Physiological sperm braking fluid as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides physiological sperm braking fluid as well as a preparation method and application thereof. The physiological sperm brake fluid comprises a basic component and a core component, the basic component comprises an inorganic salt, an energy component and protein; the core component comprises a speed limiting agent, a buffering agent and nutrient substances. The preparation method comprises the following steps: (1) dissolving the core component in water and stirring to obtain a first mixed solution; (2) dissolving the basic component in water and stirring to obtain a second mixed solution; and (3) mixing the first mixed solution and the second mixed solution, fixing the volume and filtering to obtain the physiological sperm brake fluid. According to the physiological sperm braking fluid, the problems that in the sperm braking process, exogenous substances and amphoteric organic buffering agents damage sperms and oocytes and nutrient substances support insufficient sperm-oocyte combination are solved, the components are safer, the abnormal fertilization rate can be remarkably reduced, the blastocyst rate is increased, and the utilization rate of available embryos is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a physiological sperm brake fluid and a preparation method and application thereof. Background Art

[0002] Studies have found that compared with conventional in vitro fertilization (IVF) technology, intracytoplasmic sperm injection (ICSI) technology cannot improve the live birth rate after the first transplant, the number of available embryos obtained is small, the transplantation rate is low, and the cumulative live birth rate observed subsequently is also relatively low. However, the gap between ICSI technology and IVF technology is affected by many factors, such as oocyte status, sperm motility and morphology, and the proficiency of operators. Among them, the sperm braking link is also a factor restricting ICSI technology. The sperm braking link may involve three aspects: rate limiting agent, buffer system, and nutrients. The following is a detailed introduction to the research status of these three aspects.

[0003] (1) Rate-limiting agent

[0004] Commercial sperm brake fluid or similar products add polyvinyl pyrrolidone (PVP) at a concentration of 7% and 10% as a rate-limiting agent to reduce the speed of sperm movement, thereby capturing sperm and injecting it into the oocyte. During the injection process, a small amount of PVP will be injected into the oocyte with the sperm. Since PVP cannot diffuse out and cannot be digested by lysosomal enzymes, it will stay in the oocyte for a long time. Even low concentrations of PVP will damage the sperm plasma membrane, acrosome and nucleus, delay the occurrence of calcium ion oscillation in the egg and sperm nuclear deagglutination, and thus affect fertilization and embryonic development (Chinese expert consensus on intracytoplasmic sperm injection (ICSI) technology). Therefore, it is necessary to first find a physiological alternative to PVP to reduce sperm motility in order to promote ICSI sperm capture. Ideally, the product should have the following characteristics: ① It is viscous, slows down sperm motility, and is sufficient to be sucked into the ICSI pipette; ② It can prevent sperm from sticking to the pipette; ③ This substance is a substance that exists in the human body itself and is not an exogenous substance.

[0005] However, some scholars have proposed that low-concentration or PVP-free solutions can be used for the operation. For example, Y Barak published an article titled "A physiological replacement for polyvinylpyrrolidone (PVP) in assisted reproductive technology" in Human Fertility, which found that hyaluronate (HA) solution is as effective as PVP in promoting the injection process, its effect on sperm motility is easily reversed, and its use does not affect the results of the treatment cycle in terms of fertilization, pregnancy and live birth rate. Because HA is a high-molecular-weight glycosaminoglycan, it is the main component of the matrix surrounding human oocytes. During physiological fertilization, sperm binds to the extracellular matrix of the cumulus through its HA receptor. Only mature sperm can bind and digest HA, pass through the cumulus and finally reach and fertilize the oocyte. Therefore, HA plays an important role in the sperm selection process. In vitro studies have confirmed that sperm that can bind to HA have completed their plasma membrane remodeling, cytoplasmic extrusion and nuclear maturation, and show a lower risk of chromosomal imbalance. The original ICSI process only evaluates sperm in terms of morphology and vitality, while HA, as a rate-limiting agent, can further evaluate the maturity and provide a guarantee for the next step of sperm-egg combination. At the same time, products containing HA can sufficiently slow down the movement of sperm, so that sperm can be captured by the injection pipette, easily sucked in and discharged from the pipette, prevent sperm from adhering to plastic or glassware, and do not affect the development of the fertilized egg after injection.

