Sperm sorting fluid based on sperm taxis and preparation method thereof

Abstract

The present invention discloses a sperm sorting fluid based on sperm taxis and a preparation method thereof. The sperm sorting fluid based on sperm taxis comprises the following components: Liquid A: selected from a sperm culture fluid, sperm capacitation fluid or human fallopian tube fluid; Component B: selected from a sodium alginate, hydroxyethyl cellulose, hyaluronic acid, dextran or polyethylene glycol, with a final concentration of 5-25 mg / mL; Component C: selected from a sodium bicarbonate, potassium bicarbonate or basic amino acid, with a final concentration of 2.5-12.5 mg / mL. The components work together to simulate the physicochemical properties of cervical mucus in vitro, simulate the sperm selection microenvironment in vitro, sort active sperm based on sperm taxis, effectively improve the active sperm rate and the normal sperm morphology rate, and reduce sperm DFI.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to a sperm sorting fluid based on sperm taxis and a preparation method thereof. Background Art

[0002] Assisted reproductive technology can help infertile patients reproduce, and high-quality sperm sorting is a key step in determining the success of in vitro fertilization (IVF). Currently, the main clinical methods for sperm sorting are swim-up and density gradient centrifugation. However, the centrifugation involved in these two methods can damage sperm DNA, and certain components in the separation fluid can reduce sperm motility, affecting the success rate of IVF.

[0003] Therefore, it is necessary to provide a sperm sorting fluid and a sorting method that are easy to operate and cause little damage to the sample. Summary of the Invention

[0004] The present invention aims to provide a sperm sorting fluid based on sperm taxis, which can sort active sperm based on sperm taxis, effectively improve the active sperm rate and the normal sperm morphology rate, and is easy to operate.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect of the present invention, a sperm sorting solution based on sperm taxis is provided, wherein the sperm sorting solution based on sperm taxis comprises the following components:

[0007] Fluid A: selected from sperm culture medium, sperm capacitation fluid or human fallopian tube fluid;

[0008] Component B: one selected from sodium alginate, hydroxyethyl cellulose, hyaluronic acid, dextran or polyethylene glycol, with a final concentration of 5-25 mg / mL;

[0009] Component C: one selected from sodium bicarbonate, potassium bicarbonate or basic amino acid, with a final concentration of 2.5-12.5 mg / mL.

[0010] Furthermore, for human sperm, the sperm sorting solution based on sperm taxis includes the following components:

[0011] Fluid A: selected from sperm culture medium, sperm capacitation fluid or human fallopian tube fluid;

[0012] Component B: sodium alginate at a final concentration of 9-11 mg / mL;

[0013] Component C: Sodium bicarbonate at a final concentration of 7-8 mg / mL.

[0014] Furthermore, for human sperm, the sperm sorting solution based on sperm taxis includes the following components:

[0015] Fluid A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid;

[0016] Component B: sodium alginate with a final concentration of 10 mg / mL;

[0017] Component C: Sodium bicarbonate at a final concentration of 7.5 mg / mL.

[0018] Furthermore, for mouse sperm, the sperm sorting solution based on sperm taxis includes the following components:

[0019] Liquid A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid;

[0020] Component B: sodium alginate at a final concentration of 14-16 mg / mL;

[0021] Component C: Sodium bicarbonate at a final concentration of 7-8 mg / mL.

[0022] Furthermore, for mouse sperm, the sperm sorting solution based on sperm taxis includes the following components:

[0023] Fluid A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid;

[0024] Component B: sodium alginate with a final concentration of 15 mg / mL;

[0025] Component C: Sodium bicarbonate at a final concentration of 7.5 mg / mL.

[0026] Furthermore, the sperm culture medium is COOK sperm culture medium from the United States, with the product number K-SISM-20; the sperm capacitation fluid is COOK sperm capacitation fluid from the United States, with the product number K-SIFM-20; and the human fallopian tube fluid is Vitrolife assisted reproductive IVF-sperm washing and insemination fluid, with the product number G-IVF PLUS / 60mL-10136.

[0027] In a second aspect of the present invention, a method for preparing a sperm sorting fluid based on sperm taxis is provided, the method comprising:

[0028] Measure the liquid A;

[0029] Component B is added to the solution A to a final concentration of 5-25 mg / mL, and component C is added to a final concentration of 2.5-12.5 mg / mL to obtain the sperm sorting solution based on sperm taxis.

