CN103424299A - New method for analysis of sperm survival rate - Google Patents
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- CN103424299A CN103424299A CN2012101499944A CN201210149994A CN103424299A CN 103424299 A CN103424299 A CN 103424299A CN 2012101499944 A CN2012101499944 A CN 2012101499944A CN 201210149994 A CN201210149994 A CN 201210149994A CN 103424299 A CN103424299 A CN 103424299A
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Abstract
The invention relates to a new method for analysis of a male sperm survival rate. A semen sample is stained by a gentian violet solution at a certain concentration, a staining result is observed under a microscope, survival sperms and dead sperms are determined through colored difference, and the sperm survival rate is calculated; after a same sample is stained by an eosin Y standard staining method recommended by the WHO, the sperm survival rate is calculated; a statistical difference between the two sperm survival rates obtained by the two methods is tested by statistics, a result shows that the gentian violet normal saline solution staining method can be used for distinguishing the dead sperms and the survival sperms, almost all of the sperms are colored after being stained by the gentian violet normal saline, sperm motility is decreased, so as to help to count and carry out subsequent analysis under the microscope. The result proves that the method by using the gentian violet solution to stain the semen sample can easily distinguish the survival sperms and the dead sperms, can further analyze the sperm survival rate, can also assist in the analysis of the sperm motility, and contributes to identification and analysis of male sterility.
Description
Technical field
The present invention relates to analyze the new method of mankind spermatozoon survival rate.Adopt certain density aldrich mixture to dye to the seminal fluid sample, coloration result under the microscope can be easy differentiation survival sperm and dead sperm, can further analyze the sperm survival rate, also can assist to analyze sperm motility, contribute to male sterility distinguish and analyze.
Background technology
Male sterility is one of sixty-four dollar question of human reproduction, especially the modal clinical problem of andrology and serious challenge.Because mankind spermatozoon quality since nearly 30～50 years and quantity descend, the trend that carcinoma of testis, testicular descent are bad, the incidence of disease of hypospadia and sterility constantly raises, male sterility enjoys attention.Although in recent years diagnosis and the treatment of male sterility have been obtained to obvious progress, but male sterility is still the very difficult problem that the clinician of andrology faces up to now, its reason is that people are to male genetic, particularly spermatogenetic understanding is far unclear, sterility is not that independently disease is planted simultaneously, be subject to the impact of the many factors such as environment, heredity, the actual cause of disease that surpasses 50% patient in male sterility still belongs to unknown, and this just must strengthen the difficulty in the clinical position.
Causing many factors, complexity that Mr. and Mrs are sterile, relate to men and women's reproduction and holistic health, and the clearly special cause of disease of any sick kind is not only to selection rationally with therapeutic scheme institute is essential targetedly, is also the objective basis of judging prognosis.The World Health Organization (WHO) (WHO) finds in the multicenter study of 9000 pairs of infertile couples of 25 countries, and 50% male sterility is the idiopathic cacospermia.Has carried out deeply and systematic research 1549 routine male sterilities by the diagnostic routine of WHO standard in Northern Europe in 1999, find that wherein the ratio of specific oligospermatism, necrospermia, teratozoospermia and azoospermia is respectively 27.6%, 10.7%, 1.3% and 4.2%.
Necrospermia is a kind of serious dysspermatism, is not equal to the sperm of the motion activity deficiency in asthénospermie through the Necrospermia of definition, should be than the latter more very.Because the sperm of the motion activity deficiency in asthénospermie, likely reactivate after obtaining energy, and cell membrane damage has appearred in dead sperm, can not participate in acrosome reaction and become pregnant.Therefore necrospermia has a strong impact on male reproductive health.
