CN103352025B - In vitro human sperm culture solution for improving sperm motility and application thereof - Google Patents
In vitro human sperm culture solution for improving sperm motility and application thereof Download PDFInfo
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- CN103352025B CN103352025B CN201310256093.XA CN201310256093A CN103352025B CN 103352025 B CN103352025 B CN 103352025B CN 201310256093 A CN201310256093 A CN 201310256093A CN 103352025 B CN103352025 B CN 103352025B
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- 238000000338 in vitro Methods 0.000 title claims abstract description 16
- 230000019100 sperm motility Effects 0.000 title abstract description 12
- 235000015097 nutrients Nutrition 0.000 claims description 42
- 239000007788 liquid Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 238000007670 refining Methods 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- NGSFWBMYFKHRBD-UHFFFAOYSA-N sodium;2-hydroxypropanoic acid Chemical compound [Na+].CC(O)C(O)=O NGSFWBMYFKHRBD-UHFFFAOYSA-N 0.000 claims description 3
- LKDRXBCSQODPBY-ZXXMMSQZSA-N alpha-D-fructopyranose Chemical compound OC[C@]1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-ZXXMMSQZSA-N 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 1
- 230000004899 motility Effects 0.000 abstract description 18
- 206010003883 azoospermia Diseases 0.000 abstract description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 8
- GSXOAOHZAIYLCY-HSUXUTPPSA-N keto-D-fructose 6-phosphate Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@H](O)COP(O)(O)=O GSXOAOHZAIYLCY-HSUXUTPPSA-N 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 6
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- 206010067162 Asthenospermia Diseases 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 5
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- 239000003960 organic solvent Substances 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
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- 208000008634 oligospermia Diseases 0.000 abstract 2
- 230000036616 oligospermia Effects 0.000 abstract 2
- 231100000528 oligospermia Toxicity 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 1
- 238000007865 diluting Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 41
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 210000000582 semen Anatomy 0.000 description 9
- 230000033001 locomotion Effects 0.000 description 7
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- 238000002360 preparation method Methods 0.000 description 5
- 206010021929 Infertility male Diseases 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 208000007466 Male Infertility Diseases 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
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- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 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
- 230000002159 abnormal effect Effects 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
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- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses in vitro human sperm culture solution for improving sperm motility and application thereof. The culture solution contains fructose 6-phosphoric acid (F6P). The in vitro human sperm culture solution for improving the sperm motility of human sperms is used for culturing the purified human sperms which leave from a human body; the vitality of normal human sperms is capable of maintaining sperm movement and can be obviously improved; and a meaningful reference is provided for improving and assisting the reproductive technology. The F6P is directly used for cultivating and diluting the culture solution, and does not relate to organic solvents such as ethanol or dimethylsulfoxide (DMSO) and the like; the toxic action of the ethanol or the DMSO on the sperms is avoided; by adopting the F6P added to the sperm culture solution, the vitality of the sperms of clinical patients with asthenospermia can be obviously improved; the improvement degree of the vitality is the most obvious for severe oligospermia; the activity of a part of cultured sperms reaches the normal sperm vitality range; and the improvement degree of the vitality for the severe oligospermia is the most obvious. This fully demonstrates that low motility of the sperms of the patients with asthenospermia can be improved by using of the F6P.
Description
Technical field
The present invention relates to a kind of nutrient solution, be specifically related to a kind of nutrient solution and the application thereof that improve vitro culture Sperm Motility.
Background technology
At present, infertile rate accounts for the couple at child-bearing age's 1/10th, and wherein male factor infertility accounts for half, but all can produce sperm more than 85% in Infertility male.Male sterility a kind of important behaviour is clinically azoospermia, and namely Sperm Motility is low, causes male sterility, and the motor capacity improving sperm is a kind of important method for the treatment of the type male sterility.This also illustrates the importance of the motor capacity of sperm in sperm normal function.
Treating infertilely has multiple supplementary reproduction means, as artificial insemination (IUI), in vitro fertilization-embryo transfer (IVF) and intracytoplasmic sperm injection (ICSI) etc., these technology all relate to vitro culture and the operation of sperm, in the process of cultivating in vitro and operate, maintaining the function even improving sperm has important effect for the success ratio improving supplementary reproduction.
Eupyrene sperm is present in refining, and the main source of energy is fructose.Nutrient solution such as BWW, HTF etc. of Clinical practice use glucose as energy derive.The monose of this two type is utilized Shi Junxu and is transformed into fructose 6-phosphoric acid (fructose 6-phosphate, F6P) in sperm, and final metabolism converts ATP to, for the motion of sperm provides energy.
