CN105695557A - Method for evaluating quality of sperm obtaining energy in vitro - Google Patents
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Abstract
The invention provides a method for evaluating the quality of sperm obtaining energy in vitro. The method specifically includes the following steps of A, collecting a fresh liquefied sperm specimen, washing sperm with a phosphate buffer or a BWW culture solution, sufficiently removing residual seminal plasma components, and separating out sperm in a centrifugal mode; B, adding the BWW culture solution to sperm separated out in the step A, obtaining upstream sperm through an upstream method, counting the number of sperm, incubating sperm for 3-4 hours in a CO2 cell culture box with 5% of in-vitro energy obtaining liquid, conducting centrifugal separation on the obtained supernate, and collecting supernate as a to-be-tested sample; C, measuring the activity of neuraminidase in sperm energy obtaining liquid, and finally obtaining the value of neuraminidase activity/sperm number to evaluate the quality of sperm. By detecting the activity of neuraminidase in sperm in-vitro energy obtaining liquid, diagnosis basis and clinical queries are provided for male infertility patients of unknown clinical reasons.
Description
Technical field
The invention belongs to medical detection technology, a kind of method that after being specifically related to In-vitro Capacitation, sperm quality is assessed。
Background technology
Along with the development of society's industry, the total incidence of infertile couples is significantly raised。Within 2004, European genesiology can be added up: the couple at child-bearing age pregnancy person can not account for 25% in 1 year, wherein 15% seeks treatment, and bridegroom's or husband's side factor causes and sterile accounts for 50%。The cause of disease sexual disorders (1.7%) of male sterility, varicocele (12.3%), reproductive tract infection (6.6%), congenital abnormal development (2.1%) the acquired disease day after tomorrow (2.6%), endocrine regulation (0.6%), immunity factor (3.1%), other abnormal (3%), but the patient being up to 60% ~ 75% can not find reason, being called idiopathic male infertility, it is abnormal that they only show as the sperm qualities such as few essence, azoospermia or teratozoospermia。The male sterility of unknown cause is likely to be due to many factors and causes, as long-term stress environmental factors causes endocrine regulation, active oxygen and genetic flaw etc.。
For many years, traditional semen routine analysis is always up the most basic clinical indices for judging male fertility。Clinically the real sterile reason of the male sterility patient of the overwhelming majority is still unclear。Especially nearly 1/3rd infertile patients clinically, the Sperm routine analysis result of male is all normal and close normal。Clinically this kind of patient is divided into Unexplained infertility。Therefore, for a long time, there is very big difficulty in the clinical diagnosis to male sterility。Topmost reason is the quantity that Sperm routine analysis only measures sperm, viability, activity ratio and form。These indexs can only reflect most basic semen quality, it is impossible to reflects all of sperm function, such as, the maturation of sperm nucleus and DNA damage, sperm and human oocyte zona pellucida association reaction with penetrate, acrosome situation and reaction and with vitellinae membrana binding ability etc.。Accordingly, it would be desirable to set up special sperm function tests could measure the above function。Capacitation is the essential condition that sperm ovum binding forms germ cell, although the correlational study of capacitation has some progress, but still there is a lot of still undefined problem。
In clinical position, the Infertility male patient of a part of former or secondary utilizes existing conventional semen quality to check that project prompting sperm and semen quality are without exception。Sperm detection means conventional at present is CASA (computer aided sperm analysis Computeraidedofsemenanalysis), and antisperm antibody etc.。Current the most widely used CASA technology and some morphologic coherent detections, mainly can evaluate the mobility of sperm and sperm ratio of living, thus speculating that it enters the fertility of female genital tract, and rely on current sperm function appraisement system, be there is certain defect in the evaluation of sperm quality and function, and be difficult to provide corresponding clinical consultation to patient。It is true that this is only the aspect evaluating sperm function, we are specifically contemplated that to be likely to also have affects the more of sperm reproductive performance and does not have revealed factor。