[0030] In a third aspect of the present invention, a method for sperm sorting using the sperm sorting solution based on sperm taxis is provided, the method comprising:

[0031] A microfluidic chip is obtained, wherein the microfluidic chip comprises: an integrated microfluidic chip body and an inclined base; the integrated microfluidic chip body comprises a substrate, an integrated area provided on the substrate, the integrated area comprising a treatment liquid sampling pool, a treatment liquid sampling channel, a sperm enrichment and in vitro fertilization pool, a sperm sorting channel and a sperm capacitation pool arranged in sequence, the bottom of the sperm enrichment and in vitro fertilization pool being provided with a plurality of inwardly recessed egg cell positioning and embryo culture units; the bottom of the treatment liquid sampling pool is connected to the bottom of the sperm enrichment and in vitro fertilization pool via the treatment liquid sampling channel, and the bottom of the sperm capacitation pool is connected to the bottom of the sperm enrichment and in vitro fertilization pool via the sperm sorting channel; the inclined base is detachably connected to the bottom of the integrated microfluidic chip body, the inclined base comprises an inclined lower end and an inclined upper end, the sperm capacitation pool is close to the inclined lower end, and the treatment liquid sampling pool is close to the inclined upper end; the inclined angle of the inclined base is 5°-65°;

[0032] The sperm sorting fluid is injected into the processing fluid sampling pool of the microfluidic chip until the entire sperm sorting channel is filled. Then, the processed human sperm sample is injected into the sperm sampling hole and placed in an incubator at 37°C and a carbon dioxide concentration of 5% to allow the sperm to pass through the sperm sorting fluid and swim from the sperm sampling hole to the sperm enrichment and in vitro fertilization pool to complete sperm sorting.

[0033] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0034] 1. The present invention provides a sperm sorting fluid based on sperm taxis. The components work together to simulate the physical and chemical properties of cervical mucus in vitro, simulate the sperm selection microenvironment in vitro, and sort active sperm based on sperm taxis, effectively improving the active sperm rate and the normal sperm morphology rate.

[0035] 2. The present invention provides a method for sperm sorting using a sperm sorting fluid based on sperm tropism, which can effectively maintain sperm activity in vitro and is combined with microfluidic chip technology for sperm sorting, thereby better achieving a standardized in vitro fertilization process, saving process steps, and eliminating artificial subjective interference factors. The sperm sorting process does not include the centrifugation operations of conventional swim-up method and density gradient centrifugation method, effectively reducing sperm density fraction (DFI). Furthermore, the method is simple to operate, convenient for personnel in related fields to operate, more user-friendly, and easy to industrialize, with great application prospects and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is the analysis of the viscosity and pH of the sperm sorting fluid in the embodiment of the present invention; wherein, Figure 1 A: Effect of sodium alginate concentration on the viscosity of sperm sorting fluid; Figure 1 B: Effect of sodium bicarbonate concentration on the pH of sperm sorting fluid;

[0038] Figure 2 This is the analysis of human sperm movement trajectory in Experimental Example 1 of the present invention; wherein, Figure 2 A: Analysis of sperm movement trajectory before sorting; Figure 2 B: Analysis of sperm movement trajectory after sorting; Figure 2 C: Comparison of sperm activity before and after sorting;

[0039] Figure 3 This is the human sperm morphology analysis in Experimental Example 1 of the present invention; wherein, Figure 3 A: Sperm morphology analysis before sorting; Figure 3 B: Sperm morphology analysis after sorting; Figure 3 C: Comparison of sperm morphology before and after sorting;

[0040] Figure 4 This is the DFI analysis of human sperm in Experimental Example 1 of the present invention; Figure 4 A: DFI analysis of sperm before sorting; Figure 4 B: DFI analysis of sperm after sorting; Figure 4 C: Comparison of sperm DFI before and after sorting;

[0041] Figure 5 This is the analysis of mouse sperm movement trajectory in Experimental Example 2 of the present invention; Figure 5 A: Analysis of sperm movement trajectory before sorting; Figure 5 B: Analysis of sperm movement trajectory after sorting; Figure 5 C: Comparison of sperm activity before and after sorting.

