CN113041261B - Method for improving oligospermia sperm function by using exosomes - Google Patents
Method for improving oligospermia sperm function by using exosomes Download PDFInfo
- Publication number
- CN113041261B CN113041261B CN202110280768.9A CN202110280768A CN113041261B CN 113041261 B CN113041261 B CN 113041261B CN 202110280768 A CN202110280768 A CN 202110280768A CN 113041261 B CN113041261 B CN 113041261B
- Authority
- CN
- China
- Prior art keywords
- exosomes
- sperm
- semen
- asthenospermia
- asthenozoospermia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 15
- 206010003883 azoospermia Diseases 0.000 title abstract description 8
- 208000008634 oligospermia Diseases 0.000 title abstract description 8
- 230000036616 oligospermia Effects 0.000 title abstract description 8
- 231100000528 oligospermia Toxicity 0.000 title abstract description 8
- 206010067162 Asthenospermia Diseases 0.000 claims abstract description 31
- 210000000582 semen Anatomy 0.000 claims abstract description 31
- 230000006870 function Effects 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 8
- 230000019100 sperm motility Effects 0.000 claims abstract description 7
- 230000035558 fertility Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 19
- 208000007799 Asthenozoospermia Diseases 0.000 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 9
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 230000033001 locomotion Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 231100000527 sperm abnormality Toxicity 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 230000004720 fertilization Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000007781 pre-processing Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 6
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000005002 female reproductive tract Anatomy 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 208000007466 Male Infertility Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 208000000509 infertility Diseases 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 230000009194 climbing Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 208000021267 infertility disease Diseases 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 235000010981 methylcellulose Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 229960003387 progesterone Drugs 0.000 description 2
- 239000000186 progesterone Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 206010021929 Infertility male Diseases 0.000 description 1
- 244000258044 Solanum gilo Species 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000007488 abnormal function Effects 0.000 description 1
- 230000030120 acrosome reaction Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000032692 embryo implantation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000037417 hyperactivation Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000510 mucolytic effect Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Reproductive Health (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Pregnancy & Childbirth (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gynecology & Obstetrics (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for improving the sperm function of asthenospermia by using exosomes, belonging to the technical field of medicine. The method comprises the following steps: separating normal human seminal plasma exosomes, screening and preprocessing asthenospermia sperms, and intervening the exosomes on the asthenospermia spermia. The invention improves the sperm motility and other functions by utilizing the seminal plasma exosome from the human body to interfere with sperm in vitro, so as to interfere with and treat oligospermia by simple non-invasive means such as artificial fertilization and the like, and improve the fertility outcome of patients.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a method for improving the functions of asthenospermia sperms by using exosomes.
Background
Male reproductive health has become a focus of close attention in today's human society. Based on WHO statistics, the sterility rate of the couples of the worldwide childbearing age is 10-15%, wherein the sterility caused by factors of men accounts for about half. Sperm quality problems are a major cause of male infertility. Epidemic investigation has found that asthenozoospermia has become a major factor in causing male infertility and has an increasing trend year by year. Asthenozoospermia refers to a type of infertility caused by a percentage of sperm forward movement in semen below 32% (WHO fifth edition). Meanwhile, weak sperm not only has reduced forward movement ability, but also is accompanied by abnormal functions of other sperm, such as capacitation, super-activation, acrosome reaction and the like, and the comprehensive factors lead to male sterility. However, the pathogenesis is not clear due to the complex etiology of the asthenozoospermia, and research models and means are relatively deficient, so that effective treatment means are not available at present. Although offspring can be produced by abnormal conception means (e.g. egg cytoplasmatic single sperm microinjection technique, genetic diagnosis/screening prior to embryo implantation), there is still a major risk to the health of the test tube infant. Therefore, the conventional treatment of asthenozoospermia patients makes the birth of the asthenozoospermia patients by natural means or by less invasive artificial fertilization means of great significance for human health.
