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Sperm piRNA (Piwi-interacting Ribonucleic Acid) marker combination related to male reproductive functional disturbance and application thereof

A reproductive function and marker technology, applied in the field of sperm piRNA marker combination, can solve problems such as reproductive disorders, demethylation of retrotransposons, de-inhibition of primary piRNA barriers, etc., to improve accuracy, reduce pain, and facilitate promotion Effect

Active Publication Date: 2017-10-20
NANJING YOUZHIYUAN PHARMACEUTICAL TECHNOLOGY CO LTD +1
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another protein associated with the formation of primary piRNA is the single-strand specific endonuclease Zuc (Zucchini or MitoPLD) (16-18), mutations in this protein can lead to demethylation of retrotransposons, derepression and primary piRNA production disorders, which eventually lead to reproductive disorders

Method used

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  • Sperm piRNA (Piwi-interacting Ribonucleic Acid) marker combination related to male reproductive functional disturbance and application thereof
  • Sperm piRNA (Piwi-interacting Ribonucleic Acid) marker combination related to male reproductive functional disturbance and application thereof
  • Sperm piRNA (Piwi-interacting Ribonucleic Acid) marker combination related to male reproductive functional disturbance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: High-throughput next-generation sequencing screening of piRNAs with specific changes as biomarkers of male sperm motility

[0045] (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 was analyzed, it was centrifuged at 1000 g for 10 minutes to collect the sperm.

[0046] (2) Collect the sperm samples of 10 cases of normal fertile persons and 10 cases of weak sperm respectively, and mix the samples in the groups respectively. The R...

Embodiment 2

[0050] Example 2: Screening out 10 piRNAs with significant differences from the sequencing results

[0051] According to the two principles of the level of sperm content and the magnitude of change, the screening conditions were set up: the top 10 piRNAs with the highest content in sperm, and in asthenospermia were reduced by more than 1.5 times compared with the normal control. Based on this screening condition, the following 10 piRNAs were screened out: piR-hsa-28131, piR-hsa-1207, piR-hsa-23317, piR-hsa-27493, piR-hsa-2107, piR-hsa-25783, piR- hsa-2106, piR-hsa-25781, piR-hsa-18709, piR-hsa-25780; the reduction range is shown in the table below, figure 2 b.

[0052]

Embodiment 3

[0053] Example 3: Determination of piRNA expression and specific changes in seminal plasma by TaqMan probe Real-time PCR piRNA as a biomarker of sperm motility

[0054] For the two piRNAs of piR-hsa-1207 and piR-hsa-2107, the TaqMan probe Real-time PCR quantitative detection of a single sample was carried out, and RNU6-6P was used as an internal reference; and the specific change of piRNA was further determined as the indicator of sperm motility Biomarkers.

[0055] The specific steps are: extract the total RNA in the sperm 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 fluorescent signal was recorded, and the instrument used was a Roche 480 fluorescent quantit...

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Abstract

The invention discloses sperm piRNA (Piwi-interacting Ribonucleic Acid) marker combination related to male reproductive functional disturbance and application thereof. A marker comprises ten piRNAs (mainly comprising piR-1207 and piR-2107), and the sperm piRNA marker combination can be used for detection and prediction of sperm viability. In the invention, sperm can be relatively easily obtained, other tissues are not required, and the detection belongs to non-traumatic examination; sperm piRNA and MitoPLD (Phospholipase D) protein can be used for reflecting pathological and physiological status of a whole spermatogenic process at a molecular level, the accurate level of detection can be increased by taking the sperm piRNA and the MitoPLD protein as auxiliary detection indexes, and a potential target spot is provided for treatment of the male reproductive functional disturbance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a combination of sperm piRNA markers related to male reproductive dysfunction and its application. Background technique [0002] Infertility has become a worldwide reproductive health problem. Currently, more than 15% of couples in the world suffer from infertility problems (1,2). Among them, about 20% to 30% of infertility is caused by male factors alone, and 50% of infertility is related to male factors. In recent years, this data has been on the rise (2,3), so male infertility Infertility plays an important role, however, the molecular mechanism of male infertility is not fully understood (4,5). At present, the clinical reproductive laboratory inspection items generally include biochemical inspection of semen, acid phosphatase, lactate dehydrogenase-X and other enzyme inspections, and semen immunological inspection, but these technologies have some defects and cannot directly refl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N33/68
CPCC12Q1/6883C12Q2600/158C12Q2600/178G01N33/689G01N2800/367
Inventor 陈熹洪叶挺付正
Owner NANJING YOUZHIYUAN PHARMACEUTICAL TECHNOLOGY CO LTD
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