[0042] Figure 6 This is a schematic diagram of a microfluidic chip device in an embodiment of the present invention; Figure 6 A is the overall side view; Figure 6 B is a top view; Figure 6 C is a structural diagram;

[0043] Among them, the figure markings are: 1-integrated microfluidic chip body; 10-base; 20-integration area; 201-treatment liquid sampling pool; 2011-treatment liquid sampling hole; 202-treatment liquid sampling channel; 203-sperm enrichment and in vitro fertilization pool; 2031-egg cell positioning and embryo culture unit; 2032-filter; 204-sperm sorting channel; 205-sperm capacitation pool; 2051-sperm sampling hole; 2-tilted base. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0045] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0046] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0047] In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

[0048] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0049] The applicant has analyzed that within the female reproductive tract, a series of mechanisms guide sperm toward the egg, ensuring smooth fertilization. Sperm rhizotaxis is one of the most important mechanisms. Cervical mucus is a glycoprotein gel secreted by glandular cells in the cervical mucosa, and its state is influenced by various ovarian hormones. Before ovulation, cervical mucus gradually increases in volume under the influence of estrogen; after ovulation, it gradually decreases under the influence of progesterone. Cervical mucus has a certain viscosity, creating resistance to sperm migration. Only sperm with high motility can pass through the cervix and continue toward the uterus, thus sorting active sperm.

[0050] Therefore, the present invention develops a sperm sorting fluid based on sperm taxis based on the principle of reverse engineering, simulates the physical and chemical properties of cervical mucus in vitro, optimizes the sperm selection process, and realizes the sorting of active sperm.

[0051] According to a typical embodiment of the present invention, a sperm sorting solution based on sperm taxis is provided, wherein the sperm sorting solution based on sperm taxis comprises the following components:

[0052] Fluid A: selected from sperm culture medium, sperm capacitation fluid or human fallopian tube fluid;

[0053] Component B: selected from sodium alginate, hydroxyethyl cellulose, hyaluronic acid, dextran or polyethylene glycol, with a final concentration of 5-25 mg / mL, which can make the viscosity of the mixed solution range from 0-0.5 Pa.s.

[0054] Component C: selected from sodium bicarbonate, potassium bicarbonate or basic amino acid, with a final concentration of 2.5-12.5 mg / mL, which can make the pH range of the mixed solution 7-8.

[0055] In the above technical solution,

[0056] Liquid A: used to simulate the biological characteristics of cervical mucus, provide nutrients to sperm, maintain sperm motility, and meet the needs of sperm sorting and sperm-egg combination;

[0057] Component B: used to simulate the viscosity of cervical mucus, provide a certain viscous resistance during sperm swimming, and use the sperm's rheotaxis to sort high-quality sperm;

[0058] Component C: used to adjust the pH of the sperm sorting fluid, simulate the chemical properties of cervical mucus, provide sperm with a stable weak alkaline environment, maintain their vitality, and meet the needs of sperm sorting and sperm-egg binding;

[0059] As a preferred embodiment, for human sperm, the sperm sorting fluid based on sperm taxis comprises the following components: Fluid A: selected from sperm culture fluid, sperm capacitation fluid, or human fallopian tube fluid; Component B: sodium alginate at a final concentration of 9-11 mg / mL; Component C: sodium bicarbonate at a final concentration of 7-8 mg / mL. It should be noted that for human sperm, the sperm sorting fluid based on sperm taxis can be referred to as a human sperm sorting fluid.

[0060] The above formula makes the viscosity of the mixed liquid range from 0.05-0.1 Pa.s and the pH range from 7.4-7.6, simulating the physical and chemical properties of cervical mucus in vitro, optimizing the human sperm selection process, and realizing the active sperm sorting.

[0061] As a more preferred embodiment, the human sperm sorting fluid includes the following components: Liquid A: selected from sperm culture medium, sperm capacitation fluid or human fallopian tube fluid; Component B: sodium alginate with a final concentration of 10 mg / mL; Component C: sodium bicarbonate with a final concentration of 7.5 mg / mL.

[0062] As a preferred embodiment, for murine sperm, the sperm sorting fluid based on sperm taxis comprises the following components: Fluid A: selected from sperm culture fluid, sperm capacitation fluid, or human fallopian tube fluid; Component B: sodium alginate at a final concentration of 14-16 mg / mL; Component C: sodium bicarbonate at a final concentration of 7-8 mg / mL. It should be noted that for murine sperm, the sperm sorting fluid based on sperm taxis can be referred to as a murine sperm sorting fluid.

