CN102323398B - Solution for sperm detection - Google Patents
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- CN102323398B CN102323398B CN201110130338.5A CN201110130338A CN102323398B CN 102323398 B CN102323398 B CN 102323398B CN 201110130338 A CN201110130338 A CN 201110130338A CN 102323398 B CN102323398 B CN 102323398B
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Abstract
The invention discloses a solution for sperm detection. The solution for sperm detection of the invention comprises hyaluronic acid and L-carnitine, and can be used for sperm-cervical mucus penetration tests. Thereinto, the hyaluronic acid is used as a substitute of human cervical mucus, and L-carnitine can protect sperm motility. Based on conditions that patients with oligozoospermia/asthenospermia have less sperm numbers and low sperm motility, the invention is further improved, and a sperm detection solution for patients with oligozoospermia/asthenospermia is prepared. The sperm detection solution for patients with oligozoospermia/asthenospermia has high sensitivity, and a high concentration of L-carnitine, and the L-carnitine can increase the oxidative damage resistance of sperms and protect sperm motility.
Description
Technical field
The present invention relates to sperm and detect reagent.In particular to take hyaluronic acid and L-BETAIN, be configured for the solution that sperm detects as principal ingredient.
Background technology
The method that current China evaluates male fertility clinically mainly adopts seminal fluid conventional analysis (semen routine analysis, SRA) to understand the general proterties of seminal fluid, sperm and refining.It comprises sperm concentration, motility rate, vigor and morphological examination thereof.Though SRA can provide spermatogenesis and mature condition thereof, yet very unreliable [the YUAN Yu-ying & .SHI Qi-xian:The Evaluation of sperm function.Reproduction & .Contraception18 of these conventional parameter indication sperm fertilizing abilities, 58,1998.David G, Jouannet P, Martin-Boyce A.Spitz A & Schwartz D:Sperm counts in fertile and infertile men.Fertil Steril 31:453,1979].Sperm function evaluation is to detect the state that sperm is carried out certain specific function.Comprise that sperm motility feature detection, Motility test, sperm membrane integrity test, acrosome status are measured, Mucus interacts test etc.For making egg cell fertilization, sperm, except reaching some, also must possess good motor function and the ability (abbreviation penetration power) that penetrates cervical mucus, corona radiate of ovum, oolemma and dotter haut.The penetration power of sperm is to evaluate the important parameter of sperm function.Someone reports the sperm that approximately has the normal person of 18% seminal fluid routine inspection, can not penetrate cervical mucus.
Though people's sperm in vitro zona-free hamster egg penetration test can correctly reflect fertility [the Shi QX of sperm, Zhong CL.Yuan Y Y.Ye Z.Ren Y & .Wang ZJ:Spermme inhibition of in vitro fertilizing ability of human spermatozoa and its possible mode of action.Acta Physiologica Sinica.43:480.1991], but the method technical difficulty is large, expensive, be therefore difficult to promote.Evaluate that male-contraception medicine feels difficulty most be its validity problem [Kumar TCH & Waites GMH eds:Methods of the Regulation of Male Ferfility.New Delhi:Indian Council of Medical Research 1985, p.76].Check the method for sperm to cervical mucus penetration capacity, mainly contain Huhner's test, sperm kapillary penetration test and sperm, cervical mucus slide penetration test.Huhner's test is to detect after sexual intercourse number of motile sperm in certain hour cervical mucus, to evaluate the saturating ability of wink of sperm.Its result is subject to the proterties, female incretion situation of cervical mucus etc. to be permitted multifactorial impact, and operation is very not convenient.For getting rid of female factors impact, people, by cervical mucus standardization, are sub-packed in kapillary or drip on slide, the penetration power in observation in vitro sperm to cervical mucus.Influence factor is few, can lot inspection, and than the Huhner's test practical application of being more convenient for.
Carlson etc. disclose in the semicentennial time the summary of male semen quality in last century, and mankind spermatozoon density obviously declines.Meanwhile.Domestic scholars is by also having disclosed similar male semen quality downward trend to semen data analysis.With external homogeneous data comparison, the development trend of China seems more obvious.Need to and set up the detection methods for scientific research and sperm quality clinical, molecular level and sperm function in laboratory research and development, the crucial detection technique of setting up seminal fluid/sperm quality is one of basic guarantee technology of male fertility monitoring and evaluation.
Therefore, by Mucus breakthrough experiment, measure the interaction of Mucus, be also more and more subject to researchist's attention.In the < < WHO human seminal fluid of the World Health Organization (WHO) inspection and treatment of laboratory handbook > > of new edition in 2011, not only to the method for above-mentioned experiment is clear and definite, defined, and formally released breakthrough experiment device and corresponding using method and criterion.