[0006] (2) Buffer system

[0007] The microdroplets used for ICSI injection are usually composed of a buffer system of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and 3-morpholinepropanesulfonic acid (MOPS), which provides pH stability under atmospheric conditions and allows efficient acquisition and release of free H in solution. +ions, making it easier for operators to operate for a long time. However, these zwitterionic buffers may not be the best choice for ICSI surgery. HEPES and MOPS have significant effects on cell function outside of pH control. For example, Robert J. Mendola published an article in the Journal of Assisted Reproduction and Genetics in 2024 titled "Infux of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes." The study showed that HEPES and MOPS can inhibit protein transporters and exchangers, inhibit Cl - Anion membrane channel; inhibits electron transport pathways in mitochondria; inhibits connexin channels and gap junctions; interacts with metal ions that are essential for embryonic development, such as Ca 2+ and Fe 3+ ; Interact with DNA to form complexes; Generate oxygen free radicals. MOPS and HEPES interact with lipids, increase membrane thickness, and reduce membrane potential and permeability. At the same time, injection of zwitterionic proteins after ICSI membrane puncture can change the transcriptome of human oocytes, which is beyond the control of the operator and cannot be evaluated for potential hazards. Therefore, a physiological buffer pair is needed to replace HEPES / MOPs;

[0008] Bicarbonate is a physiological buffer that maintains pH in all mammals. To combat acidosis, cells use a sodium-dependent bicarbonate / Cl exchanger (HCE) and Na + / H + Antiporters, which add and remove bicarbonate and hydrogen ions, respectively, from cells. To combat alkalosis, cells may contain bicarbonate / Cl exchangers. Zwitterionic buffers HEPES and MOPs are used for pH maintenance under atmospheric conditions and do not provide any nutritional benefit. There is literature suggesting that the presence of HEPES supports embryonic development when bicarbonate is present, but not when bicarbonate is absent.

[0009] (3) Nutrients: The ingredients of commercial sperm brake fluid-related products are simple, consisting of inorganic salts, buffer systems, rate-limiting agents, human albumin, and antibiotics. No additional nutrients are provided to support better combination of sperm and oocytes at this stage and increase subsequent blastocyst formation. Therefore, it is necessary to find a physiological nutrient to support the subsequent development of the embryo.

[0010] Leptin is encoded by the obese (ob) gene and is a 16kDa adipokine protein that is mainly secreted from adipose tissue. It plays a vital role in regulating food intake and energy expenditure. Leptin is present in human and mouse oocytes, human follicular fluid, and human granulosa cells and cumulus cells. The human endometrium and placenta are the sites where leptin is produced locally and are also the target tissues of circulating leptin, which may be related to the human implantation process and maternal-fetal interaction. For example, Craig et al. published an article titled "Leptin enhancesporcine preimplantation embryo development in vitro" in Molecular and Cellular Endocrinology in 2005, which showed the effect of leptin on the development of pig preimplantation embryos. The study found that compared with embryos without leptin, the addition of leptin to the culture medium not only significantly increased embryonic divisions, but also significantly increased blastocyst development after in vitro fertilization. For example, patent CN 117281108A discloses the use of leptin in cryopreservation of goat semen, goat semen freezing diluent and its use, and a method for cryopreservation of goat semen. The invention can improve the vitality, plasma membrane integrity, acrosome integrity and mitochondrial membrane potential of goat semen after freezing and thawing, reduce reactive oxygen species (ROS) during cryopreservation, and reduce oxidative stress damage; in addition, frozen semen with leptin added for in vitro fertilization can improve the fertilization effect and significantly improve the blastocyst development rate.

