Kit for evaluating azoospermia before operation based on seminal plasma exosome mRNA expression profile

A technique for azoospermia and kits, applied in the field of biomedicine, can solve the problems of non-invasive, comprehensive and accurate disease assessment, poor detection sensitivity, difficult to promote and other problems of azoospermia, and achieve sensitivity and stability , detect sensitive and stable effects

Pending Publication Date: 2021-11-19
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reasons why it is difficult for existing methods to predict the outcome of NOA sperm retrieval non-invasively and accurately before surgery mainly include: although the technology of sex hormone detection is mature, its detection sensitivity is not good, and it is difficult to distinguish whether there are subtle differences in sperm in the testis; Invasive examination i...

Method used

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  • Kit for evaluating azoospermia before operation based on seminal plasma exosome mRNA expression profile
  • Kit for evaluating azoospermia before operation based on seminal plasma exosome mRNA expression profile
  • Kit for evaluating azoospermia before operation based on seminal plasma exosome mRNA expression profile

Examples

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Embodiment 1

[0030] Example 1 Screening of biomolecular markers

[0031] The screening steps of biomolecular markers of the present invention are as follows:

[0032] 1. Firstly, transcriptome sequencing was performed on four parts (testis, head of epididymis, body of epididymis, tail of epididymis) of normal young male mice. The result is as figure 1 As shown, the number of specific mRNAs in the testis was the largest, reaching 2148, and the number of specific mRNAs in the three parts of the epididymis was 677 (head of the epididymis), 355 (body of the epididymis), and 272 (caudal of the epididymis).

[0033] 2. From the specific mRNAs obtained in step 1 above, 25 specific mRNAs with high expression levels in the testis, head of the epididymis, body of the epididymis, and tail of the epididymis were screened out. The result is as image 3As shown, there are 100 specific mRNAs in the testis, head of epididymis, body of epididymis, and tail of epididymis that are highly expressed in ma...

Embodiment 2

[0040] Example 2 Application of Molecular Markers for Preoperative Evaluation of Azoospermia Type

[0041] A kit for preoperative assessment of azoospermia typing can be constructed based on the molecular markers screened in the present invention, and the kit includes the six molecular markers obtained in Example 1. The specific method of use of the kit of the present invention is as follows:

[0042] 1. Extraction and purification of seminal plasma exosomes

[0043] Take semen (>0.5 mL), centrifuge at 4°C, 12,000×g for 30 minutes to remove cell debris, and filter the supernatant through a 0.22 μm filter, then ultracentrifuge at 4°C, 100,000×g for 70 minutes to collect exosome precipitates. After the pellet was resuspended in PBS, it was again purified by ultracentrifugation at 100,000×g at 4°C for 70 min to obtain exosome pellets.

[0044] 2. Extract exosomal RNA from seminal plasma and perform real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) detecti...

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Abstract

The invention provides a group of biomolecular markers and a kit for accurate typing of azoospermia, the molecular markers are seminal plasma exosome mRNA, and the mRNA comprises PRM2, ADAM28, WFDC13, WFDC9, SEMG2 and PSA. The seminal plasma exosome is used as a marker source and is focused to genital gland specific mRNA, and the specific mRNA expression of each part in the seminal plasma exosome is comprehensively detected, so that whether obstruction exists or not and obstruction parts or sperm collection outcome is determined. The kit disclosed by the invention is based on the RNA enrichment effect and the stability of exosomes, and is combined with seminal plasma exosome long-chain RNA template amplification and qRT-PCR detection technologies to amplify and acquire a genitalia specific mRNA signal in seminal plasma, so that the sensitivity and the stability of detection are realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a kit for preoperatively evaluating azoospermia based on seminal plasma exosome mRNA expression profiles. Background technique [0002] Azoospermia is the most serious type of male infertility, which can be divided into Obstructive Azoospermia (OA) and Nonobstructive Azoospermia (NOA). At present, male surgery (reversal surgery, testicular sperm retrieval, etc.) is mainly used to meet the fertility needs of patients with azoospermia, but the premise of effective treatment is to accurately determine the type of azoospermia, its obstruction site, and the outcome of sperm retrieval, etc. . Therefore, it is very important to accurately evaluate the condition of patients with azoospermia before surgery, which will help to choose the appropriate surgical method and avoid unnecessary surgery or secondary surgery. According to the standard diagnosis and treatment proces...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/166
Inventor 谢云邓春华邹学农孙祥宙姚嘉慧王竹庄锦涛涂响安
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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