CN103805559A - Novel sperm washing nutrient solution and preparation method thereof - Google Patents
Novel sperm washing nutrient solution and preparation method thereof Download PDFInfo
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- CN103805559A CN103805559A CN201410000549.0A CN201410000549A CN103805559A CN 103805559 A CN103805559 A CN 103805559A CN 201410000549 A CN201410000549 A CN 201410000549A CN 103805559 A CN103805559 A CN 103805559A
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Abstract
The invention discloses a novel sperm washing nutrient solution and a preparation method thereof. The novel sperm washing nutrient solution prepared by the method is stable and reliable and has good solubility. The novel sperm washing nutrient solution can be used for washing the sperm according to certain procedures and the sperm can be directly stored in the sperm washing nutrient solution. The comparison of activity and survival rate of the sperm in the sample proves that the novel sperm washing nutrient solution can obviously improve the activity and survival rate of the sperm in the sample in comparison with the washing solution without neurotrophic factor and shows increase of total proportion of a-grade sperm and b-grade sperm; and six hours later, the survival rate of sperm is improved. The novel sperm washing nutrient solution has effect of promoting reproduction, and is expected to provide a more effective measure to keep male fertility for assisted reproduction in clinic.
Description
Technical field
The present invention relates to a kind of Novel sperm washing nutrient solution, it is characterized in that containing certain density neurotrophic factor, and disclose the preparation method of this Sperm washing nutrient solution.The Novel sperm washing nutrient solution preparing according to the method, reliable and stable, solubleness is good, use after this Novel sperm washing nutrient solution washing seminal fluid sample, can obviously accelerate the movement velocity of sperm, improve motility of sperm, increase sperm survival rate and survival time in washing nutrient solution, show as the effect that promotes reproduction.This Sperm washing nutrient solution can, for the nutrient solution of external sperm preparation and Sperm storage, be applied to the supplementary reproduction fields such as artificial insemination.
Background technology
Infertile is the disease that has a strong impact on human life quality, and at present the whole world approximately has 10%～15% family to face a fertility difficult problem.World Health Organization's prediction, in 21 century, infertility will become the third-largest disease that is only second to tumour and cardiovascular disorder.Male sterility causes mainly due to sperm concentration and vigor decline.Modern assisted reproductive facts have proved that external sperm washing can increase the vigor promotion reproductive performance of sperm.
Must row semen washing while being expert at insémination intrautérine, and the Main Function of semen washing comprises that the prostaglandin(PG) removed in seminal fluid and other can cause the material of hysterospasm and pain, remove bacterium and the chemical substance that in seminal fluid, can cause infection and other side effects, increased the capacitation of sperm simultaneously, thereby strengthen its potentia concipiendi, and then likely remove the antibody that affects sperm function.
Semen washing method is to use Sperm washing liquid, conventional Sperm washing liquid formula is open, and the product of many commodities in useization thus, also can configure voluntarily, but in clinical practice, find to understand the vigor that reduces sperm, the potentia concipiendi that even can affect sperm having after some samples use washingss.
In addition, after washing, sometimes also need sperm nutrient solution to preserve sperm, to assist to keep motility of sperm, extend the survival time.If the washings using in washing process does not possess the function of nutrient solution, just need to be replaced by special nutrient solution and preserve sperm.If the washings using in washing process has the function of certain nutrient solution, generally do not need to change special nutrient solution, directly by sperm storage in washings.Therefore some Sperm washing liquid itself is exactly nutrient solution, therefore claim Sperm washing nutrient solution.
The invention discloses a kind of novel Sperm washing nutrient solution formula, by add the neurotrophic factor that adapts to concentration in existing Sperm washing nutrient solution.Use this Sperm washing nutrient solution to process with a collection of seminal fluid sample, result and the comparison of existing Sperm washing nutrient solution, prove that this Sperm washing nutrient solution can obviously accelerate the movement velocity of sperm, improves the survival rate of sperm, the time that extends motility of sperm, be conducive to become pregnant.
Summary of the invention
The present invention relates to the Novel sperm washing nutrient solution that one contains nerve growth factor (nerve growth factor, NGF).Contriver has proved to add the NGF of suitable concentration by experiment in Sperm washing nutrient solution, in vitro the vigor of people's sperm is had to promoter action, makes sperm continue to keep translational motion state, discloses the preparation method of this Sperm washing nutrient solution simultaneously.The Sperm washing nutrient solution obtaining by the present invention can be for Sperm washing under condition of in vitro culture and preservation, also can be used for promoting women's potentia concipiendi, and then realize dysgenesia disease that external treatment causes because mankind spermatozoon vigor declines or for human assistance reproductive technology, there is good economic benefit and social benefit.