[0063] The above formula makes the viscosity of the mixed liquid range from 0.1-0.2 Pa.s and the pH range from 7.4-7.6, simulating the physical and chemical properties of cervical mucus in vitro, optimizing the sperm selection process of mice, and realizing the sorting of active sperm.

[0064] As a more preferred embodiment, the mouse sperm sorting fluid comprises the following components: liquid A: selected from sperm culture fluid, sperm capacitation fluid or human fallopian tube fluid; component B: sodium alginate with a final concentration of 15 mg / mL; component C: sodium bicarbonate with a final concentration of 7.5 mg / mL.

[0065] According to another typical embodiment of the present invention, a method for preparing a sperm sorting fluid based on sperm taxis is provided, the method comprising:

[0066] S10, measuring the liquid A;

[0067] S20, adding component B to the solution A to make a final concentration of 5-25 mg / mL, and adding component C to make a final concentration of 2.5-12.5 mg / mL to obtain the sperm sorting solution based on sperm taxis.

[0068] As a preferred embodiment, the final concentrations of the components B and C are selected to be the most preferred concentrations according to different human or other animal sperm samples, specifically:

[0069] S1. For different human or other animal sperm samples, calculate the viscosity and pH of the corresponding sperm sorting fluid according to their motility and the biological characteristics of cervical mucus, mainly based on the characteristic parameters of human cervical mucus or mouse cervical mucus;

[0070] For different types of sperm, the sperm sorting fluid based on sperm taxis can be divided into human sperm sorting fluid, mouse sperm sorting fluid, etc. Human sperm sorting fluid has slight differences for different people. As a preferred embodiment, the viscosity and pH of the corresponding sperm sorting fluid can be calculated and then prepared.

[0071] S2. Adjust the final concentration of component B based on the viscosity measured in S1: Add solution A to a centrifuge tube, add a certain volume of component B to solution A, and mix thoroughly using a vortex shaker. Stir for 10 minutes, then adjust the viscosity to the above-mentioned value (±10%). Incubate in a 37°C water bath for 1 hour to obtain an incubated mixture.

[0072] S3. Adjust the final concentration of component C according to the pH measured in S1: add a certain volume of the component to the incubated mixture, stir for 5 minutes, and adjust to the above pH (±0.1). Then, filter the prepared liquid with a 0.22μm filter membrane and store it in a refrigerator at 4℃.

[0073] The following is a detailed description of a sperm sorting fluid based on sperm taxis of the present application in combination with examples, comparative examples and experimental data.

[0074] Example 1: Human sperm sorting fluid, preparation method, and sorting method

[0075] 1. A sperm sorting fluid based on sperm taxis (specifically, human sperm sorting fluid), comprising the following components: Solution A: selected from sperm culture fluid, sperm capacitation fluid, or human fallopian tube fluid; sodium alginate at a final concentration of 10 mg / mL; and sodium bicarbonate at a final concentration of 7.5 mg / mL.

[0076] 2. The method for preparing the sperm sorting solution based on sperm taxis comprises the following steps:

[0077] Step 1: For different human or other animal sperm samples, the viscosity and pH of the corresponding sperm sorting fluid are calculated based on their motility and the biological characteristics of cervical mucus;

[0078] Step 2: Add 100 mg of sodium alginate powder and 10 mL of human oviductal fluid to a centrifuge tube and mix thoroughly using a vortex shaker. After stirring for 10 minutes, adjust the viscosity to the above value and then incubate in a 37°C water bath for 1 hour.

[0079] Step 3: Add 75 mg of sodium bicarbonate powder to the incubated mixture, stir for 5 minutes, and adjust the pH to the above value. Then, filter the prepared liquid with a 0.22 μm filter membrane and store it in a refrigerator at 4°C.

[0080] 3. A method for sperm sorting using the sperm sorting solution based on sperm taxis combined with a microfluidic chip, the method comprising the following steps:

[0081] (1) Obtain a microfluidic chip, such as Figure 6 Shown, including:

[0082] The integrated microfluidic chip body 1 includes a substrate 10 and an integrated area 20 provided on the substrate. The integrated area 20 includes a treatment liquid injection pool 201, a treatment liquid injection channel 202, a sperm enrichment and in vitro fertilization pool 203, a sperm sorting channel 204, and a sperm capacitation pool 205, which are sequentially connected. The bottom of the sperm enrichment and in vitro fertilization pool 203 is provided with a plurality of inwardly concave egg cell positioning and embryo culture units 2031;

[0083] The tilt base 2 has an inclination angle of 20° and is detachably connected to the bottom of the integrated microfluidic chip body 1 .