[0011] However, there is no specific research on the content, environment, preparation method and joint application of HA, bicarbonate and leptin in sperm brake fluid.

[0012] In summary, it is urgent to solve the problem of damage to sperm and oocytes caused by exogenous substances PVP and amphoteric organic buffers during sperm braking and insufficient nutrients to support sperm-egg binding, and to find the preparation environment, required dosage, and preparation method of sperm brake fluid. The present invention needs to provide a physiological sperm brake fluid and its preparation method and application that can solve this problem. Summary of the invention

[0013] Based on the existing deficiencies, in order to solve the damage of exogenous substances PVP and amphoteric organic buffers to sperm and oocytes during sperm braking, the problem of insufficient nutrients supporting sperm-egg binding, and to find the preparation environment, required dosage, and preparation method of sperm brake fluid, the present invention aims to provide a physiological sperm brake fluid, a preparation method, and application thereof.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] In the first aspect, the present invention provides a physiological sperm brake fluid, which comprises a basic component, a core component and a solvent. The core component comprises a rate-limiting agent, a buffer and nutrients; the basic component comprises inorganic salts, energy components and proteins; and the solvent is purified water, water for injection or sterile water for injection in accordance with the Chinese Pharmacopoeia.

[0016] Preferably, the rate-limiting agent in the core component is HA and its salts or derivatives, more preferably sodium hyaluronate. Preferably, the final concentration of sodium hyaluronate is 1g / L-10g / L; more preferably, the final concentration is 2g / L-5g / L; further preferably, the final concentration of sodium hyaluronate is 2.5g / L.

[0017] Preferably, the buffer in the core component is bicarbonate, preferably sodium bicarbonate. Preferably, the final concentration of sodium bicarbonate is 20-30 mM; more preferably, the final concentration is 22.5-27.5 mM; further preferably, the final concentration is 25 mM.

[0018] Preferably, the nutrient in the core component is leptin. Preferably, the final concentration of leptin is 10-500 μg / L; more preferably, the final concentration is 50-200 μg / L; more preferably, the final concentration is 100 μg / L;

[0019] Preferably, the inorganic salt in the base component includes at least one of sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and derivatives thereof. Preferably, the inorganic salt in the base component includes sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate and calcium chloride. Further preferred final concentrations of the components included are: sodium chloride 81.28-121.92 mM, potassium chloride 3.75-5.63 mM, magnesium sulfate 0.16-0.24 mM, potassium dihydrogen phosphate 0.29-0.45 mM and calcium chloride 1.63-2.45 mM.

[0020] Preferably, the energy component in the basic component is selected from at least one of glucose, sodium lactate and sodium pyruvate. Preferably, sodium lactate, glucose and sodium pyruvate are selected. More preferably, the final concentration of the components is 17.12-25.68 mM sodium lactate, 2.22-3.34 mM glucose and 0.26-0.4 mM sodium pyruvate.

[0021] Preferably, the protein in the basic component is selected from at least one of human serum albumin, recombinant human serum albumin and globulin, more preferably human serum albumin, and further preferably its final concentration is 2.5-7.5 g / L.

[0022] Further preferably, the physiological sperm brake fluid comprises the following components at final concentrations:

[0023] Core components: sodium hyaluronate 2.5g / L, sodium bicarbonate 25mM and leptin 100μg / L.

[0024] Basic components: sodium chloride 101.6mM, potassium chloride 4.69mM, magnesium sulfate 0.2mM, potassium dihydrogen phosphate 0.37mM, calcium chloride 2.04mM, sodium lactate 21.4mM, glucose 2.78mM, sodium pyruvate 0.33mM, human serum albumin 5g / L;

[0025] Preferably, the solvent is water for injection.