In first patent 201310431847.0, it is proposed that a kind of sialidase detection method affecting sperm function。The first step, quantifies sialidase expression in sperm intuitively by monosperm Fluorescence Intensity Assays, or utilizes fluorescence imaging under microscope to observe sialidase expression in sperm intuitively；If sialidase is high expressed in sperm, then carry out second step, utilize In-vitro Capacitation liquid to make sperm affect the sialidase detection method capacitation of sperm function, and use Fluorometric assay sialidase activity。Although this patent discloses the detection of the expression in sperm of the sialidase protein molecule and primary activity。But method includes the detection of expression of sperm sialidase NEU1/3, it is necessary to fluorescence microscope or flow cytometer, has the disadvantage in that 1) techniqueflow complexity；2) experimental facilities and operator's skills and experience are required height；3) it is unsuitable for being applied to clinical laboratory and routine techniques librarian use；4) test kit of abcam company is used, expensive。Further, since the sperm quantity of different samples is different, the protein value of expression is different, and corresponding enzyme value alive is also different, and therefore, the enzyme of simple relatively different samples is alive is worth not science。
Summary of the invention
For above-mentioned technical problem, a kind of method that after the invention provides In-vitro Capacitation, sperm quality is assessed。By the detection of sialidase activity in capacitation liquid after sperm microcytotoxicity, the sperm quality after assessment capacitation, provide diagnosis basis and clinical consultation for clinical agnogenic male sterility patient。
In order to realize foregoing invention purpose, the present invention adopts the following technical scheme that:
A kind of method of sperm quality assessment after In-vitro Capacitation, it is characterised in that: specifically comprise the following steps that
A, sperm washing
Collect fresh liquefaction semen sample, wash sperm with phosphate buffer or BWW culture fluid, fully remove the refining composition of residual, then centrifugation goes out sperm；
The present invention first adds phosphate buffer in semen sample or BWW culture fluid washs, then is centrifugally separating to obtain Sperm pellets。This is owing to refining itself is sticky, directly it is centrifuged and all sperms in seminal fluid can not be made fully to precipitate, after it is diluted by addition phosphate buffer or BWW culture fluid, and centrifugally operated better effects if。
Preferably, seminal fluid carries out 3 times be centrifuged, it is ensured that when detection does not have refining to disturb, reduce the number of times of centrifuged sperm as far as possible, it is ensured that motility of sperm。
B, sperm In-vitro Capacitation
In the isolated sperm of step A, add BWW culture fluid, adopt upper reaches method, at the CO of 5%2Cell culture incubator is cultivated, results upstream sperm, counts sperm quantity, with In-vitro Capacitation liquid 5% CO2Cell culture incubator is hatched sperm 3 ~ 4h；Centrifugation, the supernatant recentrifuge collected separates, and collects supernatant as sample to be tested；After upper reaches method obtains the good sperm of vigor, it being carried out capacitation process, experimental result is more scientific objective at once。
Preferably, upstream sperm quantity is controlled at 1X107Above, set according to the needs of detection sensitivity。
C. the mensuration of sialidase activity in capacitation liquid
(1) being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, is sequentially added into each sialidase standard solution and sample to be tested in detection plate。
The gradient of standard solution be discharged into the amount in capacitation liquid according to sperm sialidase after capacitation and sensitivity that the inventive method can be detected by and set。
Preferably, described detection plate is 96 hole blackboards。The present invention adopts fluorescence method to measure the activity of sialidase, and sensitivity and specificity are best by the detection plate of black, it is possible to protection fluorescent dye is not by light cancellation, so that the sensitivity of detection is greatly improved。
(2) with detection buffer dilution fluorometric reagent, it is added in the hole of above-mentioned each sialidase standard solution and sample to be tested, again detection plate is placed in 37 DEG C, after lucifuge hatches 1-2 hour, each hole fluorescence intensity is read by microplate reader, calculate the sialidase activity of sample to be tested according to sialidase activity standard curve, finally draw the value of sialidase activity/sperm number。
Preferably, the concentration being diluted to fluorometric reagent with detection buffer is 0.05mM, for best working concentration。
Preferably, microplate reader reads each hole fluorescence intensity under the wavelength of excitation wavelength and transmitting wavelength respectively 365nm and 450nm。365nm is maximum excitation wavelength, and 450nm is best transmitting wavelength。
Preferably, for sample sialidase activity in the hole detected should within the scope of 0.3-5mU/ml, if beyond; the sialidase activity value of sample to be tested in hole should be adjusted within this range of linearity。
Detection buffer of the present invention is the PBS containing 0.05mmoL/L sodium acetate and 0.25 μ g/ μ l bovine serum albumin。The buffer system of detection buffer, detects specific preparation for sialidase activity。
Preferably, the pH value of described sodium acetate is 5-6, is the working environment best for ptyalin activity。
