A method for removing low-quality sperm

Abstract

This invention discloses a method for removing low-quality sperm, comprising the following steps: (1) modifying the 5' end of a nucleic acid aptamer that can specifically bind to low-quality sperm with biotin to obtain a biotin-modified nucleic acid aptamer; (2) co-incubating the biotin-modified nucleic acid aptamer with streptavidin magnetic beads to couple biotin with streptavidin; (3) blocking the uncoupled biotin sites on the streptavidin magnetic beads in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers; (4) co-incubating the magnetic bead-coupled nucleic acid aptamers with sperm cells, and then removing the sperm cells that are bound to the magnetic bead-coupled nucleic acid aptamers by magnetic field force, and performing computer-aided sperm analysis (CASA) quality detection on the remaining supernatant.

Description

Technical Field

[0001] This invention belongs to the field of aptamer technology, specifically relating to a method for removing low-quality sperm. Background Technology

[0002] Over the past 50 years, male semen quality has been declining, leading to a rising incidence of infertility. Routine semen quality analysis primarily includes parameters such as sperm count, motility, and viability. Studies have shown that approximately 15% of infertile patients have normal semen analysis results, thus limiting its effectiveness in assessing male fertility. In the clinical field of assisted reproductive technology (ART), mature sperm are traditionally prepared from human semen using Percoll gradient centrifugation or Swim-up methods. Both techniques can improve sperm motility to some extent, but current ART focuses on sperm concentration, motility, and morphology while neglecting assessment of the nucleus. Some techniques, such as sperm chromatin structure analysis, comet assays, and flow cytometry, while detecting DNA integrity, come at the cost of sperm death, failing to yield healthy sperm for artificial insemination. Therefore, a method capable of accurately detecting and separating normally morphological sperm has significant clinical application value. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for removing low-quality sperm.

[0004] The technical solution of the present invention is as follows:

[0005] A method for removing low-quality sperm includes the following steps:

[0006] (1) Biotin-modified nucleic acid aptamers are obtained by modifying the 5' end of nucleic acid aptamers that can specifically bind to low-quality sperm.

[0007] (2) The above-mentioned biotin-modified nucleic acid aptamers were co-incubated with streptavidin magnetic beads to couple biotin with streptavidin;

[0008] (3) Block the sites on the streptavidin magnetic beads that are not coupled with biotin in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers;

[0009] (4) The above-mentioned magnetic bead-coupled nucleic acid aptamer was co-incubated with sperm cells, and then the sperm cells bound to the magnetic bead-coupled nucleic acid aptamer were removed by magnetic field force. The remaining clear liquid was subjected to CASA quality test.

[0010] In a preferred embodiment of the invention, the nucleic acid aptamer includes at least one of the sequences shown in SEQ ID NO. 01 to 04.

[0011] In a preferred embodiment of the present invention, the sequence of the nucleic acid aptamer is shown in SEQ ID NO.01.

[0012] More preferably, the sequence of the nucleic acid aptamer is shown in SEQ ID NO.02.

[0013] More preferably, the sequence of the nucleic acid aptamer is shown in SEQ ID NO.03.

[0014] More preferably, the sequence of the nucleic acid aptamer is shown in SEQ ID NO.04.

[0015] In a preferred embodiment of the present invention, step (3) is: using biotin to block the uncoupled biotin sites on the streptavidin magnetic beads in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers.

[0016] In a preferred embodiment of the present invention, the co-incubation time in step (4) is 14-16 min.

[0017] More preferably, the co-incubation time in step (4) is 15 minutes.

[0018] In a preferred embodiment of the present invention, the CASA quality testing items include VCL, VSL, VAP, ALH, LIN, WOB, STR, BCF, and MAD.

[0019] The beneficial effects of this invention are: it can screen low-quality sperm that have normal morphological appearance but abnormal or potentially abnormal internal factors, which is of great significance in improving fertilization capacity and embryo development quality. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the principle of this invention.