Carry out in vitro in liquefacient duration process, sperm has departed from internal milieu, is in the nutrient solution of artificial preparation.Except needing suitable nutrient solution pH and osmotic pressure, the motion of sperm needs to consume a large amount of energy.High-quality sperm nutrient solution has extensive and important purposes in reproductive medicine field, especially along with Assisted Reproductive Technology ART carrying out clinically, receives enough attention especially.As when artificial insemination, there is the rapid upstream of sperm energy of good motor capacity to uterine tube and ovum fertilization; The sperm that the time of fertilization motor capacity is good in vitro has the ovarian cumulus of egg penetration and the enough power of zona pellucida, enters in archiblast smoothly and completes fertilization.For common are motor capacity abnormal spermium clinically, improving motility of sperm and there is very important therapeutic potential.So the sperm nutrient solution that preparation can improve sperm function is one of research emphasis of reproductive medicine worker always.Traditional nutrient solution for extracorporeal treatment sperm mainly contains Ham ' s F10; Tyrode ' s; BWW, Whitten ' nutrient solution such as s, the advantage of these nutrient solutions is through long-term clinical application to be proved sperm nontoxicity and has certain provide protection and trophism to sperm.Substantially, can meet the application carrying out extracorporeal treatment sperm in Assisted Reproductive Technology ART clinically.But significant shortcoming is inadequate in the improvement of Sperm Motility, and test-tube success ratio always not high (about hovering at about 10 %) may be also relevant therewith.Therefore, develop a kind of sperm nutrient solution that significantly can improve Sperm Motility and there is very important clinical meaning.
Summary of the invention
the technical problem solved:the object of this invention is to provide a kind of nutrient solution and the application method thereof that improve vitro culture motility of sperm, show human sperm's cultivation results of different motion ability, the motor capacity of sperm all increases significantly.
technical scheme:improve the vitro culture liquid of human spermatogoa motor capacity, containing fructose 6-phosphoric acid.
Above-mentioned often liter of nutrient solution contains following component: 5.5-6.0g NaCl, 2.0-2.5g NaHCO, 0.3-0.4g KCl, 0.04-0.06g MgSO
4 .7 (H
2o), 0.04-0.06g KH
2pO
4, 0.035-0.04g Sodium.alpha.-ketopropionate, 3.8-4.0g Sodium.alpha.-hydroxypropionate, 0.28-0.35g CaCl
2 .2 (H
2o), 0.5-1g F6P.
Improve the application of the vitro culture liquid of human spermatogoa motor capacity, incubation time is at 1-2h.Sperm used is the in vitro human spermatogoa removing refining afterwards.
beneficial effect:
1, the present invention improves the vitro culture liquid of human spermatogoa motor capacity, F6P is used in the composition of sperm nutrient solution, the human spermatogoa of in vitro rear purifying being cultivated, the vigor of human spermatogoa can also be significantly improved except maintaining sperm motility, providing significant reference for improving Assisted Reproductive Technology ART.
2, the F6P in the present invention is directly with nutrient solution dilution with cultivate, and does not relate to the organic solvent such as ethanol or DMSO, avoids ethanol or DMSO to the toxic action of sperm.
3, in the present invention, the F6P added in sperm nutrient solution, the vigor of clinical Asthenospermia sperm can be significantly improved, when severe azoospermia, vigor improvement degree is the most obvious, and the motility of sperm making part cultivate reaches eupyrene sperm vigor scope, wherein degree is improved to severe azoospermia the most obvious.This absolutely proves that the use of F6P has and improves the low effect of azoospermia patient Sperm Motility.
Accompanying drawing explanation
Fig. 1 HTF-F6P nutrient solution is to the action diagram of the motility of sperm of different motion ability;
Illustrate: X-axle is sperm incubation time in different nutrient solution, Y-axle refers to motility of sperm (Motility=A+B, with per-cent counting).Scheme A and represent that the weak sperm of severe cultivates the statistics of rear motility of sperm, scheme B and represent that mild or moderate azoospermia sperm cultivates the statistics of rear vigor data, figure C represents the statistics of the rear vigor data of normal vital sperm cultivation.In figure, data show with mean value ± standard error.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition and replacement, all belong to scope of the present invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.The reagent used in following examples and material are commercially available prod, and such as, the F6P used in experiment can buy from SIGMA-ALDRICH, MO, USA.
embodiment 1
1. preparation of samples
The sperm sample of severe Asthenospermia is selected to test.Masturbation method ejaculation, collects the sperm sample of ascetic 2-7 days with specific sterile chamber.Sample water-bath is kept 22-37 DEG C, the seminal fluid of post liquefaction, according to the judgement criteria of WHO1999, use computer-aided semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc.Beverly, MA, USA), Sperm Parameters (volume, sperm count, the per-cent of vigor and motion characteristics) is assessed.Azoospermia is defined as A level sperm number <25% or A+B level sperm number <50%, (World Health Organization, 1999), and every other parameter is all normal.The sample polluted containing white corpuscle or somatocyte is got rid of according to morphological observation.The kinematic parameter of severe azoospermia sperm is (a+ b) level <25%, wherein a level sperm <5%.