[0026] Preferably, the pH of the physiological sperm brake fluid is 7.2-8.4 in an atmospheric environment at 20-30° C. More preferably, the pH of the physiological sperm brake fluid is 7.2-7.5 in an atmospheric environment at 20-30° C.

[0027] Preferably, the pH of the physiological sperm brake fluid is 7.2-7.6 after being balanced in a CO2 incubator or a three-gas incubator containing 5-7% CO2. Further preferably, the pH of the physiological sperm brake fluid is 7.2-7.4 after being balanced in a CO2 incubator or a three-gas incubator containing 5-7% CO2.

[0028] Preferably, the osmotic pressure of the physiological sperm brake fluid is 280-360 mOsm / kg. Further preferably, the osmotic pressure of the physiological sperm brake fluid is 300-330 mOsm / kg.

[0029] Preferably, the viscosity of the physiological sperm brake fluid is 50-500 cP at 20-30° C. More preferably, the viscosity of the physiological sperm brake fluid is 100-400 cP at 20-30° C.

[0030] In a second aspect, the present invention provides a method for preparing the above-mentioned physiological sperm brake fluid, the preparation comprising the following steps:

[0031] (1) Weighing a rate-limiting agent, a buffer, and a nutrient respectively, adding them to a solvent, and stirring until they are completely dissolved, to obtain a rate-limiting agent solution, a buffer solution, and a nutrient solution respectively; and mixing the three solutions to obtain a first mixed solution;

[0032] (2) mixing the inorganic salt, energy component and protein, adding the solvent and stirring until they are completely dissolved to obtain a second mixed solution;

[0033] (3) The first mixed solution and the second mixed solution are mixed, fixed to volume, filtered, and sterilized to obtain the physiological sperm brake fluid.

[0034] Preferably, when sodium hyaluronate is weighed and dissolved in water for injection in the above step (1), the temperature of the water for injection is 50-60°C, more preferably 60°C.

[0035] Preferably, the stirring speed of the stirrer in the above step (1) is 300-600 rpm, more preferably 500 rpm.

[0036] Preferably, in the above step (1), when sodium bicarbonate is dissolved in water for injection, the stirring and heating temperature is 50°C.

[0037] Preferably, the sterilization in step (3) is performed by filtering and sterilizing with a filter membrane under Class 100 conditions and a CO2 protective gas laminar flow of 0.8-1.2 bar. More preferably, the pressure of the CO2 protective gas layer is 1 bar.

[0038] Preferably, the pore size of the filter membrane during filtration in the above step (3) is 0.2-0.3 μM, and more preferably 0.22 μM.

[0039] In a third aspect, the present invention provides the use of the above-mentioned physiological sperm brake fluid in the ICSI process.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The core components of the physiological sperm brake fluid provided by the present invention, including sodium hyaluronate, sodium bicarbonate and leptin, are all substances existing in the body, i.e., "physiological" materials, which are more natural and safer. There is no non-"physiological" substance added to conventional sperm brake or similar products, such as PVP, HEPES and MOPS.

[0042] 2. The physiological sperm brake fluid provided by the present invention can identify mature sperm, reduce the DNA fragmentation rate of sperm, significantly reduce the abnormal fertilization rate, and obtain embryos with better quality based on the hyaluronic acid receptors carried by mature sperm.

[0043] 3. The physiological sperm brake fluid provided by the present invention uses sodium bicarbonate as a buffer system, which is a substance required in the cell development process and reduces the risk of changes in the oocyte transcriptome.

[0044] 4. The leptin added to the physiological sperm brake fluid provided by the present invention can improve the blastocyst rate and increase the utilization rate of available embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The blastocyst is obtained by using the physiological sperm brake fluid of implementation 1;

[0046] Figure 2 To stain and count the blastocysts obtained using the physiological sperm brake fluid of Implementation 1. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments. The embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention is described in detail with reference to the following embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the following embodiments can still be modified, or some of the technical features can be replaced by equivalents; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0048] In the following examples, unless otherwise specified, all raw materials used are commercially available or prepared by conventional methods in the art, wherein sodium hyaluronate is injection-grade sodium hyaluronate, which is sourced from Bloomage Biotechnology Co., Ltd.; and leptin is recombinant human leptin (HY-P7232), which is sourced from MCE.

