Gradient sperm separating medium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of assisted reproduction, in particular to a sperm gradient separating medium and a preparation method and application thereof. The sperm gradient separation liquid comprises a gradient liquid A and a gradient liquid B; the gradient solution A and the gradient solution B comprise sodium chloride, potassium chloride, magnesium sulfate, monopotassium phosphate, calcium chloride, sodium pyruvate, glucose, gentamicin, a sodium lactate solution, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium bicarbonate, human serum albumin, progesterone and Percoll PLUS; the Percoll PLUS mass concentration of the gradient solution A is 70 to 85 percent; the Percoll PLUS mass concentration of the gradient solution B is 35 to 45 percent; the mass ratio of sodium chloride to potassium chloride to magnesium sulfate to calcium chloride in the gradient solution A to the gradient solution B is (5-5.5): (0.3-0.38): (0.25-0.30): (0.24-0.28); the mass ratio of the human serum albumin to the progesterone in the gradient solution A to the gradient solution B is (3-7): (0.00075-0.00085). The sperm gradient separation liquid can improve the activity of forward sperms after treatment, the activity of sperms after treatment, the recovery rate of active sperms and the improvement rate of sperm activity.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to a sperm gradient separation fluid, a preparation method thereof, and an application thereof. Background Art

[0002] Semen optimization is a crucial step in assisted reproductive technology. Its purpose is to remove seminal plasma, immotile sperm, sperm abnormalities, cell debris, and other harmful substances, while producing a high proportion of morphologically normal, motile sperm. Ideally, sperm pretreatment methods should remove seminal plasma, white blood cells, and bacteria, while enriching for sperm with normal morphology and motility.

[0003] Sperm gradients work based on the density differences of sperm. They are typically composed of solutions with varying concentrations of ions, such as sulfate or polyethylene glycol. During centrifugation, sperm are separated into layers within the gradient according to their density. Normally shaped sperm, due to their higher density, settle to the bottom of the gradient, while abnormally shaped sperm, cell debris, and other impurities remain in the upper layer.

[0004] Chinese patent CN110951679B provides a sperm density gradient centrifuge and its preparation method. The sperm density gradient centrifuge comprises solution 1 and solution 2. Solution 1 is prepared from a base solution and the following ingredients: 40-50 wt% Percoll; 0.5-2.0 mg / L Coenzyme Q10; and 5-15 mmol / L HEPES. Solution 2 is prepared from the same ingredients as solution 1, except that the mass fraction of Percoll is 80-98%. This sperm density gradient centrifuge can reduce damage to sperm caused by reactive oxygen species during centrifugation, thereby protecting sperm and maintaining sperm integrity and motility. However, its preparation method is relatively complex, and sperm motility still needs to be improved.

[0005] Wang Lihong et al. studied the recovery of poorly motile sperm using PureSperm density gradient centrifugation in their study on the separation of poorly motile semen samples. The results showed that the a+b grade (forward-pointing sperm motility) was 37.92% before treatment and 51.37% after treatment. While this represents a modest improvement, the forward-pointing sperm motility of 51.37% still has room for improvement.

[0006] CN108531446B2 discloses a protective system for a sperm gradient centrifuge, wherein the protective system comprises lecithin at a concentration of 30-100 mg / L, and further comprises MOPS and HEPES; the concentrations of MOPS and HEPES are 3.85-4.30 mmol / L and 8-12 mmol / L, respectively; the sperm gradient centrifuge further comprises a base system and Percoll, and according to the mass fraction of Percoll, the sperm gradient centrifuge is divided into two solutions, namely, Percoll 40%-45% solution and Percoll 80%-90% solution; Percoll is a silica suspension coated with polyvinyl pyrrolidone; the base system comprises sodium, potassium, calcium, magnesium ions, as well as glucose, sodium pyruvate, glycine and glutamine: with water as the matrix, sodium pyruvate 10.51 mmol / L, glucose 1 mmol / L, alanyl-glutamine 27.49 mmol / L, magnesium sulfate 1 mmol / L, gentamicin 50 mg / L, sodium bicarbonate 0 0.64mmol / L, sodium chloride 99.93mmol / L, taurine 0.48mmol / L, potassium chloride 5.53mmol / L, potassium phosphate 0.15mmol / L, glycine 0.25mmol / L, glutamine 0.73mmol / L, and calcium lactate 0.51mmol / L. Sperm a+b grade (forward sperm motility) was 90-93.56%, showing room for improvement. Summary of the Invention

