WO2017156844A1 - Kit d'évaluation de la qualité du sperme après capacitation in vitro et son procédé d'utilisation - Google Patents

Kit d'évaluation de la qualité du sperme après capacitation in vitro et son procédé d'utilisation Download PDF

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Publication number
WO2017156844A1
WO2017156844A1 PCT/CN2016/081276 CN2016081276W WO2017156844A1 WO 2017156844 A1 WO2017156844 A1 WO 2017156844A1 CN 2016081276 W CN2016081276 W CN 2016081276W WO 2017156844 A1 WO2017156844 A1 WO 2017156844A1
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sperm
sialidase
kit
vitro
solution
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PCT/CN2016/081276
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Chinese (zh)
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马芳
马黔红
潘倩
马学
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四川大学华西第二医院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase

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  • the invention belongs to the technical field of medical detection, and particularly relates to a kit for evaluating sperm quality after in vitro capacitation and a using method thereof.
  • sperm capacitation is a necessary condition for the combination of sperm and egg to form fertilized eggs.
  • CASA Computer Aided Semen Analysis
  • anti-sperm antibodies Today's most widely used CASA technology and some morphological related tests can mainly evaluate sperm activity The ratio of ability to live sperm, and thus the ability to fertilize into the female reproductive tract, and rely on the current sperm function evaluation system, there are certain defects in the evaluation of sperm quality and function, and it is difficult to provide patients with appropriate clinical consultation. In fact, this is just one aspect of evaluating sperm function, and we should consider more factors that may not be revealed that may affect sperm motility.
  • the first step is to visualize the expression of sialidase in sperm by single sperm fluorescence intensity analysis, or to visually observe the expression of sialidase in sperm by microscopic fluorescence imaging; if sialidase is highly expressed in sperm,
  • the sialidase assay for sperm motility is determined by the in vitro capacitation solution, and the sialidase activity is detected by fluorescence.
  • This patent discloses the expression of sialidase protein molecules in sperm and the detection of essential activities.
  • the method includes the expression detection of sperm sialidase NEU1/3, which requires fluorescence microscopy or flow cytometry, and has the following disadvantages: 1) complicated technical process; 2) high demand for experimental equipment and operator skills and experience; Not suitable for use in clinical laboratories and routine technicians; 4) The use of abcam kits is expensive.
  • the expressed protein values are different, and the corresponding enzyme activity values are also different. Therefore, it is not scientific to simply compare the enzyme activities of different samples.
  • the present invention provides a kit for assessing sperm quality after in vitro capacitation and a method of using the same.
  • a kit for assessing sperm quality after in vitro capacitation Through the detection of sialidase activity in the energy-receiving solution after sperm capacitation in vitro, the sperm quality after capacitation is evaluated, and the diagnosis basis and clinical consultation are provided for male infertility patients with unknown clinical causes.
  • kits for assessing sperm quality after in vitro capacitation characterized in that the kit comprises a detection buffer 1, a BWW culture solution, an in vitro energy-receiving solution, a fluorescent reagent, a sialidase standard stock solution and a detection buffer 2 .
  • the detection buffer 1 is PBS (phosphate buffer) or BWW culture solution.
  • the detection buffer 2 was PBS containing 0.05 mmoL/L sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the buffer system for assay buffer 2 was specifically formulated for detection of sialidase activity.
  • the sodium acetate has a pH of 5-6 and is the best working environment for salivary enzyme activity.
  • the in vitro capacitation solution is HTF (human oviduct fluid) containing HSA (human serum albumin) 5 mg/ml.
  • the standard stock solution of sialidase is Clostridium perfringens sialidase (AUS) at a concentration of 5 U/ml, which can be conveniently diluted to a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml.
  • AUS Clostridium perfringens sialidase
  • the method for detecting a small amount of sperm of the present invention is applied to Clostridium perfringens sialidase.
  • the fluorescent reagent is 2'-4-methylumbelliferyl- ⁇ -D-N-acetylneuraminic acid, which has high sensitivity.
  • the concentration of the fluorescent reagent is 10 mM, providing a concentrated solution of the fluorescent reagent, which can ensure stability, is not easy to be decomposed and quenched, and can reduce volume and facilitate boxing.
  • test buffer 1 Collect fresh liquefied semen samples, test buffer 1 to wash the sperm, fully remove the residual seminal plasma components, and then centrifuge to separate the sperm.
  • the detection buffer 1 is first added to the semen sample for washing, and then centrifuged to obtain a sperm precipitate. This is because the seminal plasma itself is sticky, and it can not completely precipitate all the sperm in the semen by centrifuging it directly. After adding the detection buffer 1 to dilute it, the centrifugal operation is better.
  • the semen is centrifuged 3 times to ensure that the number of spermatozoa is reduced as much as possible to ensure sperm motility without detecting interference from the seminal plasma.
  • BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO 2 cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO 2 cells.
  • the sperm was incubated for 3 to 4 hours in the chamber; the supernatant was centrifuged again, and the supernatant was collected as the sample to be tested. After getting the sperm with better vigor in the upstream method, it will be processed immediately, and the experimental results are more scientific and objective.
  • the number of upstream sperm is controlled to be 1 ⁇ 10 7 or more, which is set according to the need for detection sensitivity.
  • the gradient of the standard solution is set based on the amount of sperm sialidase released into the capacitation liquid after capacitation and the sensitivity detectable by the method of the present invention.
  • the detection plate is a 96-well blackboard.