[0049] Examples 1-3 and Comparative Examples 1-8 provide a physiological sperm brake fluid, the specific components and final concentrations of which are shown in Table 1 below.

[0050] The preparation method is:

[0051] (1) Accurately weigh sodium hyaluronate, dissolve it in 400 mL of water for injection at 60° C., stir it with a stirrer at a speed of 500 rpm, stir it evenly, and obtain a clear and transparent sodium hyaluronate solution; weigh sodium bicarbonate, dissolve it in 100 mL of water for injection, heat it to about 50° C. and stir it evenly to obtain a sodium bicarbonate solution; accurately weigh leptin, dissolve it in water for injection, prepare a concentrated stock solution (100×), draw 1× leptin concentrated stock solution, add it to water for injection, stir it evenly, and obtain a leptin solution; mix the sodium hyaluronate solution, sodium bicarbonate solution, and leptin solution to obtain a first mixed solution;

[0052] (2) Weighing sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium lactate, glucose, sodium pyruvate, and human albumin, and adding them to 200 mL of water for injection to obtain a second mixed solution;

[0053] (3) The first mixed solution and the second mixed solution are mixed, and the volume is adjusted to 1 L with injection water. Finally, the mixture is filtered and sterilized with a 0.22 μM filter membrane under Class 100 conditions and a 1 bar CO2 protective gas laminar flow to obtain the physiological sperm brake fluid.

[0054] Table 1

[0055]

[0056]

[0057] Comparative Example 1 A physiological sperm brake fluid and its preparation method (1L)

[0058] The difference compared with Example 1 is that, except that the same mass of polyvinyl pyrrolidone (PVP) is used to replace sodium hyaluronate, the concentrations of other components and the preparation methods are the same.

[0059] Comparative Example 2 A physiological sperm brake fluid and its preparation method (1L)

[0060] The difference compared with Example 1 is that taurine of the same concentration is used to replace leptin as a nutrient, and the concentrations and preparation methods of other components are consistent.

[0061] Comparative Example 3 A physiological sperm brake fluid and its preparation method (1L)

[0062] The difference compared with Example 1 is that except that 21 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is used to replace 21 mM sodium bicarbonate, the concentrations of other components and the preparation methods are consistent.

[0063] Comparative Example 4 A physiological sperm brake fluid and its preparation method (1L)

[0064] The difference compared with Example 1 is that except that 21 mM 3-morpholinepropanesulfonic acid (Mops) is used to replace 21 mM sodium bicarbonate, the concentrations of other components and the preparation methods are the same.

[0065] Comparative Example 5 A physiological sperm brake fluid and its preparation method (1L)

[0066] The difference compared with Example 1 is that except for the concentrations of sodium bicarbonate and sodium hyaluronate being different from those in Example 1, the concentrations of other components and the preparation methods are the same.

[0067] Comparative Example 6 A physiological sperm brake fluid and its preparation method (1L)

[0068] Compared with Example 1, the difference is that except for the concentrations of sodium bicarbonate and sodium hyaluronate, the concentrations of other components and the preparation methods are the same.

[0069] Comparative Example 7 A physiological sperm brake fluid and its preparation method (1L)

[0070] Compared with Example 1, the difference is that except for the leptin concentration which is different from that in Example 1, the concentrations of other components and the preparation methods are the same.

[0071] Comparative Example 8 A physiological sperm brake fluid and its preparation method (1L)

[0072] Compared with Example 1, the difference is that except for the leptin concentration which is different from that in Example 1, the concentrations of other components and the preparation methods are the same.