In-vitro Capacitation liquid of the present invention is the HTF(HOF containing HSA (human serum albumin) 5mg/ml)。
Described sialidase standard stock solution is bacillus perfringens sialidase (AUS), and concentration is 5U/ml, can easily dilute for these Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml。Bacillus perfringens sialidase is adopted to be applicable to the detection method of a small amount of sperm of the present invention。
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium, has higher sensitivity。
The concrete structure of described 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium is as follows:
The beneficial effects of the present invention is:
1, the reagent of detection method all selects routine test reagent, and combine the particularity to sperm detection, and reducing experimental cost, required instrument and equipment, consumptive material kind are few, it is only centrifuge, microplate reader, EP pipe, rifle head and detection plate etc., is Routine Test Lab configuration；And operational approach is simple, is only centrifugal, application of sample etc., is absent from the situation that need to repeatedly practise obtaining operation skill, can be widely applied to clinical laboratory and routine techniques librarian use。
2, the detection mode of the present invention is for assessing the quality of sperm after capacitation, according to the activity value of sialidase in unit sperm quantity, the crowd of making expresses the marginal value of sialidase activity, can live clinically by unit enzyme and be worth deeper level understanding sperm function, can be applicable to reproduction and assist such as technology such as test-tube babies。
3, owing to the sperm quantity of different samples is different, the protein value of expression is different, and corresponding enzyme value alive is also different, so, the enzyme of simple relatively different samples is lived and is worth not science, if because the sperm number of a sample is little, but sperm quality is significantly high, and the sperm number of another sample is a lot, but sperm quality is relatively low, if comparing merely enzyme value alive, then the former is likely to be defeated by the latter, but practical situation is, the former is higher than the latter at quality。Therefore, the detection of the present invention is that the unit enzyme of sialidase activity/sperm number is lived value, and testing result is more objective, accurately and reliably。
4, sperm is washed by the present invention fully, fully removes the refining composition of residual, makes the albumen of residual in refining and sialidase without influence on the accuracy of testing result；Optimize the standard substance protein concentration gradient arranged when surveying sperm protein concentration and survey the standard substance enzyme stepladder degree arranged when enzyme is lived, if gradient arranges unreasonable, all can affect very much sensitivity and the accuracy of result；Use black detection plate to survey enzyme to live, can obviously reduce the light negative effect to fluorometric reagent cancellation, be thus greatly improved the stability of experimental result, sensitivity and accuracy；Final testing result is designed as the value of sialidase work/sperm number, so can reduce the difference between different sample, make to have more each other comparability, it is ensured that the stability of testing result and reliability。
Accompanying drawing explanation
The method for evaluating quality that Fig. 1 is the present invention measures non-capacitated sperm and the sialidase activity of capacitated sperm (A is mice, and B is people) respectively。
Detailed description of the invention
Below in conjunction with detailed description of the invention, the essentiality content of the present invention is described in further detail。
Embodiment 1
After a kind of In-vitro Capacitation, the method for sperm quality assessment, specifically comprises the following steps that
A, sperm washing
Collect fresh liquefaction semen sample, wash sperm with phosphate buffer or BWW culture fluid, fully remove the refining composition of residual, then centrifugation goes out sperm；
B, sperm In-vitro Capacitation
In the isolated sperm of step A, add BWW culture fluid, adopt upper reaches method, at the CO of 5%2Cell culture incubator is cultivated, results upstream sperm, counts sperm quantity, with In-vitro Capacitation liquid 5% CO2Cell culture incubator is hatched sperm 3h；Centrifugation, the supernatant recentrifuge collected separates, and collects supernatant as sample to be tested；
The mensuration of sialidase activity in C, capacitation liquid
(1) being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, is sequentially added into each sialidase standard solution and sample to be tested in detection plate。
(2) with detection buffer dilution fluorometric reagent, it is added in the hole of above-mentioned each sialidase standard solution and sample to be tested, again detection plate is placed in 37 DEG C, after lucifuge hatches 1 hour, each hole fluorescence intensity is read by microplate reader, calculate the sialidase activity of sample to be tested according to sialidase activity standard curve, finally draw the value of sialidase activity/sperm number。
