Application of pyridoxamine in sperm optimization and / or freezing recovery operation of asthenospermia

Abstract

The invention provides an application of pyridoxamine in sperm optimization and / or freezing recovery operation of asthenospermia, pyridoxamine is added in sperm operation liquid or sperm freezing liquid, and the following shortages of asthenospermia patients are effectively improved: (1) the ratio of sperm forward motility sperms; (2) determining the ratio of forward motility sperms after the semen is frozen and resuscitated; (3) determining the sperm DNA fragment rate after the sperm is frozen and resuscitated; (4) determining the content of sperm acrosomal enzyme after the sperm is frozen and resuscitated; in conclusion, the pyridoxamine can be added into sperm operating fluid or sperm refrigerating fluid as a natural sperm protective agent, the sperm optimization treatment and freezing resuscitation effects of the asthenospermia patient are obviously improved, and the pyridoxamine is suitable for being applied to assisted reproductive therapy and fertility preservation of the asthenospermia patient.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, in particular to the application of pyridoxamine in the sperm optimization and / or cryopreservation operation of asthenospermia. BACKGROUND

[0002] Male infertility is on the rise, and research over the past few decades has shown that the main cause of male infertility is a decline in sperm quality, manifested as oligospermia, asthenospermia, and teratospermia. The cause of asthenospermia in men is not yet clear in clinical practice, and it is currently generally believed to be related to work pressure, environmental pollution, unhealthy lifestyle, varicocele, and reproductive tract infection. Among them, the optimization of motile sperm and semen freezing technology is a common treatment for assisted reproduction and fertility preservation for asthenospermia.

[0003] Based on clinical cohorts, the present product analyzes the content differences of metabolites in the seminal plasma of asthenospermia patients and normal men by high-performance liquid chromatography-mass spectrometry technology (equipment: Thermo; company: Suzhou Panomics), and finds that the content of pyridoxamine (PM) in the seminal plasma of asthenospermia patients is significantly reduced. Pyridoxamine is a natural component of vitamin B6 and is widely present in animal bodies. According to research reports, pyridoxamine can inhibit glycosylation and the formation of advanced glycosylation end products, and has therapeutic potential for various chronic diseases caused by oxidative stress and carbonyl compounds. Oxidative stress can damage the cytoplasmic membrane of sperm cells, causing sperm apoptosis and asthenospermia, suggesting that pyridoxamine can reduce oxidative stress in sperm cells and improve sperm motility. However, there is no drug on the market that uses pyridoxamine as a therapeutic target for semen optimization for asthenospermia.

[0004] In the process of clinical assisted reproduction, the sperm motility, cryopreservation rate, and in vitro fertilization rate of asthenospermia patients after sperm optimization treatment and semen cryopreservation are lower than those of normal people, which may be related to the lack of active ingredients in the seminal plasma of asthenospermia patients. Some existing sperm optimization and cryopreservation products on the market improve sperm motility by adding commonly used antioxidants such as superoxide dismutase and non-enzymatic antioxidants such as glutathione and resveratrol, but these substances lack specificity in the treatment of asthenospermia patients. In addition, the addition of antioxidant substances other than seminal plasma components to sperm operation fluid and cryopreservation fluid also poses potential safety risks. SUMMARY

[0005] To solve the technical problems existing in the prior art, the present application provides the following technical solutions: Pyridoxamine is used for the following applications: Improving the ratio of sperm forward motility in asthenospermia patients.

[0006] Pyridoxamine for use in: Improving the ratio of forward motility sperm after semen cryopreservation and recovery in patients with asthenospermia.

[0007] Pyridoxamine for use in: Improving the DNA fragmentation rate of sperm after semen cryopreservation and recovery in patients with asthenospermia.

[0008] Pyridoxamine for use in: Improving the acrosomal enzyme content of sperm after semen cryopreservation and recovery in patients with asthenospermia.

[0009] An asthenospermia sperm preferred and / or cryopreservation operation liquid additive, the additive comprising pyridoxamine.

[0010] Further, the concentration of the pyridoxamine is 200 μmol / L (micromoles per liter).

