CN112481197A

CN112481197A - Semen cleaning fluid and preparation method thereof

Info

Publication number: CN112481197A
Application number: CN202011272707.XA
Authority: CN
Inventors: 林小贞
Original assignee: Shenzhen Vitavitro Biotech Co ltd
Current assignee: Shenzhen Vitavitro Biotech Co ltd
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2021-03-12

Abstract

The invention discloses a human sperm cleaning solution, which comprises an inorganic salt solution containing buffer components, and is characterized in that biotin and lipoic acid are added into the inorganic salt solution.

Description

Semen cleaning fluid and preparation method thereof

Technical Field

The invention belongs to the field of human assisted reproduction, and particularly relates to a human sperm cleaning solution and a method for preparing the same.

Background

Semen optimization is a very important step in Assisted Reproductive Technology (ART) to remove seminal plasma, non-motile sperm, teratospermia, cellular debris and other harmful substances, and to produce motile sperm with a high proportion of morphologically normal sperm. Ideally the pre-treated sperm cells are those which have been added to a small volume of culture medium to remove seminal plasma, leukocytes and bacteria and to enrich for normal morphology and motility. Currently, the methods for optimizing the sperm, which are commonly used in clinical practice, mainly include an upstream method, a density gradient centrifugation method, and the like.

Sperm cells contain a large amount of polyunsaturated fatty acids and are unable to effectively repair membrane damage due to the apparent lack of cytoplasm and a lack of defense system, making sperm particularly susceptible to oxidative stress, and any in vitro manipulation may result in elevated levels of reactive oxygen species that can affect sperm survival.

The currently used sperm preparation solution or semen washing solution is a basic salt buffer solution, and the damage and the rise of the level of active oxygen brought by the operation process are difficult to avoid in the sperm in-vitro operation process. In the conventional upstream method, functional sperm are brought into close intercellular contact with defective sperm or leukocytes by centrifugation, and due to the high amount of polyunsaturated fatty acids contained in the plasma membrane of sperm, these reactive oxygens cause lipid peroxidation, resulting in a drastic decrease in sperm function (including motility), causing extensive oxidative damage of the plasma membrane of sperm by the reactive oxygens, thereby causing damage to sperm function (mortken RJ, et al. Signal of reactive oxygen species and reactive species in refining the effect of sperm production technology. J. animal) (mortier D.norm preservation techniques and synthetic factors of in-vitro fertilization. hum reproduction).

Biotin (vitamin B7 or vitamin H) is a micronutrient essential to normal growth and development of human body, and is involved in carboxylation reactions such as fatty acid biosynthesis, gluconeogenesis, branched chain amino acid metabolism, and purine nucleotide synthesis. Studies have shown that biotin plays a role in reproduction and development, and that a deficiency in gestation biotin can lead to teratogenicity in mice and hamsters (Mock dm. natural biotin specificity is common in normal human prognosis and is high purity geneticic in microorganism.j Nutr Watanabe t. dietary biotin specificity affects reproduction function and expression level in hamsters.j Nutr).

Lipoic acid (AApAa-AApAAc acadA ALA) is one of the effective biological antioxidants, and is considered to be a general antioxidant due to its solubility in water and fat. The antioxidant can easily penetrate different tissues, cells, and even cell organelles mainly generating active oxygen like mitochondria. Lipoic acid is an organic sulfur compound (dithiol) derived from caprylic acid and plays an essential auxiliary role for many enzymes.

The inventor tries to use the two components for washing semen, and hopefully, the two components can reduce the damage effect in the process of in-vitro processing of the sperm, promote the in-vitro sperm motility and reduce the active oxygen level so as to improve the sperm preparation efficiency and quality in assisted reproduction.

Disclosure of Invention

The invention aims to provide a human sperm cleaning solution. Biotin such as vitamin B7 or vitamin H is a micronutrient essential for normal growth and development of human body, and is involved in carboxylation reactions such as fatty acid biosynthesis, gluconeogenesis, branched chain amino acid metabolism and purine nucleotide synthesis; lipoic Acid (ALA), an organic sulfur compound (dithiol) derived from caprylic acid, is one of the effective biological antioxidants, and is a versatile antioxidant due to its solubility in water and fat, which can easily penetrate various tissues, cells, and even mitochondria, which are organelles in which active oxygen is mainly produced, and plays a necessary auxiliary role for many enzymes. The invention uses inorganic salt solution containing buffer component, adds biotin and lipoic acid, and obtains human sperm cleaning solution with excellent performance.

