CN103454416B - Affect the sialidase detection method of sperm function - Google Patents

Abstract

The present invention proposes a kind of sialidase detection method affecting sperm function.The method is a kind of test in laboratory method of new assessment sperm quality and function, can be clinical agnogenic male sterility patient and provides detection and on.It is characterized in that: the first step, quantize the expression of sialidase in sperm intuitively by monosperm Fluorescence Intensity Assays, or under utilizing microscope, fluorescence imaging observes the expression of sialidase in sperm intuitively; If sialidase is high expressed in sperm, then carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use Fluorometric assay sialidase activity.

Description

Sialidase Detection Method Affecting Sperm Function

technical field

The invention belongs to the technical field of medical detection, and in particular relates to a method for detecting sialidase that affects sperm function.

Background technique

With the continuous development of social industry, the total incidence of infertile couples increased significantly. In 2004, the statistics of the European Society of Reproduction: 25% of couples of childbearing age were unable to conceive within one year, 15% of them sought medical treatment, and 50% were infertility caused by male factors. The causes of male infertility are sexual dysfunction (1.7%), varicocele (12.3%), reproductive tract infection (6.6%), congenital dysplasia (2.1%) acquired disease (2.6%), endocrine disorders (0.6%), immune factors (3.1%), and other abnormalities (3%), but up to 60% to 75% of patients cannot find the cause, which is called idiopathic male infertility, and they only show oligospermia, Abnormal sperm quality such as asthenozoospermia or teratozoospermia. Unexplained male infertility may be caused by a variety of factors, such as endocrine disorders caused by long-term stress environmental factors, reactive oxygen species and gene defects.

For many years, traditional semen routine analysis has been the most basic clinical indicator for judging male fertility. Clinically, the true cause of infertility in the vast majority of male infertility patients is still unclear. In particular, there are about one-third of infertile patients clinically, and the routine semen analysis results of men are normal or close to normal. Clinically, these patients are classified as unexplained infertility. Therefore, there have been great difficulties in the clinical diagnosis of male infertility for a long time. The main reason is that routine semen analysis only measures sperm count, viability, motility and morphology. These indicators can only reflect the most basic semen quality, and cannot reflect all sperm functions, such as sperm nucleus maturation and DNA damage, sperm binding reaction and penetration with human egg zona pellucida, acrosome status and reaction, and binding to vitelline membrane ability etc. Therefore, special sperm function tests need to be established to measure these functions. Sperm capacitation is a necessary condition for the combination of sperm and eggs to form fertilized eggs. Although the research on sperm capacitation has made some progress, there are still many problems that have not been clarified.

In clinical work, some male patients with primary or secondary infertility use the existing routine semen quality inspection items to indicate that there is no abnormality in the quality of sperm and semen. Currently commonly used sperm detection means for CASA (Computeraidedofsemenanalysis), and anti-sperm antibodies. Today’s most widely used CASA technology and some morphological related tests can mainly evaluate the motility of sperm and the ratio of live sperm, so as to infer its fertilization ability entering the female reproductive tract. However, depending on the current sperm function evaluation system, the quality of sperm There are certain deficiencies in the evaluation of and function, and it is difficult to provide corresponding clinical consultation to patients. In fact, this is only one aspect of evaluating sperm function, and we should take into account that there may be more unrevealed factors affecting sperm reproductive ability.

For example, "Sialidase of mammalian sperm participates in the shedding of sialic acid during capacitation" (Sialidasesonmammalianspermmediatedeciduoussialylationduringcapacitation, "The Journal of Biological Chemistry", 2012Nov2;287(45):38073-9), the first report of sialic acid on the sperm membrane Enzymes (NEU1/3) are novel factors affecting sperm fertility. Sialic acid (sialic acid) is shed from the sperm cell membrane in the form of a single sugar molecule during the in vitro capacitation of mice and humans, and at the same time, the sialidase (NEU1/3) on the sperm membrane may be due to sperm membrane The fluidity and shedding are involved in shearing to cause the shedding of a single sialic acid molecule. The literature has confirmed that inhibiting the activity of NEU3 will reduce the expression of phosphorylated ErK in the process of sperm capacitation, and a small number of patients with unexplained infertility. These two enzymes low expression.