  • the invention adopts the fluorescence method to determine the activity of sialidase, and the black detection plate is optimal for sensitivity and specificity, and can protect the fluorescent dye from being quenched by light, thereby greatly improving the sensitivity of detection.
  • the concentration of the fluorescent reagent diluted to 0.05 mM with the detection buffer 2 is the optimum working concentration.
  • the microplate reader reads the fluorescence intensity of each well at a wavelength of excitation wavelength and emission wavelength of 365 nm and 450 nm, respectively.
  • 365 nm is the best excitation wavelength
  • 450 nm is the optimal emission wavelength.
  • the sample in the well for detection has a sialidase activity in the range of 0.3 to 5 mU/ml, and if it is exceeded, the sialidase activity value of the sample to be tested in the well should be adjusted within this linear range.
  • the reagents in the kit of the invention all select the conventional test reagent, and combine the speciality of the sperm detection to reduce the experiment cost, and the required instruments and equipments and the consumables are few, only the centrifuge, the microplate reader, the EP tube. , the gun head and the test board are all routine laboratory configurations; and the operation method is simple, only for centrifugation, sample loading, etc. There is no need to practice multiple times to obtain the operation skills, and can be widely used in clinical laboratories and routines. Used by technicians.
  • the kit of the present invention is used for assessing the quality of sperm after capacitation, according to the number of sperm in the unit.
  • the activity value of liquefaction enzyme is a critical value for expressing sialidase activity in the human population, and the sperm function can be understood deeper through the unit enzyme activity value in the clinic, and can be applied to reproductive assist such as test tube baby.
  • the invention fully cleans the sperm, completely removes the residual seminal plasma component, so that the residual protein and sialidase in the seminal plasma does not affect the accuracy of the detection result; the standard set when the sperm protein concentration is optimized is optimized. Protein concentration gradient and standard enzyme activity gradient set when the enzyme is active. If the gradient setting is unreasonable, it will affect the sensitivity and accuracy of the result.
  • Using the black detection plate to measure the enzyme activity can significantly reduce the quenching of the fluorescent reagent. The negative effects, thereby greatly improving the stability, sensitivity and accuracy of the experimental results; the final test results are designed as the value of the number of sialidase/sperm, which can reduce the difference between different samples and make them more comparable sexuality guarantees the stability and reliability of test results.
  • Figure 1 shows the sialidase activity of uncapacity sperm and capacitated sperm (A is a mouse and B is a human) in the kit of the present invention.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • Detection buffer 1 was a phosphate buffer;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the kit of the present invention measures the sialidase activity of the uncaptured sperm and the capacitated sperm (A is a mouse, B is a human), and the results show that the sperm can release the sialidase into the capacitate after capacitation.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • the detection buffer 1 was a BWW culture solution;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • Detection buffer 1 was a phosphate buffer;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
  • the standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • Detection buffer 1 was a phosphate buffer;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
  • the standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.
  • the fluorescent reagent is sodium 2'-4-methylumbelliferyl- ⁇ -D-N-acetylneuraminic acid.
  • the sodium acetate has a pH of 5.5.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • Detection buffer 1 was a phosphate buffer;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
  • the standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.
  • the fluorescent reagent is sodium 2'-4-methylumbelliferyl- ⁇ -D-N-acetylneuraminic acid.
  • the concentration of the fluorescent reagent was 10 mM.
  • the sodium acetate has a pH of 5.
  • a kit for assessing sperm quality after in vitro capacitation comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2;
  • Detection buffer 1 was a phosphate buffer;
  • the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 ⁇ g/ ⁇ l bovine serum albumin.
  • the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
  • the standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.
  • the fluorescent reagent is sodium 2'-4-methylumbelliferyl- ⁇ -D-N-acetylneuraminic acid.
  • the concentration of the fluorescent reagent was 10 mM.
  • the sodium acetate has a pH of 6.
  • BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO 2 cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO 2 cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;
  • BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO 2 cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO 2 cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;
  • the detection board is a 96-hole blackboard.
  • BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO 2 cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO 2 cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;
  • the detection board is a 96-hole blackboard.
  • BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO 2 cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO 2 cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;
  • the detection board is a 96-hole blackboard.
  • BWW was added to the sperm pellet, and the upstream method was cultured in a 37 ° C 5% CO 2 cell incubator for 30 min, the upstream sperm was harvested, the sperm count was counted (1 ⁇ 10 7 or more), and the sperm was precipitated by low speed centrifugation (1500 g/5 min).
  • sialidase standard stock solution Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0mU/ml using the detection buffer 2, and then in a 96-well assay plate (black). 50 ⁇ l of each sialidase standard solution was added.
  • sialidase activity/sperm number obtained will be calculated and the unit enzyme activity value (U AUS/10 4 sperm) will be calculated.
  • the sialidase activity of the sample used for the assay should be in the range of 0.3-5 mU/ml. If it is exceeded, the sialidase activity value of the sample to be tested in the well should be adjusted within this linear range.

