Use of pyridoxamine in sperm selection and / or cryopreservation procedures for asthenozoospermia

Abstract

The application provides application of pyridoxamine in sperm of oligospermia, preferably and / or in a freezing and resuscitation operation, pyridoxamine is added in a sperm operation liquid or a semen freezing liquid, and the following shortcomings of patients with oligospermia are effectively improved: (1) the ratio of sperm forward motility; (2) the ratio of sperm forward motility after semen freezing and resuscitation; (3) the sperm DNA fragmentation rate after semen freezing and resuscitation; (4) the sperm acrosome enzyme content after semen freezing and resuscitation; in summary, pyridoxamine can be added in the sperm operation liquid or the semen freezing liquid as a natural semen protective agent, the sperm selection treatment and the freezing and resuscitation effect of patients with oligospermia are obviously improved, and the application is suitable for application of patients with oligospermia in assisted reproductive treatment and fertility preservation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of pyridoxine in the selection and / or cryopreservation of asthenospermia sperm. Background Technology

[0002] Male infertility is on the rise. Research over the past few decades has shown that the main cause of male infertility is declining sperm quality, manifesting as oligospermia, asthenospermia, and teratospermia. Currently, the exact causes of asthenospermia in men are unclear, but it is generally believed to be related to factors such as work stress, environmental pollution, unhealthy lifestyle habits, varicocele, and reproductive tract infections. Among these, sperm motility optimization and semen freezing are common treatments for assisted reproduction and fertility preservation in patients with asthenospermia.

[0003] This study, based on a clinical cohort and using high-performance liquid chromatography-mass spectrometry (Thermo; company: Suzhou Panomick), analyzed the differences in metabolite content in the seminal plasma of patients with asthenospermia and normal men. The results showed a significant decrease in pyridoxamine (PM) levels in the seminal plasma of the asthenospermia group. Pyridoxamine is a natural component of vitamin B6, widely found in animals. Studies have reported that pyridoxamine can inhibit glycosylation and the formation of advanced glycation end products (AGEs), showing therapeutic potential for various multifactorial chronic diseases caused by oxidative stress and carbonyl compounds. Oxidative stress damages sperm cell membranes, leading to sperm apoptosis and asthenospermia, suggesting that pyridoxamine can reduce oxidative stress in sperm and improve sperm motility. However, currently, there are no commercially available semen-optimizing drugs targeting asthenospermia with pyridoxamine as the therapeutic target.

[0004] In clinical assisted reproductive technology (ART), patients with asthenospermia who undergo sperm optimization and seminal cryopreservation / thawing treatment often experience lower sperm motility, cryopreservation / thawing rates, and in vitro fertilization rates compared to the general population. This may be related to the deficiency of active components in the seminal plasma of asthenospermia patients. Some commercially available sperm optimization and cryopreservation / thawing products improve sperm motility by adding commonly used antioxidants, such as enzymatic antioxidants like superoxide dismutase (SOD) and non-enzymatic antioxidants like glutathione and resveratrol. However, these substances lack specificity for treating asthenospermia. Furthermore, adding antioxidants other than those found in seminal plasma to sperm handling and freezing solutions also poses potential safety risks. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention provides the following technical solution:

[0006] Pyridoxine is used in the following applications:

[0007] Improve the ratio of forward-moving sperm in patients with asthenospermia.

[0008] Pyridoxine is used in the following applications:

[0009] Improve the ratio of forward-moving, motile sperm after semen cryopreservation and thawing in patients with asthenospermia.

[0010] Pyridoxine is used in the following applications:

[0011] Improve sperm DNA fragmentation rate after semen cryopreservation and thawing in patients with asthenospermia.

[0012] Pyridoxine is used in the following applications:

[0013] Improve the acrosomal enzyme content of sperm after cryopreservation and thawing in patients with asthenospermia.

[0014] An additive for a preferred and / or cryopreservation solution for asthenospermia, the additive comprising pyridoxine.