[0073] Effect experiment:

[0074] Test case effect test

[0075] Evaluation content:

[0076] 1. pH of sperm brake fluid: According to the "0631 pH Value Determination Method" of the 2020 edition of the Chinese Pharmacopoeia, the pH of physiological sperm brake fluid should be tested, and the pH should be 7.2-8.4.

[0077] 2. Osmotic pressure of sperm brake fluid: The osmotic pressure of sperm brake fluid is tested according to the "0632 Osmotic pressure molar concentration determination method" of the Chinese Pharmacopoeia 2020 edition. The osmotic pressure should be 280-360mOsm / kg.

[0078] 3. Viscosity of sperm brake fluid: The viscosity of sperm brake fluid is tested according to the "0633 Viscosity Determination Method" of the 2020 edition of the Chinese Pharmacopoeia. The viscosity should be 50-500cP.

[0079] 4. Sterility of sperm brake fluid: The sperm brake fluid should be sterile according to the "1101 Sterility Test Method" of the 2020 edition of the Chinese Pharmacopoeia.

[0080] 5. Endotoxin in sperm brake fluid: Endotoxin test of sperm brake fluid is carried out according to the "1143 Bacterial Endotoxin Test Method" of the 2020 edition of the Chinese Pharmacopoeia, and the endotoxin is <0.15EU / mL.

[0081] 6. The formulations of Examples 1-3 and Comparative Examples 1-8 were subjected to ICSI tests, and the sperm braking effect was tested by the same test personnel who had been trained in the ICSI operating system:

[0082] Experimental methods:

[0083] Method of use of Examples 1-3 and Comparative Examples 1-2, 5-8: Add 1-2 μL of sperm suspension treated with gradient density separation solution and swim-up method to the physiological sperm brake fluid droplets balanced with 5-7% CO2, and culture for several minutes to allow the sperm to migrate to the outside of the droplets to achieve the purpose of separating dead and live sperm. The tails of mature sperms show a small swing in the droplets, and the tails of immature sperms curl and swing freely in the droplets. Use the ICSI injection needle to press the tails of mature sperms, break the membrane for braking, and aspirate the sperm and inject it into the oocyte. The oocyte with injected sperm is placed in the culture medium.

[0084] Comparative Examples 3 and 4: Add 1-2 μL of sperm suspension treated with gradient density separation solution and swim-up method to the sperm brake droplets incubated in an atmospheric environment at 37°C, and incubate for several minutes to allow the sperm to migrate to the outside of the droplets to achieve the purpose of separating dead and live sperm. Use the ICSI injection needle to press the tail of the mature sperm, break the membrane for braking, and aspirate the sperm and inject it into the oocyte. Place the oocyte injected with sperm in the culture medium. Observe and count the subsequent fertilization rate and blastocyst development rate.

[0085] Experimental results:

[0086] The above test results are shown in Table 2 and Table 3

[0087] Table 2

[0088]

[0089] Table 3

[0090]

[0091]

[0092] Result analysis:

[0093] It can be seen from Table 2 and Table 3 above that among the three embodiments, Embodiment 1 has the best effect, with a fertilization rate of 93.33%±2.08% and a blastocyst rate of 97.49%±0.68%, which are better than Embodiments 2-3, indicating that the component concentration ratio of Embodiment 1 is the best.

[0094] Compared with Example 1, Comparative Example 1 uses polyvinyl pyrrolidone (PVP) of the same mass to replace sodium hyaluronate, and the fertilization rate is reduced by 53.21%, and the blastocyst rate is reduced by 30.09%, indicating that the sodium hyaluronate component of Example 1 is superior to PVP currently used in the market.

[0095] Compared with Example 1, Comparative Example 2 used taurine instead of leptin as a nutrient, and the fertilization rate decreased by 57.12%, and the blastocyst rate decreased by 31.66%, indicating that the component leptin in Example 1 affects the fertilization rate and blastocyst rate, and has a significant effect on the development of available blastocysts.