Semen is composed of cells (sperm cells) and non-cellular components (seminal plasma). The seminal plasma is rich in various substances required by sperm vital activities such as lipid, sugar, growth factors, transcription factors, proteins and the like, can provide stable and comfortable environment for sperm survival and movement, can also be used as a carrier for transporting sperm to the fertilization part of female genital tract, and has several important functions: providing nutrition for sperm transport; the sperm can be protected and prevented from being infected by harmful substances; so that the sperm can overcome the chemical and immunological environment of the hostile vagina. Studies have shown that seminal plasma also contains a large number of exosomes which are excreted with the prostatic secretions during ejaculation and are involved in the maturation and functional regulation of sperm cells, and therefore exosomes have great potential in the treatment of asthenozoospermia.
At present, development of a method for treating oligospermia with small toxic and side effects and convenient clinical application is urgently needed to improve sperm function, so that a patient can naturally give birth after intervention or can give birth by using an artificial fertilization method with small wound.
Disclosure of Invention
Aiming at the technical problems that the conventional treatment means of the current asthenozoospermia are limited in clinic and most of the asthenospermia with unknown etiology do not have very effective treatment means, the invention provides a method for improving the sperm function of the asthenospermia by using exosomes.
In order to achieve the above object, the present invention adopts the following technical means:
a method for improving oligospermia sperm function using exosomes, comprising the steps of:
step 1, separation of normal human seminal plasma exosomes: collecting normal fertility semen, centrifuging 12000 g for 10 min, removing cells and impurities, sequentially filtering the seminal plasma with microporous filter membranes of 0.45 μm and 0.22 μm, removing cell debris and macromolecular aggregate impurities, adding 2.5 mL supernatant into 300 μl of sucrose pad solution containing 20 mM Tris, 30 wt% sucrose and deuterium oxide, ultracentrifugating at 100000 and 4 ℃ for 90 min, collecting supernatant, ultracentrifugating at 100000 g and 4 ℃ in sucrose pad solution containing 20 mM Tris, 25 wt% sucrose and deuterium oxide, mixing exosomes obtained by the two centrifugation, washing with HS solution once, dissolving the obtained exosomes with HS solution, adjusting 10 times of physiological concentration, and preserving at-80 ℃ for standby;
step 2, screening and pretreatment of asthenozoospermia spermia: liquefying a patient semen sample collected on the same day in a constant-temperature water bath at 37 ℃ for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human semen by using a computer-aided semen analyzer, screening samples meeting the requirements on diagnostic parameters of asthenozoospermia, and staining samples for eliminating obvious abnormal sperm morphology;
step 3, intervention of exosomes on asthenospermia sperms: washing asthenospermia with HS twice, 1000 g min each time, 5 min each time, adding 90% of HS solution or HTF solution, re-suspending sperm, adding 10% of exosomes, and incubating at 37deg.C for 10 min.
Further, the sample satisfying the diagnostic parameter requirements for asthenozoospermia in step 2 is a sample with sperm motility < 32%.
The invention improves the sperm motility and other functions by utilizing the seminal plasma exosome from the human body to interfere with sperm in vitro, so as to interfere with and treat oligospermia by simple non-invasive means such as artificial fertilization and the like, and improve the fertility outcome of patients.
Drawings
Figure 1 shows the result of the enhancement of the forward motor capacity of the asthenospermia sperm by the exosomes. Wherein: HS is solvent control group, N-exosome is normal exosome, A-exosome is exosome from oligospermia, HSA is human serum albumin positive control.
FIG. 2 shows the effect of exosomes on sperm hyperactivation ability. Wherein: HTF is the solvent control group and exosomes are normal exosomes.
FIG. 3 shows the effect of exosomes on sperm climbing capacity. Wherein: HTF is solvent control group, N-exosome is normal exosome, A-exosome is exosome from oligospermia, and P4 is progesterone.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
The formula of the HS solution adopted in the invention is as follows: 135 mM NaCl,5 mM KCl,1 mM MgSO 4 ，2 mM CaCl 2 20 mM HEPES,5 mM sucrose, 10 mM lactic acid, 1 mM sodium pyruvate, pH was adjusted to 7.4 using NaOH.
