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Kit for evaluating testis microscopic sperm extraction effect of idiopathic non-obstructive azoospermia patient

A technique for azoospermia and kits, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of few micro-TESE sperm retrieval effects, and cannot evaluate the surgical retrieval of NOA patients well. To achieve the effect of convenient material collection, stable expression, and high diagnostic value

Active Publication Date: 2021-11-02
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing variables have not been able to evaluate the effect of surgical sperm retrieval in patients with NOA well
Furthermore, few studies have investigated improving micro-TESE sperm retrieval in patients with idiopathic NOA

Method used

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  • Kit for evaluating testis microscopic sperm extraction effect of idiopathic non-obstructive azoospermia patient
  • Kit for evaluating testis microscopic sperm extraction effect of idiopathic non-obstructive azoospermia patient
  • Kit for evaluating testis microscopic sperm extraction effect of idiopathic non-obstructive azoospermia patient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 sample collection

[0022] (1) Research object and experimental design

[0023] All experiments involved in the present invention have been approved by the First Affiliated Hospital of Nanjing Medical University (ethical review number: 2018-SRFA-035). All patients included in the study gave informed consent to the study. Before operation, all patients had completed Doppler ultrasonography, sex hormone examination, chromosome karyotype examination, AZF deletion examination, etc. to clarify the etiology. Testicular tissues from 6 patients with idiopathic NOA (including 3 patients with successful sperm retrieval and 3 patients with unsuccessful sperm retrieval) were used for high-throughput transcriptomics next-generation sequencing.

[0024] From October 2018 to October 2020, a total of 52 idiopathic NOA patients were included in the study. According to the results of surgical sperm retrieval, these patients were divided into success group (20 cases) and fail...

Embodiment 2

[0028] Example 2 Extraction of RNA and sequencing of circRNA

[0029] Total RNA was extracted from testicular tissue and seminal plasma using the TRIzol kit (Invitrogen, Carlsbad, CA, USA). Then, the concentration and purity of total RNA was assessed using NanoDrop 200 (Madison, WI, USA).

[0030] The construction of the testis sample circRNA library for sequencing includes the following steps: 1. Removal of ribosomal RNA (rRNA); 2. Removal of linear RNA using RNase R; 3. Fragmentation of RNA using fragmentation buffer; 4. Reverse transcription of RNA Synthesis of complementary DNA (cDNA); 5. Processing of cDNA fragments by end repair reaction and adapter ligation after addition of a single "A" base; 6. Screening of cDNA libraries by quality inspection and selection of libraries of appropriate size; 7. Commissioning RiboBio Co, Ltd (Guangzhou, China) performed sequencing on the Illumina sequencing platform.

[0031] After obtaining raw data, implement quality control (includ...

Embodiment 3

[0036] Example 3 RT-qPCR verification of the differences in related circRNAs between the microsperm retrieval success group and failure group in idiopathic NOA

[0037] We selected five circRNAs with the most significant differences (hsa_circ_0025349, hsa_circ_0000277, hsa_circ_0060394, hsa_circ_0008226, hsa_circ_0007773) for RT-qPCR verification, according to the instructions of the reverse transcription kit (PrimeScript TM RT Master Mix, TaKaRa, Japan) total RNA (500ng) from testis and seminal plasma was reverse transcribed into cDNA.

[0038] Then, obtain the sequence of above-mentioned 5 circRNAs from circBase database (hsa_circ_0025349 nucleotide sequence is shown in SEQ ID NO:1; hsa_circ_0000277 nucleotide sequence is shown in SEQ ID NO:2; Has_circ_0060394 nucleotide sequence is shown in SEQ ID NO : 3; hsa_circ_0008226 nucleotide sequence as shown in SEQ ID NO: 4; hsa_circ_0007773 nucleotide sequence as shown in SEQ ID NO: 5), and use Primer5 software to design RT-qPCR p...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit for evaluating the testis microscopic sperm extraction effect of an idiopathic non-obstructive azoospermia patient, reagents contained in the kit are used for determining the content of three circRNAs, namely, hsa_circ_0000277, hsa_circ_0060394 and hsa_circ_0007773, in seminal plasma, the three circRNAs are combined through logistic regression to construct a model with a higher diagnostic value, and the kit is used for evaluating the testis microscopic sperm extraction effect of an idiopathic non-obstructive azoospermia patient. The kit has the beneficial effects that: the circRNA from the testis in the seminal plasma is stable in expression and convenient to obtain, and can be used for dynamically reflecting the spermatogenic state in the testis. Clinical specimens verify that circRNA from testis in seminal plasma has high diagnostic value as an evaluation marker of microscopic sperm extraction effect of idiopathic NOA patients.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cirRNA marker used for evaluating the effect of testicular microsperm extraction in patients with idiopathic non-obstructive azoospermia. Background technique [0002] Azoospermia refers to a condition in which no sperm is found in the semen ejaculated three times in a row, and is the main cause of male infertility. According to whether the testis has spermatogenesis, azoospermia can be divided into two categories: obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). At present, the pathogenesis of idiopathic NOA is still unclear, and the number of cases is increasing day by day. For patients with idiopathic NOA, obtaining sperm through surgery is the only way to help them achieve fertility. [0003] In 1993, Craft et al first used testicular sperm extraction (TESE) to obtain sperm for OA patients. Since then, various surgical methods have been develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/178C12Q2600/158Y02A50/30
Inventor 宋宁宏秦超季承建王仪春魏希夷张星雨丛戎姚亮宇张其杰周翔栾焦晨游泽斌陈杏林
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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