Biomarker for assisting diagnosis of sudden asthenozoospermia and application thereof

Abstract

The invention relates to a biomarker for assisting the diagnosis of sudden asthenozoospermia and application thereof. The biomarker is characterized in that a CpG methylation locus is in a peripheralblood leucocyte DAZ3 gene promoter area. A kit comprises quantitative PCR primers for detecting the methylation locus. The biomarker, a method and the kit have the advantages that peripheral blood leucocyte can be easily obtained, other tissue is not needed, and noninvasive examination is achieved; peripheral blood leucocyte DAZ3 gene promoter area methylation reflects the overall pathological andphysiological conditions of a human body, and diagnosis accuracy can be increased by using the CpG methylation locus as the biomarker for assisting the diagnosis of sudden asthenozoospermia; the kitsimplifies a quantitative PCR primer library, production cost and production time are reduced, targeting performance and practicality are increased, and detection sensitivity and specificity are increased; the biomarker, the method and the kit can be used for assisting the diagnosis of sudden asthenozoospermia.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a biomarker for auxiliary diagnosis of sudden weak sperm and its application. Background technique

[0002] The factors causing male asthenozoospermia are complex, including genetic factors, reproductive tract infection and varicocele, among which genetic defects are one of the important causes of male infertility and asthenozoospermia. Abnormalities in the number or structure of chromosomes can cause abnormalities in male reproductive organs, affect the production of sperm and reduce their fertility. Klinefelter syndrome is more common in this disease, and the karyotype is 47, XXY. The pathogenesis is that the X chromosome does not separate during meiosis, resulting in small and hard testes, low testosterone synthesis, and severe asthenospermia or azoospermia.

[0003] Sudden asthenospermia is a kind of asthenospermia, but its etiology is not yet clear. Men with sudden asthenosperm...

Examples

Embodiment 1

[0053] The collection of embodiment 1 sample

[0054] We collected EDTA anticoagulated whole blood samples from 17 healthy men with offspring and 13 age-matched men with sudden asthenospermia. The details are as follows:

[0055] Table 1 Sample information table

[0056]

[0057] Data are presented as mean ± SD. ***P<0.001.

Embodiment 2

[0058] Example 2 Quantitative detection of the methylation level of each CpG site in the promoter region of the DAZ1-3 gene in peripheral blood leukocytes of healthy men with offspring and men with sudden asthenospermia

[0059] In the initial screening stage, quantitative detection of CpG sites in the promoter region of the DAZ1-3 gene in peripheral blood leukocytes was performed on 17 healthy men with offspring and 13 age-matched men with sudden asthenospermia to screen for the presence of methylation CpG sites with level differences. The results showed that the methylation level of a specific CpG site (-312~-313) in the promoter region of the DAZ3 gene was different between the two groups (P figure 2 ). Table 2 CpG site (-312~-313) methylation in the promoter region of DAZ3 gene in the peripheral blood leukocytes of healthy men with offspring and men with sudden asthenospermia initially screened by the Sequenom MassARRAY methylation quantitative detection and analysis platf...

Embodiment 3

[0062] Example 3 Real-time fluorescence quantitative PCR experiment of the methylation level of the promoter region CpG site (-312~-313) of the DAZ3 gene in peripheral blood leukocytes

[0063] The quantitative PCR method was used to verify the CpG sites initially screened from the methylation quantitative detection and analysis platform for batch samples one by one. The experimental principle and experimental steps of quantitative PCR are the same as those of RT-PCR. The instrument is a Roche Cobas z 480.

[0064] (1) Use the DNA blood mini kit (Qiagen Company) to extract the total DNA of peripheral blood leukocytes

[0065] a) Add 20μL Qiagen Protease to the bottom of 1.5mL EP tube;

[0066] b) Add 200 μL EDTA anticoagulated blood sample;

[0067] c) Add 200 μL Buffer AL and vortex for 15 seconds;

[0068] d) 56°C water bath for 10 minutes;

[0069] e) Briefly centrifuge the 1.5mL EP tube;

[0070] f) Add 200 μL of absolute ethanol, vortex for 15 seconds, and centrifug...

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a biomarker for auxiliary diagnosis of sudden weak sperm and its application. Background technique

[0002] The factors causing male asthenozoospermia are complex, including genetic factors, reproductive tract infection and varicocele, among which genetic defects are one of the important causes of male infertility and asthenozoospermia. Abnormalities in the number or structure of chromosomes can cause abnormalities in male reproductive organs, affect the production of sperm and reduce their fertility. Klinefelter syndrome is more common in this disease, and the karyotype is 47, XXY. The pathogenesis is that the X chromosome does not separate during meiosis, resulting in small and hard testes, low testosterone synthesis, and severe asthenospermia or azoospermia.

[0003] Sudden asthenospermia is a kind of asthenospermia, but its etiology is not yet clear. Men with sudden asthenosperm...

Examples

Embodiment 1

[0053] The collection of embodiment 1 sample

[0054] We collected EDTA anticoagulated whole blood samples from 17 healthy men with offspring and 13 age-matched men with sudden asthenospermia. The details are as follows:

[0055] Table 1 Sample information table

[0056]

[0057] Data are presented as mean ± SD. ***P<0.001.

Embodiment 2

[0058] Example 2 Quantitative detection of the methylation level of each CpG site in the promoter region of the DAZ1-3 gene in peripheral blood leukocytes of healthy men with offspring and men with sudden asthenospermia

[0059] In the initial screening stage, quantitative detection of CpG sites in the promoter region of the DAZ1-3 gene in peripheral blood leukocytes was performed on 17 healthy men with offspring and 13 age-matched men with sudden asthenospermia to screen for the presence of methylation CpG sites with level differences. The results showed that the methylation level of a specific CpG site (-312~-313) in the promoter region of the DAZ3 gene was different between the two groups (P figure 2 ). Table 2 CpG site (-312~-313) methylation in the promoter region of DAZ3 gene in the peripheral blood leukocytes of healthy men with offspring and men with sudden asthenospermia initially screened by the Sequenom MassARRAY methylation quantitative detection and analysis platf...

Embodiment 3

[0062] Example 3 Real-time fluorescence quantitative PCR experiment of the methylation level of the promoter region CpG site (-312~-313) of the DAZ3 gene in peripheral blood leukocytes

[0063] The quantitative PCR method was used to verify the CpG sites initially screened from the methylation quantitative detection and analysis platform for batch samples one by one. The experimental principle and experimental steps of quantitative PCR are the same as those of RT-PCR. The instrument is a Roche Cobas z 480.

[0064] (1) Use the DNA blood mini kit (Qiagen Company) to extract the total DNA of peripheral blood leukocytes

[0065] a) Add 20μL Qiagen Protease to the bottom of 1.5mL EP tube;

[0066] b) Add 200 μL EDTA anticoagulated blood sample;

[0067] c) Add 200 μL Buffer AL and vortex for 15 seconds;

[0068] d) 56°C water bath for 10 minutes;

[0069] e) Briefly centrifuge the 1.5mL EP tube;

[0070] f) Add 200 μL of absolute ethanol, vortex for 15 seconds, and centrifug...

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