[0084] The specific dimensions are:

[0085] The substrate is a 6 cm cell culture dish, model NEST-704001;

[0086] The sperm sampling pool has a diameter of 4 mm and a height of 6 mm;

[0087] The sperm injection hole has a diameter of 0.2 mm and a height of 0.4 mm;

[0088] The sperm sorting channel has a width of 0.2 mm, a length of 15 mm, and a height of 100 μm;

[0089] The diameter of the treatment liquid injection pool is 4mm and the height is 6mm;

[0090] The treatment liquid injection hole has a diameter of 0.2 mm and a height of 0.4 mm;

[0091] The sperm enrichment pool has a diameter of 4 mm and a height of 6 mm;

[0092] The manufacturing method of the above-mentioned microfluidic chip is as follows:

[0093] Step 1: Draw the desired micro pattern using CAD software, and then make a mask based on the micro pattern;

[0094] Step 2: Developing the micro pattern on a silicon wafer using ultraviolet lithography to obtain a silicon wafer mold with the micro pattern;

[0095] Step 3: Using the organic material polydimethylsiloxane, pour unsolidified polydimethylsiloxane on the silicon wafer mold, and heat-bake at 80°C for 1 hour to solidify, thereby obtaining a semi-finished product with several microchannels;

[0096] Step 4: cutting the microchannel structure and punching holes with a puncher, and then bonding the microchannel structure to the substrate after surface treatment with a plasma cleaning machine.

[0097] (2) Sperm sorting is performed using the above-mentioned sperm sorting solution in combination with the above-mentioned microfluidic chip, and the specific steps are as follows:

[0098] Take 1 mL of the prepared sperm sorting solution and inject it into the treatment solution injection pool until the entire sperm sorting channel is filled. Then inject 200 μL of the processed human sperm sample into the sperm injection hole.

[0099] The entire device was placed in an incubator at 37°C and a carbon dioxide concentration of 5%, and the sperm were allowed to swim through the sperm sorting fluid from the sperm injection hole to the sperm enrichment and in vitro fertilization pool;

[0100] After 30 minutes, 5 μL of sorted sperm samples were taken from the sperm enrichment pool for sperm motility detection, sperm DFI, and sperm morphology tests.

[0101] Example 2: Human sperm sorting fluid

[0102] In this comparative example, the final concentration of component B of the sperm sorting solution is 9 mg / mL, and the final concentration of component C is 7 mg / mL. Other structures and steps are the same as those in Example 1.

[0103] Example 3: Human sperm sorting fluid

[0104] In this comparative example, the final concentration of component B of the sperm sorting solution is 11 mg / mL, and the final concentration of component C is 8 mg / mL. Other structures and steps are the same as those in Example 1.

[0105] Comparative Example 1

[0106] In this comparative example, the final concentration of component B of the sperm sorting solution is 5 mg / mL, and the final concentration of component C is 7.5 mg / mL. Other structures and steps are the same as those in Example 1.

[0107] Comparative Example 2

[0108] In this comparative example, the final concentration of component B of the sperm sorting solution was 15 mg / mL, and the final concentration of component C was 7.5 mg / mL. Other structures and steps were the same as those in Example 1.

[0109] Comparative Example 3

[0110] In this comparative example, the final concentration of component B of the sperm sorting solution was 0 mg / mL, and the final concentration of component C was 7.5 mg / mL. Other conditions were the same as those in Example 1.

[0111] Comparative Example 4

[0112] In this comparative example, the final concentration of the sperm sorting solution component B is 20 mg / mL, and the final concentration of the component C is 7.5 mg / mL. Other conditions are the same as those in Example 1.