The HTF solution formulation was: 93.8 mM NaCl,4.69 mM KCl,0.2 mM MgSO 4 ，0.37 mM KH 2 PO 4 ，2.04 mM CaCl 2 ，21.4 mM lactic acid，2.78 mM glucose，21 mM HEPES，4 mM NaHCO 3 The pH was adjusted to 7.4 and 330 mM Na-Pyruvate (1000:1), HSA (25:1), 21 mM NaHCO were added prior to use 3 。
Example 1
Step 1, separation of normal human seminal plasma exosomes
The specific separation method is as follows: collecting semen of normal fertile volunteers, centrifuging 12000 g for 10 min, and removing cells such as sperms and other impurities; sequentially filtering the refined pulp with microporous filter membranes of 0.45 μm and 0.22 μm, and removing cell fragments, macromolecular aggregate impurities, etc.; 2.5. 2.5 mL supernatant was added to 100000 g of a 300. Mu.L sucrose cushion solution containing 20 mM Tris/30% sucrose/deuterium oxide and ultracentrifuged at 4℃for 90 min; the supernatant was collected and then centrifuged at 100000 g in sucrose pad solution containing 20 mM Tris/25% sucrose/deuterium oxide at 4℃for 5 h; mixing exosomes in 30% and 25% sucrose pads, washing with HS solution once (100000 g,4 ℃ C., ultracentrifugation for 90 min); dissolving the obtained exosomes with HS; finally, BCA protein is quantified, and the concentration is adjusted to 10 times of physiological concentration and then stored at-80 ℃ for standby.
Step 2, screening and pretreatment of asthenospermia sperm
The patient semen samples collected on the same day were placed in a constant temperature water bath at 37℃for liquefaction for 60 min. Blowing and mixing evenly, taking 10 mu L of liquefied semen, analyzing various parameters of human sperms by using a computer-aided semen analyzer (CASA), screening samples meeting the requirements on the diagnostic parameters (sperm motility < 32%) of asthenozoospermia in the standard parameters of 5 th edition (WHO 5) of semen in the handbook of human semen inspection and treatment laboratory of world health organization, and staining samples for eliminating obvious abnormal sperm morphology.
Step 3, intervention of exosome on asthenospermia sperm
Washing asthenospermia sperm twice with HS (high speed) for 1000 g and 5 min each time; sperm were re-selected with HS at 90% of the initial semen (e.g., semen of 2 mL was re-suspended with 1.8 mL HS or HTF solution) and 10% exosomes were added and incubated for 10 min at 37 ℃.
And then, analyzing sperm motility parameters and super-activation motility levels treated by exosomes by using CASA, so as to evaluate the improvement level of exosomes on sperm functions. When sperm motility parameters VCL > =150 um, lin < =50%, ALH > =7 μm, we consider that super-activation occurs, the percentage of super-activation being the number of sperm/total number of sperm that have super-activated.
The ability of sperm to penetrate viscous media was tested using a climbing experiment, the specific method was as follows:
the mucolytic ability of sperm mimics the ability of sperm to traverse the female reproductive tract and uses 1% methyl cellulose to mimic the environment of the female reproductive tract, since sperm can only combine with an egg to complete fertilization after traversing the female reproductive tract. After exosomes and sperm were treated in HTF solution for 30 min, aspirated in capillary glass tubes, capillaries containing 20 μl of 1% methylcellulose were placed in the well mixed sperm samples, and after 1h the number of sperm 1, 2, 3cm in the capillaries was recorded under a microscope.
As can be seen from fig. 1, normal exosomes at physiological concentrations can significantly increase the forward motor capacity of sperm, about 60% above the group without exosomes, but exosomes derived from severe asthenozoospermia do not. As can be seen from fig. 2, exosomes can significantly increase the level of hyperactivated motility of sperm (about 60% increase). In addition, another parameter that reflects sperm fertility, the ability to cross mucus was also significantly improved by the addition of exosomes (fig. 3, about 70% increase), with an effect similar to that produced by 1 μm progesterone. These data indicate that the addition of normal exosomes has the effect of significantly improving sperm function, and is expected to be used for treating asthenozoospermia in future.