The in-vitro evaluation of Sperm penetration cervical mucus has long history [Kurzok, R.and Miller, E. (1932) Biochemical studies of human semen.III.Factors affecting migration of sperm through the cervix.Am.J.Obstet.Gynecol., 24,19-26.Botella-Llusia, J.and Ruiz-Velasco, V. (1960) Postcoital test in vitro.Int.J.Fertil., 5,301-306.].Have at present multiple sperm detection technique, the method that wherein sperm kapillary penetrates has become the most frequently used method.Sperm kapillary penetration test was founded by Kremer in nineteen sixty-five.By observing in vitro sperm, whether penetrate the cervical mucus in kapillary, to evaluate the penetration power of sperm.Simple this technology of statement is that relatively the penetrating power of different medium, comprises the quantity of assessing sperm in the nearest migration distance to 1cm and 1/2 migration distance place kapillary.Because it can cross-beta contributor's sperm and mucus, to distinguish women's (uterine neck) and male sex's (sperm) factor [Kremer, J. (1965) A simple sperm penetration test.Int.J.Fertil., 10,209-215.].The method is easy and simple to handle, and experiment condition is easy to control, and influence factor is few.Particularly can use cervical mucus or the cervical mucus substitute of donor, can identify very easily large quantities of patients' Sperm penetration power, and can identify the factor that causes Huhner's test abnormal, be on the bridegroom's or husband's side or on the wife's side.Therefore, value for clinical application is large.So far, Sperm-Cervical mucus penetration test has been widely used in evaluating the intermiscibility of sperm and cervical mucus and the ability that sperm passes through cervical mucus.
An important clinical application of sperm-mucus penetration test is exactly the diagnosis [Clarke of Sperm autoimmunity, G.N., Elliott, P.J.and Smaila, C. (1985) Detection of sperm antibodies in semen using the immunobead test:a survey of 813 consecutive patients.Am.J.Reprod.Immunol.Microbiol., 7, 118-123.Clarke, G.N., Lopata, A., McBain, J.C.et al. (1985) Effect of sperm antibodies in males on human in vitro fertilization (IVF) .Am.J.Reprod.Immunol.Microbiol., 8, 62-66.Kremer, J.and Jager, S. (1992) The significance of antisperm antibodies for sperm-cervical mucus interaction.Hum.Reprod., 7, 781-784.].By the strict coated sperm of AsAb, can not be penetrated cervical mucus.Therefore, the penetration test of cervical mucus can be used for determining that positive sperm antibody detects importance clinically.Sperm antibody tests positive, but the male sex of Mucus penetrating value > 2cm in 1h, it is rational making its companion's pregnancy, but those do not have the male sex of sperm-mucus penetration capacity, (have extremely low to conceived the predicted value) [Clarke that makes the probability of its companion's pregnancy very low, G.N., Lopata, A., McBain, J.C.et al. (1985) Effect of sperm antibodies in males on human in vitro fertilization (IVF) .Am.J.Reprod.Immunol.Microbiol., 8, 62-66.Kremer, J.and Jager, S. (1988) Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies.Hum.Reprod., 3, 69-73.].The latter will treat to improve by glucocorticoid or intracytoplasmic sperm injection.[Wang，C.，Baker，H.W.G.，Jennings，M.G.et?al.(1985)Interaction?between?human?cervical?mucus?and?sperm?surface?antibodies.Fertil.Steril.，44，484-488.]。
In Mucus breakthrough experiment process in the past, no matter for experimenter or experimenter, the collection requirement of women's cervical mucus is all higher.Concrete HCM detects for laboratory several problems that exist.Particularly, the viscoelastic changeability that the menstrual cycle is relevant and collect the variation of mucus essential component at mid-term in cycle or estrin treatment.In collecting the process of sample, vaginal fluid mixes with mucus, can cause sample unavailable, because sour environment (pH < 6.5) can make sperm lose mobile ability.It is also limited obtaining high-quality cervical mucus, because HCM measures few (0.3-0.5mL/ people), and source difficulty, its quality also varies with each individual, and is difficult for standardization.And in HCM's storage process, mucus quality easily changes, preserve for 4 ℃ and require to use in one week.In addition, cervical mucus is filled into and in kapillary, also exists certain technical difficulty.