Although the literature proposes that sialidase can be used to evaluate sperm function, and discloses the detection method of sialidase in mouse and a small amount of human frozen sperm. However, the literature only discloses the expression detection of sialidase in sperm, and does not further report the detection of sialidase activity; and the disclosed expression detection method cannot successfully detect sialidase in fresh sperm, and cannot Suitable for clinical application.

Contents of the invention

In order to solve the above technical problems, the present invention proposes a method for detecting sialidase that affects sperm function. This method is a new laboratory test method for evaluating sperm quality and function, which can provide testing and clinical consultation for male infertility patients with clinically unknown causes.

In order to realize the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

The method for detecting sialidase that affects sperm function is characterized in that: the first step is to visually quantify the expression of sialidase in sperm by analyzing the fluorescence intensity of single sperm, or to visually observe the expression of sialidase in sperm by using fluorescence imaging under a microscope. If the sialidase is highly expressed in the sperm, proceed to the second step, using the in vitro capacitation solution to capacious the sperm, and detect the sialidase activity with a fluorescent method.

The in vitro capacitation fluid is HSA (human serum albumin) 5 mg/ml HTF (human fallopian tube fluid).

In the first step, according to the following steps, the expression detection of sperm sialidase NEU1 and NEU3 is carried out respectively:

A Collect fresh liquefied semen samples, centrifuge at low speed to separate the sperm, wash the sperm with PBS (phosphate buffer solution), and fully remove the residual seminal plasma components;

B Count the number of sperm, put it on ice for 20-30 minutes, to immobilize the sperm, and facilitate the subsequent operation;

C The sperm were pretreated with 0.1% Triton X-100 (polyethylene glycol octyl phenyl ether). After washing the sperm with PBS, the sperm was pretreated again with 3% PFA (paraformaldehyde);

D When detecting NEU1, use 1% BSA (bovine serum albumin)-PBS solution for blocking; or, when detecting NEU3, use methanol to fix;

E. Incubate with 1 μg/μl anti-human NEU1 or NEU3 antibody for 1-3 hours to fully ensure the antigen-antibody binding time, and wash the sperm with PBS;

F: At room temperature, the sperm were incubated with conventional fluorescent secondary antibodies at a ratio of 1:1000 for 1-2 hours;

G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

In the second step, the specific steps are as follows:

A. Fresh liquefied semen samples are collected, and the sperm are separated by low-speed centrifugation to maintain the integrity and vitality of the sperm;

B Using the upstream method, culture the sperm in a 5% CO ₂ cell incubator, harvest the upstream sperm, and further obtain the sperm with high vitality, wash the sperm with BWW (Biggers, Whitten, and Whittinghamreedit) culture medium, and fully remove the remaining sperm in the seminal plasma Protein and other components, avoid increasing non-specific fluorescence background and remove some "decapacitating factors".

Since the sperm will release sialidase after death, the interference experiment will increase non-specific expression, and the upstream method can ensure the capacitation of sperm with good vitality and avoid dead sperm.

C Count the number of sperm, and incubate the sperm in a 5% CO ₂ cell incubator for 3-4 hours with in vitro capacitation solution HAS5mg/mlHTF;

D. First, centrifuge the solution obtained through the above operations, then centrifuge the collected supernatant again, collect the supernatant, and fully remove the formed components such as sperm, so as to avoid increasing the non-specific fluorescent background;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

In step A of the first and second steps, centrifuge at a low speed of 500-600 g/min for 5-6 minutes to ensure that the sperm morphology is complete and separated from the seminal plasma.

In step B of the second step, the upstream method is used to culture in a 5% CO ₂ cell incubator at 36-37° C. for 10-30 min.

In step C of the second step, the number of sperm is counted and adjusted to more than 1×10 ⁷ /ml, which not only ensures a sufficient in vitro capacitation environment close to physiology, but also allows the sialidase activity to be detected;

In step C of the second step, incubate with in vitro capacitation solution HAS 5 mg/ml HTF at 36-37° C. in a 5% CO ₂ cell incubator for 3-4 hours, with a reaction volume of 100-200 μl.

Because the enzyme activity of sperm is very low compared with that of bacteria, in order to ensure the highest sensitivity, the reaction volume must be controlled to 100-200 μl to ensure both capacitation and a measurable concentration of enzyme.