[0007] The present invention provides a sperm gradient separation solution and a preparation method and application thereof. The sperm gradient separation solution of the present invention can improve the forward sperm motility after treatment, the sperm motility after treatment, the recovery rate of active sperm and the sperm motility improvement rate, and the preparation method is simple and efficient.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sperm gradient separation solution, comprising, by mass concentration, a gradient solution A and a gradient solution B; the gradient solution A comprises sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium pyruvate, glucose, gentamicin, sodium lactate solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium bicarbonate, human serum albumin, progesterone, and Percoll PLUS, wherein the mass concentration of Percoll PLUS is 750-850 g / L; The gradient solution B comprises sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium pyruvate, glucose, gentamicin, sodium lactate solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium bicarbonate, human serum albumin, progesterone and Percoll PLUS, wherein the mass concentration of Percoll PLUS is 350-450 g / L; the mass ratio of sodium chloride, potassium chloride, magnesium sulfate and calcium chloride in gradient solution A and gradient solution B is (5-5.5): (0.3-0.38): (0.25-0.30): (0.24-0.28); the mass ratio of human serum albumin and progesterone in gradient solution A and gradient solution B is (3-7): (0.00075-0.00085).

[0009] Furthermore, the solvent of the sperm gradient separation fluid is water.

[0010] Furthermore, the pH of the sperm gradient separation fluid is 7.45-7.55.

[0011] Preferably, the mass ratio of sodium chloride, potassium chloride, magnesium sulfate and calcium chloride is (5.3-5.5): (0.35-0.38): (0.29-0.30): (0.24-0.25).

[0012] Preferably, the mass ratio of human serum albumin to progesterone is (4-6): (0.00079-0.00085).

[0013] Furthermore, the gradient solution A comprises, by mass concentration, 5-5.5 g / L of sodium chloride, 0.3-0.38 g / L of potassium chloride, 0.25-0.30 g / L of magnesium sulfate, 0.15-0.18 g / L of potassium dihydrogen phosphate, 0.24-0.28 g / L of calcium chloride, 0.028-0.04 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.02 g / L of gentamicin, 4.8-5.0 g / L of sodium lactate solution, 4.5-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-05 g / L of sodium bicarbonate, 3-7 g / L of human serum albumin, 0.00075-0.00085 g / L of progesterone, and 750-850 g / L of Percoll PLUS.

[0014] Preferably, the gradient solution A comprises, by mass concentration, 5.3-5.5 g / L of sodium chloride, 0.35-0.38 g / L of potassium chloride, 0.29-0.30 g / L of magnesium sulfate, 0.16-0.17 g / L of potassium dihydrogen phosphate, 0.24-0.25 g / L of calcium chloride, 0.028-0.03 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.015 g / L of gentamicin sulfate, 4.9-5.0 g / L of sodium lactate solution, 4.7-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.4 g / L of sodium bicarbonate, 4-6 g / L of human serum albumin, 0.00079-0.00085 g / L of progesterone, and 800-815 g / L of Percoll PLUS.

[0015] Furthermore, the gradient solution B comprises, by mass concentration, 5-5.5 g / L of sodium chloride, 0.3-0.38 g / L of potassium chloride, 0.25-0.30 g / L of magnesium sulfate, 0.15-0.18 g / L of potassium dihydrogen phosphate, 0.24-0.28 g / L of calcium chloride, 0.028-0.04 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.02 g / L of gentamicin, 4.8-5.0 g / L of sodium lactate solution, 4.5-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-05 g / L of sodium bicarbonate, 3-7 g / L of human serum albumin, 0.00075-0.00085 g / L of progesterone, and 350-450 g / L of Percoll PLUS.