[0015] Furthermore, the concentration of the pyridoxine is 200 μmol / L.

[0016] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0017] This invention adds pyridoxine to sperm manipulation fluid or semen freezing solution, effectively improving the ratio of forward-moving sperm in patients with asthenospermia; improving the ratio of forward-moving sperm after semen cryopreservation and thawing; improving sperm DNA fragmentation rate after semen cryopreservation and thawing; and improving sperm acrosomal enzyme content after semen cryopreservation and thawing. In summary, pyridoxine can be added to sperm manipulation fluid or semen freezing solution as a natural semen protectant, significantly improving the sperm selection and cryopreservation effects in patients with asthenospermia, and is suitable for use in assisted reproductive treatment and fertility preservation for patients with asthenospermia. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figures 1-4 This is a schematic diagram illustrating the enrichment of pyridoxine expression levels in the seminal plasma of patients with asthenospermia, provided in an embodiment of the present invention. Figures 1-4 These include: statistical information on different expressed metabolites; a heatmap comparing the metabolic rates of metabolites; a schematic diagram illustrating the effects of differentially expressed metabolites on amino acid metabolism pathways; and a comparison of the logarithmic intensity of pyridoxine expression levels in the seminal plasma of patients with asthenospermia.

[0020] Figure 5 This is a schematic diagram illustrating the effect of pyridoxine on sperm forward motility and DNA integrity, provided in an embodiment of the present invention.

[0021] Figure 6 This is a schematic diagram illustrating the effect of pyridoxine on the forward motility and DNA integrity of sperm after semen cryopreservation and thawing, provided by an embodiment of the present invention.

[0022] Figure 7 This is a schematic diagram illustrating the effect of pyridoxine on the fertilization capacity of sperm after semen cryopreservation and thawing, provided in an embodiment of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0024] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0025] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning.

[0026] In this embodiment of the invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meaning they express is the same.

[0027] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0028] The reagents or equipment used in this embodiment can be provided by the laboratory or purchased from the market.

[0029] This invention adds a certain amount of pyridoxine to sperm handling fluid or semen freezing fluid, which can effectively improve sperm motility (dynamics). Pyridoxine can be added to sperm handling fluid or semen freezing fluid as a natural semen protectant, which significantly improves the sperm selection and cryopreservation effects in patients with asthenospermia. It is suitable for use in assisted reproductive treatment and fertility preservation for patients with asthenospermia.

[0030] The following examples and experiments will be provided for illustration.

[0031] Example 1: The expression level of pyridoxine in the seminal plasma of patients with asthenospermia was significantly reduced.

[0032] Methods: Twenty patients with asthenospermia and 20 healthy male controls were recruited clinically (there were no significant differences in age and BMI between the healthy and asthenospermia groups). After abstinence for 2 to 7 days, semen samples were collected by masturbation into sterile containers. The semen samples were centrifuged (3000 rpm, 10 minutes, 4°C). The supernatant seminal plasma was then transferred to centrifuge tubes.

[0033] Non-targeted metabolomics analysis of seminal plasma was performed using high performance liquid chromatography-mass spectrometry (Suzhou Panomics).

[0034] The detection principle is as follows: High performance liquid chromatography-mass spectrometry (HPLC-MS) is used to perform non-targeted metabolomics detection in seminal plasma. It combines the separation capability of HPLC with the detection capability of mass spectrometry to comprehensively analyze metabolites in seminal plasma.

[0035] 1. Principle of high performance liquid chromatography (HPLC) separation

[0036] High-performance liquid chromatography (HPLC) separates different metabolites based on the differences in their partition coefficients between the stationary and mobile phases. Various metabolites in seminal plasma samples possess different chemical properties, such as polarity, molecular size, and charge. When the sample is injected into the HPLC system, as the mobile phase (solvent) flows, the metabolites continuously partition between the stationary phase (column packing material) and the mobile phase. More polar metabolites interact weakly with the stationary phase, resulting in shorter retention times and earlier elution; conversely, less polar metabolites interact strongly with the stationary phase, leading to longer retention times and later elution. Thus, the various metabolites in seminal plasma are separated into different peaks over time according to their differences in chemical properties.

