In-vitro culture solution for improving sperm motility of asthenospermia and application of in-vitro culture solution

Abstract

The invention relates to an in-vitro culture solution for enhancing the motility of human sperms and application of the in-vitro culture solution. The culture solution contains 3-phosphoglyceric acid (3-PG). The culture solution can be used for culturing purified asthenospermia sperms under an in-vitro condition, and the forward movement capability and the total activity of the sperms are remarkably improved. The 3-PG in the culture solution is directly diluted and cultured by adopting the sperm culture solution, so that the possible toxic influence of organic solvents such as ethanol or DMSO (Dimethylsulfoxide) on sperms is avoided. Experimental results show that the sperm motility of clinical asthenospermia patients can be obviously enhanced by adding the 3-PG into the culture solution, the enhancement effect on moderate and severe asthenospermia is particularly obvious, and the sperm motility of most mild asthenospermia patients is recovered to a normal range. It is fully proved that 3-PG has the effect of effectively improving the sperm motility function of a patient with asthenospermia in in-vitro culture.

Description

TECHNICAL FIELD

[0001] The present application relates to a sperm culture medium, in particular to a culture medium for improving the sperm motility of asthenospermia in vitro and application thereof. BACKGROUND

[0002] The incidence of infertility has been increasing in recent years. According to the report of the World Health Organization, the global infertility rate has reached 17.5%, of which about 50% of the infertility cases are related to male factors. Male infertility is often manifested in clinical practice as a decrease in sperm count, a decrease in motility and morphological abnormalities. Asthenospermia is one of the main causes of male infertility, accounting for about 81% of male infertility cases, and its diagnostic criteria is that the proportion of sperm forward movement is less than 32% set by the World Health Organization. Globally, the quality of male semen has generally declined, and the prevalence of asthenospermia has increased significantly. Improving sperm motility is not only a key point for the treatment of male infertility, but also has crucial clinical significance for improving the overall fertility outcome of couples of childbearing age.

[0003] Normal sperm exists in seminal plasma, and its energy is mainly derived from fructose. The culture medium commonly used in clinical practice (such as HTF) uses glucose as the energy substrate. Mature sperm produces energy through glycolysis and mitochondrial oxidative phosphorylation. Due to the special structure of sperm, its motility function is highly dependent on glycolysis for energy. The sperm of asthenospermia patients often has problems of insufficient glycolytic flux or decreased metabolic enzyme activity, leading to energy supply shortage and thus low sperm motility.

[0004] The current clinical treatment methods for asthenospermia include lifestyle intervention, antioxidant therapy, hormone therapy, traditional Chinese medicine conditioning, and assisted reproductive technology (such as intrauterine insemination, in vitro fertilization-single sperm injection, etc.). These methods can improve sperm motility and increase the chances of pregnancy to some extent, but still have obvious limitations. For example, drug treatment has large individual differences, long cycle and side effects that cannot be ignored; assisted reproductive technology can bypass natural fertilization barriers, but has high cost, complex operation and certain ethical and psychological pressure. Therefore, the development of a new type of in vitro culture medium that can specifically improve the energy metabolism of asthenospermia sperm and safely and effectively improve its motility has important clinical significance for improving the success rate of assisted reproduction for asthenospermia patients. SUMMARY

[0005] In view of the above technical problems, the present application provides an in vitro culture medium for improving the sperm motility of asthenospermia and application thereof.

[0006] The purpose of the present application can be achieved by the following technical solutions:

[0007] An in-vitro culture solution for improving sperm motility of asthenospermia, wherein the in-vitro culture solution contains 3-phosphoglycolate.

[0008] In the technical solution, the concentration of 3-phosphoglycolate in the sperm culture solution is 0.1-5 mM. In some preferred technical solutions, the concentration of 3-phosphoglycolate in the sperm culture solution is 0.2-2 mM.

[0009] In the technical solution, the culture time of the in-vitro culture solution is 1-5 hours. In some preferred technical solutions, the culture time is 1-2 hours.

[0010] In the technical solution, the sperm used for culture is human sperm after removal of seminal plasma.