[0096] Compared with Example 1, Comparative Example 3 used 21 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) to replace 21 mM sodium bicarbonate, and the fertilization rate decreased by 6.06%, and the blastocyst rate decreased by 21.69%, indicating that HEPES reduced the fertilization rate and blastocyst rate, and the sodium bicarbonate component of Example 1 was superior to HEPES.

[0097] Compared with Example 1, Comparative Example 4 used 21 mM 3-morpholinepropanesulfonic acid (MOPS) to replace 21 mM sodium bicarbonate, and the fertilization rate decreased by 21.07% and the blastocyst rate decreased by 16.91%, indicating that Mops reduced the fertilization rate and blastocyst rate, and the sodium bicarbonate component of Example 1 was better than MOPS.

[0098] Compared with Example 1, the sodium hyaluronate in Comparative Example 5 is lower than the concentration range limited by the present invention, and the sodium bicarbonate is higher than the concentration range limited by the present invention. The fertilization rate is reduced by 47.85%, and the blastocyst rate is reduced by 19.93%, indicating that the concentrations of the rate limiter and the buffer in the sperm brake fluid must be within the range limited by the present invention to improve the fertilization rate and the blastocyst rate.

[0099] Compared with Example 1, the sodium hyaluronate in Comparative Example 6 is higher than the concentration range limited by the present invention, and the sodium bicarbonate is lower than the concentration range limited by the present invention. The fertilization rate is reduced by 48.92%, and the blastocyst rate is reduced by 20.54%, indicating that the concentrations of the rate limiter and the buffer in the sperm brake fluid must be within the range limited by the present invention to improve the fertilization rate and the blastocyst rate.

[0100] Compared with Example 1, the leptin in Comparative Example 7 was higher than the concentration range limited by the present invention, the fertilization rate was reduced by 20.36%, and the blastocyst rate was reduced by 14.16%, indicating that the concentration of leptin in the sperm brake fluid must be within the range limited by the present invention to improve the fertilization rate and blastocyst rate.

[0101] Compared with Example 1, the leptin in Comparative Example 8 was lower than the concentration range limited by the present invention, the fertilization rate was reduced by 37.14%, and the blastocyst rate was reduced by 16.16%, indicating that the concentration of leptin in the sperm brake fluid must be within the range limited by the present invention to improve the fertilization rate and blastocyst rate.

[0102] The above shows that the physiological sperm brake fluid provided by the present invention is more natural and safer, can reduce the DNA fragmentation rate and abnormal fertilization rate of sperm, can reduce the risk of changes in the oocyte transcriptome, and can increase the blastocyst rate and increase the utilization rate of available embryos, thereby obtaining embryos of better quality.

Claims

1. A physiological sperm brake fluid, characterized in that: The physiological sperm brake fluid comprises a basic component, a core component and a solvent; the core component comprises a rate-limiting agent, a buffer and nutrients; the basic component comprises inorganic salts, energy components and proteins; the solvent is purified water or water for injection; the final concentration of the rate-limiting agent is 1g / L-10g / L, the final concentration of the buffer is 20-30mM, and the final concentration of the nutrients is 10-500μg / L.

2. The physiological sperm brake fluid according to claim 1, characterized in that: The rate-limiting agent is hyaluronic acid and its salt or derivative, and the buffer is bicarbonate.

3. The physiological sperm brake fluid according to claim 1, characterized in that: The rate-limiting agent is sodium hyaluronate, the buffer is sodium bicarbonate, and the nutrient is leptin.

4. The physiological sperm brake fluid according to claim 3, characterized in that: The final concentration of the sodium hyaluronate is 2g / L-5g / L, the final concentration of the sodium bicarbonate is 22.5-27.5mM, and the final concentration of the leptin is 50-200μg / L.

