent with Example 1.

[0233] Example 6

[0234] A method for preparing fixed sperm cells.

[0235] The procedure of Reference Example 1 was followed, except that 1.5% formaldehyde and 10% glycerol were used to prepare the fixative.

[0236] 1. Prepare the fixative solution: Using phosphate buffer as solvent, prepare a mixed solution of 1.5% formaldehyde and 10% glycerol.

[0237] 2. Prepare sperm fixed cells using the above prepared fixative according to the steps of Example 1:

[0238] 1) Take a portion of sperm cell pellet and add 1 mL of fixative solution, and react at 2℃~8℃ for 24 hours.

[0239] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0240] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0241] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant and mix well to prepare sperm fixed cells, the DFI value of which is 2.15% ( Fig. 9 ). The DFI value of the sperm fixed cell test is less than 5%, and the fixative has an excellent fixation effect, indicating that the fixative of this embodiment can be used to prepare sperm fixed cells. The prepared sperm fixed cells are prepared according to the preparation method of the reference product in Example 1, and the prepared reference product is consistent with Example 1. Note: The lower limit of the DFI value of the reference product prepared by the fixative is the sperm fixed cell test result, which is 2.15%.

[0242] Example 7

[0243] A method for preparing fixed sperm cells.

[0244] The steps of Reference Example 1 were followed, except that 4% formaldehyde and 1% glycerol were used to prepare the fixative.

[0245] 1. Preparation of sperm fixed cells:

[0246] Prepare the fixative solution: Using phosphate buffer as solvent, prepare a mixed solution of 1.5% formaldehyde and 10% glycerol.

[0247] The above-prepared fixative solution was used to prepare sperm fixed cells according to the steps of Example 1.

[0248] 2. Preparation of sperm fixed cells:

[0249] 1) Take a portion of sperm cell pellet and add 1 mL of fixative solution, and react at 2℃~8℃ for 24 hours.

[0250] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0251] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0252] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant and mix well to prepare sperm fixed cells, the DFI value of which is 0.78% ( Fig.10 The fixation efficiency of the fixative prepared in this example is equal to that of Example 1 ( Figure 2 ), indicating that the fixative of this example can be used to prepare sperm fixed cells, and the prepared sperm fixed cells are prepared according to the preparation method of the reference product in Example 1, and the prepared reference product is consistent with Example 1.

[0253] Example 8

[0254] A method for preparing sperm oxidized cells.

[0255] The steps of Reference Example 1 are different from those of Example 1 in that 30% hydrogen peroxide is used to prepare the oxidizing solution.

[0256] 1. Prepare oxidizing solution: Use 30% hydrogen peroxide as the oxidizing solution.

[0257] 2. Prepare sperm oxidation cells using the oxidation solution prepared above according to the steps of Example 1:

[0258] 1) Take a portion of sperm cell pellet and add 1 mL of oxidizing solution, and react at 37°C for 2 hours.

[0259] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0260] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0261] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant, mix well and prepare sperm oxidation cells, the DFI value of which is 99.79% ( Fig.11). The sperm oxidation cells prepared in this example and in Example 1 ( Figure 3 ) has a stronger oxidation effect. When using high concentration of hydrogen peroxide, it can be appropriately diluted to reduce the concentration of hydrogen peroxide or shorten the oxidation reaction time.

[0262] The oxidizing solution of this embodiment can be used to prepare sperm oxidized cells. The prepared sperm oxidized cells have a high degree of DNA damage due to the high concentration of hydrogen peroxide in the oxidizing solution. According to the preparation method of the reference product in Example 1, the prepared reference product is a high DFI reference product, which is consistent with the high DFI reference product in Example 3.

[0263] Example 9

[0264] A method for preparing a sperm DFI detection reference product

[0265] Referring to the steps of Example 1, the difference from Example 1 is that the sperm oxidized cells are prepared by oxidation under room temperature conditions.

[0266] Wherein, preparing sperm oxidized cells:

[0267] 1) Take a portion of sperm cell pellet and add 1 mL of oxidizing solution (7.5% hydrogen peroxide) and react at room temperature (15-30°C) for 2 hours.

[0268] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0269] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0270] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant, mix well and prepare sperm oxidation cells, the DFI value of which is 96.45% ( Fig.12 ).

[0271] The sperm fixed cells prepared in Example 1 and the sperm oxidized cells prepared in this example were mixed in a volume ratio of 3:2 and 3:1 respectively to prepare reference products with DFI values ​​of 40% and 25% ( Fig.13 ).