This pharmaceutical composition comprises NGF and other additives.NGF as main ingredient in the present invention is generally mouse, can be also humanized or gene engineering product etc.Their content in composition can be 0.1 μ M～100 μ M (concentration), preferably 0.1 μ M～10 μ M (concentration), more preferably 1 μ M～50 μ M (concentration).
Preparation method
Material
Nerve growth factor, the mouse NGF that mouse submaxillary gland extracts, can be solution or lyophilized powder, is produced effect phase safety by the pharmaceutical factory that meets GCP authentication.
Basis Sperm washing nutrient solution: Sperm washing nutrient solution that can commodity in use, produced effect phase safety by the pharmaceutical factory that meets GCP authentication.Also can configure voluntarily, its preparation method is as follows: F-104.9g, KHCO
30.225g, NaHCO
30.84g, calcium lactate 0.142g, MgSO
47H
2o0.062g, benzylpenicillin potassium 0.052mg, mentioned reagent is dissolved in respectively in 500ml distilled water, surveys pH value 7.3～7.5, and adjusting osmotic pressure is 280～290mmol/L (mOsm/kg) H
2o, then with 0.22 μ m millipore filter filtration, for subsequent use in 4 ℃ of refrigerators.Before washing nutrient solution uses, it is clear that 90ml washing nutrient solution adds 10ml bleeding of the umbilicus, after 37 ℃ of pre-temperature, uses.
Wash the preparation of nutrient solution containing the Novel sperm of NGF: in basic Sperm washing nutrient solution, add a certain amount of NGF, control final concentration, use and use again 0.22 μ m millipore filter filtration sterilization, for subsequent use in 4 ℃ of refrigerators.Use front 90ml washing nutrient solution to add 10ml bleeding of the umbilicus clear, use after 37 ℃ of pre-temperature.
Compliance test result
Adopt Sperm washing nutrient solution to wash seminal fluid sample, semen washing method is to use Sperm washing nutrient solution, will put in sterilized container with masturbation method extracting extract, and 37 ℃ of liquefaction, repeat conventional semen efamination.Get 1ml seminal fluid and add 3ml washing nutrient solution, mix centrifugal 1000 revs/min with suction pipe, 5 minutes, abandon supernatant liquor, retain sperm and add again washing nutrient solution 3ml, after repeated centrifugation, abandon again supernatant liquor, add 0.8～1ml washing nutrient solution and mix, be pumped in the syringe that connects a thin plastic pipe.
After washing, check vigor and survival rate and the survival time of sperm in washing nutrient solution, process the result with a collection of seminal fluid sample by more different Sperm washing nutrient solutions, evaluate the movement velocity that it can obviously accelerate sperm, improve the survival rate of sperm, the effect of the aspects such as the time of prolongation motility of sperm.
Found that, the Novel sperm that the contains NGF washing nutrient solution obtaining according to preparation method disclosed by the invention, reliable and stable, solubleness is good, use after this Novel sperm washing nutrient solution washing seminal fluid sample, with the comparison of basic Sperm washing nutrient solution, can obviously accelerate the movement velocity of sperm, the vigor that improves sperm, the ratio that shows as a level sperm and b level sperm significantly improves.
Conclusion
The involved in the present invention Sperm washing nutrient solution that contains NGF can fine maintenance even increase the vigor of sperm, if can apply, will have good economic benefit and social benefit.
Embodiment one:
Containing Sperm washing nutrient solution and the impact contrast of basic Sperm washing nutrient solution on motility of sperm of 0.1 μ M NGF
1, material:
(1) nerve growth factor, the mouse NGF dry powder that Shutaishen (Beijing) Pharmaceutical Co., Ltd. produces, the accurate word S20060023 of traditional Chinese medicines, is the albumen of about molecular weight 26.5kD, years 09 month 11 days effect phase to 2014.
(2) basic Sperm washing nutrient solution: the H-MOPS sperm nutrient solution of producing with Mariya company of Korea S, years 08 month 10 days effect phase to 2015.