Because these reasons are impelled researcher and are striven to find suitable HCM's substitute.Blandan RJ report application ox cervical mucus in estrus (BCM) in 1978 conduct penetrates medium and substitutes HCMC[Blandan Rj, Goddum-Rosse P & Lee WI:Letter to the Editor.Fertil Steril 30 (7): 707,1978], ox cervical mucus is a lot [Urry of product research as an alternative, R.L., Middleton, R.G.and Mayo, D. (1986) A comparison of the penetration of human sperm into bovine and artificial cervical mucus.Fertil.Steril., 45, 135-137.Kummerfeld, H.L., Vosburgh, J.K., Lorton, S.P.et al. (1981) Influence of bovine cervical mucus samples and storage conditions on sperm migration in vitro.Fertil.Steril., 35, 218-221.].This improvement, has promoted the clinical research that external male fertility is evaluated.Hanson in 1979 etc. have proposed to substitute with egg white the penetration capacity [Hanson KB second Hjort T:The penetration of antibody covered spermatozoa into cerical mucus and egg white Arch Androl 3:245.1979] that cervical mucus is carried out appraiser's sperm.Also there is research to adopt hyaluronic acid and polyacrylamide gel [Lorton, S.P., Kummerfeld, H.L.and Foote, R.H. (1981) Polyacrylamide as a substitute for cervical mucus in sperm migration tests.Fertil.Steril., 35, 222-225.Mortimer, D., Mortimer, S.T., Shu, M.A.et al. (1990) A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test.Hum.Reprod., 5, 835-841.Aitken, R.J., Bowie, H., Buckingham, D.et al. (1992) Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence.J.Androl., 13, 44-54.].In general, desirable mucus substitute should be cheap, and easily preparation is stable, the most important thing is to have the Penetration Signature identical with HCM.In addition the clinical treatment that can be used for, male sterility.The penetration test of the sperm that antagonist is coated, substitute must have similar susceptibility to cervical mucus.Although substitute and women's cervical mucus function have certain similarity, cheap, easily obtain, most substitutes have in test significant deficiency.Such as, Countryside Egg is clear, as penetrating medium, experimental repeatability is bad, and time of break-through and penetration range and cervical mucus differ larger; The physicochemical property of sperm nutrient solution and cervical mucus differs larger, and as penetrating medium, time of break-through and penetration range and cervical mucus differ larger.Degree of recognition is very low in the world for the result of study of the cervical mucus substitute gained of many kinds, can not effectively be promoted.
Improve sperm detection sensitivity and convenience for this reason, improve sperm detection solution and have great importance.The present invention has selected hyaluronic acid as penetrating medium, in conjunction with unique prolection of L-BETAIN, has invented a kind of sperm and has detected solution; And continue on this basis to improve, invented and be applicable to very much the solution that few/azoospermia patient sperm detects.
Summary of the invention
The invention discloses a kind of solution detecting for sperm, the hyaluronic acid that the solution that described sperm detects comprises 3-7.5mg/mL and the L-BETAIN of 2.5-10mM/L.
Described hyaluronic molecular weight is 1.5-2.5 * 10
6.
Described L-BETAIN can be protected the vigor of sperm.
The invention also discloses a kind of sperm for few/azoospermia patient and detect solution, the hyaluronic acid that comprises 4-6mg/mL and the L-BETAIN of 10-20mM/L.The sperm that described detection solution is applicable to few/azoospermia patient detects.
Described hyaluronic molecular weight is 1.5-2.5 * 10
6.
Described L-BETAIN can strengthen the ability of sperm anti-oxidative damage, the vigor of protection sperm.
The solution that sperm of the present invention detects and traditional detection solution phase ratio, can better simulate the performance of sperm in HCM.
Sperm number based on few/azoospermia patient is few, the situation that sperm motility is low, and the present invention, through further improving, has prepared a kind of sperm for few/azoospermia patient and has detected solution.Few/azoospermia patient's sperm detects solution, highly sensitive, and can effectively evaluating few/azoospermia patient's the order of severity, for certain directive function is played in corresponding drug therapy.
Embodiment 1 sperm detects the research of solution
One, test material
The collection of seminal fluid: seminal fluid was from the contributor who has fecundity of ascetic 2-5 days, and masturbation produces.According to World Health Organization's handbook, according to the strict standard of revision, seminal fluid is carried out to concentration (91 ± 72 * 10 dying in wink on slide
6/ ml, range 5.0-271), the analysis of total motility (55 ± 16%, range 6.0-81) and eupyrene sperm form (20 ± 7.8%, range 1.0-42).