[0011] In the technical solution, the in-vitro culture solution is used for improving sperm motility of asthenospermia.

[0012] In the technical solution, the in-vitro culture solution is HTF in-vitro fertilization culture solution and modified HTF in-vitro fertilization culture solution.

[0013] Beneficial effects

[0014] 1. In the present application, supplementing 3-phosphoglycolate in the sperm culture solution can enhance the motility of sperm of patients with different degrees of asthenospermia.

[0015] 2. In the present application, supplementing 3-phosphoglycolate in the sperm culture solution can restore the motility of sperm of patients with mild asthenospermia to normal level.

[0016] 3. In the present application, supplementing 3-phosphoglycolate in the sperm culture solution can improve the motility of sperm of patients with severe asthenospermia by 2 times. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Normal sperm lactate proteomics research; illustration: Figure A shows the schematic diagram of lactate proteomics analysis of normal sperm samples, Figure B shows the number of identified sperm lactate proteins and sites, and Figure C is the KEGG pathway enrichment analysis of sperm lactate proteins.

[0018] Figure 2 PGK2 lactate modification and enzyme activity analysis in asthenospermia patients and healthy control group sperm; illustration: Figure A shows the lactate modification site of PGK2, Figure B is the IP experiment verifying the PGK2 lactate modification in asthenospermia sperm, and Figure C is the enzyme activity of PGK2 in asthenospermia sperm.

[0019] Figure 3Figure showing the effect of different concentrations of mHTF-3-PG medium on the sperm motility of mild asthenozoospermia; Figure A shows the sperm progressive motility of mild asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium, and Figure B shows the total sperm motility of mild asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium.

[0020] Figure 4 Figure showing the effect of different concentrations of mHTF-3-PG medium on the sperm motility of moderate asthenozoospermia; Figure A shows the sperm progressive motility of moderate asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium, and Figure B shows the total sperm motility of moderate asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium.

[0021] Figure 5 Figure showing the effect of different concentrations of mHTF-3-PG medium on the sperm motility of severe asthenozoospermia; Figure A shows the sperm progressive motility of severe asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium, and Figure B shows the total sperm motility of severe asthenozoospermia after 1-2 hours of culture in different concentrations of mHTF-3-PG medium. DETAILED DESCRIPTION

[0022] The following examples further illustrate the present application but should not be construed as limiting the application. Modifications and variations of the methods, steps and conditions of the application are possible without departing from the spirit and scope of the application. Unless otherwise indicated, the technical means used in the examples are conventional means known to those skilled in the art. The reagents and materials used in the following examples are commercially available, for example, the modified HTF (mHTF) used in the experiments was purchased from Irvine Scientific, USA, with the item number 90126; and 3-PG was purchased from Sigma-Aldrich, USA, with the item number P7788.

[0023] Example 1 3-PG promotes sperm motility of mild asthenozoospermia

[0024] 1 Normal semen samples were used for the experiments. Patients were asked to abstain (no sexual intercourse, masturbation, and ejaculation) for 2-7 days. Too short abstinence time would affect semen volume and sperm concentration; too long abstinence time would increase the proportion of dead and abnormal sperm. Semen was collected by masturbation and immediately placed in a specific sterile container. The container was immediately sent to the laboratory within 30 minutes at 37°C (e.g., placed in a constant temperature transport box or close to the body). After the sample was liquefied, the computer-aided semen analyzer CASA (IVOS, USA) was used to evaluate the sperm parameters (volume, sperm count, percentage of motility and movement characteristics). Normal sperm was obtained by 60% Percoll solution. After normal sperm was lysed, the protein was reduced, alkylated, trypsin digested, and desalted using OASIS HLB Vac device. The lactic acidified peptide was enriched using agarose beads coupled with anti-L-lactyl lysine antibody (PTM Bio), eluted, dried, desalted, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using Orbitrap Fusion Lumos mass spectrometer. The raw MS / MS data was analyzed using Spectronaut (Biognosys 19.0). The data was searched in the UniProt database. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and protein visualization were performed by ClusterProfiler software, with an annotation threshold of adjusted P value < 0.05.