In step D of the second step, first centrifuge at a low speed of 500-600 g/min for 5-6 min, and then centrifuge at a low speed of 5000-6000 g/min for 10-15 min to fully remove formed components such as sperm.

The beneficial effects of the present invention are:

1. The present invention provides a sialidase detection method that affects sperm function. It is a new laboratory detection method for evaluating sperm quality and function. It is non-invasive to patients and provides more detection indicators and methods for patients with primary infertility. clinical interpretation.

2. The present invention uses a fluorescent method to detect and evaluate sialidase in sperm, because the sialidase shed from the sperm participates in the sialic acid shearing on the sperm surface during the capacitation process, because the activity theory of detecting sialidase The basis is to use sialidase to cut the fluorescent sialic acid complex. If there is activity, the separation of the fluorescent group can generate fluorescence, so its activity can be detected and evaluated. Therefore, the present method is a highly sensitive assay required for the sialidase activity of spermatozoa.

3. When the present invention detects the expression of sialidase in sperm, the sperm is placed on ice for 20-30 minutes, which is used to immobilize the sperm and make the fresh liquefied sperm move static, which is convenient for subsequent operations.

4. When the present invention detects sialidase activity, the upstream method is used to obtain sperm with excellent vitality. Since the sperm will release sialidase after death, the interference experiment increases non-specific expression, and the upstream method can ensure sperm capacitation with good vitality. Avoid dead sperm; at the same time, use BWW culture medium to wash sperm, fully remove residual protein and other components in seminal plasma, avoid increasing non-specific fluorescent background and remove some "capacitance factors", and ensure the accuracy of the experiment.

5. When detecting the activity of sialidase, the present invention adopts the method of centrifugation twice to fully remove the formed components such as sperm and avoid increasing the non-specific fluorescent background. Moreover, the present invention strictly controls the centrifugal speed, first centrifuges at a low speed of 500~600g/min for 5~6min, and then centrifuges at a low speed of 5000~6000g/min for 10~15min, further fully removes the formed components such as sperm, and avoids Detection interferes.

6. When collecting sperm, the present invention centrifuges at a low speed of 500-600g/min for 5-6 minutes to ensure that the sperm is morphologically complete and completely separated from the seminal plasma.

7. When detecting the sialidase activity, the present invention counts the number of sperm and adjusts it to more than 1×10 ⁷ /ml, which not only ensures a sufficient capacitation environment in vitro close to the physiology, but also allows the sialidase activity to be detected.

8. When detecting sialidase activity in the present invention, in vitro capacitation liquid HAS5mg/mlHTF is used to incubate in a 5% _CO2 cell incubator at 36-37°C for 3-4 hours, and the reaction volume is controlled to be 100-200 μl. Because the enzyme activity of sperm is very low compared with that of bacteria, in order to ensure the highest sensitivity, the reaction volume must be controlled to ensure both capacitation and a measurable concentration of enzyme.

Description of drawings

Figure 1 shows the expression of sialidase (NEU1/3) in patients with unexplained male infertility.

detailed description

The substantive content of the present utility model will be further described in detail below in conjunction with specific embodiments.

Example 1

The method for detecting sialidase that affects sperm function is characterized in that: the first step is to visually quantify the expression of sialidase in sperm by analyzing the fluorescence intensity of single sperm, or to visually observe the expression of sialidase in sperm by using fluorescence imaging under a microscope. If the sialidase is highly expressed in the sperm, proceed to the second step, using the in vitro capacitation solution to capacious the sperm, and detect the sialidase activity with a fluorescent method.

Example 2

The method for detecting sialidase that affects sperm function is characterized in that: the first step is to visually quantify the expression of sialidase in sperm by analyzing the fluorescence intensity of single sperm, or to visually observe the expression of sialidase in sperm by using fluorescence imaging under a microscope. If the sialidase is highly expressed in the sperm, proceed to the second step, using the in vitro capacitation solution to capacious the sperm, and detect the sialidase activity with a fluorescent method.

The in vitro capacitation solution is HSA5mg/mlHTF.