2. sperm in vitro is cultivated
1) density gradient centrifugation is from sorting sperms in the middle of seminal fluid.
Place 1mL 60%(v/v in vitro) Percoll parting liquid, spread the seminal fluid of 1mL-1.5mL thereon, centrifugal 5-10 minute under the centrifugal force of 300g, abandoning supernatant.Resuspended sperm suspension is in the HTF-S nutrient solution (commercialization HTF nutrient solution is called for short HTF-S) of 2mL, and 300g centrifugal force 5-10 minute, repeats this step once.Sperm suspension is transferred to gently in the various sperm nutrient solutions used by experiment.
2) sperm is cultivated
The sperm (5 × 10 of suitable quantity
6left and right) transfer in various sperm nutrient solution after, be placed in 37 DEG C, 5% CO
2incubator in 1-2 hour.
Sperm nutrient solution used is: business-like HTF-S nutrient solution and HTF-F6P nutrient solution (formula is in table 1).Lower same.
Table 1. HTF-F6P nutrient solution is filled a prescription
| Component | g/L |
| NaCl | 5.9 |
| NaHCO? | 2.1 |
| KCl | 0.35 |
| MgSO 4 .7(H 2O) | 0.05 |
| KH 2PO 4 | 0.05 |
| Sodium.alpha.-ketopropionate | 0.036 |
| Sodium.alpha.-hydroxypropionate | 3.998 |
| CaCl 2 .2(H 2O) | 0.294 |
| F6P | 0.78 |
3. the detection of motility of sperm
According to the judgement criteria of WHO1999, use computer-assisted semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc.Beverly, MA, USA), motility of sperm is assessed.For each sample, choose 6-10 the visual field, the sperm number count down to is not less than 500.
embodiment 2
1. preparation of samples
The sperm sample of mild or moderate Asthenospermia is selected to test.Masturbation method ejaculation, collects the sperm sample of ascetic 2-7 days with specific sterile chamber.Sample water-bath is kept 22-37 DEG C, the seminal fluid of post liquefaction, according to the judgement criteria of WHO1999, use computer-aided semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc. Beverly, MA, USA), Sperm Parameters (volume, sperm count, the per-cent of vigor and motion characteristics) is assessed.According to the standard of the World Health Organization in 1999, the sperm motility parameters of mild or moderate azoospermia is 25%< (a+b) level <50% and a level sperm <25%.
2. sperm in vitro is cultivated
1) density gradient centrifugation is from sorting sperms in the middle of seminal fluid.
Place 1mL 60%(v/v in vitro) Percoll parting liquid, spread the seminal fluid of 1mL-1.5mL thereon, centrifugal 5-10 minute under the centrifugal force of 300g, abandoning supernatant.Resuspended sperm suspension is in the HTF-S nutrient solution of 2mL, and 300g centrifugal force 5-10 minute, repeats this step once.Sperm suspension is transferred to gently in the various sperm nutrient solutions used by experiment.
2) sperm is cultivated
The sperm (5 × 10 of suitable quantity
6left and right) transfer in various sperm nutrient solution after, be placed in 37 DEG C, 5% CO
2incubator in 1-2 hour.
Sperm nutrient solution used is: business-like HTF-S nutrient solution and HTF-F6P nutrient solution (formula is in table 1).Lower same.
3. the detection of motility of sperm
According to the judgement criteria of WHO1999, use computer-assisted semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc.Beverly, MA, USA), motility of sperm is assessed.For each sample, choose 6-10 the visual field, the sperm number count down to is not less than 500.
embodiment 3
1. preparation of samples
The sperm sample of the sperm donors of healthy male is selected to test.Masturbation method ejaculation, collects the sperm sample of ascetic 2-7 days with specific sterile chamber.Sample water-bath is kept 22-37 DEG C, the seminal fluid of post liquefaction, according to the judgement criteria of WHO1999, use computer-aided semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc.Beverly, MA, USA), Sperm Parameters (volume, sperm count, the per-cent of vigor and motion characteristics) is assessed.
2. sperm in vitro is cultivated
1) density gradient centrifugation sorting sperms from seminal fluid.
Place 1mL 60%(v/v in vitro) Percoll parting liquid, slowly spread the seminal fluid of 1mL-1.5mL thereon, centrifugal 5-10 minute under the centrifugal force of 300g, abandoning supernatant.Resuspended sperm suspension is in the HTF-S nutrient solution of 2mL, and 300g centrifugal force 5-10 minute, repeats this step once.Sperm suspension is transferred to gently in the various sperm nutrient solutions used by experiment.