[0011] The technical scheme provided by the embodiment of the present application brings at least the following beneficial effects: The present application adds pyridoxamine in the sperm operation liquid or semen freezing liquid, effectively improves the ratio of forward motility sperm in patients with asthenospermia; improves the ratio of forward motility sperm after semen cryopreservation and recovery; improves the DNA fragmentation rate of sperm after semen cryopreservation and recovery; improves the acrosomal enzyme content of sperm after semen cryopreservation and recovery; in summary, pyridoxamine can be added to the sperm operation liquid or semen freezing liquid as a natural semen protective agent, significantly improves the sperm preferred treatment and cryopreservation recovery effect of patients with asthenospermia, and is suitable for application in assisted reproductive therapy and fertility preservation of patients with asthenospermia. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figures 1-4 is a schematic diagram of enrichment of pyridoxamine expression level in the seminal plasma of the asthenospermia patient group provided by the embodiment of the present application Figures 1-4 are respectively: statistical information of different expression metabolites; metabolic rate control heat map of metabolites; schematic diagram of influence of differential metabolites on amino acid metabolism pathway; logarithmic intensity comparison diagram of pyridoxamine expression level in the seminal plasma of the asthenospermia patient group; Figure 5 is a schematic diagram of influence of pyridoxamine on forward motility ability and DNA integrity of sperm; Figure 6is a pyridoxamine effect diagram on sperm forward motility and DNA integrity of frozen-thawed sperm provided by an embodiment of the present application. Figure 7 is a pyridoxamine effect diagram on sperm fertilization capacity of frozen-thawed sperm provided by an embodiment of the present application. DETAILED DESCRIPTION

[0014] The technical solutions in the present application will be described below with reference to the drawings.

[0015] In the embodiments of the present application, the words such as "example", "for example" are used to represent as an example, illustration or description. Any embodiment or design scheme described as "example" in the present application should not be interpreted as more preferred or more advantageous than other embodiments or design schemes. Rather, the word "example" is intended to present the concept in a specific manner. In addition, in the embodiments of the present application, the meaning expressed by "and / or" can be both, or can be one of the two.

[0016] In the embodiments of the present application, "image" and "picture" can be used interchangeably at times, and it should be pointed out that the meanings expressed are consistent when the distinction is not emphasized. "Of", "corresponding" and "corresponding" can be used interchangeably at times, and it should be pointed out that the meanings expressed are consistent when the distinction is not emphasized.

[0017] In the embodiments of the present application, sometimes the subscript such as W1 may be mistakenly used in the form of non-subscript such as W1, and the meanings expressed are consistent when the distinction is not emphasized.

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present application more clear, specific embodiments will be described in detail below with reference to the drawings.

[0019] The reagents or devices used in the present embodiment can be provided by a laboratory or purchased from the market.

[0020] The present application adds a certain amount of pyridoxamine in the sperm manipulation liquid or the semen freezing liquid, which can effectively improve the sperm motility (dynamic force). Pyridoxamine can be added to the sperm manipulation liquid or the semen freezing liquid as a natural semen protective agent, which can significantly improve the sperm optimal treatment and freezing recovery effect of patients with asthenospermia, and is suitable for application of patients with asthenospermia in assisted reproductive therapy and fertility preservation.

[0021] The following will provide several groups of embodiment experiments for illustration.

[0022] Example 1: The expression level of pyridoxamine in the seminal plasma of the asthenospermia patient group is significantly reduced Methods: 20 patients with asthenospermia and 20 healthy male controls were recruited (no significant difference in age and BMI between the healthy group and the asthenospermia group). After 2-7 days of abstinence, semen samples were collected by masturbation into sterile containers. The semen samples were centrifuged (3000 rpm for 10 minutes at 4°C). The supernatant seminal plasma was then transferred to centrifuge tubes.

[0023] Non-targeted metabolomics detection of seminal plasma was performed by high-performance liquid chromatography-mass spectrometry (Suzhou Panomics).

[0024] The detection principle is as follows: non-targeted metabolomics detection of seminal plasma by high-performance liquid chromatography-mass spectrometry (HPLC-MS) combines the separation ability of high-performance liquid chromatography and the detection ability of mass spectrometry for comprehensive analysis of metabolites in seminal plasma.