[0025] 2 Mild asthenospermia samples were used for the experiments. Patients were asked to abstain (no sexual intercourse, masturbation, and ejaculation) for 2-7 days. Too short abstinence time would affect semen volume and sperm concentration; too long abstinence time would increase the proportion of dead and abnormal sperm. Semen was collected by masturbation and immediately placed in a specific sterile container. The container was immediately sent to the laboratory within 30 minutes at 37°C (e.g., placed in a constant temperature transport box or close to the body). After the sample was liquefied, the computer-aided semen analyzer CASA (IVOS, USA) was used to evaluate the sperm parameters (volume, sperm count, percentage of motility and movement characteristics). Asthenospermia was defined as the number of grade a sperm (PR) < 32% or the number of grade a+b sperm (PR+NP) < 40% (World Health Organization, 5th edition, 2010), while all other parameters were normal. The sperm motility parameter of mild asthenospermia was 20% < a < 32%. Sperm was sorted from semen by density gradient centrifugation. In a 15 mL centrifuge tube, 1 mL of 60% (v / v) Percoll separation solution was placed, and an equal volume of semen was slowly added on top, centrifuged at 300g for 10 minutes, and the supernatant was aspirated. The sperm suspension was resuspended in 2 mL of mHTF culture solution, centrifuged at 300g for 5-10 minutes, and the step was repeated once. The sperm suspension was gently transferred to various sperm culture solutions used in the experiment.

[0026] 3 Collection of normal male and oligospermia semen specimens, after separation of sperm, add IP solubilizer (Thermo) to the sperm sample, lyse on ice for 10 minutes, then centrifuge to collect the supernatant. Respectively add 10 pg / mg of PGK2 antibody (Proteintech) and IgG antibody (Bi Yun Tian) solution, and incubate overnight in a 4°C circulating mixer to obtain antigen-antibody complex. Mix the magnetic beads well, take 30 pL and wash with washing buffer. Add the antigen-antibody complex to the magnetic beads and incubate in a 4°C rotating mixer for 2 hours. After 4 washes, add 1x loading buffer and incubate at 95°C for 5 minutes. Separate the magnetic beads and collect the supernatant to obtain the sample. Then add an equal amount of protein sample to a 12% SDS-PAGE gel for electrophoresis, and transfer to a 0.22 pm PVDF membrane (Borel). Block with 5% milk solution at room temperature for 1 hour. Add anti-Pan-Kla and anti-PGK2 antibodies and incubate at 4°C overnight. The next day, incubate the corresponding HRP-conjugated secondary antibody at room temperature for 1 hour. Then develop the protein bands using an ECL chemiluminescence kit (Vazyme) and save the image for analysis.

[0027] 4 Collection of normal male and oligospermia semen specimens, after separation of sperm, use PGK2 activity detection kit to detect sperm PGK2 enzyme activity, follow the kit instructions for test detection. The kit is purchased from Sorlabio Biotechnology Co., Ltd., with the item number BC2250.

[0028] 5 The sorted mild oligospermia sperm sample is divided into five groups: control group, 0.25 mM 3-PG group, 0.5 mM 3-PG group, 1 mM 3-PG group and 2 mM 3-PG group. An appropriate amount of sperm (5 x 10 6 left and right) is transferred to mHTF sperm culture medium, and then placed in a 37°C, 5% CO2 incubator for 1-2 hours.

[0029] The sperm culture medium used is: commercial mHTF culture medium and 3-PG-containing mHTF culture medium, with 3-PG concentrations of 0.25, 0.5, 1 and 2 mM.

[0030] 6 According to the evaluation criteria of the World Health Organization in 2010, the sperm motility is evaluated using a computer-aided semen analyzer CASA (IVOS, USA). For each specimen, 6-10 fields are selected, and the number of counted sperm is not less than 500.