(3) seminal fluid sample: the seminal fluid sample of employing, for volunteer provides, therefore needs healthy male volunteers some, provides seminal fluid sample according to requirement below:
1) sample is the sample of normal masturbation ejaculation, can not be for obtaining in condom
2) acquisition of sample, must be strict controlled in and ejaculate last time in 3～7 days
3) seminal fluid sample is directly collected and is entered in the sample cup providing specially, fills out ejaculation time and volunteer's numbering, covers tightly after sample bowl cover, delivers to laboratory or appointed place in specific time
4) once the rear all samples of ejaculation must all be collected in sample cup, must not have omission, also must not have any non-seminal fluid ingredients substance to sneak into
5) leave after health should intact preservation in sample cup for seminal fluid sample, prevents from polluting.Attention: human seminal fluid belongs to potential source biomolecule hazardous product, please disposes according to international organization and national relevant laws and regulations.Advise that insulation next to the skin carries, temperature is too low or too highly may affect motility of sperm result of determination
6) guaranteeing that seminal fluid sample has liquefied well: can within half an hour, complete liquefaction under seminal fluid sample room temperature, therefore advise that seminal fluid leaves after health and start test after half an hour.Meanwhile, seminal fluid sample is not suitable for room temperature and preserves for a long time, therefore advises that seminal fluid leaves after health to complete test in the longest 24 hours
(4) 0.9% physiological saline: Shijiazhuang Siyao Co., Ltd produces, the accurate word H13023200 of traditional Chinese medicines, 500ml, 29, on January of effect phase to 2015.
(5) human serum albumin (HAS): QUINN'S company of the U.S., ART-3001,5ml, 1, on June of effect phase to 2014
(6) prepare containing the Sperm washing nutrient solution of 0.1 μ M NGF: the H-MOPS sperm nutrient solution of producing take Mariya company of Korea S, as basis, adds 0.1 μ MNGF
2, method:
(1) Sperm washing: get 1ml seminal fluid and add 3ml sperm nutrient solution (before use must with the CO2 balance to 37 ℃ of 6% concentration), mix with suction pipe, centrifugal 1000 revs/min, 5 minutes, abandon supernatant liquor, retain sperm and add again nutrient solution 3ml, after repeated centrifugation, abandon again supernatant liquor, add 0.8～1ml nutrient solution and mix, be pumped in the syringe that connects a thin plastic pipe.
For same seminal fluid sample, get respectively 1ml parallel laboratory test, a copy of it washs with physiological saline, and another part is with Korea S's sperm nutrient solution washing, last a with the sperm nutrient solution washing containing 0.1 μ M NGF.
(2) motility of sperm analysis:
CASA measures motility of sperm: the seminal fluid after 37 ℃ of liquefaction is splashed on sperm count Marker plate, detect the vigor of sperm with full-automatic sperm image analysis system, sectional drawing is preserved.According to ABCD ranking method, according to spermatozoa motility under microscope, sperm is divided into a level sperm, b level sperm, c level sperm and d level sperm, demarcate motility of sperm with the summation of a level sperm per-cent and b level sperm per-cent.
3, result:
In the three kinds of rear washing of sperm nutrient solution washing nutrient solutions, sperm is as Fig. 1.
Vigor analytical results is as Fig. 2.
Can find out, adopt containing after washing, a level sperm and b level sperm ratio significantly increase, and the ratio of corresponding c, d level sperm reduces, and illustrate in washing nutrient solution that motility of sperm is apparently higher than basis washing nutrient solution group and physiological saline group.
4, conclusion
The above results proves, the Sperm washing nutrient solution that contains 0.1 μ M NGF, according to correct method washing seminal fluid sample, has the effect that promotes motility of sperm.
Embodiment two:
The impact contrast on motility of sperm of 10 μ M NGF and sperm nutrient solution
1, method:
(1) the H-MOPS sperm nutrient solution of producing take Mariya company of Korea S is as basal component, using 0.9% physiological saline as blank group, with Korea S's sperm nutrient solution as a control group, using the sperm nutrient solution that adds 10 μ M NGF as experimental group, observe the percentile impact of the percentage of different ingredients sperm nutrient solution on motility of sperm a level sperm, the shared sperm sum of b level sperm, b level sperm, the shared sperm sum of c level sperm by CASA method.
(2), CASA measures motility of sperm:
Seminal fluid after 37 ℃ of liquefaction is splashed on sperm count Marker plate, detect the vigor of sperm with full-automatic sperm image analysis system, sectional drawing is preserved.According to ABCD ranking method, according to spermatozoa motility under microscope, sperm is divided into a level sperm, b level sperm, c level sperm and d level sperm, demarcate motility of sperm with the summation of a level sperm per-cent and b level sperm per-cent.