[0037] 2. Mass spectrometry detection principle

[0038] Mass spectrometry analyzes the structure and composition of substances by measuring the mass and relative abundance of ions. Metabolites separated from high-performance liquid chromatography (HPLC) enter a mass spectrometer and are first ionized, converting neutral metabolite molecules into charged ions. Various ionization methods exist, such as electrospray ionization (ESI), which ionizes metabolites under mild conditions while preserving their molecular structure. The ionized metabolite ions are then deflected under electric and magnetic fields, the degree of deflection depending on the ion's mass-to-charge ratio (m / z). By detecting the ion's mass-to-charge ratio and relative abundance, a mass spectrum of the metabolite can be obtained. Each metabolite has a characteristic mass spectrum, like a fingerprint; by comparing it with a mass spectrometry database of known metabolites, the types of metabolites in seminal plasma can be identified.

[0039] Non-targeted metabolomics detection principle

[0040] The goal of untargeted metabolomics is to detect all metabolites in seminal plasma as comprehensively as possible, without pre-setting specific metabolites to be detected. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) allows for the unbiased separation and detection of metabolites in seminal plasma. During the detection process, the chromatographic peaks and mass spectrometric 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 identified, thereby identifying metabolic biomarkers related to physiological states, diseases, etc.

[0041] 3. Testing Steps

[0042] (1) Sample collection and preprocessing

[0043] Sample Collection: Semen samples are collected using professional semen collection containers and following standard semen collection methods. Abstinence for 2-7 days is generally required to ensure sample quality and stability. Collected semen samples should be sent to the laboratory for processing within 30 minutes.

[0044] Liquefaction process: The collected semen sample is incubated at 37°C to allow it to liquefy naturally. The liquefaction time is generally 15-60 minutes, but the specific time varies depending on individual differences.

[0045] Centrifugation: The liquefied semen sample is centrifuged under specific conditions (e.g., 3000-5000 rpm, 10-15 minutes) to separate the seminal plasma from sperm and other components. The supernatant is collected, which is the seminal plasma sample.

[0046] Filtration and Preservation: The seminal plasma sample is filtered through a 0.22 μm or 0.45 μm filter membrane to remove impurities and particles. The filtered seminal plasma sample can be stored at -80℃ for subsequent testing.

[0047] (2) High performance liquid chromatography separation

[0048] Column selection: Choose a suitable chromatographic column based on the properties of the metabolites in seminal plasma. Commonly used columns include reversed-phase columns (such as C18 columns) and normal-phase columns. Reversed-phase columns are suitable for separating polar and moderately polar metabolites, while normal-phase columns are suitable for separating highly polar metabolites.

[0049] Mobile phase preparation: The mobile phase generally consists of an organic solvent (such as methanol, acetonitrile, etc.) and water, with a certain proportion of buffer salts (such as ammonium formate, ammonium acetate, etc.) added to adjust the pH value and improve the separation effect. A suitable mobile phase gradient is prepared according to the properties of the metabolites and the requirements of the chromatographic column. For example, linear gradient elution can be used, gradually increasing the proportion of organic solvent from low to high to achieve the separation of metabolites of different polarities.

[0050] Sample injection: The pretreated plasma sample is injected into the injector of the high-performance liquid chromatography (HPLC) system, with an injection volume typically ranging from 1 to 10 μL. After injection, the sample enters the chromatographic column along with the mobile phase for separation.

[0051] Chromatographic separation process: Under certain flow rate (e.g., 0.2 - 1 mL / min) and column temperature (e.g., 25 - 40℃), metabolites in seminal plasma are separated in a chromatographic column. Chromatograms of the metabolites are obtained by monitoring the absorbance or other physical properties of the column effluent.