[0025] 1. High-performance liquid chromatography separation principle High-performance liquid chromatography separates different metabolites based on their differences in partition coefficients between the stationary phase and the mobile phase. Various metabolites in seminal plasma have different chemical properties, such as polarity, molecular size, and charge. When the sample is injected into the high-performance liquid chromatography system, as the mobile phase (solvent) flows, the metabolites continuously partition between the stationary phase (chromatographic column packing material) and the mobile phase. Metabolites with stronger polarity interact less with the stationary phase, have shorter retention times in the chromatographic column, and flow out earlier; while metabolites with weaker polarity interact more with the stationary phase, have longer retention times, and flow out later. In this way, various metabolites in seminal plasma are separated into different peaks according to their differences in chemical properties in the time dimension.

[0026] 2. Mass spectrometry detection principle Mass spectrometry analyzes the structure and composition of substances by measuring the mass and relative abundance of ions. After being separated by high-performance liquid chromatography, the metabolites enter the mass spectrometer, where they are first ionized, converting neutral metabolite molecules into charged ions. Ionization methods such as electrospray ionization (ESI) can ionize metabolites under mild conditions, preserving their molecular structure information. Ionized metabolite ions are deflected under the action of electric and magnetic fields, and the degree of deflection depends on the mass-to-charge ratio (m / z) of the ions. By detecting the mass-to-charge ratio and relative abundance of ions, the mass spectrum of the metabolite can be obtained. Each metabolite has its characteristic mass spectrum, like a fingerprint, which can be identified by comparing it with the mass spectrum database of known metabolites to identify the types of metabolites in seminal plasma.

[0027] Non-targeted metabolomics detection principle The goal of non-targeted metabolomics is to detect as many metabolites in the seminal plasma as possible without pre-selecting specific metabolites to be detected. By using high-performance liquid chromatography-mass spectrometry technology, metabolites in seminal plasma can be separated and detected without bias. During the detection process, the chromatographic peak and mass spectrometry information of all detected metabolites are recorded, and then these data are analyzed using bioinformatics methods. By analyzing and comparing a large number of samples, differences in metabolites between different samples can be found, and metabolite markers related to physiological status, disease, etc. can be identified.

[0028] 3. Detection step (1) Sample collection and pretreatment Sample collection: Use a professional semen collection container to collect the semen sample according to the standard semen collection method. Generally, 2-7 days of abstinence is required to ensure the quality and stability of the sample. The semen sample collected should be sent to the laboratory for processing within 30 minutes.

[0029] Liquefaction treatment: Incubate the collected semen sample at 37°C to allow it to liquefy naturally. The liquefaction time is generally 15-60 minutes, and the specific time varies depending on individual differences.

[0030] Centrifugal separation: Centrifuge the liquefied semen sample under certain conditions (such as 3000-5000 rpm for 10-15 minutes) to separate the seminal plasma from sperm and other components. Collect the supernatant, which is the seminal plasma sample.

[0031] Filtering and preservation: Filter the seminal plasma sample through a 0.22 μm or 0.45 μm filter to remove impurities and particles. The filtered seminal plasma sample can be stored in a -80°C freezer for subsequent detection.

[0032] (2) High-performance liquid chromatography separation Column selection: According to the properties of metabolites in seminal plasma, select appropriate chromatographic columns. Commonly used chromatographic columns include reverse-phase chromatographic columns (such as C18 columns) and normal-phase chromatographic columns. Reverse-phase chromatographic columns are suitable for separating polar and moderately polar metabolites, while normal-phase chromatographic columns are suitable for separating strongly polar metabolites.

[0033] Mobile phase preparation: The mobile phase is generally composed of organic solvents (such as methanol, acetonitrile, etc.) and water, with the addition of a certain proportion of buffer salt (such as ammonium formate, ammonium acetate, etc.) to adjust the pH value and improve the separation effect. According to the properties of metabolites and the requirements of the chromatographic column, prepare appropriate mobile phase gradients. For example, a linear gradient elution can be used, gradually increasing the proportion of organic solvents from low to high, to separate metabolites of different polarities.

[0034] Sample injection: The pretreated seminal plasma sample is injected into the autosampler of the high-performance liquid chromatography system, with a typical injection volume of 1-10 μL. After injection, the sample enters the chromatographic column with the mobile phase for separation.