2, result:
Result shows that experimental group can significantly increase the quantity of a level motile sperm, reduces the ratio of c, d level sperm, and its promoter action to motility of sperm is better than sperm nutrient solution group and physiological saline group (P<0.01).See Fig. 3, Fig. 4.
3, conclusion
The above results shows, the NGF sperm nutrient solution that adds concentration and be 10 μ M can improve motility of sperm, and along with the prolongation of incubation time, the vigor of sperm, motility rate occur that difference is better than the control group without NGF.
Accompanying drawing explanation:
Fig. 1. three kinds of Sperm washing nutrient solution sperms after treatment, A: the sperm after physiological saline washing; B: sperm after processing containing the Sperm washing nutrient solution of 0.1 μ M NGF; C:H-MOPS sperm nutrient solution is processed rear sperm.Magnification: 400 times
Fig. 2: three kinds of Sperm washing nutrient solution motility of sperm analytical resultss after treatment, A:a level sperm per-cent, B:b level sperm per-cent,
C:c level sperm per-cent, D:d level sperm per-cent.
*: P<0.05, with the comparison of physiological saline group, n=12
Fig. 3. three kinds of Sperm washing nutrient solution sperms after treatment, A: the sperm after physiological saline washing; B: sperm after processing containing the Sperm washing nutrient solution of 10 μ M NGF; C:H-MOPS sperm nutrient solution is processed rear sperm.Magnification: 400 times
Fig. 4: three kinds of Sperm washing nutrient solution motility of sperm analytical resultss after treatment, A:a level sperm per-cent, B:b level sperm per-cent, C:c level sperm per-cent, D:d level sperm per-cent.
*: P<0.05, with the comparison of physiological saline group, n=12.
Claims (7)
1. cultivate for the washing of seminal fluid sample or sperm a Sperm washing nutrient solution of preserving, it is characterized in that containing certain density neurotrophic factor (NGF).
2. contain a preparation method for the Sperm washing nutrient solution of NGF, its main process is in basic Sperm washing liquid or sperm nutrient solution, to add the NGF that final concentration is 0.1 μ M～100 μ M (concentration).
3. Sperm washing nutrient solution as claimed in claim 1, can, separately for the washing of seminal fluid sample, also can be applied to separately sperm and cultivate preservation, also can be applied to washing and follow-up cultivation and preserve.
4. Sperm washing nutrient solution as claimed in claim 1, can be liquid, can be also freeze-dried, tablet that can be rapidly dissolvable in solution and water that comprises main component etc.
5. NGF as claimed in claim 1, can mouse or humanized's tissue extraction, also can gene engineering product.
6. basic Sperm washing liquid as claimed in claim 2, can be market-oriented commercially produced product, can be also the Non-standard preparation that laboratory configures voluntarily.
7. basic sperm nutrient solution as claimed in claim 2, can be market-oriented commercially produced product, can be also the Non-standard preparation that laboratory configures voluntarily.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724666A (en) * | 2019-10-29 | 2020-01-24 | 力妲康生命科学(上海)有限公司 | Sperm cleaning solution and preparation method and application thereof |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
| CN113005077A (en) * | 2021-03-19 | 2021-06-22 | 吉林大学第一医院 | Preparation method of in-vitro testicular tissue living single sperm suspension |
-
2014
- 2014-01-02 CN CN201410000549.0A patent/CN103805559A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| C.LI 等: "Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function", 《THERIOGENOLOGY》 * |
| 任晚琼 等: "冻干鼠神经生长因子中试制备及几点问题的探讨", 《药物生物技术》 * |
| 周晓东 等: "神经生长因子对精子活动力的影响", 《医学研究生学报》 * |
| 周晓东 等: "神经生长因子对精子活动力的影响", 《医学研究生学报》, vol. 25, no. 4, 30 April 2012 (2012-04-30), pages 1 - 1 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724666A (en) * | 2019-10-29 | 2020-01-24 | 力妲康生命科学(上海)有限公司 | Sperm cleaning solution and preparation method and application thereof |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
| CN113005077A (en) * | 2021-03-19 | 2021-06-22 | 吉林大学第一医院 | Preparation method of in-vitro testicular tissue living single sperm suspension |
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Application publication date: 20140521 |