Embodiment 2
After a kind of In-vitro Capacitation, the method for sperm quality assessment, specifically comprises the following steps that
A, sperm washing
Collect fresh liquefaction semen sample, wash sperm with phosphate buffer or BWW culture fluid, fully remove the refining composition of residual, then centrifugation goes out sperm；
B, sperm In-vitro Capacitation
In the isolated sperm of step A, add BWW culture fluid, adopt upper reaches method, at the CO of 5%2Cell culture incubator is cultivated, results upstream sperm, counts sperm quantity, with In-vitro Capacitation liquid 5% CO2Cell culture incubator is hatched sperm 4h；Centrifugation, the supernatant recentrifuge collected separates, and collects supernatant as sample to be tested；
The mensuration of sialidase activity in C, capacitation liquid
(1) being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, is sequentially added into each sialidase standard solution and sample to be tested in detection plate。
(2) with detection buffer dilution fluorometric reagent, it is added in the hole of above-mentioned each sialidase standard solution and sample to be tested, again detection plate is placed in 37 DEG C, after lucifuge hatches 2 hours, under the wavelength of excitation wavelength and transmitting wavelength respectively 365nm and 450nm, each hole fluorescence intensity is read by microplate reader, calculate the sialidase activity of sample to be tested according to sialidase activity standard curve, finally draw the value of sialidase activity/sperm number。
Embodiment 3
After a kind of In-vitro Capacitation, the method for sperm quality assessment, specifically comprises the following steps that
A, sperm washing
Collect fresh liquefaction semen sample, wash sperm with phosphate buffer or BWW culture fluid, fully remove the refining composition of residual, then centrifugation goes out sperm；
B, sperm In-vitro Capacitation
In the isolated sperm of step A, add BWW culture fluid, adopt upper reaches method, at the CO of 5%2Cell culture incubator is cultivated, results upstream sperm, counts sperm quantity, with In-vitro Capacitation liquid 5% CO2Cell culture incubator is hatched sperm 3.5h；Centrifugation, the supernatant recentrifuge collected separates, and collects supernatant as sample to be tested；
The mensuration of sialidase activity in C, capacitation liquid
(1) being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, is sequentially added into each sialidase standard solution and sample to be tested in detection plate。
(2) being diluted to the concentration of fluorometric reagent with detection buffer is 0.05mM, it is added in the hole of above-mentioned each sialidase standard solution and sample to be tested, again detection plate is placed in 37 DEG C, after lucifuge hatches 1.5 hours, under the wavelength of excitation wavelength and transmitting wavelength respectively 365nm and 450nm, each hole fluorescence intensity is read by microplate reader, calculate the sialidase activity of sample to be tested according to sialidase activity standard curve, finally draw the value of sialidase activity/sperm number。
Embodiment 4
The present embodiment is on the basis of embodiment 1:
Described detection plate is 96 hole blackboards。
Embodiment 5
The present embodiment is on the basis of embodiment 1:
Described detection plate is 96 hole blackboards。
Described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
Embodiment 6
The present embodiment is on the basis of embodiment 2:
Described detection plate is 96 hole blackboards。
Described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
The pH value of described sodium acetate is 5。
Embodiment 7
The present embodiment is on the basis of embodiment 2:
Described detection plate is 96 hole blackboards。
Described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
The pH value of described sodium acetate is 6。
Described In-vitro Capacitation liquid is the HTF containing HSA5mg/ml。
Embodiment 8
The present embodiment is on the basis of embodiment 3:
Described detection plate is 96 hole blackboards。
Described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
The pH value of described sodium acetate is 5.5。
Described In-vitro Capacitation liquid is the HTF containing HSA5mg/ml。
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml。
Embodiment 9
The present embodiment is on the basis of embodiment 3:
Described detection plate is 96 hole blackboards。
Described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
The pH value of described sodium acetate is 5。
Described In-vitro Capacitation liquid is the HTF containing HSA5mg/ml。
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml。
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium。
Embodiment 10
Consumptive material (equal sterilizing): 15mlEP manages, and 1.5mlEP manages, 3 kinds of specification rifle heads (1ml/200 μ l/10 μ l), 96 hole lamella lucidas, 96 hole blackboards
Instrument: high speed low temperature centrifugal machine, microplate reader
After a kind of In-vitro Capacitation, the method for sperm quality assessment, specifically comprises the following steps that
A, sperm washing
1. collect fresh liquefaction semen sample 4-5ml(to adjust according to patient's sperm quantity, preserve on ice and transport).