[0035] Chromatographic separation: Under certain flow rates (e.g., 0.2-1 mL / min) and column temperatures (e.g., 25-40°C), the metabolites in the seminal plasma are separated in the chromatographic column. By monitoring the absorbance or other physical properties of the chromatographic column effluent, the chromatogram of the metabolites is obtained.

[0036] (3) Mass spectrometry detection Ion source selection and parameter setting: According to the properties of metabolites and detection requirements, select appropriate ion sources, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), etc. For most metabolites in biological samples, ESI is the most commonly used ion source. Set the parameters of the ion source, such as spray voltage, capillary temperature, nebulizer gas flow, etc., to ensure the effective ionization of metabolites.

[0037] Mass spectrometry scanning mode selection: Full scan mode or selected ion monitoring (SIM) mode can be selected. Full scan mode can detect all ionized metabolites, suitable for non-targeted metabolomics detection; while SIM mode only monitors ions of specific mass-to-charge ratios, suitable for quantitative analysis.

[0038] Data acquisition: After the metabolites flow out of the chromatographic column and enter the mass spectrometer, the mass spectrometer performs data acquisition according to the set scanning mode and parameters. Record the mass spectrum at each time point to obtain the mass spectrometry data of the seminal plasma metabolites.

[0039] (4) Data processing and analysis Data preprocessing: Preprocess the collected mass spectrometry data, including baseline correction, peak identification, peak alignment, etc. Baseline correction can remove background noise and improve data quality; peak identification can determine the peak position and peak area in the chromatogram; peak alignment can align the same metabolite peaks in different samples for subsequent comparison and analysis.

[0040] Metabolite identification: Compare the preprocessed data with known metabolite mass spectrometry databases, such as HMDB (Human Metabolome Database), METLIN, etc. By comparing mass-to-charge ratios, retention times, etc., identify the types of metabolites in the seminal plasma. For metabolites that cannot be matched in the database, further structural analysis methods (such as tandem mass spectrometry) can be used for identification.

[0041] Statistical analysis: Statistical analysis is performed on the metabolite data from different samples, such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), etc. These analysis methods can help find the differences in metabolites between different samples and identify potential metabolic markers.

[0042] Bioinformatics analysis: Bioinformatics tools are used to annotate the functions of the identified metabolites and analyze their metabolic pathways. Understanding the biological processes and metabolic pathways in which these metabolites are involved can help us better understand the relationship between changes in seminal plasma metabolome and physiological status, disease, etc.

[0043] The results showed that compared with the healthy group, the expression levels of 177 metabolites in the semen of the asthenospermia group were significantly abnormal, of which 149 were significantly up-regulated and 28 were down-regulated (p<0.05) Figure 1 and Figure 2 KEGG functional analysis showed that the differential metabolites were enriched in amino acid metabolism, protein digestion and absorption, lipid metabolism, etc. Figure 3 Among them, pyridoxamine had a significantly lower expression level in the seminal plasma of the asthenospermia patient group Figure 4 , suggesting that pyridoxamine can be used as a natural seminal active substance to improve the clinical treatment effect of semen selection and semen freezing in asthenospermia patients.

[0044] Among them, KEGG (Kyoto Encyclopedia of Genes and Genomes) functional analysis is an important method for systematic analysis of gene function, genomic information and biological molecular network. It reveals the biological functions and metabolic processes in which these metabolites are involved by mapping the differential metabolites to the metabolic pathways in the KEGG database. The basic process and principle are as follows: 1. Data collection and processing: When performing KEGG functional analysis, the first step is to obtain the relevant data of metabolites in the sample. This usually involves the use of advanced metabolomics techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to detect samples and obtain information such as the types and contents of metabolites. Then standardize the raw data to eliminate differences caused by experimental conditions, instrument errors, etc. in order to facilitate subsequent analysis.

[0045] 2. Differential metabolite screening: Use statistical methods such as t-test, variance analysis, etc. to compare the content differences of metabolites between different sample groups. Set appropriate thresholds (such as p-value, fold change, etc.) to screen out metabolites with significant differences between different groups. These differential metabolites may be related to different physiological states, disease states or external stimuli of the sample.