[0031] 7 Results: As Figure 1As shown, a proteomic study of lysine lactation in normal male sperm identified 220 sperm lactation-modified proteins and 557 lactation modification sites. KEGG pathway enrichment analysis of the 220 sperm lactation proteins revealed that these proteins are enriched in pathways such as glycolysis, amino acid biosynthesis, and pyruvate metabolism. The glycolysis pathway was of particular interest, with PGK2 protein being a key enzyme in this pathway. Figure 2 As shown, there are 10 lactation modification sites for PGK2 in sperm. IP assay revealed that the level of PGK2 lactation modification in sperm with asthenospermia was lower than that in normal males. The enzyme activity of PGK2 in normal male sperm was 352.58±0.97 U / mg, while the enzyme activity in sperm with asthenospermia was 31.25±4.72 U / mg, significantly reduced (P<0.0001), a decrease of more than 10-fold. Figure 3 As shown, after culturing sperm with mild asthenospermia in different concentrations of 3-PG (0, 0.25, 0.5, 1 and 2 mM) in mHTF culture medium for 1-2 hours, the forward motility and total vitality of sperm showed significant changes. After 1 hour of culture, the progressive motility of sperm in the control group with mild asthenospermia was 24.64±0.82 (mean ± standard error), and the total motility was 32.73±1.88 (mean ± standard error). In the 1 mM 3-PG-mHTF culture group, the progressive motility of sperm in the control group with mild asthenospermia increased to 30.10±0.70 (P<0.0001), an increase of 1.2 times, and the total motility increased to 39.98±1.49 (P<0.05), an increase of 1.3 times. In the 2 mM 3-PG-mHTF culture group, the progressive motility of sperm in the control group with mild asthenospermia increased to 32.64±1.16 (P<0.0001), an increase of 1.3 times, and the total motility increased to 42.11±1.67 (P<0.001), an increase of 1.3 times. After 2 hours of culture, the progressive motility of sperm in the control group with mild asthenospermia was 23.87±0.77, and the total motility was 32.59±1.89. In the 1mM 3-PG-mHTF culture group, the progressive motility of sperm in the control group with mild asthenospermia increased to 30.13±0.54 (P<0.0001), an increase of 1.3 times, and the total motility increased to 40.13±1.26 (P<0.01), an increase of 1.2 times. In the 2mM 3-PG-mHTF culture group, the progressive motility of sperm in the control group with mild asthenospermia increased to 32.99±1.13 (P<0.0001), an increase of 1.4 times, and the total motility increased to 42.85±1.31 (P<0.001), an increase of 1.3 times.

[0032] Example 2: 3-PG promotes sperm motility in patients with moderate asthenospermia

[0033] 1. Sperm samples from patients with moderate asthenospermia are collected for testing. Patients are required to abstain from ejaculation (no sexual activity, masturbation, or nocturnal emission) for 2-7 days. Too short abstinence time will affect semen volume and sperm concentration; too long abstinence will lead to an increase in the proportion of dead and abnormal sperm. Using the masturbation ejaculation method, the semen is collected in a specific sterile container and immediately transported to the laboratory within 30 minutes at 37°C (e.g., in a constant temperature transport box or placed close to the body). After liquefaction, the sperm parameters (volume, sperm count, percentage of motility and viability) are assessed using a computer-assisted semen analyzer (CASA, IVOS, USA). Asthenospermia is defined as grade a sperm (PR) <32% or grade a+b sperm (PR+NP) <40% (World Health Organization Fifth Edition, 2010), while all other parameters are normal. For moderate asthenospermia, sperm motility parameters are 10% ≤ a < 20%.

[0034] 2. Sperm separation from semen by density gradient centrifugation. Place 1 mL of 60% (v / v) Percoll separation medium in a 15 mL tube, and slowly add an equal volume of semen. Centrifuge at 300 g for 10 minutes, and discard the supernatant. Resuspend the sperm suspension in 2 mL of mHTF medium (commercially modified HTF medium, abbreviated as mHTF), and centrifuge at 300 g for 5-10 minutes. Repeat this step once. Gently transfer the sperm suspension to the various sperm culture media used in the experiment.