[0052] (3) Mass spectrometry detection

[0053] Ion source selection and parameter settings: Based on the properties of the metabolites and detection requirements, select a suitable ion source, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). For most metabolites in biological samples, ESI is the most commonly used ion source. Set the ion source parameters, such as spray voltage, capillary temperature, and nebulizer gas flow rate, to ensure effective ionization of the metabolites.

[0054] Mass spectrometry scanning mode selection: You can choose between full scan mode or ion monitoring (SIM) mode. Full scan mode can detect all ionized metabolites and is suitable for non-targeted metabolomics detection; while SIM mode only monitors ions with a specific mass-to-charge ratio and is suitable for quantitative analysis.

[0055] Data Acquisition: After the metabolites elute from the chromatographic column and enter the mass spectrometer, the mass spectrometer acquires data according to the set scanning mode and parameters. Mass spectra are recorded at each time point to obtain the mass spectrometric data of the seminal plasma metabolites.

[0056] (4) Data processing and analysis

[0057] Data preprocessing: The acquired mass spectrometry data are preprocessed, including baseline correction, peak identification, and peak alignment. Baseline correction removes background noise and improves data quality; peak identification determines the peak position and peak area in the chromatogram; peak alignment aligns peaks of the same metabolite from different samples for subsequent comparative analysis.

[0058] Metabolite identification: The preprocessed data is compared with mass spectrometry databases of known metabolites, such as HMDB (Human Metabolomics Database) and METLIN. By comparing information such as mass-to-charge ratio and retention time, the types of metabolites in seminal plasma are identified. For metabolites that cannot be matched in the database, further structural analysis methods (such as tandem mass spectrometry) can be used for identification.

[0059] Statistical analysis: Statistical analysis is performed on metabolite data from different samples, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). These analytical methods can help discover differences in metabolites between different samples and identify potential metabolic biomarkers.

[0060] Bioinformatics analysis: Utilizing bioinformatics tools, functional annotation and metabolic pathway analysis are performed on identified metabolites. Understanding the biological processes and metabolic pathways involved by these metabolites leads to a deeper understanding of the relationship between changes in the seminal plasma metabolome and physiological states, diseases, etc.

[0061] 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 upregulated and 28 were downregulated. Figure 1 and Figure 2 KEGG functional analysis showed that differentially metabolites were enriched in amino acid metabolism, protein digestion and absorption, and lipid metabolism. Figure 3 Pyridoxine expression levels were significantly reduced in the seminal plasma of patients with asthenospermia. Figure 4 The suggestion is that pyridoxine can be used as a natural semen-activating substance as an additive in semen preparation solutions for patients with asthenospermia to improve the clinical efficacy of semen optimization and semen freezing.

[0062] KEGG (Kyoto Encyclopedia of Genes and Genomes) functional analysis is an important method for systematically analyzing gene function, genomic information, and biomolecular networks. It reveals the biological functions and metabolic processes involved by mapping differentially expressed metabolites to metabolic pathways in the KEGG database. Its basic process and principles are as follows:

[0063] 1. Data Collection and Processing: Before performing KEGG functional analysis, it is necessary to obtain relevant data on metabolites in the sample. This typically involves using advanced metabolomics techniques, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), to detect the sample and obtain information such as the types and amounts of metabolites. These raw data are then standardized to eliminate differences caused by experimental conditions, instrument errors, and other factors, for subsequent analysis.

[0064] 2. Screening for Differential Metabolites: Statistical methods, such as t-tests and analysis of variance, are used to compare the differences in metabolite content between different sample groups. Appropriate thresholds (such as p-values, fold changes, etc.) are set to screen for metabolites that show significant differences between different groups. These differential metabolites may be related to different physiological states, disease states, or external stimuli in the samples.