Example 3

The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

Follow the steps below to detect the expression of sperm sialidase NEU1:

A. Collect fresh liquefied semen samples, centrifuge to separate the sperm, and wash the sperm twice with PBS;

B Count the number of sperm and place on ice for 20 minutes;

C pre-treat the sperm with 0.1% Triton X-100, wash the sperm twice with PBS, and pre-treat the sperm again with 3% PFA;

D uses 1% BSA-PBS solution to block;

E was incubated with 1 μg/μl anti-human NEU1 antibody for 1 h, and the sperm was washed twice with PBS;

F At room temperature, the sperm were incubated with conventional fluorescent secondary antibodies at a ratio of 1:1000 for 1 h;

G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

Example 4

The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

Follow the steps below to detect the expression of sperm sialidase NEU3:

A. Collect fresh liquefied semen samples, centrifuge to separate the sperm, and wash the sperm twice with PBS;

B Count the number of sperm and place on ice for 30 minutes;

C pre-treat the sperm with 0.1% Triton X-100, wash the sperm twice with PBS, and pre-treat the sperm again with 3% PFA;

D is fixed with methanol;

E was incubated with 1 μg/μl anti-human NEU3 antibody for 3 hours, and the sperm was washed twice with PBS;

F At room temperature, the sperm were incubated in 1:1000 conventional fluorescent secondary antibody for 2 hours;

G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

Example 5

The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

Follow the steps below to detect sialidase activity:

A Collect fresh liquefied semen samples and centrifuge to separate sperm;

B Using the upstream method, culture the sperm in a 5% CO ₂ cell incubator, harvest the upstream sperm, and wash the sperm with BWW culture medium.

C Count the number of sperm, use in vitro capacitation solution HSA5mg/mlHTF, and incubate the sperm for 3h in a 5% CO ₂ cell incubator;

D first centrifuge the solution obtained through the above operations, then centrifuge the collected supernatant again, and collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Example 6

The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

Follow the steps below to detect sialidase activity:

A Collect fresh liquefied semen samples and centrifuge to separate sperm;

B Use the upstream method to culture in a 5% CO ₂ cell incubator for 10 minutes at 36°C, harvest the upstream sperm, and wash the sperm 3 times with BWW medium.

C Count the number of sperm, use in vitro capacitation solution HSA5mg/mlHTF, and incubate the sperm for 4h in a 5% CO ₂ cell incubator;

D first centrifuge the solution obtained through the above operations, then centrifuge the collected supernatant again, and collect the supernatant;

E The supernatant after capacitation was immediately detected for sialidase activity by fluorescent blue method.

Sialidase activity in the capacitation supernatant was detected using ABCAM's Sialidase Assay Kit (Fluorometric-Blue) (ab138888).

Use Ex/Em=~320/~450nm fluorescence microplate reader to detect, calculate the sialic acid activity according to the positive control standard curve, the unit is: uUAUS/1×10 ⁶ sperm (microunit/1×10 ⁶ sperm).

Example 7

In the first step, the expression detection of sperm sialidase NEU1 is carried out according to the following steps:

A Collect fresh liquefied semen samples, centrifuge to separate the sperm, centrifuge at a low speed of 500g/min for 6 minutes, wash the sperm 3 times with PBS;

B Count the number of sperm and place on ice for 25 minutes;

C The sperm were pretreated with 0.1% Triton X-100, the sperm were washed with PBS three times, and the sperm was pretreated again with 3% PFA;

D uses 1% BSA-PBS solution to block;

E was incubated with 1 μg/μl anti-human NEU1 antibody for 2 hours, and the sperm was washed 3 times with PBS;

F At room temperature, the sperm were incubated for 1.5h in a conventional fluorescent secondary antibody at a ratio of 1:1000;

G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

Then follow the steps below to detect the expression of sperm sialidase NEU3:

A Collect fresh liquefied semen samples, centrifuge to separate the sperm, centrifuge at a low speed of 500g/min for 6 minutes, wash the sperm 3 times with PBS;

B Count the number of sperm and place on ice for 25 minutes;

C The sperm were pretreated with 0.1% Triton X-100, the sperm were washed with PBS three times, and the sperm was pretreated again with 3% PFA;

D is fixed with methanol;

E was incubated with 1 μg/μl anti-human NEU1 antibody for 2 hours, and the sperm was washed 3 times with PBS;

F At room temperature, the sperm were incubated for 1.5h in a conventional fluorescent secondary antibody at a ratio of 1:1000;

G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

The fluorescence intensity of single sperm was analyzed by flow cytometry, and two comparison methods were used: a. Absolute comparison: the isotype control between the tested sample and the antibody (the isotype control used the same species source, the same subtype, the same dose and The same immunoglobulins and subtypes of immunoglobulins are used to eliminate the background caused by the non-specific binding of antibodies to cells) b Relative comparison: compare the fluorescence intensity of single sperm among the same batch of test samples.