Claims (1)
1. A method for improving the functions of asthenospermia sperms by utilizing exosomes for non-diagnostic treatment purposes, which is characterized by comprising the following steps of: the method comprises the following steps:
step 1, separation of normal human seminal plasma exosomes: collecting normal fertility semen, centrifuging 12000 g for 10 min, removing cells and impurities, sequentially filtering the seminal plasma with microporous filter membranes of 0.45 μm and 0.22 μm, removing cell debris and macromolecular aggregate impurities, adding 2.5 mL supernatant into 300 μl of sucrose pad solution containing 20 mM Tris, 30 wt% sucrose and deuterium oxide, ultracentrifugating at 100000 and 4 ℃ for 90 min, collecting supernatant, ultracentrifugating at 100000 g and 4 ℃ in sucrose pad solution containing 20 mM Tris, 25 wt% sucrose and deuterium oxide, mixing exosomes obtained by the two centrifugation, washing with HS solution once, dissolving the obtained exosomes with HS solution, adjusting 10 times of physiological concentration, and preserving at-80 ℃ for standby;
step 2, screening and pretreatment of asthenozoospermia spermia: liquefying a patient semen sample collected on the same day in a constant-temperature water bath at 37 ℃ for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human semen by using a computer-aided semen analyzer, screening samples meeting the requirements on diagnostic parameters of asthenozoospermia, and staining samples for eliminating obvious abnormal sperm morphology;
the sample meeting the requirements of the diagnostic parameters for asthenozoospermia is a sample with sperm forward movement sperm motility < 32%;
step 3, intervention of exosomes on asthenospermia sperms: washing asthenospermia with HS twice, 1000 g min each time, 5 min each time, adding 90% of HS solution or HTF solution, re-suspending sperm, adding 10% of exosomes, and incubating at 37deg.C for 10 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110280768.9A CN113041261B (en) | 2021-03-16 | 2021-03-16 | Method for improving oligospermia sperm function by using exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110280768.9A CN113041261B (en) | 2021-03-16 | 2021-03-16 | Method for improving oligospermia sperm function by using exosomes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113041261A CN113041261A (en) | 2021-06-29 |
| CN113041261B true CN113041261B (en) | 2023-12-01 |
Family
ID=76512641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110280768.9A Active CN113041261B (en) | 2021-03-16 | 2021-03-16 | Method for improving oligospermia sperm function by using exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113041261B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113171378A (en) * | 2021-04-30 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Stem cell exosome preparation for preventing and treating male sexual dysfunction |
| CN114621337B (en) * | 2022-03-31 | 2023-08-01 | 南通大学 | Polypeptide containing IDGR sequence for improving sperm function and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6716812B1 (en) * | 1998-12-02 | 2004-04-06 | King's College London | Modulation of sperm function |
| CN103352025A (en) * | 2013-06-25 | 2013-10-16 | 南京医科大学 | In vitro human sperm culture solution for improving sperm motility and application thereof |
| EP3543358A1 (en) * | 2018-03-22 | 2019-09-25 | Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) | Methods and markers for azoospermia characterisation |
| CN111808809A (en) * | 2020-06-05 | 2020-10-23 | 广州医科大学附属第二医院 | Exosome derived from human umbilical cord blood and preparation method and application thereof |
| CN112251508A (en) * | 2020-09-25 | 2021-01-22 | 徐州医科大学 | Seminal plasma exosome tsRNA marker related to non-obstructive azoospermia diagnosis and application thereof |
-
2021
- 2021-03-16 CN CN202110280768.9A patent/CN113041261B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6716812B1 (en) * | 1998-12-02 | 2004-04-06 | King's College London | Modulation of sperm function |
