Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof

A technology of asthenozoospermia and markers, applied in the fields of genetic engineering and reproductive medicine

Active Publication Date: 2015-11-11
NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is no report on the application of mitochondrial-associated miRNAs in the diagnosis of severe asthenozoospermia. If the mitochondrial-associated miRNAs that are susceptible to severe asthenospermia can be screened out as biomarkers, and the corresponding diagnostic kits are developed, the diagnosis of severe asthenospermia will be greatly improved. The status quo of diagnosis will definitely be a powerful impetus, and it will also open up a new way for its drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof
  • Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof
  • Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof

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Embodiment 1

[0081] Embodiment 1 Research Object Selection and Grouping Basis

[0082] The inventor collected semen samples from severe asthenospermia cases and normal fertile control adult males who met the requirements from July 2007 to October 2012 from the Reproductive Medicine Center of Nanjing Medical University. 80 cases of normal fertile controls (average age: 22.15±3.45 years old) and 80 cases of severe asthenospermia (average age: 22.32±3.51 years old) were used as experimental subjects for qRT-PCR detection of miRNA expression. Specific sample classification criteria are as follows:

[0083] (1) Repeat the semen quality test and be diagnosed with severe asthenozoospermia;

[0084] (2) Normal sexual function, excluding patients with known causes such as cryptorchidism, history of vascular trauma, orchitis, vas deferens obstruction, vasectomy, polychromosomal abnormalities, and Y chromosome azoospermia factor microdeletion

[0085] (3) Healthy male controls matched with the age ...

Embodiment 2

[0089] Example 2 Mitochondria-related miRNATaqManarray screening

[0090]Preparation of cDNA samples: a) Take 100 μl seminal plasma; b) Add 900 μl Trizol, shake and mix, centrifuge at 12,000 rpm for 15 minutes at 4°C, discard the waste liquid in the lower layer; c) Add 1.5 times the volume of supernatant ethanol, shake and mix, transfer to the spin column, centrifuge at 12000rpm for 15 seconds, and discard the lower layer of waste liquid; d) add 700 μl of RWT buffer to the spin column, centrifuge at 10000 rpm for 15 seconds, and discard the lower layer of waste liquid. e) Add 500 μl of RPE buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer. f) Repeat e. g) Add the spin column to a new 2ml tube and centrifuge at 10000rpm for 1 minute to remove the RPE buffer. h) Add 50 μl of DEPC-treated water to the column and centrifuge at 12000 rpm to collect RNA. i) Then cDNA is obtained by RNA reverse transcription reaction. The reverse tran...

Embodiment 3

[0100] Example 3 qRT-PCR method to measure the expression of mitochondria-related miRNA in seminal plasma

[0101] The primers (Table 1) were designed for the quantitative Real-time PCR detection of each miRNAs in the seminal plasma of 80 cases of normal fertility controls and 80 cases of severe asthenospermia.

[0102] (1) Preparation of cDNA samples: a) Take 100 μl of seminal plasma; b) Add 900 μl of Trizol, shake and mix, centrifuge at 12,000 rpm at 4°C for 15 minutes, discard the lower layer of waste liquid; c) add 1.5 times the volume of supernatant absolute ethanol, shake and mix Mix well, transfer to the spin column, centrifuge at 12000rpm for 15 seconds, discard the lower waste liquid; d) add 700 μl RWT buffer to the spin column, centrifuge at 10000 rpm for 15 seconds, discard the lower waste liquid. e) Add 500 μl of RPE buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer. f) Repeat e. g) Add the spin column to a new 2ml tu...

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Abstract

The invention belongs to the field of gene engineering and reproductive medicine, and discloses mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof. The markers are mitochondria-related miRNAs selected from hsa-miR-101-3p, hsa-miR-151a-5p, and hsa-let-7b-5p. The markers can be used for separating patients with severe asthenospermia from a control group with normal fertility, and can be used for preparing human severe asthenospermia diagnosis or monitor kits.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to mitochondria-associated seminal plasma microRNA as a marker for the occurrence of severe human asthenospermia and its application. Background technique [0002] Infertility has always been a major scientific problem to be solved in the field of reproductive health. In our country, about 9% of couples of childbearing age face this kind of predicament. Due to the large population base in our country, the number of couples of childbearing age is particularly considerable, and the number of infertile patients among newlyweds far exceeds one million. There are many causes of infertility, among which about 50% are caused by male factors. Research by the WHO Infertility Prevention and Treatment Task Force shows that reproductive endocrine dysfunction, especially asthenospermia, is one of the main causes of male infertility or decreased fertility. [0003...

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 陆春城周冉秦玉峰王嵘傅广波王晓燕夏彦恺王心如
Owner NANJING MEDICAL UNIV
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