A sperm motility enhancing liquid and a method for enhancing sperm motility

Abstract

The present invention provides a sperm motility enhancing solution and a method for enhancing sperm motility. A sperm motility enhancing solution, the sperm motility enhancing solution comprising a stimulant, recombinant human insulin and a balancing solution, wherein the stimulant comprises 2-deoxyadenosine, pentoxifylline or caffeine. The balancing solution is selected from one or more inorganic salt solutions; the inorganic salt is selected from NaCl, KCl, CaCl2·2H2O, KH2PO4 and MgSO4·7H2O. The present invention selects stimulants 2-deoxyadenosine, pentoxifylline and caffeine respectively for improving sperm motility, by increasing the cAMP level in cryopreserved sperm cells, under the synergistic effect of recombinant human insulin, further promoting the cell substance metabolism level, and effectively promoting the sperm motility of normal sperm and asthenozoospermia patients, which has a promoting effect on improving the success rate of in vitro fertilization.

Description

Technical Field

[0001] The present invention belongs to the technical field of assisted reproduction, and in particular relates to an enhancing liquid for improving sperm motility and a method for improving sperm motility. Background Art

[0002] At present, due to the intensification of social competition, men are facing increasing work pressure, coupled with the intensification of environmental pollution, more chemicals that affect sperm production and maturation are absorbed by men through various channels, resulting in a decrease in the number of sperm produced by men and a decrease in sperm activity; according to statistics, male infertility accounts for 35-50% of all infertility, and oligoasthenozoospermia is an important factor leading to male infertility. Oligozoospermia refers to sperm density in semen less than 20*10 / ml; asthenozoospermia refers to sperm a+b less than 50% or a sperm less than 25% in semen.

[0003] Asthenozoospermia patients can freeze their own sperm in a medical institution with relevant qualifications to prevent fertility risks. Sperm freezing technology is relatively mature, and sperm freezing can be used as an assisted reproductive method for infertile people. However, freezing sperm will reduce the vitality of human sperm.

[0004] US Patent US09861656B2 uses a mixture of inulin and phospholipid compounds to improve sperm motility, and uses phospholipids to modify the lipid composition of sperm membranes to repair cell membrane damage caused by peroxidation. However, this method does not solve the problem of low sperm metabolism after freezing, and the metabolic stimulation of sperm is insufficient. Moreover, only the effect of modifiers on sperm motility of normal sperm was studied, and the effect of modifiers on sperm motility of asthenozoospermia was not explored. As asthenozoospermia makes fertilization more difficult, it is increasingly important to explore the effect of modifiers on sperm motility of asthenozoospermia after freezing.

[0005] Based on this, the present invention provides a technical means to solve the deficiencies of the above technical solutions. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a sperm motility enhancing liquid and a method for enhancing sperm motility, so as to solve the problem that the prior art cannot effectively promote the sperm motility of patients with asthenozoospermia, and has a positive effect on improving the success rate of in vitro fertilization.

[0007] One embodiment of the present invention provides a sperm motility enhancing solution.

[0008] A sperm motility enhancing solution, comprising a stimulant, recombinant human insulin and a balancing solution.

[0009] Wherein, the stimulant includes one or more of 2-deoxyadenosine, pentoxifylline or caffeine.

[0010] In one embodiment, the balancing solution is selected from one or more inorganic salt solutions;

[0011] The inorganic salt is selected from NaCl, KCl, CaCl 2 ·2H 2 O.KH 2 PO 4 and MgSO 4 7H 2 One or more of O.

[0012] In one embodiment, the sperm motility enhancing solution comprises the following components in mass fractions:

[0013]

[0014] In one embodiment, the pH of the sperm motility enhancing solution is 7.2-7.6.

[0015] One embodiment of the present invention further provides a method for improving sperm motility by using a sperm motility enhancing solution, the method comprising the following steps:

[0016] S1: adding sperm, stimulant, recombinant human insulin and balancing solution to the culture medium, culturing to obtain culture medium;

[0017] S2: The culture solution and cryoprotectant solution are mixed evenly, placed in a cryotube and allowed to stand, then transferred to liquid nitrogen and placed for 6-8 days, then placed in a water bath until completely thawed and taken out.