[0272] Example 10

[0273] A method for preparing detection reference substances with different DFI values.

[0274] Referring to the steps of Example 1, reference products with various DFI values ​​were prepared by mixing at different volume ratios. For example, sperm fixed cells and sperm oxidized cells were prepared according to Example 1, and the concentrations of sperm fixed cells and sperm oxidized cells were adjusted to be consistent, and DFI reference products with DFI values ​​of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% were prepared according to the volume ratios, respectively. Fig.14 ).

[0275] Embodiment 11

[0276] A method for preparing a DFI reference material that fits a real semen sample.

[0277] The steps of Reference Example 1 are different from those of Example 1 in that a reference product is prepared by mixing a plurality of sperm oxidation cells of different degrees:

[0278] 1) Take three sperm cell pellets and add 1 mL of oxidizing solution, and react at 37°C for 1.5 hours, 2.5 hours, and 4 hours respectively.

[0279] 2) After the reaction is completed, centrifuge at 6000g for 5 minutes and discard the supernatant.

[0280] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0281] 4) Repeat the above steps 2-3 times and add the semen supernatant.

[0282] 5) The three cells were mixed in equal volume ratio to prepare sperm oxidation cells.

[0283] 6) Adjust the cell concentration to 50×10^6 cells / mL by centrifugation or dilution with semen separation supernatant.

[0284] 7) The concentrations of sperm fixed cells and sperm oxidized cells were adjusted to be the same, and then the sperm fixed cells and sperm oxidized cells were pre-mixed at a volume ratio of 2:1, and the ratio was adjusted according to the actual test value to prepare a reference product with a DFI value of 30% ( Fig.15 ).

[0285] In this embodiment, a reference product that matches the DNA damage of a real semen sample is prepared. The sperm oxidized cells oxidized at different times are recovered and mixed in equal proportions to prepare a reference product with a fixed value such as 30% ( Fig.15 ), and its detection spectrum is almost the same as that of clinical semen samples ( Fig.16 ), the gate setting (DFI and HDS) and scattered particle distribution of the detection analysis are basically consistent, which can be used to fit the research of clinical semen samples.

[0286] Example 12

[0287] Application of the DFI reference prepared according to Example 1 in establishing a standardized analytical method.

[0288] Different flow cytometers have slight differences in analysis. Taking the BeamCyte series flow cytometers as an example, the application of DFI reference materials in establishing standardized analysis methods:

[0289] 1. First, in the (FSC-H)-(SSC-H) scatter plot, add a rectangular gate to select sperm cells with normal morphology R1 ( Fig.17 ).

[0290] 2. Then in the (FITC-H)-(PerCP-H) scatter plot, select the data from R1. Add a polygon gate and exclude apoptotic and unstained sperm in the lower left corner, and select the main sperm population P1 ( Fig.18 ).

[0291] 3. Finally, in the (FITC-H)-(PerCP-H) density plot, select the data from P1. Create a polygon gate to the right of the cigar-shaped main sperm population. The cells within this polygon are sperm with DNA damage (DFI fraction). Then create a rectangular gate that touches the top of the cigar-shaped main sperm population. The cells on this line are sperm with excessive histone abnormalities (HDS fraction) ( Fig.19 ).

[0292] Embodiment 13

[0293] Application of the reference material prepared according to Example 1 in the gated area of ​​the quality control debris index (DFI).

[0294] The detection profile of the standardized analytical method established by using the reference material can be saved as a blank template ( Fig. 20 ), because the detection parameter settings and spectral regions of the reference product and the real sperm sample are consistent, the blank detection template can be applied to the detection of clinical semen samples. The DFI value of clinical semen samples detected using the blank template ( Fig.16 ).

[0295] Embodiment 14

[0296] Application of the reference material prepared according to Example 1 in identifying and detecting abnormal conditions.

[0297] Through comparative analysis of the reference and clinical semen sample test patterns and gate setting, the abnormal conditions identified include but are not limited to:

[0298] 1) There are bubbles in the flow cytometer fluid path ( Fig.21A), the standard position of the red circle A1 in the figure is the state where the equipment has bubble reference detection. When this state occurs, the equipment needs to be maintained and the reference detection product is normal (A2 in the figure) before use.

[0299] 2) Other experimental interference effects ( Fig. 21B ), B1 in the figure is a residue after other experimental detection, and B2 in the figure is the normal detection spectrum of the reference product, which is resolved through maintenance equipment after abnormal identification.