The specific technical scheme of the invention is as follows:

a human sperm cleaning solution comprises inorganic salt solution containing buffer component, biotin 8-20nM/L, thioctic acid 0.01-0.05 mM/L; wherein the formulation of the inorganic salt solution containing the buffer component comprises 100-120 mM/L of sodium chloride, 4.10-5.28 mM/L of potassium chloride, 0.25-0.45 mM/L of monopotassium phosphate, 1.70-2.38 mM/L of calcium chloride dihydrate, 0.10-0.30 mM/L of magnesium sulfate heptahydrate, 1-7 mM/L of sodium bicarbonate, 12-15.5mM/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 5-9mM/L of 4-hydroxyethyl piperazine ethanesulfonic acid, 0.10-0.56 mM/L of sodium pyruvate, 5-16.4 mM/L of sodium lactate, 0.5-5.06 mM/L of glucose, 0-1.0 mM/L of propylamine glutamine, 0-0.02 mM/L of disodium ethylenediamine tetraacetate, 0.01-0.19 mM/L of taurine, 0.01-0.02 g/L of gentamycin sulfate and 5-10 g/L of human serum protein.

The invention also aims to provide the application of the human sperm cleaning solution in the in-vitro preparation process of human sperm.

It is still another object of the present invention to provide a method for preparing a human sperm washing solution, comprising the steps of:

a. weighing all solid components according to a formula and placing the solid components separately, wherein the biotin and the lipoic acid are weighed by 100 times;

b. adding and dissolving all components except biotin, lipoic acid, gentamicin sulfate and human serum albumin in ultrapure water to prepare a buffer salt solution;

c. dissolving biotin and lipoic acid in a proper amount of water to prepare a 100 multiplied concentrated stock solution;

d. b, adding gentamicin sulfate, one percent of biotin and lipoic acid into the buffer salt solution obtained in the step b, and dissolving to obtain a base solution;

e. measuring and adjusting the pH value of the basic solution to 7.4 +/-0.1, and the osmotic pressure to 280-290 mOsm/kg;

f. filtering the basic solution with 0.22 μm filter, adding human serum albumin at a certain proportion, packaging, sealing, and storing at 2-8 deg.C.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the present invention, and the materials, reagents, and equipment used in the examples are commercially available without specific reference.

[ example 1 ] sperm washing solution component content and preparation method:

A. the sperm cleaning solution comprises the following components:

B. the preparation method comprises the following steps:

a. weighing all solid components according to a formula and placing the solid components separately, wherein the weight of biotin and lipoic acid is 100 times;

Example 2 quality control detection of in vitro administered semen

1. pH value detection

Taking a proper amount of a sample to be measured, measuring the sample by using a pH meter for three times, taking the average value of the obtained three data as a result, and judging the sample to be qualified at 7.4 +/-0.1.

2. Osmolarity detection

After the osmometer is corrected, taking a 500 mu A sample to be tested to a test tube to start testing, reading data after the numerical value is stable, measuring 3 data according to the method, taking the average value as a result, and judging the result as qualified at 280-290 mOsm/kg.

3. Bacterial endotoxin detection

According to the requirements of pharmacopoeia 2015 edition of the people's republic of China, a limulus reagent gel method is used for detection, and the result is less than or equal to 0.1EU/mA, which is regarded as qualified.

4. Cytotoxicity

The cytotoxicity score should not exceed 1 point, as measured according to the regulations of GB/T16886.5.

5. Sensitization detection

No sensitization should be detected according to the regulations of GB/T16886.10.

6. Sterility test

According to the requirements of pharmacopoeia 2015 edition of the people's republic of China, a sample to be tested is filtered by a filter membrane and then directly inoculated into a culture medium for culture for 14 days by using a membrane filtration method, and the result is observed every day and is aseptic.

7. Intradermal stimulation

No subcutaneous irritation should be detected according to the regulations of GB/T16886.10.

8. Human sperm survival test

The human sperm survival test is carried out according to YY/T1535-2017 human sperm survival test evaluated by medical apparatus biology for human in vitro assisted reproduction technology, and in the three effective tests, the sperm motility coefficient (SMI) of at least two test group samples is more than or equal to 0.7, and the relative sperm motility coefficient is more than or equal to 0.9.

9. The result of the detection

[ example 3 ] sperm motility test

(I) Effect of different pairing sperm motility

Sperm motility test was performed on sperm cells prepared according to the formulation of example 2, as follows:

1. taking the normal liquefied semen into 2mL of sperm cleaning solution without biotin and lipoic acid, and centrifuging 10mAn at 300 g;

2. the sperm sediment at the bottom of the test tube is resuspended in sperm washing solution of each mixture ratio in example 2, and centrifuged at 300g for 10 mAn;

3. taking out the test tube, discarding the supernatant, and adding 2mL of sperm cleaning solution with each proportion respectively to resuspend the sperm;

4. each group of sperm cells was incubated at 37 ℃ for 1 hour, 4 hours, and 24 hours;

(II) Effect of Biotin and lipoic acid on sperm motility

Sperm wash was prepared according to formulation 3 of example 2 and sperm motility test was performed according to the following procedure:

2. the sperm pellet at the bottom of the tube was resuspended in the above-described wash solution and divided into two equal portions, one control and the other test. Two sperm suspensions 300g were centrifuged 10 mAn;

3. taking out the test tube, discarding the supernatant, adding 2mL of sperm cleaning solution without biotin and lipoic acid into the control group, and adding the sperm cleaning solution containing biotin and lipoic acid into the test group to resuspend the sperm;

4. two groups of sperm were incubated at 37 ℃ for 1 hour, 4 hours and 24 hours;

5. at different time points, 10 μ A of the upstream sperm were taken for viability in counting plates.