In the present invention, the single sperm fluorescence intensity analysis (isotype control/batch control) can relatively intuitively quantify the expression of sialidase in sperm; or the expression of sialidase can be visually observed by using fluorescence imaging under a microscope, so the present invention adopts It is a method of visual imaging and relative quantitative detection method to evaluate the expression of sialidase (NEU1/3) in sperm.

The above detection found the high expression of sialidase NEU1 and sialidase NEU3 in sperm. In the second step, the sialidase activity was detected as follows:

A Collect fresh liquefied semen samples, centrifuge to separate sperm, and centrifuge at a low speed of 500g/min for 6min;

B Use the upstream method to culture in a 5% CO ₂ cell incubator for 30 minutes at 37°C, harvest the upstream sperm, and wash the sperm 3 times with BWW medium.

C Count the number of sperm, use in vitro capacitation solution HSA5mg/mlHTF, and incubate the sperm for 3.5h in a 5% CO ₂ cell incubator;

D first centrifuge the solution obtained through the above operations, then centrifuge the collected supernatant again, and collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Example 8

This embodiment is basically the same as Embodiment 7, on this basis:

Detect sialidase activity as follows:

A Collect fresh liquefied semen samples, centrifuge to separate the sperm, and centrifuge at a low speed of 600g/min for 5min;

B Use the upstream method to culture in a 5% CO ₂ cell incubator for 20 minutes at 36.5°C, harvest the upstream sperm, and wash the sperm 3 times with BWW medium.

C Count the number of sperm, adjust it to be more than 1×10 ⁷ /ml, use in vitro capacitation solution HSA5mg/mlHTF, and incubate the sperm in a 5% CO ₂ cell incubator for 3.5 hours;

D first centrifuge the solution obtained through the above operations, then centrifuge the collected supernatant again, and collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Example 9

This embodiment is basically the same as Embodiment 7, on this basis:

Detect sialidase activity as follows:

A Collect fresh liquefied semen samples, centrifuge to separate the sperm, and centrifuge at a low speed of 550g/min for 5.5min;

B Use the upstream method to culture at 36°C for 15 minutes in a 5% CO ₂ cell incubator, harvest the upstream sperm, and wash the sperm 3 times with BWW medium.

C Count the number of spermatozoa, adjust to more than 1×10 ⁷ /ml, incubate with in vitro capacitation solution HSA5mg/mlHTF at 36°C, 5% CO ₂ cell incubator for 3.5h, the reaction volume is 100μl.

D first centrifuge the solution obtained through the above operations, centrifuge at a low speed of 500g/min for 6min, centrifuge the collected supernatant again, centrifuge at a low speed of 5000g/min for 15min, and collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Example 10

This embodiment is basically the same as Embodiment 7, on this basis:

Detect sialidase activity as follows:

A Collect fresh liquefied semen samples, centrifuge to separate the sperm, and centrifuge at a low speed of 580g/min for 5min;

B Use the upstream method to culture in a 5% CO ₂ incubator for 2 minutes at 37°C, harvest the upstream sperm, and wash the sperm 3 times with BWW medium.

C Count the number of spermatozoa, adjust to more than 1×10 ⁷ /ml, incubate with in vitro capacitation solution HAS5mg/mlHTF at 37°C, 5% CO ₂ cell incubator for 3.5h, and the reaction volume is 200μl.

D first centrifuge the solution obtained through the above operations, centrifuge at a low speed of 600g/min for 5min, and centrifuge the collected supernatant again, centrifuge at a low speed of 6000g/min for 10min, collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Example 11

This embodiment is basically the same as Embodiment 7, on this basis:

Detect sialidase activity as follows:

A Collect fresh liquefied semen samples, centrifuge to separate sperm, and centrifuge at a low speed of 520g/min for 5min;

B Use the upstream method to culture in a 5% CO ₂ cell incubator for 18 minutes at 36-37°C, harvest the upstream sperm, and wash the sperm three times with BWW culture medium.