[0016] Preferably, the gradient solution B comprises, by mass concentration, 5.3-5.5 g / L of sodium chloride, 0.35-0.38 g / L of potassium chloride, 0.29-0.30 g / L of magnesium sulfate, 0.16-0.17 g / L of potassium dihydrogen phosphate, 0.24-0.25 g / L of calcium chloride, 0.028-0.03 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.015 g / L of gentamicin sulfate, 4.9-5.0 g / L of sodium lactate solution, 4.7-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.4 g / L of sodium bicarbonate, 4-6 g / L of human serum albumin, 0.00079-0.00085 g / L of progesterone, and 400-410 g / L of Percoll PLUS.

[0017] In a second aspect, the present invention provides a method for preparing the sperm gradient separation solution, comprising the following steps: Percoll PLUS, sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, glucose, 4-hydroxyethylpiperazineethanesulfonic acid, calcium chloride, sodium pyruvate, gentamicin, sodium lactate, sodium bicarbonate, human serum albumin, and progesterone were dissolved in 1000 mL of water according to the mass concentrations and the pH was adjusted to prepare gradient solutions B and A, respectively.

[0018] In a third aspect, the present invention provides the use of the sperm gradient separation fluid and / or the sperm gradient separation fluid prepared by the preparation method in the preparation of assisted reproductive products.

[0019] Furthermore, the assisted reproductive product is an assisted reproductive kit.

[0020] In a fourth aspect, the present invention provides an assisted reproductive product, comprising the sperm gradient separation fluid.

[0021] Furthermore, the assisted reproductive product is an assisted reproductive kit.

[0022] The beneficial effects of the present invention are: (1) The present invention adopts a one-step method to directly prepare the sperm gradient separation solution, which simplifies the operation steps and shortens the time for preparing the separation solution.

[0023] (2) The present invention improves the forward sperm motility after treatment, sperm motility after treatment, the recovery rate of active sperm and the sperm motility enhancement rate by adjusting the specific permanent relationship of sodium chloride, potassium chloride, magnesium sulfate and calcium chloride and cooperating with other components, which is beneficial to the separation of sperm and subsequent fertilization in assisted reproduction.

[0024] (3) The present invention adds progesterone and improves the forward sperm motility after treatment, the sperm motility after treatment, the recovery rate of active sperm and the sperm motility improvement rate by adjusting the specific mass ratio of progesterone and human serum albumin. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials available from commercial sources.

[0026] Percoll PLUS was purchased from Cytiva, catalog number 10346748.

[0027] Human serum albumin was purchased from Shenzhen Weiguang Biological Products Co., Ltd.

[0028] 1. Examples and Comparative Examples The material tables of Examples 1-3 and Comparative Examples 1-3 are shown in Tables 1 and 2.

[0029] Table 1

[0030] Table 2

[0031] Comparative Example 4 SAGE gradient centrifugation medium ART-2080 (gradient medium A) and ART-2040 (gradient medium B).

[0032] The preparation method of Example 1: 1) Turn on a digital heating magnetic stirrer (hereinafter referred to as the "stirrer"), set the temperature to 25°C, and the speed to 300 r / min. Stir using these parameters throughout the entire process. Place a 1000 mL empty beaker on a zero-calibrated balance, weigh 406.8000 g of Percoll PLUS, and weigh the balance on the zero-calibrated balance. Place the beaker on a rotor, add water for injection to 600 mL, and place the beaker in the rotor. This is referred to as Liquid A1. Place another 1000 mL empty beaker on a zero-calibrated balance, weigh 813.600 g of Percoll PLUS, and weigh the balance on the zero-calibrated balance. Place the beaker on the rotor, and place the beaker in the rotor. This is referred to as Liquid A2.

[0033] 2) Weigh 5.3297 g of sodium chloride twice using a 10,000-degree balance. Slowly and carefully add each to the 1000-mL beaker and stir until clear and free of solid particles, forming mixed solutions B1 and B2.

