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Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof

A technology of azoospermia and markers, applied in the fields of genetic engineering and reproductive medicine, can solve the problems of large fluctuations in individual semen quality, failure to reflect results in time, and fluctuations in results

Active Publication Date: 2013-06-05
NANJING MEDICAL UNIV
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Problems solved by technology

At present, the evaluation of the degree of spermatogenesis obstacle mainly relies on conventional sperm density judgment (manual counting or computer-aided system analysis, namely CASA), and there is no other effective means
However, conventional sperm density analysis also has the following disadvantages: Individual semen quality fluctuates greatly, especially susceptible to factors such as abstinence days, temperature, semen collection method, etc., resulting in inaccurate sperm density measurement; it cannot reflect the results after treatment in time, And it is not easy to carry out dynamic monitoring, and the effect of early diagnosis of corresponding diseases is far from meeting the needs; and non-obstructive azoospermia, which is one of the most important manifestations of male infertility, often requires testicular biopsy for its definite diagnosis. Great pain for people seeking fertility help
However, seminal plasma, which is also a body fluid, has not received corresponding attention. If we can find stable and specific seminal plasma miRNAs related to spermatogenesis disorders as biomarkers, and develop diagnostic and monitoring kits for corresponding diseases, it will not only be in the leading position in this field. The leading position in the world can create remarkable economic benefits, and it will also be a strong impetus to the male reproductive health in my country
[0007] At present, the evaluation of the degree of spermatogenesis disorder is mostly limited to routine semen analysis, which is prone to fluctuations in results, and is difficult for early diagnosis and dynamic monitoring. The diagnosis of azoospermia still depends on testicular biopsy
So far, there have been no reports on the use of seminal plasma miRNA in the evaluation and treatment of semen quality and male reproductive function, especially in the evaluation, diagnosis and treatment of seminal plasma miRNA in human non-obstructive azoospermia

Method used

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  • Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof
  • Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof
  • Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0086] Example 1 Research Object Selection and Grouping Basis

[0087] The inventor collected semen samples of adult males who met the requirements from the First Affiliated Hospital of Nanjing Medical University and the Nanjing Maternal and Child Health Hospital affiliated to Nanjing Medical University from September 2006 to September 2008. 80 cases of healthy fertile male controls (average age: 29.32±3.13) and 80 patients with non-obstructive azoospermia (average age: 28.76±4.07) were selected as the experimental subjects for Real-time PCR detection of miRNA expression ( figure 1 ). Specific sample classification criteria are as follows:

[0088] Group A: healthy fertile male control group (n=80, 20 persons for microarray screening, 20 persons for first-stage verification, 40 persons for independent population verification):

[0089] 1. Between 24 and 34 years old;

[0090] 2. No reproductive and endocrine system diseases;

[0091] 3. No other systemic diseases;

[0092...

Embodiment 2

[0103] Example 2 Semen Collection and Routine Analysis of Semen Quality of Research Objects

[0104] After at least 2 days of abstinence, subjects were asked to masturbate semen in a sterile wide-mouth plastic container in the room. After the semen samples were incubated at 37°C for about 30 minutes to liquefy, we performed routine semen analysis according to the WHO Human Semen Analysis Laboratory Manual (World Health Organization, 1999), including semen volume, sperm concentration, total number of ejaculates, sperm motility, Sperm motility and forward sex parameters, etc., mainly use μ-cell plate and computer-aided semen analysis system (CASA, WLJY 9000, Weili New Century Science & Tech Dev.). The rate of motile sperm was WHO standard "A" grade sperm (fast moving, velocity ≥ 25 μm / sec at 37°C) plus "B" grade sperm (slow advancing, velocity between 5 μm / sec and 25 μm / sec), while Grade "C" sperm (not advancing, velocity 6 / ml), the total number of one ejaculate (40×10 6 ) an...

Embodiment 3

[0105] Example 3 Taqman miRNA array screening

[0106] Preparation of cDNA samples: a) Take 500 μl of seminal plasma; b) Add an equal volume of Trizol, shake and mix, centrifuge at 15,000 rpm for 30 minutes at 4°C, and take the supernatant; c) Add an equal volume of chloroform to the supernatant, shake and mix Evenly, centrifuge at 12,000 rpm for 30 minutes at 4°C, and take the supernatant; d) repeat steps b) and c) twice, and centrifuge at 12,000 rpm for 20 minutes. Take the supernatant as an RNA sample; e) Then obtain cDNA through RNA reverse transcription reaction. The reverse transcription reaction system includes 4 μl 5×AMV buffer, 2 μl 10mM dNTP mixture (Takara), 0.5ul RNase inhibitor (Takara), 1ul AMV (Takara) and 1.5μl loop reverse transcription primer (URP, see Table 1). The reaction steps are incubation at 16°C for 15 minutes, reaction at 42°C for 1 hour, and incubation at 85°C for 5 minutes;

[0107] The cDNA after reverse transcription was pre-amplified accordin...

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Abstract

The invention which belongs to the medical field of genetic engineering and reproduction discloses a seminal plasma miRNA marker associated with human non-obstructive azoospermia and an application thereof. The maker is selected from several of hsa-miR-141, hsa-miR-193a-5p, hsa-miR-590-5p and hsa-miR-7-1*. The maker which has specificities and sensitivities to the non-obstructive azoospermia can be used for the preparation of a reagent for non-obstructive azoospermia diagnosis or monitoring, so invasive diagnosis is avoided, repeated detection is realized, and the dynamic monitoring of the obstacle degree of sperm generation can be easily achieved.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to seminal plasma microRNA (microRNA, miRNA) markers related to human non-obstructive azoospermia and applications thereof. Background technique [0002] Infertility is a common reproductive disease, the incidence rate of couples of childbearing age in my country is about 10%. Nearly half of them are related to the man's factor, which is called male infertility. Among all the causes of male infertility, spermatogenesis disorder is the most common cause, and it is also one of the most important threats to the reproductive health of adult males in my country. The occurrence of spermatogenesis disorder is a multi-factor and multi-stage process. The main clinical manifestation is the decrease of sperm density in ejaculated semen. The specimen without sperm was centrifuged at 3000g for 15 minutes, but still no sperm). At present, the evaluation of the deg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/113C12N15/11
Inventor 王心如夏彦恺胡志斌沈洪兵吴炜
Owner NANJING MEDICAL UNIV
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