The collection of cervical mucus: cervical mucus comes from the women after spontaneous ovulation after much different artificial insemination.All samples is all before ovulation, and collect for 3 days at interval.Mucus is drawn into from cervical canal a polyethylene pipe.After collecting, assess cervical mucus, comprise (WHO, 2001) such as ropiness, fernlike crystal, stickiness and pH values of measuring mucus.Normal cervical mucus (pH > 7.0) is distributed into 4 ℃ of preservations in tubule, in 7 days, uses.Only come from and have the contributor's of fecundity the mucus that has normal penetrating power to be used to detect.
The preparation that Countryside Egg is clear: egg white is around to draw from Fresh Egg egg yolk layer with syringe.
Hyaluronic acid is from Sigma company.It is extracted with separated as hyaluronic sodium salt from rooster combs (cockscomb).Its molecular weight is 1.5-2.5 * 10
6, part obtains by chemical degradation.Hyaluronic working concentration is 3-7.5mg/ml, in Tyrode damping fluid, adds 10-50mg/ml bovine serum albumin(BSA) (BSA Commonwealth Serum Laboratories, Melbourne, Australia) before use again.L-BETAIN is bought from Sigma company.
Hyaluronic acid solution (solution A 1) preparation containing L-BETAIN: in 60min, L-BETAIN is dissolved in Tyrode damping fluid before test, mixes with hyaluronic acid solution, form the hyaluronic acid solution containing L-BETAIN.L-BETAIN concentration is 2.5-10mM/L.
HCM's (solution A 2) preparation containing L-BETAIN: in 60min, L-BETAIN is dissolved in Tyrode damping fluid before test, mixes with HCM, form the HCM containing L-BETAIN.L-BETAIN concentration is 2.5-10mM/L.
Contrast migration medium: the Tyrode solution (Commonwealth Serum Laboratories Melboume Australia) containing BSA is used as contrast migration medium.
Two, test method
1) by 37 ℃ of preheatings of All Media.Use circular section kapillary, 100mm is long, internal diameter 0.798mm (50 μ l Drummond microcaps; Edwards Instruments, Melbourne, Australia).Each effective 1ml syringe pump, plastic tube of upper termination, and lower end is inserted in medium.The Cylindric medium of 7-8cm is inhaled in pipe, so the semilune liquid level that medium forms is 2-3cm to the distance of managing top.
In operation, answer extreme care, prevent that bubble from entering in pipe.The top of pipe seals with rubber cement, and all media of coming in are trapped to produce a flat interphase in bottom.
2) seminal fluid of about 100 μ l is added into a little plastics tapered tube bottom, and one contains a kind of its openend of kapillary that moves medium and is placed in seminal fluid.Medium detects simultaneously in same seminal fluid.Seminal fluid storage pond and kapillary are installed in shown in microslide Shang Ru World Health Organization handbook (WHO, 2001).Then microslide is put into a double dish that contains moist sponge, keep humidity, prevent that seminal fluid and medium are dry.Horizontal with capillary tube is hatched.
3) hatch after 60min, microslide is taken out from double dish.Under a bright visual field, observe the object lens of kapillary * 20, * 10 eyepiece.Adjust objective table, select focal plane and the visual field that kapillary axis is combined, and amplify at this, the width of field of microscope is approximately equal to internal diameter capillaceous.Then scan the length of pipe, to determine the maximum distance of the free sperm bank of antherozoid.
The ultimate range of hatching antherozoid migration after 60min is defined as migration distance, and bibliographical information approaches 1cm.At 1/2 migration distance place, the penetration density of sperm is assessed, under same focal length from bottom capillaceous to top, with identical enlargement ratio and sperm count.The volume of estimating dielectric sample during counting is 0.34 μ l.The number that 1/2 migration distance is multiplied by 1/2 migration distance place sperm obtains permeability.By detecting, at 1/2 migration distance place, have 200 sperms at least, to assess the mobility of sperm.
Experiment is divided into two parts, and first carries out breakthrough experiment with the multiple medium that penetrates to be selected simultaneously, confirms whether hyaluronic acid can replace HCM to carry out Sperm-Cervical mucus penetration test; Second portion adds L-BETAIN in hyaluronic acid, confirms whether L-BETAIN can protect the vigor of sperm.
Three, experimental result
First experiment shows that hyaluronic acid, as penetrating medium, comparing with HCM aspect sperm migration distance, penetration density and energy, there is no significant difference (table 1), can replace HCM to carry out Sperm-Cervical mucus penetration test.With contain albuminous Tyrode solution phase ratio, cervical mucus and hyaluronic acid have very high penetrating power, and egg white penetrating power a little less than.