[0113] Experimental Example 1: Human sperm sorting

[0114] 1. Human sperm was sorted using the chips of each embodiment and each comparative example, and the sperm sorting effect of each group was statistically analyzed (including sperm motility test, sperm DFI, and sperm morphology test). The sperm sorting steps included:

[0115] Step 1: Inject 1 mL of sperm sorting fluid into the processing fluid injection pool, then pass it through the sperm enrichment pool until it fills the entire sperm sorting channel, and then inject 200 μL of processed human sperm sample into the sperm injection port for capacitation;

[0116] Step 2: Place the microfluidic chip on the base and place the entire device in an incubator at 37°C and 5% carbon dioxide concentration, allowing sperm to swim upstream from the sperm injection port to the sperm enrichment pool;

[0117] Step 3: After 30 minutes, take 5 μL of sorted sperm sample from the sperm enrichment pool for sperm motility detection, sperm DFI, and sperm morphology test.

[0118] The sorting effects of the chips of the above embodiments and comparative examples are statistically analyzed, as shown in Table 1.

[0119] Table 1

[0120]

[0121]

[0122] As shown in Table 1, for human sperm, the sperm sorting fluid based on sperm taxis includes the following components: Liquid A: selected from sperm culture fluid, sperm capacitation fluid or human fallopian tube fluid; sodium alginate with a final concentration of 10 mg / mL; sodium bicarbonate with a final concentration of 7.5 mg / mL, which has the best sorting effect.

[0123] Example 4, Mouse Sperm Sorting Fluid

[0124] 1. For mouse sperm, the sperm sorting solution based on sperm taxis includes the following components:

[0125] Solution A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid; component B has a final concentration of 15 mg / mL sodium alginate; component C has a final concentration of 7.5 mg / mL sodium bicarbonate.

[0126] 2. The method for preparing the sperm sorting solution based on sperm taxis comprises the following steps:

[0127] 150 mg of sodium alginate powder and 10 mL of human oviduct fluid were added to a centrifuge tube and mixed thoroughly using a vortex shaker. After stirring for 10 min, the viscosity was adjusted to the above value and then incubated in a 37 °C water bath for 1 hour.

[0128] 75 mg of sodium bicarbonate powder was added to the incubated mixture, stirred for 5 minutes, and then adjusted to the above pH. The prepared liquid was then filtered with a 0.22 μm filter membrane and stored in a refrigerator at 4°C.

[0129] Example 5, Mouse Sperm Sorting Fluid

[0130] In this comparative example, the final concentration of component B of the sperm sorting solution is 14 mg / mL, and the final concentration of component C is 7 mg / mL. Other structures and steps are the same as those in Example 4.

[0131] Example 6, Mouse Sperm Sorting Fluid

[0132] In this comparative example, the final concentration of component B of the sperm sorting solution is 16 mg / mL, and the final concentration of component C is 8 mg / mL. Other structures and steps are the same as those in Example 4.

[0133] Experimental Example 2: Sperm sorting in mice

[0134] The results of mouse sperm sorting using the sperm sorting solution described in Example 4 are as follows: Figure 5 As shown in C, the active sperm rate of mice was significantly improved after sorting.

[0135] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0136] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0137] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A human sperm sorting fluid based on sperm taxis, characterized in that: The separation fluid comprises the following components: Fluid A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid; Component B: sodium alginate with a final concentration of 10 mg / mL; Component C: sodium bicarbonate with a final concentration of 7.5 mg / mL; The sperm culture medium is the American COOK sperm culture medium, with the product number K-SISM-20; the sperm capacitation fluid is the American COOK sperm capacitation fluid, with the product number K-SIFM-20; and the human fallopian tube fluid is the Vitrolife assisted reproductive IVF-sperm washing and insemination fluid, with the product number G-IVF PLUS / 60 mL-10136.

2. A mouse sperm sorting fluid based on sperm taxis, characterized in that: The separation fluid comprises the following components: Liquid A: selected from sperm culture medium, sperm capacitation medium or human fallopian tube fluid; Component B: sodium alginate with a final concentration of 15 mg / mL; Component C: sodium bicarbonate with a final concentration of 7.5 mg / mL; The sperm culture medium is the American COOK sperm culture medium, with the product number K-SISM-20; the sperm capacitation fluid is the American COOK sperm capacitation fluid, with the product number K-SIFM-20; and the human fallopian tube fluid is the Vitrolife assisted reproductive IVF-sperm washing and insemination fluid, with the product number G-IVF PLUS / 60 mL-10136.

3. A method for preparing the sperm sorting fluid according to any one of claims 1 to 2, comprising: Measure the liquid A; Component B was added to the solution A to a final concentration of 10 mg / mL or 15 mg / mL, and component C was added to a final concentration of 7.5 mg / mL to obtain the sperm sorting solution.

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