[0035] 3. The sorted sperm samples with moderate asthenospermia were divided into five groups: control group, 0.25mM 3-PG group, 0.5mM 3-PG group, 1mM 3-PG group, and 2mM 3-PG group. An appropriate number of sperm (5 × 10⁻⁶) was required. 6 After transferring the sperm (left and right) to mHTF sperm culture medium, place it in an incubator at 37°C and 5% CO2 for 1-2 hours.

[0036] The sperm culture media used were commercial mHTF culture medium and mHTF culture medium containing 3-PG, with 3-PG concentrations of 0.25, 0.5, 1 and 2 mM.

[0037] 4. Sperm motility was assessed using a computer-assisted semen analyzer (CASA, IVOS, USA) according to the 2010 evaluation criteria of the World Health Organization. For each specimen, 6-10 fields of view were selected, and the number of sperm counted was no less than 500.

[0038] 5. Results: Figure 4As shown, the forward motility and total motility of the in vitro cultured moderate oligospermic sperm were significantly changed after 1-2 hours of culture in the mHTF medium with different concentrations of 3-PG (0, 0.25, 0.5, 1 and 2 mM). After 1 hour of culture, the forward motility of the moderate oligospermic sperm in the control group was 12.62 ± 1.11 (mean ± standard error), and the total motility was 22.54 ± 1.49 (mean ± standard error). The forward motility of the moderate oligospermic sperm in the 2 mM 3-PG-mHTF culture group increased to 19.37 ± 2.27 (P < 0.01), which was 1.5 times higher, and the total motility increased to 31.44 ± 2.70 (P < 0.001), which was 1.4 times higher. After 2 hours of culture, the forward motility of the moderate oligospermic sperm in the control group was 12.53 ± 1.05, and the total motility was 22.21 ± 1.44. The forward motility of the moderate oligospermic sperm in the 1 mM 3-PG-mHTF culture group increased to 18.21 ± 1.48 (P < 0.05), which was 1.5 times higher, and the total motility increased to 29.65 ± 1.55 (P < 0.01), which was 1.3 times higher. The forward motility of the moderate oligospermic sperm in the 2 mM 3-PG-mHTF culture group increased to 21.95 ± 2.25 (P < 0.0001), which was 1.8 times higher, and the total motility increased to 34.05 ± 2.28 (P < 0.0001), which was 1.5 times higher.

[0039] Example 3 3-PG promotes severe oligospermic sperm motility

[0040] 1 Severe oligospermic sperm samples were taken for the experiment. Patients were asked to abstain (no sexual intercourse, masturbation and ejaculation) for 2-7 days. Too short abstinence time would affect the semen volume and sperm concentration; too long would increase the proportion of dead and abnormal sperm. Semen was collected by masturbation and immediately sent to the laboratory within 30 minutes at 37°C (such as placed in a constant temperature transport box or close to the body). After the sample was liquefied, the computer-aided semen analyzer CASA (IVOS, USA) was used to evaluate the sperm parameters (volume, sperm count, motility and movement characteristics percentage). Oligospermia was defined as the number of a-grade sperm (PR) <32% or the number of a+b-grade sperm (PR+NP) <40% (World Health Organization, Fifth Edition, 2010), and all other parameters were normal. The sperm motility parameters of severe oligospermia were a <10%.

[0041] 2 Sperm were isolated from semen by density gradient centrifugation. 1 mL of 60% (v / v) Percoll was placed in a 15 mL centrifuge tube, and an equal volume of semen was slowly added on top. The tube was centrifuged at 300 g for 10 min, and the supernatant was aspirated. The sperm pellet was resuspended in 2 mL of mHTF medium (commercially modified HTF medium, mHTF), and centrifuged at 300 g for 5-10 min. This step was repeated once. The sperm suspension was gently transferred to the various sperm culture media used in the experiments.

[0042] 3 The sorted severe asthenozoospermia sperm samples were divided into five groups: control, 0.25 mM 3-PG, 0.5 mM 3-PG, 1 mM 3-PG, and 2 mM 3-PG. An appropriate number of sperm (5 x 10 6 to the right) were transferred to mHTF sperm culture medium, and incubated at 37 °C in a 5% CO2 incubator for 1-2 h.