[0034] 3) Weigh 0.3563 g of potassium chloride twice on a 10,000-degree balance and slowly and carefully add each of the two mixtures B1 and B2. Ultrasonicate for 40 minutes until the mixture is clear and free of solid particles, forming mixtures C1 and C2.

[0035] 4) Weigh 0.2933 g of magnesium sulfate heptahydrate twice on a 10,000-degree balance and slowly and carefully add to mixed solutions C1 and C2, respectively. Stir until clear and free of solid particles, to form mixed solutions D1 and D2.

[0036] 5) Weigh 0.1619 g of potassium dihydrogen phosphate twice on a 10,000-degree balance and slowly and carefully add to mixed solutions D1 and D2, respectively, stirring until clear and free of solid particles, to form mixed solutions E1 and E2.

[0037] 6) Weigh 0.9999 g of anhydrous glucose twice on a 10,000-degree balance and slowly and carefully add to mixed solutions E1 and E2, respectively. Stir until clear and free of solid particles, to form mixed solutions F1 and F2.

[0038] 7) Weigh 4.7660 g of 4-hydroxyethylpiperazineethanesulfonic acid twice on a 10,000-point balance and slowly and carefully add to mixed solutions F1 and F2, respectively, stirring until clear and free of solid particles, to form mixed solutions G1 and G2.

[0039] 8) Take two 25 mL empty beakers and place them on a zero balance. Take 0.2499 g of calcium chloride dihydrate in each beaker and weigh it on a ten-thousandth balance. Add water for injection until all the calcium chloride is fully dissolved. Slowly and carefully add the fully dissolved calcium chloride to mixed solutions G1 and G2, respectively. Rinse any residual calcium chloride in the beaker with water for injection at least three times. Add mixed solutions G1 and G2, and stir until clear and free of solid particles to form mixed solutions H1 and H2.

[0040] 9) Weigh 0.0297 g of sodium pyruvate twice on a 10,000-degree balance and slowly and carefully add each of the mixed solutions H1 and H2. Stir until clear and free of solid particles, to form mixed solutions I1 and I2.

[0041] 10) Weigh 0.0100 g of gentamicin sulfate twice on a 10,000-point balance and slowly and carefully add to mixed solutions I1 and I2, respectively. Stir until clear and free of solid particles, to form mixed solutions J1 and J2.

[0042] 11) Take two 25 mL empty beakers and place them on a zero balance. Pipette 4.9210 g of sodium lactate solution into each beaker and weigh accurately on a ten-thousandth balance. Slowly and carefully add the mixed solutions J1 and J2 to each beaker. Rinse the remaining sodium lactate solution in the beaker with water for injection at least three times. Add the mixed solutions J1 and J2 and stir until clear to form mixed solutions K1 and K2.

[0043] 12) Add water for injection to 800 mL, take 0.3663 g of sodium bicarbonate twice, weigh it on a balance (0.3626-0.3700 g), and slowly and carefully add the mixtures K1 and K2, respectively. Stir until clear and free of solid particles, to form mixtures L1 and L2.

[0044] 13) Take two 100 mL empty beakers and place them on a zeroed balance. Weigh 5.0247 g of human albumin into each beaker and weigh accurately. Slowly and carefully add the mixed solutions L1 and L2 to each beaker. Rinse any residual human albumin in the beaker three times with water for injection. Add the mixed solutions L1 and L2 and stir until clear to form mixed solutions M1 and M2.

[0045] 14) Weigh 2 progesterone solutions (0.00079 g) on ​​a 10,000 ppm balance and slowly and carefully add to mixed solutions O1 and O2, respectively. Stir until clear and free of solid particles, to form mixed solutions N1 and N2.

[0046] 15) Add water for injection to 900 mL of mixed solutions N1 and N2 to form mixed solutions O1 and O2. Perform pH and osmotic pressure tests.