[0018] In one embodiment, the components in the cryoprotectant solution include one or more of serum replacement, HTF-HEPES culture medium, and glycerol mixture.

[0019] In one embodiment, in step S1, the sperm is screened into high-motility sperm and low-motility sperm, and the screening method is to use double density gradient centrifugation for screening.

[0020] The term “highly motile sperm” was defined as a percentage of total motile sperm ≥40% and a percentage of progressively motile sperm ≥32%.

[0021] The term “hypomotile sperm” was defined as a percentage of total motile sperm <40% and a percentage of progressively motile sperm <32%.

[0022] In step S2, the standing temperature is 2-6°C, and the standing time is 28-32 minutes.

[0023] In one embodiment, in step S2, the temperature of the freezing tube is maintained at 23-27°C.

[0024] In one embodiment, in step S2, the temperature of the water bath is 35-39°C.

[0025] In one embodiment, in step S2, the volume ratio of the culture solution to the cryoprotectant solution is 1:1-1:3.

[0026] The sperm motility enhancing solution and preparation method thereof provided by the present invention have the following beneficial effects:

[0027] 1. The present invention selects stimulants 2-deoxyadenosine, pentoxifylline and caffeine respectively for improving sperm motility, and further promotes the cell substance metabolism level under the synergistic effect of recombinant human insulin by increasing the cAMP level in the cryopreserved sperm cells, and effectively promotes the sperm motility of normal spermatozoospermia patients and asthenozoospermia patients, which has a promoting effect on improving the success rate of in vitro fertilization.

[0028] 2. The present invention further promotes sperm cell metabolism by adding a recombinant human insulin promoter after adding a stimulant, thereby increasing the fertilization success rate. At the same time, the pH of the culture medium is adjusted to 7.2-7.6 by adding a balanced salt solution to provide a living environment suitable for sperm, thereby helping to improve sperm motility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A comparison of the progressive motility of high-motility and low-motility spermatozoa in different culture media in the test example is shown;

[0030] Figure 2 A comparison of the total motility of high-motility and low-motility sperm in different culture media in the test example is shown. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] A sperm motility enhancing solution, comprising a stimulant, recombinant human insulin and a balancing solution.

[0034] Wherein, the stimulant includes 2-deoxyadenosine, pentoxifylline or caffeine.

[0035] The balancing solution is selected from one or more inorganic salt solutions;

[0036] The inorganic salt is selected from NaCl, KCl, CaCl 2 ·2H 2 O.KH 2 PO 4 and MgSO 4 7H 2 O.

[0037] The enhancement solution for improving sperm motility comprises the following components by mass fraction:

[0038]

[0039]

[0040] The pH of the enhancement solution for improving sperm motility is 7.4.

[0041] The method for improving sperm motility by using the sperm motility enhancing liquid of this embodiment comprises the following steps:

[0042] S1: adding sperm, stimulant, recombinant human insulin and balancing solution to the culture medium, culturing to obtain culture medium;

[0043] S2: The culture solution and cryoprotectant solution are mixed evenly, placed in a cryotube and allowed to stand, then transferred to liquid nitrogen and placed for 6-8 days, then placed in a water bath until completely thawed and taken out.

[0044] The components in the cryoprotectant solution include one or more of a serum substitute, HTF-HEPES culture medium, and a glycerol mixture.

[0045] In step S1, the sperm is screened into highly motile sperm, and the screening method is to use double density gradient centrifugation for screening;

[0046] In step S2, the standing temperature is 4° C., and the standing time is 30 min.

[0047] In step S2, the temperature of the freezing tube is maintained at 25°C.

[0048] In step S2, the temperature of the water bath is 37°C.

[0049] In step S2, the volume ratio of the culture solution to the cryoprotectant solution is 1:1.

[0050] Example 2

[0051] A sperm motility enhancing solution, comprising a stimulant, recombinant human insulin and a balancing solution.

[0052] Wherein, the stimulant includes 2-deoxyadenosine, pentoxifylline or caffeine.

[0053] The balancing solution is selected from one or more inorganic salt solutions;

[0054] The inorganic salt is selected from NaCl, KCl, CaCl 2 ·2H 2 O.KH 2 PO 4 and MgSO 4 7H 2 O.