| CN103352025A (en) * | 2013-06-25 | 2013-10-16 | 南京医科大学 | In vitro human sperm culture solution for improving sperm motility and application thereof |
| EP3543358A1 (en) * | 2018-03-22 | 2019-09-25 | Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) | Methods and markers for azoospermia characterisation |
| CN111808809A (en) * | 2020-06-05 | 2020-10-23 | 广州医科大学附属第二医院 | Exosome derived from human umbilical cord blood and preparation method and application thereof |
| CN112251508A (en) * | 2020-09-25 | 2021-01-22 | 徐州医科大学 | Seminal plasma exosome tsRNA marker related to non-obstructive azoospermia diagnosis and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 男性不育患者精浆miR-551b水平变化及其临床价值;朱媛媛;顾万建;徐天;邵永;王成;张春妮;;现代检验医学杂志(06);1-4+8 * |
| 精浆外泌体miRNA作为男性不育症新型分子标志物的临床研究;卞玉莹;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》(第02期);E067-273 * |
| 精液外泌体在生殖系统中的研究进展;彭佳丽;肖卓妮;;中国性科学(03);11-14 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113041261A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Murdica et al. | Seminal plasma of men with severe asthenozoospermia contain exosomes that affect spermatozoa motility and capacitation | |
| CN113041261B (en) | Method for improving oligospermia sperm function by using exosomes | |
| Meyers et al. | Capacitation in vitro of stallion spermatozoa: comparison of progesterone‐induced acrosome reactions in fertile and subfertile males | |
| Curi et al. | Asthenozoospermia: analysis of a large population | |
| Davies et al. | Aqueous humour glucose concentration in cataract patients and its effect on the lens | |
| Pinto et al. | Sperm selection strategies and their impact on assisted reproductive technology outcomes | |
| Bendikson et al. | The outcome of intracytoplasmic sperm injection using occasional spermatozoa in the ejaculate of men with spermatogenic failure | |
| CN108531446B (en) | Sperm gradient centrifugate and preparation method thereof | |
| Gambera et al. | Effects of antioxidant treatment on seminal parameters in patients undergoing in vitro fertilization | |
| CN104046590A (en) | In-vitro culture medium for sperm from oligospermia and asthenospermia patients | |
| Liu et al. | Relationship between seminal plasma zinc concentration and spermatozoa–zona pellucida binding and the ZP-induced acrosome reaction in subfertile men | |
| Agarwal et al. | Filtration of spermatozoa through L4 membrane: a new method | |
| Romero et al. | Coenzyme Q10 improves the in vitro maturation of oocytes exposed to the intrafollicular environment of patients on fertility treatment | |
| Nordhoff et al. | Sperm preparation for therapeutic IVF | |
| Liu et al. | Defective protein kinase A and C pathways are common causes of disordered zona pellucida (ZP)–induced acrosome reaction in normozoospermic infertile men with normal sperm-ZP binding | |
| Hindal et al. | Reactive Oxygen Species Levels in Seminal Plasma in a Sample of Iraqi Infertile Men using Advanced Stimulatory Method for Activation of Spermatozoa | |
| Soygur et al. | Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm | |
| Orsolini et al. | Characterization of sperm cell membrane charge and selection of high-quality sperm using microfluidics in stallions | |
| Chan et al. | TEST‐egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro | |
| AL-Marayaty et al. | Effect of swim up techniques on sperm motility and DNA integrity versus unprepared semen | |
| CN105748531A (en) | Method for preparing animal model for salmonella typhimurium enteritis of people | |
| RU2536235C1 (en) | Method for assessing inducing action of aggravated cytomegalovirus infection in pregnant woman in third trimester of gestation on methaemoglobin and haemoglobin oxygenation in newborn's peripheral blood | |
| WO2017156844A1 (en) | Kit for evaluating sperm quality after in vitro capacitation and method of use thereof | |
| RU2751007C1 (en) | Method for predicting the development of extraretinal vasoproliferation in experimental retinopathy of prematurity (rop) | |
| Shirai et al. | Electrophoretic separation of X-and Y-Chromo-Some-Bearing sperm in human semen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |