Seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof

A reproductive function and marker technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as semen quality that cannot directly reflect testicular spermatogenesis

Active Publication Date: 2016-04-13
NANJING YOUZHIYUAN PHARMACEUTICAL TECHNOLOGY CO LTD +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The current routine seminal plasma detection items include fructose, α-glucosidase, acid phosphatase, zinc and carnitine, and some immunological indicators (including anti-sperm antibodies and anti-toxoplasma antibodies, etc.). It is helpful for the diagnosis of limited male accessory gonadal dysfunction and individual immunological factors, and cannot directly reflect the spermatogenic function of the testis and comprehensively evaluate the quality of semen (4)

Method used

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  • Seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof
  • Seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof
  • Seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1: High-throughput next-generation sequencing screening of specifically altered piRNAs as biomarkers of reproductive dysfunction

[0057] (1) The subjects of the study were infertile persons who did not take any contraceptive measures within 2 years after marriage. Age-matched males who had given birth for less than 2 years were used as normal controls. Semen was collected from all subjects after 3 to 5 days of abstinence. WLJY -9000 Weili color sperm quality detection system (Beijing Weili Company) for sperm quality and function analysis. The analysis standards were carried out according to the World Health Organization (WHO Laboratory Manual for Human Semen Examination and Processing (5th Edition)). After the semen analysis, it was centrifuged at 1000g for 10min to collect the supernatant, and then centrifuged at 12000g for 5min to collect the supernatant (seminal plasma).

[0058] (2) Collect seminal plasma samples from 21, 24, and 17 cases of normal fertili...

Embodiment 2

[0067] Example 2: TaqMan Probe Real-time PCR method to determine the absolute concentration of piRNA in seminal plasma and to determine the specific change of piRNA as a biomarker of reproductive dysfunction

[0068] For the piRNAs screened in Table 1, select some of them for quantitative detection of TaqMan probe Real-time PCR in a single sample, so as to determine the absolute concentration of piRNAs in seminal plasma, and further determine the specific changes of piRNAs as biomarkers of reproductive dysfunction things.

[0069]The specific steps are: extracting 100 μL of total RNA in the seminal plasma of a single sample. For each piRNA, design a specific reverse primer containing the same stem-loop structure, use the piRNA-specific reverse primer for reverse transcription, and obtain a cDNA that contains a common stem-loop structure but belongs to a specific piRNA. The Real-time PCR reaction based on TaqMan probe was carried out, each piRNA was amplified and the fluoresce...

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Abstract

The invention discloses seminal plasma piRNA markers or their combination for detecting and/or predicting male reproductive dysfunction and application thereof. The piRNA markers or their combination are 61 in quantity and mainly include piR-31068, piR-31925, piR-43771, piR-43773 and piR-30198; this combination can be used to detect and/or predict asthenospermia and azoospermia. In the invention, seminal plasma is easy to obtain, no other tissues are required, and examinations are noninvasive; seminal plasma piRNA can indicate pathological and biological conditions of a whole spermatogenic stage, accuracy level of detection can be increased using the seminal plasma piRNA as auxiliary detection indexes, the pathological and biological conditions the whole spermatogenic process are indicated on molecular level, and a latent target is provided for the diagnosis and treatment of male reproductive dysfunction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to seminal plasma piRNA markers or combinations thereof for detecting and / or predicting male reproductive dysfunction and applications thereof. Background technique [0002] Male infertility has become a reproductive health problem worldwide. According to estimates from the World Health Organization, there are currently nearly 80 million infertile (pregnant) couples in the world, accounting for about 8% to 12% of couples of childbearing age, of which male factors account for about 30%. % to 40% (1,2), and 60% to 75% of male factor infertility the etiology is unknown (3). In recent years, the proportion of male infertility has been on the rise, and people have higher and higher requirements for prenatal and postnatal care. Therefore, in order to effectively diagnose and treat male infertility, it is urgent to find male infertility markers with higher clinical value. [0003] However, ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 陈熹张春妮洪叶挺张辰宇梁宏伟王成曾科
Owner NANJING YOUZHIYUAN PHARMACEUTICAL TECHNOLOGY CO LTD
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