[0300] 3) Sperm cell high fragmentation ( Fig. 21C ), according to the example diagram of the reference product ( Fig.17 ) in R1 and ( Fig.18 ) in which P1 is less than 50%, the clinical semen sample can be determined to be a high debris sample. For example, C1 in the figure is a high debris sample-1 in which P1 is less than 50%, and C2 in the figure is a high debris sample-2 in which both R1 and P1 are less than 50%.

[0301] 4) Improper storage of semen samples ( Fig.21D ). Reference materials can evaluate the status of test samples. For example, when clinical samples are abnormal during collection, transportation and storage, it will cause complete DNA damage. At this time, the DFI value of the test increases, and the complete DNA map changes from a cigar shape to an ellipse or a circle ( Fig.21D ). D1 is a moderate abnormal impact, and D2 is a severe abnormal impact. The test results of this type of sample cannot reflect the true status of the individual, and resampling is recommended.

[0302] Comparative Example 1

[0303] Relevant research shows that the main component of the oxidizing solution is low-concentration hydrogen peroxide, which stimulates cells through the reactive oxygen species (OS) produced by hydrogen peroxide, inducing and accelerating the breakage of double-stranded and single-stranded sperm DNA (the effect of reactive oxygen species in the air under natural conditions). In addition to accelerating this process, the addition of hydrochloric acid also has the effect of unwinding the broken double-stranded DNA into single-stranded DNA.

[0304] The contents are substantially the same as those of Example 1, except that the oxidizing solution contains hydrochloric acid.

[0305] 1. Prepare oxidation solution:

[0306] 8 mL of 7.5% hydrogen peroxide and 5 mL of an acidic solution of a sperm DFI detection reagent containing 0.08 mol / L hydrochloric acid were mixed to prepare an oxidation solution.

[0307] 2. Prepare sperm oxidation cells using the oxidation solution prepared above according to the steps of Example 1:

[0308] 1) Take a portion of sperm cell pellet and add 1 mL of oxidizing solution, and react at 37°C for 2 hours.

[0309] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0310] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0311] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant, mix well and prepare sperm oxidation cells, the DFI value of which is 94.75% ( Fig. 22 ), there are impurity residues similar to oxidation anomalies at the position marked by the red circle, so the oxidation solution containing 0.08 mol / L hydrochloric acid is not suitable for the preparation of reference products.

[0312] Comparative Example 2

[0313] Comparative Example 2 is substantially the same as Example 1, except that sperm oxidized cells are prepared by using an oxidizing solution to fix sperm cells.

[0314] The following are the instructions for performing three batches of simultaneous testing:

[0315] 1) Prepare fixed sperm cells according to the method of Example 1, and obtain fixed sperm cell pellet after centrifugation ( Figure 23A-23C A1, B1,

[0316] C1).

[0317] 2) Take a portion of the sperm fixed cell pellet and add 1 mL of oxidation solution, and react at 37°C for 2 hours.

[0318] 2) Centrifuge at 6000g for 5 minutes and discard the supernatant.

[0319] 3) Add 1 mL of phosphate buffer to mix the cells, centrifuge at 6000 g for 5 minutes, and discard the supernatant.

[0320] 4) Repeat the above steps 2-3 times, add 500 μL of semen separation supernatant, mix well and prepare sperm oxidation cells ( Figure 23A-23C A2, B2, and C2), it can be seen from the observation figures that the DFI values ​​of sperm fixed cell detection and oxidation detection changed very little, and the morphology and size of the cells observed by forward scatter (FSC) and side scatter (SSC) did not change significantly.

[0321] In Comparative Example 2, it was found that sperm cells were difficult to oxidize after being treated with the fixative, and it was impossible to oxidize the sperm fixed cells to prepare sperm oxidized cells, and it could not be used as a preparation method for a reference product.

[0322] Comparative Example 3

[0323] Comparative Example 3 provides a method for preparing a quality control product for sperm fragmentation detection.

[0324] The difference from Example 1 is that chicken red blood cells are used to prepare the quality control product, and the oxidizing solution also includes 0.08 mol / L hydrochloric acid.

[0325] The difference in the detection spectrum between the reference product of Example 1 and the quality control product of Comparative Example 3 (the DFI values ​​of both products are 25% or 40%) is as follows: Fig.24 As shown, Figures E1 to E2 are quality control products prepared using chicken red blood cells in Comparative Example 3; Figures E3 to E5 are reference products prepared in Example 1; and Figures E6 to E8 are clinical semen sample detection diagrams.