[0043] The sperm culture media used were: commercial mHTF medium and 3-PG-containing mHTF medium, with 3-PG concentrations of 0.25, 0.5, 1, and 2 mM.

[0044] 4 Sperm motility was evaluated according to the World Health Organization's 2010 evaluation criteria, using a computer-assisted sperm analysis instrument CASA (IVOS, USA). For each sample, 6-10 fields were selected, and the number of sperm counted was not less than 500.

[0045] 5 Results: As shown in Table 1, the sperm motility of the control group was 3.5%, and the sperm motility of the 0.25 mM 3-PG group was 5.2%, which was significantly higher than that of the control group (P < 0.05). The sperm motility of the 0.5 mM 3-PG group was 6.3%, which was significantly higher than that of the control group (P < 0.05). The sperm motility of the 1 mM 3-PG group was 7.2%, which was significantly higher than that of the control group (P < 0.05). The sperm motility of the 2 mM 3-PG group was 8.1%, which was significantly higher than that of the control group (P < 0.05). Figure 5As shown, after 1-2 hours of incubation in mHTF medium with different concentrations of 3-PG (0, 0.25, 0.5, 1 and 2 mM), the sperm forward motility and total motility of severe oligospermia sperm in vitro were improved. After 1 hour of incubation, the forward motility of the control group of severe oligospermia sperm was 8.30 ± 0.17 (mean ± standard error), and the total motility was 15.70 ± 0.58 (mean ± standard error). The forward motility of the severe oligospermia sperm in the 1 mM 3-PG-mHTF culture group increased to 11.82 ± 0.41 (P < 0.0001), an increase of 1.4 times, and the total motility increased to 18.64 ± 0.81 (P < 0.05), an increase of 1.2 times. The forward motility of the severe oligospermia sperm in the 2 mM 3-PG-mHTF culture group increased to 14.30 ± 0.31 (P < 0.0001), an increase of 1.7 times, and the total motility increased to 20.94 ± 0.86 (P < 0.0001), an increase of 1.3 times. After 2 hours of incubation, the forward motility of the control group of severe oligospermia sperm was 7.97 ± 0.67, and the total motility was 15.73 ± 0.62. The forward motility of the severe oligospermia sperm in the 1 mM 3-PG-mHTF culture group increased to 15.13 ± 0.51 (P < 0.0001), an increase of 1.9 times, and the total motility increased to 21.19 ± 1.04 (P < 0.0001), an increase of 1.3 times. The forward motility of the severe oligospermia sperm in the 2 mM 3-PG-mHTF culture group increased to 18.40 ± 0.99 (P < 0.0001), an increase of 2.3 times, and the total motility increased to 26.38 ± 1.53 (P < 0.0001), an increase of 1.7 times.

Claims

1. An in vitro culture medium for improving sperm motility in patients with asthenospermia, characterized in that, The in vitro culture medium contains 3-phosphoglycerate.

2. The in-vitro culture solution for improving sperm motility in asthenozoospermia according to claim 1, characterized by The concentration of 3-phosphoglycerate in the sperm culture medium is 0.1-5 mM.

3. The in-vitro culture solution for improving sperm motility in asthenozoospermia according to claim 2, characterized by The concentration of 3-phosphoglycerate in the sperm culture medium is 0.2-2 mM.

4. The in vitro culture solution for improving sperm motility in asthenozoospermia according to claim 1 or 2, characterized by The culture time is 1-5 hours.

5. The in vitro culture solution for improving sperm motility in asthenozoospermia according to claim 4, characterized in that The culture time is 1-2 hours.

6. The in-vitro culture solution for improving sperm motility in asthenozoospermia according to claim 1, wherein The sperm used is human sperm after removal of seminal plasma in vitro.

7. The in-vitro culture solution for improving sperm motility in asthenozoospermia according to claim 1, wherein The in vitro culture medium is HTF in vitro fertilization culture medium and modified HTF in vitro fertilization culture medium.

8. Use of the in vitro culture medium of claim 1 in improving the motility of sperm in asthenospermia.

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