[0047] 16) pH meter calibration: Perform a two-point calibration at room temperature using 4.01 and 9.18 standard buffers. The slope should be 95%-105%. Repeat the calibration with the standard solution of 6.86, and the test value should be 6.86±0.05.

[0048] 17) Add 1 M sodium hydroxide solution to the mixed solutions O1 and O2 to adjust the pH and record the consumption of 1 M sodium hydroxide solution. After stirring evenly, measure the pH using a pH meter. The measured value is 7.55 (25 ± 2°C), forming mixed solutions P1 and P2.

[0049] 18) Slowly pour the mixed solutions P1 and P2 in the 1000mL beaker into a 1000mL volumetric flask, rinse the beaker three times with water for injection, pour the mixture into the 1000mL volumetric flask, and adjust the volume to 1000mL to form mixed solutions Q1 and Q2.

[0050] 19) Osmometer Calibration: Perform a two-point calibration using purified water and a 300mOsm / kg sodium chloride standard solution. Repeated osmotic pressure readings of the 300mOsm / kg sodium chloride standard solution should be between 298-302mOsm / kg. The values ​​of the mixed solutions Q1 and Q2 should be 300mOsm / kg.

[0051] After the osmotic pressure test is passed, it enters sterile filtration and filling.

[0052] Examples 2-3 and Comparative Examples 1 and 3 were prepared according to the materials in Table 1 and Table 2, and the preparation method was the same as that of Example 1.

[0053] Preparation method of comparative example 2: 1) Turn on a digital heating magnetic stirrer (hereinafter referred to as the "stirrer"), set the temperature to 25°C, and the speed to 300 r / min. Stir using these parameters throughout the entire process. Place a 1000 mL empty beaker on a zero-calibrated balance, weigh 406.8000 g of Percoll PLUS, and weigh the balance on the zero-calibrated balance. Place the beaker on a rotor, add water for injection to 600 mL, and place the beaker in the rotor. This is referred to as Liquid A1. Place another 1000 mL empty beaker on a zero-calibrated balance, weigh 813.600 g of Percoll PLUS, and weigh the balance on the zero-calibrated balance. Place the beaker on the rotor, and place the beaker in the rotor. This is referred to as Liquid A2.

[0054] 2) Weigh 5.3297 g of sodium chloride twice using a 10,000-degree balance. Slowly and carefully add each to the 1000-mL beaker and stir until clear and free of solid particles, forming mixed solutions B1 and B2.

[0055] 3) Weigh 0.3563 g of potassium chloride twice on a 10,000-degree balance and slowly and carefully add each of the two mixtures B1 and B2. Ultrasonicate for 40 minutes until the mixture is clear and free of solid particles, forming mixtures C1 and C2.

[0056] 4) Weigh 0.2933 g of magnesium sulfate heptahydrate twice on a 10,000-degree balance and slowly and carefully add to mixed solutions C1 and C2, respectively. Stir until clear and free of solid particles, to form mixed solutions D1 and D2.

[0057] 5) Weigh 0.9999 g of anhydrous glucose twice on a 10,000-degree balance and slowly and carefully add to mixed solutions D1 and D2, respectively. Stir until clear and free of solid particles, to form mixed solutions F1 and F2.

[0058] 6) Weigh 4.7660 g of 4-hydroxyethylpiperazineethanesulfonic acid twice, weigh it on a 10,000-point balance, and slowly and carefully add it to mixed solutions F1 and F2, respectively. Stir until clear and free of solid particles, to form mixed solutions G1 and G2.

[0059] 7) Take two 25 mL empty beakers and place them on a zero balance. Take 0.2499 g of calcium chloride dihydrate in each beaker and weigh it on a ten-thousandth balance. Add water for injection until all the calcium chloride is fully dissolved. Slowly and carefully add the fully dissolved calcium chloride to mixed solutions G1 and G2, respectively. Rinse any residual calcium chloride in the beaker with water for injection at least three times. Add mixed solutions G1 and G2, and stir until clear and free of solid particles to form mixed solutions H1 and H2.