[0021] Figure 2 The diagram shows the blocking results of magnetic beads coupled with nucleic acid aptamers in Example 2 of this invention, where: Aptamer represents the binding of streptavidin magnetic beads with biotin-modified nucleic acid aptamers; Aptamer + Biotin represents the binding of streptavidin magnetic beads with biotin-modified nucleic acid aptamers followed by biotin blocking; Biotin represents the binding of streptavidin magnetic beads with biotin; and Biotin + Aptamer represents the binding of streptavidin magnetic beads with biotin after biotin blocking.

[0022] Figure 3 This is a photograph of the magnetic bead-coupled nucleic acid aptamer binding to sperm cells in Embodiment 3 of the present invention.

[0023] Figure 4 This is one of the experimental results of removing low-quality sperm using nucleic acid aptamers in Example 4 of the present invention. In the figure: Blank is the blank control group, DS is the magnetic bead control group, and Aptamer-DS is the nucleic acid aptamer experimental group; A represents sperm cell viability, B represents the ratio of grade a viable sperm cells, and C represents the ratio of grade a+b viable sperm cells. GraphPad statistical data analysis showed that the nucleic acid aptamer group had extremely significant differences from both the blank control group and the magnetic bead group in terms of sperm viability, the ratio of grade a viable sperm cells, and the ratio of grade a+b viable sperm cells (P < 0.001). There was no statistically significant difference between the blank control group and the magnetic bead group.

[0024] Figure 5 This is one of the experimental results of removing low-quality sperm using nucleic acid aptamers in Example 4 of the present invention. In the figure: Blank represents the blank control group, DS represents the magnetic bead control group, and Aptamer-DS represents the nucleic acid aptamer experimental group; A represents VSL, B represents VCL, C represents STR, D represents ALH, E represents WOB, F represents LIN, G represents VAP, H represents MAD, and I represents BCF. GraphPad statistical data analysis shows that in terms of VSL, VCL, STR, and ALH, the nucleic acid aptamer group showed extremely significant differences compared to the blank group and the magnetic bead group (P < 0.001), while there was no statistically significant difference between the blank group and the magnetic bead group. In terms of WOB and LIN, the nucleic acid aptamer group showed significant differences compared to the blank group (P < 0.05), while there was no statistically significant difference between the blank group and the magnetic bead group. In terms of STR, BCF, and MAD, there was no statistically significant difference between the nucleic acid aptamer group and the blank group and the magnetic bead group. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0026] Human semen quality standards

[0027] According to the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, the normal semen reference values ​​are: ① Semen pH ≥ 7.2, ② Semen volume ≥ 1.5 mL, ③ Sperm concentration ≥ 15 × 10⁻⁶. 6 ④ Total sperm motility ≥40%, ⑤ Forward motility sperm ≥32%, ⑥ Sperm cell viability ≥58%, ⑦ Sperm morphology (normal morphology) ≥4%, ⑧ Liquefaction time ≤60min. All of the above conditions must be met for semen to be considered normal.

[0028] Example 1

[0029] like Figure 1 As shown, a method for removing low-quality sperm includes the following steps:

[0030] (1) A specified sequence of a nucleic acid aptamer modified with biotin at the 5' end was synthesized by Shanghai Sangon Biotech Co., Ltd.; the nucleic acid aptamer sequence is as follows (where B is biotin located at the 5' end):

[0031] SEQ ID NO.01: B-TGCGCATACGAGCTTGTTCAATACTGCGCATACGAGCTTGTTCAATACAAGCTGCTGAGCTGGCACCATGTAATATAAGTGCAATCTAGCCA

[0032] SEQ ID NO.02: B-TGCGCATACGAGCTTGTTCAATAGGCTGAGTGTCTGTACTAATTGTATGTGGGGCCCTGGGGTGATAGTAAGTGCAATCTAGCCA

[0033] SEQ ID NO.03: B-GCGCATACGAGCTTGTTCAATATGAGGCTTGTCGAATAACGTGTCTAATCGAGTCCCCGCCGTGATAGTAAGTGCAATCTAGCC

[0034] SEQ ID NO.04: B-GGCTAGATTGCACTTACTATCA

[0035] (2) The above-mentioned biotin-modified nucleic acid aptamers were co-incubated with streptavidin magnetic beads to couple biotin with streptavidin;

[0036] (3) Block the sites on the streptavidin magnetic beads that are not coupled with biotin in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers;

[0037] (4) The above-mentioned magnetic bead-coupled nucleic acid aptamer was incubated with sperm cells for 15 min. Then, the sperm cells that were bound to the magnetic bead-coupled nucleic acid aptamer were removed by magnetic field force, and the remaining clear liquid was subjected to CASA quality test.