C Count the number of spermatozoa, adjust to more than 1×10 ⁷ /ml, incubate with in vitro capacitation solution HAS5mg/mlHTF at 36.5°C, 5% CO ₂ cell incubator for 3.5h, and the reaction volume is 150ul.

D first centrifuge the solution obtained through the above operations, centrifuge at a low speed of 540g/min for 5.5min, and centrifuge the collected supernatant again, centrifuge at a low speed of 5500g/min for 12min, collect the supernatant;

E Immediately after the capacitation, the supernatant was detected by fluorescence method for sialidase activity.

Claims (7)

1. affect the sialidase detection method of sperm function, it is characterized in that: the first step, quantize the expression of sialidase in sperm intuitively by monosperm Fluorescence Intensity Assays, or under utilizing microscope, fluorescence imaging observes the expression of sialidase in sperm intuitively; If sialidase is high expressed in sperm, then carry out second step, utilize In-vitro Capacitation liquid to make capacitation, and use Fluorometric assay sialidase activity;

The described first step, according to following step, carry out the detection of expression of sperm sialidase NEU1 and NEU3 respectively:

A collects fresh liquefaction semen sample, and with the speed low-speed centrifugal 5 ~ 6min of 500 ~ 600g/min, isolate sperm, PBS washs sperm;

B counts sperm quantity, puts 20 ~ 30min on ice;

C employing 0.1% tritonx-100 carries out pre-service to sperm, and PBS washs sperm, and the PFA with 3% carries out pre-service to sperm again;

D, when detecting NEU1, adopts the BSA-PBS solution of 1% to close; Or, when detecting NEU3, adopt methyl alcohol to fix;

E adopts the anti-human NEU1 antibody incubation 1 ~ 3h of 1 μ g/ μ l, and PBS washs sperm, or adopts anti-human NEU3 antibody incubation 2h or 3h, the PBS of 1 μ g/ μ l to wash sperm;

At normal temperatures, sperm hatches 1 ~ 2h to F in the conventional fluorescent two of 1:1000 is anti-;

G adopts the expression of flow cytomery sialidase in sperm, or adopts fluorescent microscope to make the cell smear of sperm, observes the expression of sialidase in sperm.

2. the sialidase detection method affecting sperm function according to claim 1, is characterized in that: described In-vitro Capacitation liquid is the HTF containing HSA5mg/ml.

3. the sialidase detection method affecting sperm function according to claim 1, it is characterized in that: described second step, concrete operation step is as follows:

A collects fresh liquefaction semen sample, with the speed low-speed centrifugal 5 ~ 6min of 500 ~ 600g/min, isolates sperm;

B adopts upper reaches method, at the CO of 5% ₂cultivate sperm in cell culture incubator, results upstream sperm, adopt BWW nutrient solution washing sperm;

C counts sperm quantity, with the HTF containing HSA5mg/ml as In-vitro Capacitation liquid, at the CO of 5% ₂sperm 3 ~ 4h is hatched in cell culture incubator;

The solution centrifugal obtained through aforesaid operations is first separated by D, and the supernatant collected centrifuging again, collects supernatant;

E is immediately by the Fluorometric assay sialidase activity of the supernatant after capacitation.

4. the sialidase detection method affecting sperm function according to claim 3, is characterized in that: described step B, adopts upper reaches method in 36 ~ 37 DEG C, at the CO of 5% ₂10 ~ 30min is cultivated in cell culture incubator.

5. the sialidase detection method affecting sperm function according to claim 3, is characterized in that: described step C, and counting sperm quantity, is adjusted to 1 × 10 ⁷/ more than ml.

6. the sialidase detection method affecting sperm function according to claim 3, is characterized in that: described step C, with containing the HTF of HSA5mg/ml as In-vitro Capacitation liquid in 36 ~ 37 DEG C, the CO of 5% ₂3 ~ 4h is hatched, reaction volume 100 ~ 200 μ l in cell culture incubator.

7. the sialidase detection method affecting sperm function according to claim 3, is characterized in that: described D step, first with the speed low-speed centrifugal 5 ~ 6min of 500 ~ 600g/min, then with the speed low-speed centrifugal 10 ~ 15min of 5000 ~ 6000g/min.