5. The physiological sperm brake fluid according to claim 3, characterized in that: The final concentration of the sodium hyaluronate is 2.5 g / L, the final concentration of the sodium bicarbonate is 25 mM, and the final concentration of the leptin is 100 μg / L.

6. The physiological sperm brake fluid according to claim 1, characterized in that: The inorganic salt is selected from at least one of sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate, and calcium chloride; the energy component is selected from at least one of glucose, sodium lactate, and sodium pyruvate; and the protein is selected from at least one of human albumin, recombinant human albumin, and globulin.

7. The physiological sperm brake fluid according to claim 1, characterized in that: The inorganic salt is a mixture of sodium chloride, potassium chloride, magnesium sulfate, potassium dihydrogen phosphate and calcium chloride; the energy component is a mixture of sodium lactate, glucose and sodium pyruvate; and the protein is human serum albumin.

8. The physiological sperm brake fluid according to claim 7, characterized in that: The final concentration of sodium chloride is 81.28-121.92mM; the final concentration of potassium chloride is 3.75-5.63mM; the final concentration of magnesium sulfate is 0.16-0.24mM; the final concentration of potassium dihydrogen phosphate is 0.29-0.45mM; the final concentration of calcium chloride is 1.63-2.45mM; the final concentration of sodium lactate is 17.12-25.68mM; the final concentration of glucose is 2.22-3.34mM; the final concentration of sodium pyruvate is 0.26-0.4mM; and the final concentration of human albumin is 2.5-7.5g / L.

9. The physiological sperm brake fluid according to claim 1, characterized in that: The pH of the physiological sperm brake fluid is 7.2-8.4 under an atmospheric environment of 20-30°C; after being balanced in a CO2 incubator or a three-gas incubator containing 5-7% CO2, the pH is 7.2-7.6; the osmotic pressure of the physiological sperm brake fluid is 280-360mOsm / kg; the viscosity of the physiological sperm brake fluid is 50-500cP at 20-30°C; the endotoxin of the physiological sperm brake fluid is <0.15EU / mL.

10. The physiological sperm brake fluid according to claim 1, characterized in that: The pH of the physiological sperm brake fluid is 7.2-7.5 under an atmospheric environment of 20-30°C; after being balanced in a CO2 incubator or a three-gas incubator containing 5-7% CO2, the pH is 7.2-7.4; the osmotic pressure of the physiological sperm brake fluid is 300-330mOsm / kg; and the viscosity of the physiological sperm brake fluid is 100-400cP at 20-30°C.

11. The method for preparing the physiological sperm brake fluid according to any one of claims 1 to 10, characterized in that: The steps include: (1) Weighing a rate-limiting agent, a buffer, and a nutrient respectively, adding them to a solvent, and stirring until they are completely dissolved, to obtain a rate-limiting agent solution, a buffer solution, and a nutrient solution respectively; The three solutions are mixed to obtain a first mixed solution; (2) mixing the inorganic salt, energy component and protein, adding the solvent and stirring until they are completely dissolved to obtain a second mixed solution; (3) The first mixed solution and the second mixed solution are mixed, fixed to volume, filtered, and sterilized to obtain the physiological sperm brake fluid.

12. The preparation method according to claim 11, characterized in that: When the rate-limiting agent is added to the solvent, the temperature of the solvent is 50-60° C., and the stirring speed is 300-600 rpm.

13. The preparation method according to claim 11, characterized in that: After the buffer is added into the solvent, it is necessary to stir under heating conditions, and the heating temperature is 45-55°C.

14. The preparation method according to claim 11, characterized in that: The sterilization described in step (3) is as follows: sterilization is performed by filtering with a 0.22 μM filter membrane under Class 100 conditions and a CO2 protective gas laminar flow of 0.8-1.2 bar.

15. Use of the physiological sperm brake fluid according to any one of claims 1 to 10 in intracytoplasmic sperm injection or assisted reproduction.

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