[0038] Example 2

[0039] This embodiment is a concretization of step (3) of embodiment 1: After streptavidin magnetic beads bind to nucleic acid aptamers, there are still vacant sites. In order to avoid non-specific binding of biotin on the surface of sperm cells, this embodiment blocks the sites on streptavidin magnetic beads that are not coupled with biotin by incubating with biotin (10 mg / mL, Sangon Biotech, A100340) to obtain magnetic beads coupled with nucleic acid aptamers; the details are as follows.

[0040] 1) Nucleic acid aptamer processing: The synthesized nucleic acid aptamer powder was centrifuged at 10,000 rpm for 5 min (3-18K, Sigma, Germany), and then 100 µL of RNase-free water was added. The mixture was vortexed to obtain a concentration of 100 µm. It was diluted to 1 µm before use. High temperature can open the primary structure of the nucleic acid aptamer, while slow cooling can restore its natural folding. In this invention, a PCR gradient cooling method was used for pretreatment of the nucleic acid aptamers. Annealing program: 95℃ for 10 min, decreasing by 1℃ per second, including holding at 60℃ for 1 min, 10℃ for 30 s, and 4℃ for 30 s. The annealing temperature was then reduced to room temperature before use.

[0041] 2) Magnetic bead preparation: Take 8 µL of streptavidin magnetic beads (10 mg / mL, Invitrogen, 65601), wash three times with 100 µL binding washing buffer (BWB) (1 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 100 mM NaCl, 20 mM Tris-HCl, 0.1% NP40, pH 7.5), divide into four equal portions, and resuspend in 10 µL binding buffer (BB) (2 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 100 mM NaCl, 20 mM Tris-HCl, pH 7.4). Nucleic acid aptamers (3' end modified with 6-FAM, 5' end modified with Biotin), concentration 0.5 µM. Incubate in the following groups at room temperature on a circular mixer at 10 rpm for 30 min.

[0042] a: Incubation of streptavidin magnetic beads with nucleic acid aptamers

[0043] b: Incubation of streptavidin magnetic beads with nucleic acid aptamers

[0044] c: Incubation of streptavidin magnetic beads with biotin

[0045] d: Incubation of streptavidin magnetic beads with biotin

[0046] 3) After incubation, wash three times with 100 µL BWB to remove excess unbound molecules, and resuspend in 10 µL BB. The entire process should be kept away from light. Do not continue processing of a and c, and keep them away from light.

[0047] 4) Incubate the following groups at room temperature on a circular mixer at a speed of 10 rpm for 30 min;

[0048] b: Incubate with biotin

[0049] d: Incubation with nucleic acid aptamers

[0050] 5) After incubation, wash three times with 100 µL BWB to remove excess unbound molecules, and resuspend in 10 µL BB.

[0051] 6) Observation of smears a, b, c, and d: Take 10 µL of each group and drop it onto a glass slide, slowly cover it with a coverslip, place it flat in a light-proof humidified chamber, and observe the binding of magnetic beads and nucleic acid aptamers using an upright fluorescence microscope. First, acquire a white light image, then acquire a fluorescence image, and save the images after taking pictures. Figure 2 ).

[0052] Example 3

[0053] Streptavidin magnetic beads are coupled to nucleic acid aptamers with biotin modified at the 3' end, and then incubated with sperm cells. The coupling status between the magnetic beads and the nucleic acid aptamers is indicated by the fluorescent group of FAM modified at the 5' end.