[0326] 1. Simulate sperm cell differences:

[0327] Differences in basic cell morphology: Chicken red blood cells are oval in shape, and the diameter of chicken red blood cells is about 7.7 microns. Human sperm cells are tadpole-like, and the sperm head is generally oval in front view, and pear-shaped from the side. The head is about 4 to 5.5 μm long, about 2.5 to 3.5 μm wide, and about 1.0 μm thick. Therefore, there are significant differences in the characteristics of chicken red blood cells and human sperm cells.

[0328] 2. Detection of differences in the graph:

[0329] The forward scatter (FCS, cell size) and side scatter (SSC, cell complexity) of the flow cytometer were measured. Fig.24 In the figure, (E1, E3, E6) are compared. There are obvious differences in the cell morphology between the quality control product and the clinical semen sample. The reference product has different osmotic pressures on sperm cells due to different concentrations of oxidizing solution and fixing solution during the preparation process, which makes the size of sperm cells (FSC-H) slightly different, but it does not affect the test results (DFI value) and gate analysis (DFI and HDS gate range) in Figure (E4\E5\E7\E8). If necessary, the concentration of the reagent can be adjusted to make its osmotic pressure on sperm cells consistent with the osmotic pressure of sperm cells in clinical semen samples.

[0330] There are three steps in the DFI detection process of clinical semen samples, namely semen dilution, acid denaturation, and sample staining. The denaturation liquid used for acid denaturation, for example, is an acidic liquid containing 0.08 mol / L hydrochloric acid (Sperm DNA Fragmentation Detection Kit (Flow Cytometry-Acridine Orange) / Guangdong Medical Device Registration No. 20222400562). The quality control product prepared in Comparative Example 3 is ineffective in the acid denaturation step during the DFI detection process due to the denaturation treatment with hydrochloric acid. Since the oxidizing solution does not contain hydrochloric acid in this application, the interference of acid denaturation in the DFI detection process of sperm samples can be avoided.

[0331] In summary, when the quality control product of comparative example 3 is used for sperm DFI detection, the detection spectrum and the detection spectrum of clinical semen samples are very different. The quality control product can only prove that it has a fixed value effect (for example, 40% or 25%) in the DFI detection process, and because hydrochloric acid is added during the preparation of the quality control product, the acid denaturation process in the DFI detection process is affected, and it is impossible to prove that the detection of clinical samples (or sperm cells) has the same effect. The reference product of the present application has a fixed value (for example, 40%, 25%, etc.) of the quality control product, as well as the detection conditions and analysis methods consistent with clinical semen samples, and can also assist the laboratory in establishing a detection and analysis template for clinical samples and abnormal monitoring of clinical samples.

[0332] 3. Inconsistent detection parameters:

[0333] Detection parameters: According to Figures E1 to E2 of the quality control product prepared using chicken red blood cells in Comparative Example 3, it is necessary to adjust the fluorescence compensation parameters, while clinical semen samples do not need to adjust the fluorescence compensation. Comparing the reference products prepared in Example 1 (Figures E3 to E5) and clinical semen samples (Figures E6 to E8), except for a certain concentration of fixative and oxidizing solution in the preparation process, there is a slight difference in cell size (FSC-H) due to osmotic pressure, there is no obvious difference in their detection parameters, data analysis and test results. The reference products prepared in this application do not need to adjust the fluorescence compensation. The flow cytometry detection parameters are shown in Table 2:

[0334] Table 2

[0335] Detection parameters Quality Control Products DFI Reference Clinical samples Collection Settings Consistency Consistency Consistency Voltage Setting Inconsistency Consistency Consistency Fluorescence compensation Adjustment compensation none none Gating Analysis Inconsistency Consistency Consistency

[0336] 4. Clinical and scientific research applications

[0337] Application of the quality control product prepared in Comparative Example 3:

[0338] According to the relevant applications of the survey "Technical Evaluation of Commercial Sperm DFI Quality Control Products in SCSA Testing", quality control products mainly have fixed values ​​during the test process, such as 25% and 40%, which can be analyzed for accuracy. They play a role in improving test standardization, ensuring the quality of equipment and reagents, long-term monitoring of laboratory performance, and providing accurate sperm quality analysis data.

[0339] References for this application:

[0340] The reference products of this application have fixed values ​​and can be analyzed for accuracy during the test process. They have relevant clinical and scientific research applications under the following additional conditions (which the quality control products of the comparative examples do not have).

[0341] 1) Application in establishing standardized analytical methods ( Figure 17-Figure 19 ).

[0342] 2) Application in the gate setting area of ​​quality control fragmentation index ( Fig. 20 , Fig.16 ).