[0065] 3. Pathway Mapping: The screened differentially identified metabolites are compared with metabolic pathways in the KEGG database. The KEGG database is a comprehensive database integrating various biological information, including genes, metabolites, and metabolic pathways. Its metabolic pathway maps depict detailed chemical reactions and interactions between various biomolecules. By mapping differentially identified metabolites onto these pathway maps, the biological processes they participate in can be intuitively understood.

[0066] 4. Enrichment Analysis: To determine which metabolic pathways a differentially metabolite is significantly enriched in, enrichment analysis is required. Commonly used methods include the hypergeometric test or Fisher's exact test. These methods calculate the difference between the frequency of the differentially metabolite in each pathway and its 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 typically set; when the p-value for enrichment in a pathway is less than this threshold, the differentially metabolite is considered significantly enriched in that pathway.

[0067] The metabolic enrichment in various aspects is explained as follows:

[0068] (1) Enrichment of differential metabolites in amino acid metabolism

[0069] Amino Acid Synthesis and Catabolism: Amino acids are the basic building blocks of proteins and also participate in many important biochemical reactions. The enrichment of differentially metabolites in amino acid metabolic pathways may indicate that these metabolites are involved in amino acid synthesis or degradation. For example, some differentially metabolites may be substrates or products of key enzymes in amino acid synthesis pathways, and changes in their levels may affect the rate of amino acid synthesis. Conversely, in amino acid catabolism, some differentially metabolites may be amino acid degradation products, and changes in their levels reflect the catabolistic state of the amino acid.

[0070] Amino Acid Metabolism and Physiological Function: 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 accumulate in branched-chain amino acid metabolic pathways, it may indicate changes in the body's energy metabolism or insulin signaling pathway. Furthermore, some amino acids, such as tryptophan and tyrosine, participate in the synthesis of neurotransmitters, and abnormalities in their metabolism may affect the function of the nervous system.

[0071] (2) Accumulation of differential metabolites in protein digestion and absorption

[0072] The process of protein digestion: In the gastrointestinal tract, proteins are broken down into amino acids and small peptides by various digestive enzymes, including pepsin, trypsin, and chymotrypsin. The accumulation of differential metabolites in the protein digestion and absorption pathway may be related to the activity or secretion of digestive enzymes. For example, some differential metabolites may be activators or inhibitors of digestive enzymes, and changes in their levels can affect the efficiency of protein digestion. Furthermore, some intermediate products generated during digestion, such as oligopeptides, may also be differential metabolites, and changes in their levels reflect the degree of protein digestion.

[0073] Absorption of Amino Acids and Small Peptides: Digested amino acids and small peptides are absorbed into the bloodstream via transport proteins in intestinal epithelial cells. The accumulation of differential metabolites in the protein digestion and absorption pathway may also be related to the function of transport proteins. Some differential metabolites may affect the expression or activity of transport proteins, thereby affecting the absorption efficiency of amino acids and small peptides. For example, certain nutrients or drugs may affect protein absorption by modulating the function of transport proteins, and the metabolites of these substances may be detected as differential metabolites.

[0074] (3) Accumulation of differential metabolites in lipid metabolism

[0075] Lipid Synthesis and Catabolism: Lipids are essential components of organisms, including fats, phospholipids, and cholesterol. The accumulation of differentially metabolites in lipid metabolic pathways may involve both lipid synthesis and breakdown. In lipid synthesis, some differentially metabolites may be substrates or products of fatty acid synthases; changes in their levels affect the rate of fatty acid synthesis, and consequently, the synthesis of fats and phospholipids. In lipid breakdown, β-oxidation of fatty acids is the primary pathway, and differentially metabolites may participate in key steps of this process, such as fatty acid activation and transport.

[0076] Lipid Metabolism and Disease: Abnormalities in lipid metabolism are closely related to many diseases such as atherosclerosis and hyperlipidemia. The accumulation of differentially metabolites in lipid metabolism pathways may indicate an alteration in the body's lipid metabolism balance. For example, abnormal cholesterol metabolism leads to elevated blood cholesterol levels, increasing the risk of atherosclerosis. Differentially metabolites may reflect abnormalities in cholesterol synthesis, transport, or excretion, providing important clues for disease diagnosis and treatment.

