Method for extracorporeally detecting survival rate of sperms

An in vitro detection and survival rate technology, applied in biochemical equipment and methods, biological testing, material inspection products, etc., can solve problems that affect the reliability of results, complex operations, and low efficiency

Inactive Publication Date: 2010-06-30
SHANGHAI INST OF PLANNED PARENTHOOD RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high price of the kit, the fluorescent staining method has not been popularized in domestic clinical andrology laboratories
[0006] The integrity of the plasma membrane is evaluated by manually counting the ratio of red and green sperm under a fluorescent microscope, which is affected by subjective factors and limited by the field of view. Only a very small amount of sperm in each sample can be detected, and dead sperm agglutination and other conditions can cause life and death. Uneven distribution of spermatozoa seriously affects the reliability of the results
Qualified laboratories can use flow cytometry to detect more reliably, but flow cytometry is expensive and difficult to popularize, it is limited to a few laboratories, and cannot become a routine inspection item in hospitals; and the operation is complex and requires high technology , the efficiency of the practical application is low

Method used

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  • Method for extracorporeally detecting survival rate of sperms
  • Method for extracorporeally detecting survival rate of sperms
  • Method for extracorporeally detecting survival rate of sperms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Survival count method under fluorescence microscope

[0075] According to the WHO standard (from: Zhao Hongxin, Yuan Yao, Hua Minmin, Zhang Huiqin, Shi Huijuan using Transgreen / PI fluorescent counterstaining method to detect the survival rate of spermatozoa Chinese Journal of Andrology 22(4): 1-4), 37 Place in a water bath at ℃ until completely liquefied, take 50ul into an Eppendorf tube, add 1ul of A solution (the composition is Transgreen) and B solution (the composition is PI) to mix, let it stand at room temperature for 20min, take 10ul from the tube to a Macro sperm counting plate, and use fluorescence Observe under a microscope, count the number of green sperm and red sperm, count at least 200 sperm in total, sperm survival rate = number of green sperm / (number of green sperm + number of red sperm) × 100%.

[0076] see results figure 1 , Schematic diagram of Transgreen / PI counterstaining method under a fluorescence microscope. The results showed that under the ...

Embodiment 2

[0078] Using a fluorescent microplate reader to detect the time curve of Transgreen entering sperm and binding to DNA

[0079] Collect semen according to the WHO standard, put it in a water bath at 37°C until it is completely liquefied, and do upstream processing. After the sperm is optimized, take 50ul of the sperm suspension and add it to a 96-well microtiter plate, add 1ul of A solution (the composition is Transgreen), and use the enzyme The standard instrument continuously detects the fluorescence intensity of the sample every 1 min, sets the excitation light wavelength to 485nm, and the absorption wavelength to 515nm, and adds 1ul A solution with 50ul HTF as a blank control.

[0080] see results figure 2 , The change curve of the fluorescence intensity during the process of Transgreen penetrating the membrane and entering the sperm.

[0081] Viable sperm dye has membrane permeability, but it takes a certain time for it to enter the plasma membrane, and the accurate surv...

Embodiment 3

[0083] Detection of sperm viability coefficient with fluorescence microplate reader

[0084]Prepare a portion of semen according to the method in Example 1, take 50ul and add the sample to a 96-well microtiter plate, add 1ul of A solution (the composition is Transgreen), and place it at 37°C for 30min to detect the fluorescence intensity, set the excitation light wavelength to 485nm, and the absorption wavelength 515nm. Add 1ul solution A to the other well with 50ul HTF as a blank control. After detection, add 1ul B solution (component is PI) to both wells. After 10 min, the fluorescence intensity was detected again under the same parameter conditions.

[0085] Define the sperm viability coefficient (Viability Coefficient) = (the fluorescence intensity of the second detection - the fluorescence intensity of the control well for the second detection) / (the fluorescence intensity of the first detection - the fluorescence intensity of the control well for the first detection)....

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Abstract

The invention discloses a method for extracorporeally detecting the survival rate of sperms, which is characterized in that the method comprises the following steps that: (a) a suspending liquid of the sperms is mixed with membrane-permeable nuclear dyes to produce a sample to be detected 1, and the membrane-permeable nuclear dyes are selected from Transgreen, SYTO or SYBR; (b) a luciferase reader is used to detect the sample to be detected 1 to obtain the first-time fluorescence intensity of the sample; (c) the sample to be detected 1 and propidium iodide PI are mixed to produce a sample to be detected 2; (d) the luciferase reader is used to detect the sample to be detected 2 to obtain the second-time fluorescence intensity of the sample; and (e) the bigger the obtained survival coefficient is, the higher the survival rate of sperms is.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for detecting the viability of sperm in vitro, in other words, a new method for detecting damage to the plasma membrane of sperm. Background technique [0002] At present, the main methods for evaluating sperm plasma membrane integrity include conventional staining, hypoosmotic swelling test (HOST) and fluorescent molecular probe staining. Conventional staining methods are still commonly used in clinical andrology laboratories in China, mainly including eosin staining, eosin-nigrosin stain, typan blue stain and eosin-nigrosin-Gimsa staining. Dyeing, etc. These methods are complicated to operate and overestimate the proportion of dead sperm. More importantly, the test results vary greatly between different laboratories. [0003] The hypotonic swelling test was reported by Jeyendran et al. in 1984. When the sperm is in a hypotonic solution, the water molecules outside...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N21/64C12Q1/02
Inventor 施惠娟赵洪鑫贾晓峰袁瑶史庭燕刁华林菊芳宋丽伟时伟丽徐彦
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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