[0046] 3. Pathway mapping: The identified differential metabolites are mapped to the metabolic pathways in the KEGG database. KEGG is a comprehensive database that integrates various biological information such as genes, metabolites, and metabolic pathways. The metabolic pathway maps in KEGG depict the chemical reactions and interactions between various biological molecules in detail. By mapping the differential metabolites to these pathway maps, one can intuitively understand the biological processes they are involved in.

[0047] 4. Enrichment analysis: To determine which metabolic pathways the differential metabolites are significantly enriched in, enrichment analysis is performed. Common methods include hypergeometric test or Fisher's exact test, which calculate the difference between the frequency of differential metabolites in each pathway and the frequency in the entire database to determine whether the enrichment of these metabolites in a specific pathway is statistically significant. A significance threshold (e.g., p-value < 0.05) is usually set, and when the enrichment p-value of a pathway is less than the threshold, it is considered that the differential metabolites are significantly enriched in that pathway.

[0048] The aspects of metabolic enrichment are explained as follows: (1) Enrichment of differential metabolites in amino acid metabolism Synthesis and catabolism of amino acids: Amino acids are the basic units of protein synthesis and also participate in many important biochemical reactions. The enrichment of differential metabolites in the amino acid metabolism pathway may indicate that these metabolites are involved in the synthesis or catabolism of amino acids. For example, some differential metabolites may be substrates or products of key enzymes in the amino acid synthesis pathway, and changes in their content may affect the synthesis rate of amino acids. Conversely, in the process of amino acid catabolism, some differential metabolites may be amino acid degradation products, and changes in their content reflect the catabolic state of amino acids.

[0049] Amino acid metabolism and physiological functions: Abnormalities in amino acid metabolism are closely related to many physiological processes and diseases. For example, abnormal metabolism of branched-chain amino acids (leucine, isoleucine, and valine) is associated with metabolic diseases such as diabetes and obesity. When differential metabolites are enriched in the branched-chain amino acid metabolism pathway, it may indicate that the body's energy metabolism or insulin signaling pathway has changed. In addition, some amino acids such as tryptophan and tyrosine are also involved in the synthesis of neurotransmitters, and their metabolic abnormalities may affect the function of the nervous system.

[0050] (2) Enrichment of differential metabolites in protein digestion and absorption Digestion of proteins: Proteins are broken down into amino acids and small peptides by various digestive enzymes in the gastrointestinal tract, including pepsin, trypsin, chymotrypsin, etc. The enrichment of differential metabolites in the protein digestion and absorption pathway can be related to the activity or secretion of digestive enzymes. For example, certain differential metabolites can be activators or inhibitors of digestive enzymes, and changes in their content can affect the efficiency of protein digestion. In addition, some intermediates produced during digestion, such as oligopeptides, etc., can also be differential metabolites, and changes in their content reflect the degree of protein digestion.

[0051] Absorption of amino acids and small peptides: Amino acids and small peptides after digestion are absorbed into the blood circulation through transport proteins of the intestinal epithelial cells. The enrichment of differential metabolites in the protein digestion and absorption pathway can also be related to the function of transport proteins. Some differential metabolites can affect the expression or activity of transport proteins, thereby affecting the efficiency of amino acid and small peptide absorption. For example, certain nutrients or drugs can affect protein absorption by regulating the function of transport proteins, and the metabolites of these substances can be detected as differential metabolites.

[0052] (3) Enrichment of differential metabolites in lipid metabolism Synthesis and catabolism of lipids: Lipids are important components of organisms, including fats, phospholipids, cholesterol, etc. The enrichment of differential metabolites in the lipid metabolism pathway can involve the synthesis and catabolism of lipids. In terms of lipid synthesis, some differential metabolites can be substrates or products of fatty acid synthase, and changes in their content affect the rate of fatty acid synthesis, and thus affect the synthesis of fats and phospholipids. In terms of lipid catabolism, β-oxidation of fatty acids is the main catabolic pathway, and differential metabolites can be involved in key steps in this process, such as activation, transport, etc.

[0053] Lipid metabolism and disease: Abnormalities in lipid metabolism are closely related to many diseases such as atherosclerosis, hyperlipidemia, etc. When differential metabolites are enriched in the lipid metabolism pathway, it can indicate that the body's lipid metabolism balance has changed. For example, abnormal cholesterol metabolism can lead to elevated cholesterol levels in the blood, increasing the risk of atherosclerosis. Differential metabolites can reflect abnormalities in the synthesis, transport, or excretion of cholesterol, providing important clues for the diagnosis and treatment of diseases.