[0077] Example 2: The correlation between pyridoxine in seminal plasma and outcomes such as sperm forward motility and DNA integrity.

[0078] Method: Pyridoxine was added to the semen processing solution (200 μmol / L, FUJIFILM Irvine Scientific brand), and mixed with an equal volume of semen. The mixture was incubated at room temperature for about 90 minutes. After incubation, the sample was mixed again and the forward motility of the sperm was evaluated.

[0079] Compared with the group without pyridoxine, the addition of pyridoxine significantly increased the proportion of progressively motile sperm in patients with asthenospermia (p=0.051). However, the addition of pyridoxine had no effect on the proportion of progressively motile sperm in normal men (p=0.0956). Figure 5 (a) and (b) in the text). Additionally, the DNA fragmentation rate of sperm before and after incubation was examined, and it was found that adding pyridoxine for 90 minutes had no effect on the DFI level of sperm in patients with asthenospermia and normal men (p=0.1205; p=0.8534). Figure 5 (c) and (d) in the text.

[0080] The results showed that adding pyridoxine to the sperm manipulation fluid of patients with asthenospermia could improve sperm forward motility.

[0081] Example 3: The correlation between pyridoxine in seminal plasma and sperm forward motility and DNA integrity after cryopreservation and thawing.

[0082] Methods: Pyridoxine was added to the semen cryopreservation solution (200 μmol / L, ORIGIO brand), mixed with an equal volume of semen, and incubated at room temperature for approximately 30 minutes, followed by routine semen freezing. Sperm were thawed after 48 hours. Results showed that the group with added pyridoxine significantly increased the proportion of progressively motile sperm in thawed sperm compared to the group without added pyridoxine (p=0.0064; p=0.0097); and the difference was significantly higher in asthenospermia patients than in normal men. Figure 6 (a) and (b) in the text). Compared with the group without pyridoxine in the cryoprotectant, the sperm DNA fragmentation rate of patients with asthenospermia was significantly reduced after recovery (p=0.0322), while the DNA fragmentation rate of normal male patients also decreased, but the difference was not statistically significant (p=0.0689). Figure 6 (c) and (d) in the text.

[0083] The results showed that adding pyridoxine to the cryopreservation solution during assisted reproductive treatment for patients with asthenospermia could improve the forward motility of sperm after thawing.

[0084] Example 4: Correlation between pyridoxine in seminal plasma and sperm fertilization capacity after cryopreservation and thawing.

[0085] Methods: Pyridoxine was added to the semen cryopreservation solution (200 μmol / L, ORIGIO brand), mixed with an equal volume of semen, and incubated at room temperature for approximately 30 minutes, followed by routine semen freezing. Sperm were thawed after 48 hours. Results showed that the pyridoxine-added group significantly increased the acrosomal enzyme content in thawed sperm compared to the group without pyridoxine (p=0.0262; p=0.0316); and the improvement was significantly greater in asthenospermia patients than in normal men. Figure 7 ).

[0086] The results showed that adding pyridoxine to the cryopreservation solution during assisted reproductive treatment for patients with asthenospermia could increase the acrosomal enzyme content of sperm after thawing.

[0087] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of pyridoxine in the preparation of compositions for the in vitro preferred treatment of sperm in patients with asthenospermia.

2. Application of pyridoxine in the preparation of compositions for cryopreservation and thawing of sperm in patients with asthenospermia.

3. The application according to claim 1 or 2, characterized in that, The composition is used to improve the forward motility ratio of sperm in patients with asthenospermia.

4. The application according to claim 2, characterized in that, The composition is used to reduce the DNA fragmentation rate after cryopreservation and thawing of sperm with asthenospermia.

5. The application according to claim 2, characterized in that, The composition is used to increase the acrosomal enzyme content of sperm frozen and thawed due to asthenospermia.

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