The male reproductive health guide of the World Health Organization (WHO) (WHO) up-to-date announcement in 2011, necrospermia has been provided to clear and definite criterion, be original seminal fluid in leaving human body one hour, room temperature (more than 22 ℃) to 37 ℃ of (98.6 °F) preservation conditions, in seminal fluid, the number percent of survival sperm is lower than 58%.That is to say, when the number percent of dead sperm surpasses 42% in individual specimen, can be judged to be necrospermia.According to clinical observation, necrospermia may be relevant with many factors, as sperm nutritional deficiency, and genital system infection, poisoning and drug abuse etc., if diagnoses and treatment not in time may cause sterile.
The diagnosis of necrospermia must be in strict laboratory, by the clinical examination personnel with qualification and experience, according to science, strict method checks qualified semen sample, according to assay and duplicate test result, according to the standard determination of the World Health Organization (WHO).The dead sperm decision method of the up-to-date announcement of the World Health Organization (WHO) has three kinds, and wherein the most frequently used is the Eosin Y decoration method.
Eosin Y is a kind of acid dyes of chemosynthesis, become electronegative negative ion at dissociation in water, be combined with the kation of the amino positive charge of protein and make endochylema dyeing, cytoplasm, red blood cell, muscle, connective tissue, eosinophil granule etc. are dyed in various degree red or pink, with blue nucleus, form sharp contrast.Yihong is cytoplasmic good dyestuff.
2011 human seminal fluids of the World Health Organization (WHO) check the normal process of the Eosin Y dyeing of recommending with the treatment of laboratory handbook, its foundation is exactly water-soluble Eosin Y (Eosin Y, cas:17372-87-1, molecular weight: 691.9, color index 45380) can't pass the intact cell film, can not dye to living cells, but can enter the incomplete cell of cell membrane, and dead sperm is dyed to pink, the survival sperm is not painted or painted very shallow, therefore can distinguish under the microscope sperm alive and Necrospermia, and then through calculating the sperm survival rate.The working concentration of dyeing liquor is 5/1000ths, and dyeing time is no more than 30 minutes.
The coloration result of Eosin Y is more clearly, can distinguish at the appointed time sperm anyway, can calculate survival rate.But in the result of the method, the survival chromospermism is very shallow, therefore distinguish under the microscope comparatively difficulty, need to repeatedly focus and could recognize, bring certain inconvenience to counting.In addition, because the survival sperm is not painted or painted very shallow, the survival sperm motility is violent, the situation in " swim into " or " swimming out of " visual field often occurs, to survival rate analysis, brings certain inconvenience.
Gentian violet (Gentian violet) has another name called crystal violet, and methyl is pake purpke, gentian violet, and methyl violet, alkaline purple 3s etc., be a kind of alkaline cationic dye, because of its kation, can be combined with the carboxyl of bacterioprotein, affects its metabolism and produce bacteriostasis.It can suppress gram-positive bacteria, particularly staphylococcus, corynebacterium diphtheriae, and Candida albicans is also had to antibacterial action preferably.Its sterilizing power is strong, and tissue is not had to pungency, also there is no Side effect, therefore as coloring agent, seminal fluid is dyeed.Through retrieval, also do not use the gentian violet decoration method to analyze the standard test guide of sperm survival rate at present.
Summary of the invention
The present invention relates to analyze the new method of mankind spermatozoon survival rate.Adopt certain density aldrich mixture to dye to the seminal fluid sample, coloration result under the microscope can be easy differentiation survival sperm and dead sperm, can further analyze the sperm survival rate, also can assist to analyze sperm motility, contribute to male sterility distinguish and analyze.
