A kit for predicting sperm retrieval outcome in patients with non-obstructive azoospermia

A technology for azoospermia and patients, applied in the field of biomedicine, can solve the problems of poor stability, poor sensitivity, and difficult clinical application of free RNA detection, and achieve fully scientific and practical effects

Active Publication Date: 2022-07-15
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +1
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

A large-scale clinical study involving 1371 patients with NOA showed that the area under the curve (AUC) of the clinical prediction model based on FSH, LH and testosterone was only 0.67; The detection sensitivity is poor; the detection stability of free RNA in semen is poor; testicular tissue samples can be obtained by testicular biopsy, but as an invasive examination method, it is difficult to achieve a wide range of clinical applications

Method used

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  • A kit for predicting sperm retrieval outcome in patients with non-obstructive azoospermia
  • A kit for predicting sperm retrieval outcome in patients with non-obstructive azoospermia
  • A kit for predicting sperm retrieval outcome in patients with non-obstructive azoospermia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Seminal plasma exosome purification and long-chain RNA sequencing of normal volunteers and NOA patients

[0026] Semen (>0.5mL) was taken from a single sample, centrifuged at 4°C, 12,000×g for 30min to remove cell debris, and the supernatant was collected, filtered through a 0.22μm filter, and then ultracentrifuged at 4°C, 100,000×g for 70min to collect exocrine secretions. body precipitation. The pellets were resuspended in PBS and then ultracentrifuged at 100,000 × g for 70 min at 4°C to obtain purified exosome pellets.

[0027] The purified exosomes were precipitated, added to the RNA lysis solution, and the total exosome RNA was obtained by phenol-chloroform extraction and a library was established. Followed by long-chain RNA (lncRNA) sequencing.

Embodiment 2

[0028] Example 2 Objective Gene Screening

[0029] The sequencing data in Example 1 was analyzed, and testis-specific highly expressed genes were screened according to the NCBI database information, and at the same time, the top 20 genes showing a downward trend in the sequencing results and the largest log2(FC) multiples were selected.

Embodiment 3

[0030] Example 3 Determination of lncRNA expression in seminal plasma exosomes of NOA patients

[0031] The seminal plasma exosomal RNA of NOA patients was extracted and detected by real-time quantitative polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0032] (1) Precipitate the purified exosomes obtained in Example 1, add RNA lysis solution, obtain total exosome RNA by phenol-chloroform extraction, and use Nanodrop2000 to detect the RNA concentration and purity. Perform reverse transcription reaction with 500ng RNA / 10μL system to obtain reverse transcription reaction solution and detect its concentration. The reverse transcription reaction was diluted to 10 ng / μL with DEPC water, aliquoted at 20 μL / tube and stored at -80°C. For the reverse transcription reaction solution used for qRT-PCR detection, the concentration of the reverse transcription reaction solution should be adjusted to 2.5ng / μL.

[0033](2) Using β-Actin as the internal reference gen...

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Abstract

The invention relates to the field of biomedicine, in particular to a kit for predicting the outcome of sperm extraction for patients with non-obstructive azoospermia. The kit of the present invention includes a reagent for detecting the expression level of lncRNA in the sample, the lncRNA includes SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC100505685, LOC101929088 (XR_927561.2), LOC101929088 (XR_001745218.1), LINC00343, LINC00343, LINC00343, LINC00343 at least one of them. The invention can realize the non-invasive and accurate prediction of the sperm retrieval outcome of NOA patients before operation, and has sufficient scientificity and practicability.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a kit for predicting the outcome of sperm extraction in patients with non-obstructive azoospermia. Background technique [0002] Non-obstructive azoospermia (NOA) is one of the most serious male infertility, and its treatment has always been a hot and difficult point in the field of reproductive research at home and abroad. Although sperm cannot be found in the semen, some patients with NOA can still meet their fertility needs by testicular aspiration. However, the existing technology is difficult to predict the outcome of sperm retrieval before surgery, resulting in patients not receiving optimal clinical treatment (screening of patients who are truly suitable for surgery). [0003] At present, the preoperative markers for predicting the outcome of sperm retrieval in patients with NOA are mainly derived from blood, semen and testicular tissue. Among the above samples, the detection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883G16B25/10
CPCC12Q1/6883G16B25/10C12Q2600/158C12Q2600/178
Inventor 刘贵华谢云邹学农邓春华
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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