[0055] The enhancement solution for improving sperm motility comprises the following components by mass fraction:

[0056]

[0057]

[0058] The pH of the enhancement solution for improving sperm motility is 7.4.

[0059] The method for improving sperm motility by using the sperm motility enhancing liquid of this embodiment comprises the following steps:

[0060] S1: adding sperm, stimulant, recombinant human insulin and balancing solution to standard culture medium for culturing to obtain culture medium;

[0061] S2: The culture solution and cryoprotectant solution are mixed evenly, placed in a cryotube and allowed to stand, then transferred to liquid nitrogen and placed for 7 days, then placed in a water bath until completely thawed and taken out.

[0062] The components in the cryoprotectant solution include one or more of a serum substitute, HTF-HEPES culture medium, and a glycerol mixture.

[0063] In step S1, the sperm is screened into low-motility sperm, and the screening method is to use double density gradient centrifugation for screening;

[0064] In step S2, the standing temperature is 4° C., and the standing time is 30 min.

[0065] In step S2, the temperature of the freezing tube is maintained at 25°C.

[0066] In step S2, the temperature of the water bath is 37°C.

[0067] In step S2, the volume ratio of the culture solution to the cryoprotectant solution is 1:1.

[0068] Comparative Example 1

[0069] The difference between this comparative example and Example 1 is that no recombinant human insulin is added, and other conditions remain unchanged.

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 2 is that no recombinant human insulin is added, and the other conditions remain unchanged.

[0072] Comparative Example 3

[0073] A sperm motility enhancing solution, comprising a stimulant, recombinant human insulin and a balancing solution.

[0074] Wherein, the stimulant includes 2-deoxyadenosine, pentoxifylline or caffeine.

[0075] The balancing solution is selected from one or more inorganic salt solutions;

[0076] The inorganic salt is selected from NaCl, KCl, CaCl 2 ·2H 2 O.KH 2 PO 4 and MgSO 4 7H 2 O.

[0077] The enhancement solution for improving sperm motility comprises the following components by mass fraction:

[0078]

[0079]

[0080] The pH of the enhancement solution for improving sperm motility is 7.4.

[0081] This comparative example also provides a method for improving sperm motility by using a sperm motility enhancing liquid, the method comprising the following steps:

[0082] S1: adding sperm, stimulant, recombinant human insulin and balancing solution to the culture medium, culturing to obtain culture medium;

[0083] S2: The culture solution and cryoprotectant solution are mixed evenly, placed in a cryotube and allowed to stand, then transferred to liquid nitrogen and placed for 7 days, then placed in a water bath until completely thawed and taken out.

[0084] The components in the cryoprotectant solution include one or more of a serum substitute, HTF-HEPES culture medium, and a glycerol mixture.

[0085] In step S1, the sperm is screened into highly motile sperm, and the screening method is to use double density gradient centrifugation for screening;

[0086] In step S2, the standing temperature is 4° C., and the standing time is 30 min.

[0087] In step S2, the temperature of the freezing tube is maintained at 25°C.

[0088] In step S2, the temperature of the water bath is 37°C.

[0089] In step S2, the volume ratio of the culture solution to the cryoprotectant solution is 1:1.

[0090] Comparative Example 4

[0091] A sperm motility enhancing solution, comprising a stimulant, recombinant human insulin and a balancing solution.

[0092] Wherein, the stimulant includes 2-deoxyadenosine, pentoxifylline or caffeine.

[0093] The balancing solution is selected from one or more inorganic salt solutions;

[0094] The inorganic salt is selected from NaCl, KCl, CaCl 2 ·2H 2 O.KH 2 PO 4 and MgSO 4 7H 2 O.

[0095] The enhancement solution for improving sperm motility comprises the following components by mass fraction:

[0096]

[0097]

[0098] The pH of the enhancement solution for improving sperm motility is 7.4.