2. adding equal-volume PBS, piping and druming is uniformly。
3. low-speed centrifugal (1500g/5min), abandoning supernatant。
4. adding PBS to 3-4ml, piping and druming is uniformly。
5. low-speed centrifugal (1500g/5min), abandoning supernatant, supernatant is remained to 300 μ about l, piping and druming is uniformly。
6. add PBS to 1ml, high speed centrifugation (4 degree, 12000rpm/5min), abandoning supernatant as far as possible。
B, sperm In-vitro Capacitation
1. adding BWW in Sperm pellets, upper reaches method cultivates 30min in 37 DEG C of 5%CO2 cell culture incubators, and results upstream sperm, counting sperm quantity (needs 1X107Above), low-speed centrifugal (1500g/5min), obtain Sperm pellets。
2. add In-vitro Capacitation liquid, in 37 DEG C of 5%CO2 cell culture incubators, hatch 4 hours。
3. low-speed centrifugal (500g/5min), collects supernatant。
4. low-speed centrifugal (5000g/15min) again, collects supernatant。
C. the mensuration of sialidase activity in capacitation liquid
1. being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, detects in plate (black) to 96 holes and is sequentially added into the 50 each sialidase standard solution of μ l。
2. it is 0.05mM with being diluted to the concentration of fluorometric reagent with detection buffer, is added in above-mentioned each hole, 50 microlitres/hole。
3. plate (black) is detected in 96 holes and be placed in 37 DEG C, lucifuge hatches 1-2 hour, then it is under 365/450nm wavelength, read each hole fluorescence intensity by microplate reader at excitation wavelength/transmitting wavelength, calculates the sialidase activity of sample to be tested according to sialidase activity standard curve。
4. sialidase activity/the sperm number that finally will obtain, calculates unit enzyme value (UAUS/10 alive4sperm)。
For sample sialidase activity in the hole detected should within the scope of 0.3-5mU/ml, if beyond; the sialidase activity value of sample to be tested in hole should be adjusted within this range of linearity。
Claims (9)
1. the method for sperm quality assessment after an In-vitro Capacitation, it is characterised in that: specifically comprise the following steps that
A, sperm washing
Collect fresh liquefaction semen sample, wash sperm with phosphate buffer or BWW culture fluid, fully remove the refining composition of residual, then centrifugation goes out sperm；
B, sperm In-vitro Capacitation
In the isolated sperm of step A, add BWW culture fluid, adopt upper reaches method, at the CO of 5%2Cell culture incubator is cultivated, results upstream sperm, counts sperm quantity, with In-vitro Capacitation liquid 5% CO2Cell culture incubator is hatched sperm 3 ~ 4h；Centrifugation, the supernatant recentrifuge collected separates, and collects supernatant as sample to be tested；
The mensuration of sialidase activity in C, capacitation liquid
(1) being diluted by the Concentraton gradient of 5/2.5/1.25/0.625/0.312/0mU/ml by sialidase standard stock solution with detection buffer is sialidase standard solution, is sequentially added into each sialidase standard solution and sample to be tested in detection plate；
(2) with detection buffer dilution fluorometric reagent, it is added in the hole of above-mentioned each sialidase standard solution and sample to be tested, again detection plate is placed in 37 DEG C, after lucifuge hatches 1-2 hour, each hole fluorescence intensity is read by microplate reader, calculate the sialidase activity of sample to be tested according to sialidase activity standard curve, finally draw the value of sialidase activity/sperm number。
2. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: described detection plate is 96 hole blackboards。
3. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: the concentration being diluted to fluorometric reagent with detection buffer is 0.05mM。
4. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: microplate reader reads each hole fluorescence intensity under the wavelength of excitation wavelength and transmitting wavelength respectively 365nm and 450nm。
5. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: described detection buffer is the phosphate buffer containing 0.05mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin。
6. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 5, it is characterised in that: the pH value of described sodium acetate is 5-6。
7. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: described In-vitro Capacitation liquid is the HTF containing HSA5mg/ml。
8. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml。
9. the method for sperm quality assessment after a kind of In-vitro Capacitation according to claim 1, it is characterised in that: described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112014367A (en) * | 2020-08-14 | 2020-12-01 | 四川大学华西第二医院 | Application of AuNPs1-2 pH probe in male infertility and product |
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| CN1405562A (en) * | 2002-10-24 | 2003-03-26 | 肖洪武 | Sialidase detection reagent |
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| CN103454416A (en) * | 2013-09-22 | 2013-12-18 | 四川大学华西第二医院 | Method for detecting neuraminidase influencing sperm functions |
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| CN1503908A (en) * | 2001-02-15 | 2004-06-09 | Enzymatic test for the determination of the risk of pathologies related to the pressure of sialidase or prolidase activity in women body fluid samples | |
| CN1405562A (en) * | 2002-10-24 | 2003-03-26 | 肖洪武 | Sialidase detection reagent |
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| CN112014367A (en) * | 2020-08-14 | 2020-12-01 | 四川大学华西第二医院 | Application of AuNPs1-2 pH probe in male infertility and product |
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