Applicant of the present invention uses the gentian violet normal saline solution of variable concentrations, a plurality of Chinese male seminal fluid original samples are dyeed, examined under a microscope subsequently coloration result, by painted difference, judge survival sperm and dead sperm, and calculate the sperm survival rate.Found that the gentian violet normal saline solution can make chromospermism, the survival sperm is light blue, and dead sperm darkviolet surpasses 200 sperms by sum in a plurality of visuals field of technology, calculates the sperm survival rate that obtains sample.Same sample calculates the sperm survival rate after adopting Eosin Y dyeing equally, analyze the significant difference of the sperm survival rate of two kinds of methods acquisitions with paired t-test, result shows, the sperm survival rate that gentian violet normal saline solution decoration method obtains and Eosin Y dyeing do not have difference, illustrate that gentian violet normal saline solution decoration method can be used for distinguishing dead sperm and survival sperm, calculate the sperm survival rate, and assist to estimate sperm motility.The more important thing is, after the dyeing of gentian violet physiological saline, nearly all sperm all is colored, and motility of sperm descends, and is conducive to counting and subsequent analysis under microscope.
Method of the present invention comprises following content:
At first carry out the preparation of sample and dyestuff
1. the suggestion according to the World Health Organization (WHO) obtains the seminal fluid sample
2. Eosin Y, water-soluble: according to the suggestion of the World Health Organization (WHO), adopt the sterling of CAS 17372-87-1, molecular weight 691.9, color index 45380, be dissolved in physiological saline, brown bottle keeps in Dark Place.
3. gentian violet, be dissolved in physiological saline, and brown bottle keeps in Dark Place.
Dyeing and counting
The seminal fluid sample that liquefaction used is good, mix fully, adds a certain proportion of dyeing liquor, makes humidity strip, at optical microphotograph Microscopic observation coloration result, and calculates all sperms in each visual field, according to survival sperm and dead sperm differential count.
Survival rate is calculated and statistical analysis
Adopt Eosin Y and gentian violet, same seminal fluid sample is dyeed, result of determination under ordinary optical microscope in 30～60min.After gentian violet dyeing, light blue is the survival sperm, and darkviolet is dead sperm; After Eosin Y dyeing, water white sperm is the survival sperm, and pink is dead sperm.Choose at random 10 left and right view fields, count respectively survival sperm count and dead sperm count in each visual field, guarantee in all visuals field that the sperm sum is over 200.
Calculate the sperm survival rate according to following formula:
Each sample dual-staining once, calculates respectively the sperm survival rate twice, if, in the disparity range accepted that the two difference is determined in the two mean value, the two mean value can be used as the sperm survival rate of this sample; If the two poor definite disparity range accepted of the two mean value that surpasses, the two mean value can not be as the sperm survival rate mean value that is this sample, need to resample, dyeing again, until have in the disparity range accepted that twice mean value difference determines in the two mean value.
Effective sperm survival rate that two kinds of colouring methods are obtained for same sample is carried out statistical study, statistical method is paired t-test, be that effective sperm survival rate that same sample obtains is subtracted each other, difference and 0 relatively, t >=0.05 just can think that two kinds of methods are not having difference aspect analytic sample sperm survival rate, t<0.05 two kinds of methods of explanation are having aspect analytic sample sperm survival rate obviously, because the Eosin Y colouring method is one of standard method of world health organisation recommendations, t>0.05 an o'clock proof gentian violet can not be as the colouring method of analyzing the sperm survival rate.
Conclusion
The gentian violet normal saline solution can make chromospermism, and the survival sperm is light blue, and dead sperm darkviolet, can obtain by calculating the sperm survival rate of sample.
The sperm survival rate that gentian violet normal saline solution decoration method obtains and Eosin Y dyeing do not have the statistics difference, t >=0.05, illustrate that gentian violet normal saline solution decoration method can be used for distinguishing dead sperm and survival sperm, calculate the sperm survival rate, and assist to estimate sperm motility.