Second portion experiment is presented in hyaluronic acid, to add L-BETAIN (solution A 1) and in HCM, add L-BETAIN (solution A 2) and all can completes Sperm penetration experiment, compare with the medium that does not add L-BETAIN, indices deviation in test is less, and experimental result stability is high.
The penetration capacity comparison (n=60) of table 1 eupyrene sperm in several media
The impact (n=60) of table 2L-carnitine penetration capacity in two media on eupyrene sperm
Embodiment 2 lack/azoospermia patient sperms detect the research of solution
One, test material
The collection of seminal fluid: seminal fluid is from few/weak smart patient, and after ascetic 2-5 days, masturbation produces.
The collection of cervical mucus: cervical mucus comes from the women after spontaneous ovulation after much different artificial insemination.All samples is all before ovulation, and collect for 3 days at interval.
The preparation that Countryside Egg is clear: egg white is around to draw from Fresh Egg egg yolk layer with syringe.
Hyaluronic acid solution (solution B) preparation containing L-BETAIN: in 60min, L-BETAIN is dissolved in Tyrode damping fluid before test, mixes with hyaluronic acid solution, form the hyaluronic acid solution containing L-BETAIN.L-BETAIN concentration is 10-20mM/L.
Contrast migration medium: the Tyrode solution (Commonwealth Serum Laboratories Melbourne Australia) containing BSA is used as contrast migration medium.
Two, test method
1) by 37 ℃ of preheatings of All Media.Use circular section kapillary, each effective 1ml syringe pump, plastic tube of upper termination, and lower end is inserted in medium.The Cylindric medium of 7-8cm is inhaled in pipe, so the semilune liquid level that medium forms is 2-3cm to the distance of managing top.
2) seminal fluid of about 100 μ l is added into a little plastics tapered tube bottom, and one contains a kind of its openend of kapillary that moves medium and is placed in seminal fluid.Five kinds of media detect simultaneously in same seminal fluid.Then microslide is put into a double dish that contains moist sponge, keep humidity, prevent that seminal fluid and medium are dry.Horizontal with capillary tube is hatched.
3) hatch after 60min, microslide is taken out from double dish.Under a bright visual field, observe the object lens of kapillary * 20, * 10 eyepiece.Adjust objective table, select focal plane and the visual field that kapillary axis is combined, and amplify at this, the width of field of microscope is approximately equal to internal diameter capillaceous.Then scan the length of pipe, to determine the maximum distance of the free sperm bank of antherozoid.
Three, experimental result
In cervical mucus and hyaluronic acid, average migration distance and sperm concentration do not have significant difference (table 3,4 and 5).But solution B and cervical mucus significant difference.Compare with hyaluronic acid with HCM, increased L-BETAIN in solution B, strengthened the anti-oxidative damage ability of sperm, sperm motility is protected, in Table 3,4 and 5.Wherein solution B indices is in test more obvious, and deviation is less, and experimental result stability is high, is suitable for the practical application that few/weak sperm patient sperm detects.
Table 3 aspermia or oligospermia patient's Mucus and the interactional penetration test of replacement liquid (n=60) thereof
Table 4 azoospermia patient's Mucus and the interactional penetration test of replacement liquid (n=60) thereof
Table 5 oligospermia patient's Mucus and the interactional penetration test of replacement liquid (n=60) thereof
Claims (3)
1. the solution detecting for sperm, the hyaluronic acid that comprises 3-7.5mg/mL and the L-BETAIN of 2.5-10mM/L; Wherein said hyaluronic molecular weight is 1.5-2.5 * 10
6; Wherein said L-BETAIN can be protected the vigor of sperm.
2. the sperm for few/azoospermia patient detects a solution, the hyaluronic acid that comprises 4-6mg/mL and the L-BETAIN of 10-20mM/L; Wherein said hyaluronic molecular weight is 1.5-2.5 * 10
6; Wherein said L-BETAIN can strengthen the ability of sperm anti-oxidative damage, the vigor of protection sperm.
3. sperm according to claim 2 detects solution, it is characterized in that, described sperm detects solution and contains 10-20mM/L L-BETAIN, the sperm anti-oxidative damage ability of potential fertility is strengthened, improved the sensitivity of sperm motility Function detection, can detect for few/azoospermia patient's sperm.
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| CN112114123A (en) * | 2020-09-02 | 2020-12-22 | 上海市计划生育科学研究所 | Application of HA in Replacing Human Cervical Mucus to Detect Sperm Penetration |
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| CN102323398A (en) | 2012-01-18 |
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