2) sperm is cultivated
Suitable quantity (5 × 10
6left and right) sperm transfer in various sperm nutrient solution after, be placed in 37 DEG C, 5% CO
2incubator in 1-2 hour.
Sperm nutrient solution used is: business-like HTF-S nutrient solution, HTF-F6P nutrient solution (formula is in table 1).
3. the detection of motility of sperm
According to the judgement criteria of WHO1999, use computer-assisted semen analysis instrument CASA(IVOS; Hamilton-Thorn Research, Inc.Beverly, MA, USA), motility of sperm is assessed.For each sample, choose 6-10 the visual field, the sperm number count down to is not less than 500.
Experimental result shows: after the sperm of vitro culture is cultivated 1 hour and 2 hours in HTF-S nutrient solution, and motility does not all produce significance change.Severe azoospermia sperm cultivate in HTF-F6P nutrient solution after 1 hour between vigor raise and have significant difference (P<0.05).Mild or moderate azoospermia sperm and eupyrene sperm vigor after HTF-F6P cultivates 1 hour raises no difference of science of statistics.The sperm of three types is all increased significantly compared with before cultivation in motility of sperm after cultivating 2 hours in HTF-F6P nutrient solution.Wherein severe azoospermia motility of sperm rises to 30.00 ± 6.17% (P<0.001) from 13.25 ± 2.96% (mean value ± standard errors) after cultivating, and raises 2.26 times before reaching cultivation; Mild or moderate azoospermia motility of sperm rises to 48.31 ± 3.40 (P<0.001) from 34.19 ± 1.68 (mean value ± standard errors) after cultivating, and raises 1.41 times before reaching cultivation; And eupyrene sperm vigor rises to 63.38 ± 2.03(P<0.001 from cultivation front 54.88 ± 1.27), Detailed Experimental data are shown in Fig. 1.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these done without departing from theon the basis of the spirit of the present invention amendments are lived and are improved, and all belong to the content of application claims protection.
Claims (3)
1. fructose 6-phosphoric acid is preparing the application improved in the vitro culture liquid of human spermatogoa motor capacity.
2. application according to claim 1, is characterized in that often liter of nutrient solution contains following component: 5.5-6.0g NaCl, 2.0-2.5g NaHCO, 0.3-0.4g KCl, 0.04-0.06g MgSO
4 .7 (H
2o), 0.04-0.06g KH
2pO
4, 0.035-0.04g Sodium.alpha.-ketopropionate, 3.8-4.0g Sodium.alpha.-hydroxypropionate, 0.28-0.35g CaCl
2 .2 (H
2o), 0.5-1g F6P.
3. application according to claim 1, is characterized in that described sperm is the in vitro human spermatogoa removing refining afterwards.
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| CN103352025B true CN103352025B (en) | 2015-04-15 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10182999B2 (en) | 2014-08-18 | 2019-01-22 | Francisco PAN-MONTOJO | Glycolic acid enhances sperm mobility |
| CN110129258A (en) * | 2018-02-02 | 2019-08-16 | 兰州大学 | A kind of composition containing astragalin and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027741B (en) * | 2017-05-24 | 2020-12-29 | 四川大学华西第二医院 | Human sperm freezing protection solution without yolk and preservation method |
| CN113041261B (en) * | 2021-03-16 | 2023-12-01 | 南通大学 | Method for improving oligospermia sperm function by using exosomes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6596310B1 (en) * | 2000-08-23 | 2003-07-22 | Board Of Trustees Operating Michigan State University | Method of artificial insemination by timed release of sperm from capsules or solid beads |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6596310B1 (en) * | 2000-08-23 | 2003-07-22 | Board Of Trustees Operating Michigan State University | Method of artificial insemination by timed release of sperm from capsules or solid beads |
Non-Patent Citations (2)
| Title |
|---|
| Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa;G Kamp et al;<Reproduction>;20070131;第133卷;29-40 * |
| 果糖二磷酸锶盐对雷公藤多甙所致的大鼠少精子症的治疗作用;胡莹莹等;《中华男科学杂志》;20111231;第17卷(第5期);396-400 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10182999B2 (en) | 2014-08-18 | 2019-01-22 | Francisco PAN-MONTOJO | Glycolic acid enhances sperm mobility |
| CN110129258A (en) * | 2018-02-02 | 2019-08-16 | 兰州大学 | A kind of composition containing astragalin and its application |
| CN110129258B (en) * | 2018-02-02 | 2021-01-01 | 兰州大学 | Composition containing astragalin and application thereof |
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| CN103352025A (en) | 2013-10-16 |
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