[0060] 8) Weigh 0.0297 g of sodium pyruvate twice on a 10,000-degree balance and slowly and carefully add each of the mixed solutions H1 and H2. Stir until clear and free of solid particles, to form mixed solutions I1 and I2.

[0061] 9) Weigh 0.0100 g of gentamicin sulfate twice on a 10,000-degree balance and slowly and carefully add to mixed solutions I1 and I2, respectively. Stir until clear and free of solid particles, to form mixed solutions J1 and J2.

[0062] 10) Place two 25 mL empty beakers on a zero balance. Pipette 4.9210 g of sodium lactate solution into each beaker and weigh accurately on a ten-thousandth scale. Slowly and carefully add the mixtures J1 and J2 to each beaker. Rinse any remaining sodium lactate solution in the beakers at least three times with water for injection. Add the mixtures J1 and J2 and stir until clear to form mixtures K1 and K2.

[0063] 11) Add water for injection to 800 mL, take 0.3663 g of sodium bicarbonate twice, weigh it on a balance (0.3626-0.3700 g), and slowly and carefully add the mixtures K1 and K2, respectively. Stir until clear and free of solid particles, to form mixtures L1 and L2.

[0064] 12) Take two 100 mL empty beakers and place them on a zeroed balance. Weigh 26.1675 g of human albumin into each beaker and weigh accurately. Slowly and carefully add the mixed solutions L1 and L2 to each beaker. Rinse any residual human albumin in the beaker three times with water for injection. Add the mixed solutions L1 and L2 and stir until clear to form mixed solutions M1 and M2.

[0065] 13) Weigh 2 progesterone solutions (0.00079 g) on ​​a 10,000 ppm balance and slowly and carefully add to mixed solutions O1 and O2, respectively. Stir until clear and free of solid particles, to form mixed solutions N1 and N2.

[0066] 14) Add water for injection to 900 mL of mixed solutions N1 and N2 to form mixed solutions O1 and O2. Perform pH and osmotic pressure tests.

[0067] 15) pH meter calibration: Perform a two-point calibration at room temperature using 4.01 and 9.18 standard buffer solutions. The slope should be 95%-105%. Repeat the calibration with a 6.86 standard solution. The test value should be 6.86±0.05.

[0068] 16) Add 1 M sodium hydroxide solution to the mixed solutions O1 and O2 to adjust the pH and record the consumption of 1 M sodium hydroxide solution. After stirring evenly, measure the pH using a pH meter. The measured value is 7.55 (25° C.), forming mixed solutions P1 and P2.

[0069] 17) Slowly pour the mixed solutions P1 and P2 in the 1000mL beaker into a 1000mL volumetric flask, rinse the beaker 2-3 times with water for injection, pour the mixture into the 1000mL volumetric flask, and dilute to 1000mL to form mixed solutions Q1 and Q2.

[0070] 18) Osmometer Calibration: Perform a two-point calibration using purified water and a 300mOsm / kg sodium chloride standard solution. Repeated measurements of the 300mOsm / kg sodium chloride standard solution should yield values ​​between 298 and 302mOsm / kg. The values ​​of the mixed solutions Q1 and Q2 should be 300mOsm / kg.

[0071] After the osmotic pressure test is passed, it enters sterile filtration and filling.

[0072] 2. Effect Example Test 1. Clinical sample information The semen sample meets the sperm donor standard requirements. The criteria for selecting semen samples are: PM sperm percentage ≥ 35%, normal sperm morphology rate ≥ 4%, sperm concentration ≥ 60×10 6 Sperm / mL, round cell density <1×10 6 The test was performed using at least three qualified semen samples from different sperm donors.

[0073] 2. Density gradient centrifugation experiment of test sample Experimental materials: Examples 1-3 and Comparative Examples 1-3.

[0074] Experimental Method: Three qualified semen samples from different donors were used for the test. 1.5 mL of Gradient Solution A was added to the bottom of a sterile 15 mL sperm centrifuge tube. Then, using a sterile Pasteur pipette, 1.5 mL of Gradient Solution B was slowly added along the tube wall onto Gradient Solution A to form a density gradient.

[0075] Slowly add 0.5 mL of the corresponding liquefied semen to the top layer of each prepared gradient, ensuring a clear interface between the semen and the upper gradient layer. Centrifuge at 350g for 20 minutes at room temperature. If the pellet is no longer visible, centrifuge for an additional 3 minutes to ensure that the pellet is visible. (If the pellet is not visible, the test is invalid.)

[0076] Remove the supernatant and leave the sperm sediment at the bottom of the centrifuge tube. Use a Pasteur pipette to draw 5 mL of sperm washing solution and add it to the upper test tube. Mix thoroughly and centrifuge again at 300 g for 10 minutes. Discard the supernatant and allow the sperm pellet to settle at the bottom of the centrifuge tube. Add 1 mL of sperm incubation solution and measure the forward sperm motility (PM%), post-treatment sperm motility (PM + NM%), motile sperm recovery rate, and sperm motility enhancement rate using a computerized CASA system. The specific method is as follows: ① Sperm count: Using a counting plate and under a microscope, count the fast progressively moving and slow progressively moving (collectively recorded as PM), non-progressively moving (NM) and immotile (IM) sperm in the semen separately, counting at least 200 sperm in total and repeating the count once.

[0077] The above counting was performed before and after treatment, where the "before" refers to the semen sample before the experiment and the "after" refers to the semen sample after the density gradient centrifugation experiment.

[0078] ②Processing counting results: Post-treatment forward sperm motility = ×100%.

[0079] Sperm motility after treatment = ×100%.

[0080] Motile sperm recovery rate = (total number of motile sperm after treatment / total number of motile sperm before treatment) × 100%.

[0081] Sperm motility improvement rate = forward sperm motility after treatment - forward sperm motility before treatment.

[0082] Total motile sperm count = PM + NM.

[0083] The calculation results are shown in Table 3.

[0084] Table 3

[0085] Comparative Example 1 changed the ratio of sodium chloride, potassium chloride, magnesium sulfate and calcium chloride. After treatment with Samples 1 to 3, the forward sperm motility, sperm motility, motile sperm recovery rate and sperm motility improvement rate after treatment all decreased.

[0086] In Comparative Example 2, potassium dihydrogen phosphate was not added, and its sperm motility after forward treatment, sperm motility after treatment, motile sperm recovery rate and sperm motility improvement rate all decreased.

[0087] Comparative Example 3 changed the mass ratio of human blood protein and progesterone, and the forward sperm motility, sperm motility after treatment, motile sperm recovery rate and sperm motility improvement rate all decreased.

[0088] Comparative Example 4 is an existing product, and its sperm motility after forward treatment, sperm motility after treatment, motile sperm recovery rate and sperm motility improvement rate are all inferior to those of the present invention.

[0089] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A sperm gradient separation solution, characterized in that: Includes gradient solution A and gradient solution B; The gradient solution A comprises sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium pyruvate, glucose, gentamicin, sodium lactate solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium bicarbonate, human serum albumin, progesterone and Percoll PLUS, wherein the mass concentration of Percoll PLUS is 750-850 g / L; The gradient solution B comprises sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium pyruvate, glucose, gentamicin, sodium lactate solution, 4-hydroxyethylpiperazineethanesulfonic acid, sodium bicarbonate, human serum albumin, progesterone and Percoll PLUS, wherein the mass concentration of Percoll PLUS is 350-450 g / L; The mass ratios of sodium chloride, potassium chloride, magnesium sulfate and calcium chloride in gradient solution A and gradient solution B are (5-5.5): (0.3-0.38): (0.25-0.30): (0.24-0.28); The mass ratio of human serum albumin to progesterone in gradient solution A and gradient solution B is (3-7): (0.00075-0.00085).

2. The sperm gradient separation solution according to claim 1, characterized in that The solvent is water; and / or The pH is 7.45-7.

55.

3. The sperm gradient separation solution according to claim 1, characterized in that The mass ratio of the sodium chloride, potassium chloride, magnesium sulfate and calcium chloride is (5.3-5.5): (0.35-0.38): (0.29-0.30): (0.24-0.25).

4. The sperm gradient separation solution according to any one of claims 1 to 3, characterized in that The gradient solution A comprises, by mass concentration, 5-5.5 g / L of sodium chloride, 0.3-0.38 g / L of potassium chloride, 0.25-0.30 g / L of magnesium sulfate, 0.15-0.18 g / L of potassium dihydrogen phosphate, 0.24-0.28 g / L of calcium chloride, 0.028-0.04 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.02 g / L of gentamicin, 4.8-5.0 g / L of sodium lactate solution, 4.5-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.5 g / L of sodium bicarbonate, 3-7 g / L of human serum albumin, 0.00075-0.00085 g / L of progesterone, and 750-850 g / L of Percoll PLUS.

5. The sperm gradient separation solution according to claim 4, characterized in that The gradient solution A comprises, by mass concentration, 5.3-5.5 g / L of sodium chloride, 0.35-0.38 g / L of potassium chloride, 0.29-0.30 g / L of magnesium sulfate, 0.16-0.17 g / L of potassium dihydrogen phosphate, 0.24-0.25 g / L of calcium chloride, 0.028-0.03 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.015 g / L of gentamicin sulfate, 4.9-5.0 g / L of sodium lactate solution, 4.7-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.4 g / L of sodium bicarbonate, 4-6 g / L of human serum albumin, 0.00079-0.00085 g / L of progesterone, and 800-815 g / L of Percoll PLUS.

6. The sperm gradient separation solution according to any one of claims 1 to 3, characterized in that: The gradient solution B comprises, by mass concentration, 5-5.5 g / L of sodium chloride, 0.3-0.38 g / L of potassium chloride, 0.25-0.30 g / L of magnesium sulfate, 0.15-0.18 g / L of potassium dihydrogen phosphate, 0.24-0.28 g / L of calcium chloride, 0.028-0.04 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.02 g / L of gentamicin, 4.8-5.0 g / L of sodium lactate solution, 4.5-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.5 g / L of sodium bicarbonate, 3-7 g / L of human serum albumin, 0.00075-0.00085 g / L of progesterone, and 350-450 g / L of Percoll PLUS.

7. The sperm gradient separation solution according to claim 6, characterized in that The gradient solution B comprises, by mass concentration, 5.3-5.5 g / L of sodium chloride, 0.35-0.38 g / L of potassium chloride, 0.29-0.30 g / L of magnesium sulfate, 0.16-0.17 g / L of potassium dihydrogen phosphate, 0.24-0.25 g / L of calcium chloride, 0.028-0.03 g / L of sodium pyruvate, 0.90-1.00 g / L of glucose, 0.01-0.015 g / L of gentamicin sulfate, 4.9-5.0 g / L of sodium lactate solution, 4.7-4.8 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, 0.3-0.4 g / L of sodium bicarbonate, 4-6 g / L of human serum albumin, 0.00079-0.00085 g / L of progesterone, and 400-410 g / L of Percoll PLUS.

8. The method for preparing the sperm gradient separation solution according to any one of claims 1 to 7, characterized in that: The following steps are involved: Gradient solutions A and B were prepared by dissolving Percoll PLUS, sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, glucose, 4-hydroxyethylpiperazineethanesulfonic acid, calcium chloride, sodium pyruvate, gentamicin, sodium lactate, sodium bicarbonate, human serum albumin, and progesterone in 1000 mL of water according to their mass concentrations and adjusting the pH.

9. Use of the sperm gradient separation solution according to any one of claims 1 to 7 and / or the sperm gradient separation solution prepared by the preparation method according to claim 8 in the preparation of assisted reproductive products.

10. An assisted reproductive product, characterized in that: It comprises the sperm gradient separation fluid described in any one of claims 1 to 7 and / or the sperm gradient separation fluid prepared by the preparation method described in claim 8; the assisted reproductive product is an assisted reproductive kit.

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