Embodiment
Sperm survival rate in the simple aldrich mixture staining analysis seminal fluid sample of embodiment mono-
Materials and methods:
1. obtain human seminal fluid's sample
The seminal fluid sample adopted provides for the volunteer, therefore needs healthy male volunteers some, according to following requirement, provides the seminal fluid sample:
1) sample that sample is normal masturbation ejaculation, can not be for obtaining in sheath
2) acquisition of sample, must be strict controlled in and ejaculate 3～7 days in last time
3) the seminal fluid sample is directly collected and is entered in the sample cup provided specially, fills out ejaculation time and volunteer's numbering, after covering tightly the sample bowl cover, delivers to laboratory or appointed place in official hour
4) once the rear all samples of ejaculation must all be collected in sample cup, and omission must not be arranged, and also must not have any non-seminal fluid ingredients substance to sneak into
5) leave after health should intact preservation in sample cup for the seminal fluid sample, prevents from polluting.Attention: the human seminal fluid belongs to the potential source biomolecule dangerous goods, please according to international organization and national relevant laws and regulations, disposes.Advise that insulation next to the skin carries, temperature is too low or too highly may affect sperm survival rate result of determination
2. water-soluble Eosin Y: Chemical Reagent Co., Ltd., Sinopharm Group provide (lot number: 20110426), No. CAS: 17372-87-1, molecular weight: 691.9, color index 45380.Take 500mg, be dissolved in 100ml physiological saline, brown bottle keeps in Dark Place.
3. gentian violet, Chemical Reagent Co., Ltd., Sinopharm Group provide (lot number: 20081211), No. CAS: 548-62-9, molecular formula: C
25H
30ClN
3, molecular weight: 407.98, color index 42555.Be dissolved in 100ml physiological saline, brown bottle keeps in Dark Place.
4. microscope: Olympus BX53F (Tokyo, Japan), differential blood-cell calculator, from Chinese Shanghai.
5. colouring method: get the good seminal fluid sample of liquefaction, add 200 μ l dye liquors, mix latter standing 1 minute, take out 20 μ l points to microslide, with cover glass, push open and make humidity strip, examine under a microscope.Count all sperms under 400 times of visuals field, calculate survival rate.For the bad sample that liquefies, do not use the auxiliary liquefaction of enzyme method, directly give up.
6. survival rate can be accepted disparity range: same dye liquor dual-staining twice for each seminal fluid sample, calculate respectively the sperm survival rate, and calculate the mean value of twice survival rate.Determine and can accept disparity range by mean value, if the difference of twice mean value in accepting disparity range, the two mean value can be used as the sperm survival rate of this sample; Can accept disparity range if the difference of twice mean value surpasses, the two mean value can not be as the sperm survival rate mean value that is this sample.Need to resample, dyeing again, until have in the disparity range accepted that twice mean value difference determines in the two mean value.For example, after Eosin Y dyes for No. 22 sample two sub-samplings, the survival rate obtained is respectively 75.7% and 47.9%, the two mean value is 61.8% so, consult " human seminal fluid of the World Health Organization (WHO) check and the treatment of laboratory handbook in " determining the difference the accepted form between two percent of given mean value from 200 sperms of repeat count (altogether counting 400 sperms) ", learn that the accepted difference of average bits in 35～65% is 10%, be that can to accept disparity range while being 61.8% be 51.8%～71.8% to mean value, the survival rate 75.7% and 47.9% that above-mentioned twice dyeing obtains all exceeds this scope, therefore assert that the two mean value 61.8% can not be as effective sperm survival rate, necessary repeated sampling, dyeing, counting.Again for example, after the gentian violet normal saline solution dyes for No. 7 sample two sub-samplings, the survival rate obtained is respectively 81.4% and 79.6%, the two mean value is 80.5% so, consult " human seminal fluid of the World Health Organization (WHO) check and the treatment of laboratory handbook in " determining the difference the accepted form between two percent of given mean value from 200 sperms of repeat count (altogether counting 400 sperms) ", learn that the accepted difference of average bits in 77～83% is 8%, be that can to accept disparity range while being 80.5% be 72.5%～88.5% to mean value, the survival rate 81.4% and 79.6% that above-mentioned twice dyeing obtains does not all exceed this scope, therefore assert that the two mean value 80.5% can be used as effective sperm survival rate.