[0099] This comparative example also provides a method for improving sperm motility by using a sperm motility enhancing liquid, the method comprising the following steps:

[0100] S1: adding sperm, stimulant, recombinant human insulin and balancing solution to standard culture medium for culturing to obtain culture medium;

[0101] S2: The culture solution and cryoprotectant solution are mixed evenly, placed in a cryotube and allowed to stand, then transferred to liquid nitrogen and placed for 7 days, then placed in a water bath until completely thawed and taken out.

[0102] The components in the cryoprotectant solution include one or more of a serum substitute, HTF-HEPES culture medium, and a glycerol mixture.

[0103] In step S1, the sperm is screened into low-motility sperm, and the screening method is to use double density gradient centrifugation for screening;

[0104] In step S2, the standing temperature is 4° C., and the standing time is 30 min.

[0105] In step S2, the temperature of the freezing tube is maintained at 25°C.

[0106] In step 2, the temperature of the water bath is 37°C.

[0107] In step S2, the volume ratio of the culture solution to the cryoprotectant solution is 1:1.

[0108] Comparative Example 5

[0109] Compared with Comparative Example 3, this comparative example differs in that the stimulant is replaced by pentoxifylline, and the other conditions remain unchanged.

[0110] Comparative Example 6

[0111] Compared with Comparative Example 4, this comparative example differs in that the stimulant is replaced by pentoxifylline, and the other conditions remain unchanged.

[0112] Comparative Example 7

[0113] The difference between this comparative example and comparative example 3 is that the stimulant is replaced by caffeine, and the other conditions remain unchanged.

[0114] Comparative Example 8

[0115] The difference between this comparative example and comparative example 3 is that the stimulant is replaced by caffeine, and the other conditions remain unchanged.

[0116] The components and mass fractions of the culture medium described in Examples 1-2 and Comparative Examples 1-8 of the present invention are as shown in Table 1 below.

[0117] Table 1 Composition and mass fraction of culture medium

[0118]

[0119] Test Case

[0120] The samples of Examples 1-2 and Comparative Examples 1-8 were tested for the progressive motility and total motility of high-motility sperm and low-motility sperm.

[0121] The method used in the test is: Computer-Assisted Sperm Analysis (CASA instrument). Experimental steps: Slowly shake the culture dish to mix the sperm suspension. Use a 1ml quantitative liquid adder to draw a certain amount of sperm suspension, add it to the culture medium at a temperature of 37°C, draw the sperm suspension into the preheated stage of the microscope, and observe the sperm morphology with a microscope. The microscope preheats the stage to maintain a temperature of 37°C. Use the CASA instrument to test the movement trajectory of sperm and analyze the forward movement (progressive motility) and total vitality (total motility).

[0122] The results are shown in Table 2.

[0123] Table 2 Test results of progressive motility and total motility of high-motility sperm and low-motility sperm

[0124]

[0125]

[0126] Table 2 shows that the addition of stimulants in Example 1 and Example 2 can significantly improve the progressive motility and total motility of sperm.

[0127] Specifically, it can be seen from Table 2 that the improvement effect is more obvious, especially for low-motility sperm. At the same time, compared with Comparative Examples 1 and 2, since no stimulants were added in Comparative Examples 1 and 2, the activity of both high-motility sperm and low-motility sperm is lower. In Example 1 and Example 2, on the basis of adding stimulants, adding recombinant human insulin can further increase the progressive motility and total motility of sperm.

[0128] At the same time, in Comparative Examples 3 and 4, 2-deoxyadenosine was added to the culture medium as a stimulant, compared with Comparative Examples 5 and 6 adding pentoxifylline as a stimulant, and Comparative Examples 7 and 8 adding caffeine as a stimulant, the activity of high-motility sperm or low-motility sperm was optimal.

[0129] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0130] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A sperm motility enhancing solution, characterized in that: The sperm motility enhancing solution comprises the following components by mass fraction: composition: 2-Deoxyadenosine 0.02~0.12% Pentoxifylline 0.03~0.14% Caffeine 0.04~0.12% Recombinant human insulin 0.0017~0.0025% NaCl 0.46~0.69% KCl 0.01~0.07% CaCl2·2H2O 0.01~0.15% KH2PO4 0.01~0.07% MgSO4·7H2O 0.01~0.12% Water balance; The pH of the enhancement solution for improving sperm motility is 7.2-7.6.

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