[0054] 1) Sperm cell treatment: Take fresh semen, mix thoroughly, and take 200 µL. Add PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·12H2O, 1.4 mM KH2PO4, pH adjusted to 7.4) to a volume of 1 mL. Centrifuge at 2000×g for 5 min at 4℃. Discard the supernatant, repeat the washing 5 times, and finally resuspend the sperm cells in 1 mL of BB to achieve a cell density of 102. 6 1 / mL, keep on ice for later use;

[0055] 2) Processing of nucleic acid aptamers: The procedure is the same as in Example 2;

[0056] 3) Preparation of magnetic bead-coupled nucleic acid aptamers: Take 2 µL of streptavidin magnetic beads (10 mg / mL), wash 3 times with 100 µL BB, divide into two equal portions, and resuspend in 10 µL BB. Add random single-stranded nucleic acid (FAM-ATACGAGCTTGTTCAATACCGATAGGCGCGTCAGGGAGACTGAATCTCTGCCTCAGCATGATAGTAAGTGCAATCT-B, SEQ ID NO.05) and nucleic acid aptamer respectively, with a concentration of 0.5 µM. Incubate at room temperature on a circular mixer at 10 rpm for 30 min. After incubation, wash 3 times with 100 µL BWB to remove excess unbound molecules. After blocking the magnetic beads as in Example 2, wash 3 times with 100 µL BWB to remove excess unbound molecules, and resuspend in 10 µL BB. Avoid light during the entire process.

[0057] 4) Incubation of sperm cells with magnetic bead-coupled nucleic acid aptamers:

[0058] Control group: 10 µL of treated sperm cells and treated Control magnetic beads were added;

[0059] Nucleic acid aptamer group: 10 µL of processed sperm cells and processed Aptamer magnetic beads were added;

[0060] Incubate at room temperature on a shaker for 30 minutes.

[0061] 5) After incubation, wash three times with 100 µL BWB to remove excess unbound molecules, resuspend in 10 µL BB, and observe the binding of sperm cells to nucleic acid aptamers on a smear. Figure 3 ).

[0062] Example 4: Experiment on the removal of low-quality sperm by nucleic acid aptamers

[0063] CASA is a new technology utilizing camera and computer video technology. Its principle is to connect a camera to a microscope to track the movement of individual sperm cells, and input the acquired electronic signals into a computer. The computer analyzes these signals and provides various parameters to evaluate sperm concentration and sperm motility, among others. CASA has two advantages: ① high accuracy, and ② the ability to provide sperm kinetic parameters. Based on information from the WHO Fifth Edition, these parameters are described below:

[0064] 1) VCL: Curve velocity (µm / s), the time-averaged velocity of the sperm head trajectory, reflecting sperm motility;

[0065] 2) VSL: Linear velocity (µm / s), the time-averaged linear velocity of the sperm head between its initial and final positions during detection;

[0066] 3) VAP: Average path velocity (µm / s), the time-averaged rate of the sperm head as it moves along its average path;

[0067] 4) ALH: Sperm head lateral displacement amplitude (µm), the lateral displacement amplitude of the sperm head about its average path, expressed as the maximum or average value of the lateral displacement;

[0068] 5) LIN: Linearity, the linearity of a curved trajectory, VSL / VCL;

[0069] 6) WOB: Oscillation, the oscillation value of the actual curved path with respect to the average path, VAP / VCL.

[0070] 7) STR: Forwardness, Linearity of the Average Path, VSL / VAP;

[0071] 8) BCF: Whiplash frequency (Hz), the average frequency at which the sperm curve path crosses its mean path;

[0072] 9) MAD: Mean angular displacement (degrees), the time-averaged absolute value of the instantaneous turning angle of the sperm head along its curved trajectory.

[0073] The nucleic acid aptamers from Example 1 were used to test semen samples from 24 different individuals, with three technical replicates for each sample.

[0074] 1) Sperm cell treatment: Mix the entire sample of sperm cells thoroughly;

[0075] 2) Preparation of magnetic bead-coupled nucleic acid aptamers: Take 10 µL of streptavidin magnetic beads (10 mg / mL), wash three times with 100 µL of BWB, divide into two equal portions, and add BB and nucleic acid aptamers to the other portion to a concentration of 0.5 µM. Incubate at room temperature on a circular mixer at 10 rpm for 30 min. After incubation, wash three times with 100 µL of BWB to remove excess unbound molecules, and resuspend in 100 µL of BB.