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Abstract

L'invention concerne un kit pour évaluer la qualité du sperme après la capacitation in vitro et un procédé d'utilisation de celui-ci. Le kit comprend un tampon de détection 1, un milieu de culture Biggers-Whitten-Whittingham (BWW), une solution de capacitation in vitro, un réactif fluorescent, une solution mère standard de sialidase et un tampon de détection 2. Le tampon de détection 1 est un tampon phosphate ou un milieu de culture BWW. Le tampon de détection 2 est un tampon phosphate contenant 0,05 mmol/l d'acétate de sodium et 0,25 μg/μl d'albumine de sérum bovin.
PCT/CN2016/081276 2016-03-16 2016-05-06 Kit d'évaluation de la qualité du sperme après capacitation in vitro et son procédé d'utilisation WO2017156844A1 (fr)

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CN201610147270.4A CN105779568B (zh) 2016-03-16 2016-03-16 一种体外获能后精子质量评估的试剂盒及其使用方法
CN201610147270.4 2016-03-16

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CN103454416A (zh) * 2013-09-22 2013-12-18 四川大学华西第二医院 影响精子功能的唾液酸酶检测方法

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DE60136388D1 (de) * 2001-02-15 2008-12-11 Unibio S R L Enzymtest zur bestimmung von krankheitsrisiken in verbindung mit der anwesenheit von sialidase oder prolidase aktivität in körperflüssigkeit von frauen
CN1405562A (zh) * 2002-10-24 2003-03-26 肖洪武 一种唾液酸酶测定试剂
AU2007311444B2 (en) * 2006-10-18 2012-11-29 Periness Ltd. Method and pharmacological composition for the diagnosis and treatment of male sub-fertility

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MA F. ET AL.: "Sialidases on Mammalian Sperm Mediate Deciduous Sialylation during Capacitation", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, no. 45, 2 November 2012 (2012-11-02), pages 38073 - 38079, XP055239622 *

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