7. statistical study: effective sperm survival rate that two kinds of colouring methods are obtained for same sample is carried out statistical study, adopt SAS statistical analysis software (8.01 version), the row paired t-test, be that effective sperm survival rate that same sample obtains is subtracted each other, difference and 0 relatively, t >=0.05 just can think that two kinds of methods are not having difference aspect analytic sample sperm survival rate, and there were significant differences aspect analytic sample sperm survival rate for t<0.05 two kinds of methods of explanation.
Result
1. coloration result:
The Eosin Y coloration result is as Fig. 1, and gentian violet physiological saline coloration result is as Fig. 2.
2. sperm survival rate analysis result, as table 1.
The sperm survival rate that two kinds of colouring methods of table 1. obtain relatively
*：t＝0.8617
Conclusion:
The gentian violet normal saline solution can make chromospermism, and the survival sperm is light blue, and dead sperm darkviolet, can obtain by calculating the sperm survival rate of sample; T=0.8617, be far longer than 0.05, illustrates that gentian violet normal saline solution decoration method can be used for distinguishing dead sperm and survival sperm, calculates the sperm survival rate, and assist to estimate sperm motility.
The accompanying drawing explanation
Fig. 1: normal semen sample Eosin Y coloration result, left for amplifying 400 times, right for amplifying 1000 times.S: survival sperm, D: dead sperm
Fig. 2: normal semen sample aldrich mixture coloration result, left for amplifying 400 times, right for amplifying 1000 times.S: survival sperm, D: dead sperm.
Claims (5)
1. gentian violet is being differentiated survival sperm and dead sperm and in the application of analyzing aspect the sperm survival rate as coloring agent separately.
2. gentian violet is being differentiated survival sperm and dead sperm and in the application of analyzing aspect the sperm survival rate as additive as coloring agent.
Gentian violet separately or as adjuvant as coloring agent in the application of analyzing aspect the survival sperm motility.
4. according to the described application of claim 1,2 and 3, wherein the concentration range lower limit of gentian violet is 0.01%, and the upper limit is 5.0%.
5. according to the described application of claim 1,2 and 3, wherein related sperm should be for comprising the sperm of the mankind in interior mammal seminal fluid.
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Cited By (3)
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| CN109064469A (en) * | 2018-10-31 | 2018-12-21 | 北京新网视信传媒科技有限公司 | Sperm quality detector and sperm quality detection system |
| CN110967230A (en) * | 2019-11-22 | 2020-04-07 | 珠海高瑞特医疗科技有限公司 | Method and kit for measuring content of active oxygen in sperm |
| CN113564105A (en) * | 2021-08-19 | 2021-10-29 | 内蒙古自治区妇幼保健院 | Sperm washing upstream method |
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| CN1385700A (en) * | 2001-05-10 | 2002-12-18 | 华中科技大学同济医学院 | Sperm fluorescent staining computer detection system |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109064469A (en) * | 2018-10-31 | 2018-12-21 | 北京新网视信传媒科技有限公司 | Sperm quality detector and sperm quality detection system |
| CN109064469B (en) * | 2018-10-31 | 2023-08-15 | 北京新网视信传媒科技有限公司 | Sperm quality detector and sperm quality detection system |
| CN110967230A (en) * | 2019-11-22 | 2020-04-07 | 珠海高瑞特医疗科技有限公司 | Method and kit for measuring content of active oxygen in sperm |
| CN110967230B (en) * | 2019-11-22 | 2022-05-31 | 珠海高瑞特医疗科技有限公司 | Method and kit for measuring content of active oxygen in sperm |
| CN113564105A (en) * | 2021-08-19 | 2021-10-29 | 内蒙古自治区妇幼保健院 | Sperm washing upstream method |
| CN113564105B (en) * | 2021-08-19 | 2022-12-27 | 内蒙古自治区妇幼保健院 | Sperm washing upstream method |
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