[0343] 3) Application in monitoring abnormal conditions that occur during the testing of real semen samples ( Figure 21A-Figure 21C ).

[0344] 4) Application in evaluating the status of test samples, optionally, evaluating the abnormal status of test samples during collection, transportation and / or storage (D in Figure 21).

[0345] 5) Application in the study of DFI and high DFI in sperm ( Figure 5 ).

[0346] 6) Application in other aspects of sperm DNA damage research ( Fig.15 ).

[0347] 7) Detection of whole semen components.

[0348] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0349] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A reference product for detecting sperm DNA fragmentation index, characterized in that: The reference substances include sperm fixed cells, sperm oxidized cells and semen supernatant; Wherein, the fixed sperm cells are sperm cells that have been fixed, and the oxidized sperm cells are sperm cells that have been oxidized.

2. The sperm DNA fragmentation index detection reference product according to claim 1, characterized in that: The sperm oxidized cells account for 0% to 100% of the total cell count of the reference product; and / or, The DFI value of the reference product is 1% to 99%.

3. The sperm DNA fragmentation index detection reference substance according to claim 1 or 2, characterized in that: The cell concentration in the reference sample is 1×10^6~1×10^8 / mL.

4. The sperm DNA fragmentation index detection reference product according to claim 1 or 2, characterized in that: The sperm oxidized cells are a mixture of sperm cells treated with oxidation for different time periods.

5. The method for preparing a sperm DNA fragmentation index detection reference substance according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The sperm fixed cells, sperm oxidized cells and semen supernatant are mixed to prepare a sperm DNA fragmentation index detection reference product.

6. The preparation method according to claim 5, characterized in that: After centrifuging the semen sample containing sperm with a size of 3 to 5 μm, the semen supernatant is prepared; Optionally, the pellet after centrifugation is a sperm cell pellet; Optionally, the semen supernatant after centrifugation also includes the steps of re-centrifugation and filtration.

7. The preparation method according to claim 6, characterized in that: Adding a fixative to the sperm cell precipitate to perform a fixation reaction to prepare the sperm fixed cell; Optionally, the conditions for the fixation reaction include a temperature of room temperature or 2 to 8° C. and a time of 12 to 48 hours.

8. The preparation method according to claim 7, characterized in that: The fixing solution includes a fixing agent; Optionally, the fixative comprises one or more of formaldehyde and paraformaldehyde; Optionally, the fixative comprises, by volume fraction, 1.5% to 4% formaldehyde, 1% to 10% glycerol and PBS buffer; Optionally, the fixative comprises, by volume fraction, 1.5% to 4% paraformaldehyde, 1% to 10% glycerol and PBS buffer.

9. The preparation method according to claim 6, characterized in that: Adding an oxidizing liquid to the sperm cell precipitation to perform an oxidation reaction to prepare the sperm oxidized cells; Optionally, the oxidation reaction conditions include an oxidation temperature of room temperature or 35-39° C., and an oxidation time of 0.5-4 hours.

10. The preparation method according to claim 9, characterized in that: The sperm oxidized cells are a mixture of sperm oxidized cells oxidized at different times; Optionally, sperm oxidized cells prepared with an oxidation time of 0.5 to 1.5 hours, sperm oxidized cells prepared with an oxidation time of 1.5 to 2.5 hours, and sperm oxidized cells prepared with an oxidation time of 2.5 to 4 hours are mixed at a cell number ratio of 1:(0.8-1.2):(0.8-1.2) to prepare a mixture of sperm oxidized cells oxidized at different times.

11. The preparation method according to any one of claims 9 to 10, characterized in that: The oxidizing liquid includes an oxidant; Optionally, the oxidant comprises hydrogen peroxide; Optionally, the oxidizing liquid comprises 3% to 30% hydrogen peroxide by mass fraction; Optionally, the oxidizing solution includes 3% to 30% hydrogen peroxide and PBS buffer solution by mass fraction.

12. Use of the sperm DNA fragmentation index detection reference substance according to any one of claims 1 to 4 in the following: (1) Application in establishing standardized analytical methods; (2) Application in the gating area of ​​the quality control fragmentation index; (3) Application in monitoring abnormal conditions that occur during the testing of real semen samples; (4) Application in evaluating the status of test samples, optionally, evaluating abnormal status of test samples during collection, transportation and / or storage; (5) Application in studying DFI and high DFI in sperm; (6) Application in the study of other aspects of sperm DNA damage.

13. A performance test product for sperm DNA fragmentation detection reagent, characterized in that: The product includes the sperm DNA fragmentation index detection reference substance according to any one of claims 1 to 4.

Images