[0054] Example 2, pyridoxamine in seminal plasma and sperm forward motility and other outcomes such as DNA integrity are related Methods: Add pyridoxamine to the seminal plasma (200 micromoles per liter, brand FUJIFILM Irvine Scientific), and mix with the same volume of semen, incubate at room temperature for about 90 minutes, mix the sample after incubation, and then evaluate the forward motility of sperm.

[0055] The addition of pyridoxamine significantly improved the percentage of progressively motile sperm in asthenozoospermia patients (p=0.051) compared to the group without pyridoxamine. In addition, the addition of pyridoxamine had no effect on the percentage of progressively motile sperm in normal men (p=0.0956) (Fig. 1 (a) and (b)). In addition, the DNA fragmentation rate of sperm was detected before and after incubation, and it was found that the addition of pyridoxamine had no effect on the DFI level of asthenozoospermia patients and normal men after 90 minutes of incubation (p=0.1205; p=0.8534) (Fig. 1 (c) and (d)). Figure 5 Figure 5

[0056] The results show that the addition of pyridoxamine to the sperm manipulation solution of asthenozoospermia patients can improve the forward motility of sperm.

[0057] Example 3: Correlation between pyridoxamine in seminal plasma and the forward motility and DNA integrity of sperm after freezing and thawing of semen Method: Add pyridoxamine to the semen freezing solution (200 micromoles per liter, freezing solution brand ORIGIO), mix with an equal volume of semen, incubate at room temperature for about 30 minutes, and then perform the conventional semen freezing process. After 48 hours, the sperm is thawed, and it is found that the group with pyridoxamine added to the freezing solution significantly improves the percentage of progressively motile sperm in asthenozoospermia patients and normal men after thawing (p=0.0064; p=0.0097); and the difference in asthenozoospermia patients is more significant than that in normal men (Fig. 2 (a) and (b)). Figure 6 Figure 6 The addition of pyridoxamine to the freezing solution significantly reduces the DNA fragmentation rate of sperm in asthenozoospermia patients after thawing (p=0.0322), while the DNA fragmentation rate of normal men also decreases, but the difference is not significant (p=0.0689) (Fig. 2 (c) and (d)).

[0058] The results show that the addition of pyridoxamine to the freezing solution of asthenozoospermia patients during assisted reproductive treatment can improve the forward motility of sperm after thawing.

[0059] Example 4: Correlation between pyridoxamine in seminal plasma and the fertilization ability of sperm after freezing and thawing of semen Method: Add pyridoxamine to the semen freezing solution (200 micromoles per liter, freezing solution brand ORIGIO), mix with an equal volume of semen, incubate at room temperature for about 30 minutes, and then perform the conventional semen freezing process. After 48 hours, the sperm is thawed, and it is found that the group with pyridoxamine added to the freezing solution significantly improves the acrosin content of sperm in asthenozoospermia patients and normal men after thawing (p=0.0262; p=0.0316); and the improvement in asthenozoospermia patients is more significant than that in normal men (Fig. 3). Figure 7 ​​​​

[0060] The results show that adding pyridoxamine in the freezing operation liquid during the assisted reproductive treatment of the oligospermia patient can improve the content of sperm acrosome enzyme after recovery.

[0061] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Pyridoxine, used in the following applications: Improve the ratio of forward-moving sperm in patients with asthenospermia.

2. Pyridoxine, used in the following applications: Improve the ratio of forward-moving, motile sperm after semen cryopreservation and thawing in patients with asthenospermia.

3. Pyridoxine, used in the following applications: Improve sperm DNA fragmentation rate after semen cryopreservation and thawing in patients with asthenospermia.

4. Pyridoxine, used in the following applications: Improve the acrosomal enzyme content of sperm after cryopreservation and thawing in patients with asthenospermia.

5. An additive for sperm selection and / or cryopreservation / thawing solution in patients with asthenospermia, characterized in that, The additives include pyridoxine.

6. The additive according to claim 5, characterized in that, The concentration of pyridoxine is 200 μmol / L.

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