[0076] 3) Blocking of magnetic bead-coupled nucleic acid aptamers: 5 µL of biotin of equal concentration was added to each of the magnetic beads treated in the previous step. The mixture was incubated at room temperature on a circular mixer at 10 rpm for 30 min. After incubation, the beads were washed three times with 100 µL of BB and resuspended in 10 µL of BB. At this point, the magnetic bead concentration was 5 mg / mL.

[0077] 4) Incubation of sperm cells with magnetic beads coupled with nucleic acid aptamers: Groups were set up: Blank was the blank control group, DS was the magnetic bead control group, and Aptamer-DS was the nucleic acid aptamer experimental group.

[0078] Blank is the control group: 200 µL of well-mixed semen sample and 10 µL of PBS were added.

[0079] DS served as the magnetic bead control group: 200 µL and 10 µL of well-mixed semen sample were added;

[0080] Aptamer-DS was used as the nucleic acid aptamer experimental group: 200 µL of well-mixed semen sample and 10 µL of Aptamer-DS were added.

[0081] Incubate at room temperature on a shaker for 15 minutes;

[0082] 5) Collect the supernatant by magnetic enrichment and perform CASA analysis;

[0083] 6) The precipitate after magnetic enrichment was washed with 100 µL BWB (final DAPI staining).

[0084] 7) Resuspend in 20 µL of BB and observe on a smear. Figure 4 and Figure 5 ).

[0085] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention. sequence list <110> Huaqiao University <120> A method for removing low-quality sperm <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 92 <212> DNA <213> Artificial Sequence <400> 1 tgcgcatacg agcttgttca atactgcgca tacgagcttg ttcaatacaa gctgctgagc 60 tggcaccatg taatataagt gcaatctagc ca 92 <210> 2 <211> 85 <212> DNA <213> Artificial Sequence <400> 2 tgcgcatacg agcttgttca ataggctgag tgtctgtact aattgtatgt ggggccctgg 60 ggtgatagta agtgcaatct agcca 85 <210> 3 <211> 84 <212> DNA <213> Artificial Sequence <400> 3 gcgcatacga gcttgttcaa tatgaggctt gtcgaataac gtgtctaatc gagtccccgc 60 cgtgatagta agtgcaatct agcc 84 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ggctagattg cacttactat ca 22 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <400> 5 [[ID=**26**]]atacgagctt gttcaatacc gataggcgcg tcagggagac tgaatctctg cctcagcatg 60 atagtaagtg caatct 76

Claims

1. A method for removing low-quality sperm, characterized in that: Includes the following steps: (1) The 5' end of a nucleic acid aptamer that can specifically bind to low-quality sperm is modified with biotin to obtain a biotin-modified nucleic acid aptamer, the sequence of which is shown in SEQ ID NO.02; (2) The above-mentioned biotin-modified nucleic acid aptamers were co-incubated with streptavidin magnetic beads to couple biotin with streptavidin; (3) Block the sites on the streptavidin magnetic beads that are not coupled with biotin in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers; (4) The above-mentioned magnetic bead-coupled nucleic acid aptamer was co-incubated with sperm cells, and then the sperm cells bound to the magnetic bead-coupled nucleic acid aptamer were removed by magnetic field force. The remaining clear liquid was subjected to CASA quality test.

2. The method as described in claim 1, characterized in that: Step (3) involves using biotin to block the uncoupled biotin sites on the streptavidin magnetic beads in the material obtained in step (2) to obtain magnetic bead-coupled nucleic acid aptamers.

3. The method as described in claim 1, characterized in that: The co-incubation time in step (4) is 14-16 minutes.

4. The method as described in claim 3, characterized in that: The co-incubation time in step (4) is 15 minutes.

5. The method as described in claim 1, characterized in that: The CASA quality inspection items include VCL, VSL, VAP, ALH, LIN, WOB, STR, BCF, and MAD.

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