6. And (3) test results:

the results show that the biotin and the lipoic acid can obviously improve the total activity and the forward movement ability of the sperms.

Claims

1. The human sperm cleaning solution comprises an inorganic salt solution containing buffer components, and is characterized in that biotin and lipoic acid are added into the inorganic salt solution.

2. The human sperm washing solution of claim 1, wherein the biotin content is 8-20nM/L and the lipoic acid content is 0.01-0.05 mM/L.

3. The human sperm cleaning solution as claimed in claim 2, wherein the formulation of the inorganic salt solution containing the buffer component is 100 to 120mM/L sodium chloride, 4.10 to 5.28mM/L potassium chloride, 0.25 to 0.45mM/L potassium dihydrogen phosphate, 1.70 to 2.38mM/L calcium chloride dihydrate, 0.10 to 0.30mM/L magnesium sulfate heptahydrate, 1 to 7mM/L sodium bicarbonate, 12 to 15.5 mM/L4-hydroxyethylpiperazine ethanesulfonic acid, 5 to 9 mM/L4-hydroxyethylpiperazine ethanesulfonic acid, 0.10 to 0.56mM/L sodium pyruvate, 5 to 16.4mM/L sodium lactate, 0.5 to 5.06mM/L glucose, 0 to 1.0mM/L propylamine glutamine, 0 to 0.02mM/L disodium ethylenediaminetetraacetate, 0.01 to 0.19mM/L taurine, 0.01 to 0.02mM/L gentamicin sulfate, 0.01 to 0.02g/L gentamicin sulfate, 5-10 g/L of human serum protein.

4. The human sperm cleaning solution of claim 3 wherein the human sperm cleaning solution is formulated as: 110mM/L of sodium chloride, 4.69mM/L of potassium chloride, 0.35mM/L of monopotassium phosphate, 2.04mM/L of calcium chloride dihydrate, 0.2mM/L of magnesium sulfate heptahydrate, 4mM/L of sodium bicarbonate, 12mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid, 9mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid sodium salt, 0.33mM/L of sodium pyruvate, 10.7mM/L of sodium lactate, 2.78mM/L of glucose, 0.5mM/L of propylamine glutamine, 0.01mM/L of ethylene diamine tetraacetic acid disodium salt, 0.10mM/L of taurine, 0.01g/L of gentamicin sulfate, 5g/L of human serum albumin, 10nM/L of biotin and 0.04mM/L of lipoic acid.

5. The human sperm cleaning solution of claim 3 wherein the human sperm cleaning solution is formulated as: 105mM/L of sodium chloride, 4.4mM/L of potassium chloride, 0.3mM/L of monopotassium phosphate, 1.87mM/L of calcium chloride dihydrate, 0.15mM/L of magnesium sulfate heptahydrate, 2.5mM/L of sodium bicarbonate, 15.5mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid, 6.5mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid sodium sulfonate, 0.22mM/L of sodium pyruvate, 7.85mM/L of sodium lactate, 1.64mM/L of glucose, 0.25mM/L of propylaminoylglutamine, 0.01mM/L of ethylene diamine tetraacetic acid disodium, 0.06mM/L of taurine, 0.01g/L of gentamicin sulfate, 5g/L of human serum protein, 9nM/L of biotin and 0.03mM/L of lipoic acid.

6. The human sperm cleaning solution of claim 3 wherein the human sperm cleaning solution is formulated as: 115mM/L of sodium chloride, 4.99mM/L of potassium chloride, 0.4mM/L of monopotassium phosphate, 2.21mM/L of calcium chloride dihydrate, 0.25mM/L of magnesium sulfate heptahydrate, 5.5mM/L of sodium bicarbonate, 13.5mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid, 7mM/L of 4-hydroxyethylpiperazine ethanesulfonic acid sodium salt, 0.45mM/L of sodium pyruvate, 13.55mM/L of sodium lactate, 3.92mM/L of glucose, 0.75mM/L of propylaminoylglutamine, 0.01mM/L of ethylene diamine tetraacetic acid disodium, 0.15mM/L of taurine, 0.01g/L of gentamicin sulfate, 8.75g/L of human serum albumin, 15nM/L of biotin and 0.04mM/L of lipoic acid.

7. Use of the human sperm washing solution of claim 1 in a process for the in vitro production of human sperm.

8. A method of preparing a human sperm cell washing solution according to claim 1 comprising the steps of:

9. Human sperm cleansing fluid obtained by the method of claim 8.

10. Use of a human sperm washing solution obtained by the method of claim 8 in a process for the in vitro production of human sperm.