Seminal plasma micro ribonucleic acid marker related to spermatogenesis deficiency and application thereof

A technology of sperm production and markers, which is applied in the fields of genetic engineering and reproductive medicine, can solve the problems that the early diagnosis of diseases cannot meet the needs, the quality of individual semen fluctuates greatly, and it is not easy to monitor dynamically, so as to delay and prevent the progress of diseases and improve Sensitivity and specificity, effects of avoiding invasive diagnostics

Inactive Publication Date: 2011-08-31
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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; more importantly, the definite diagnosis of azoospermia often requires testicular biopsy, which brings great benefits to the crowd seeking fertility help. pain
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 spermatogenesis disorders such as oligospermia and azoospermia

Method used

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  • Seminal plasma micro ribonucleic acid marker related to spermatogenesis deficiency and application thereof
  • Seminal plasma micro ribonucleic acid marker related to spermatogenesis deficiency and application thereof
  • Seminal plasma micro ribonucleic acid marker related to spermatogenesis deficiency and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1 Research Object Selection and Grouping Basis

[0075] 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. A total of 96 healthy fertile male controls (average age: 29.32±3.13), 96 cases of oligospermia patients (average age: 28.96±4.32), and 96 cases of azoospermia patients (average age: 28.76±4.07) were selected as Real-time PCR detection of miRNA expression experimental subjects ( figure 1 ). Specific sample classification criteria are as follows:

[0076] Group A: healthy fertile male control group (n=96)

[0077] 1. Between 24 and 34 years old

[0078] 2. No reproductive and endocrine system diseases

[0079] 3. No other systemic diseases

[0080] 4. Normal sexual function

[0081] 5. Normal semen quality

[0082] 6. Have a norm...

Embodiment 2

[0098] Example 2 Research object semen collection and routine analysis of semen quality

[0099]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 ) and spe...

Embodiment 3

[0100] Embodiment 3Real-time PCR method measures miRNA expression in seminal plasma

[0101] Primers (Table 1) were designed for quantitative Real-time PCR detection of miRNAs in the seminal plasma of 96 healthy fertile male controls, 96 oligospermia, and 96 azoospermia patients.

[0102] (1) Preparation of cDNA samples: a) Take 500 μl of seminal plasma; b) Add an equal volume of water-saturated phenol, shake and mix, centrifuge at 15,000 rpm at 4°C for 30 minutes, and take the supernatant; c) Add an equal volume of phenol to the supernatant Shake and mix with chloroform, 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 10 mM dNTP mixture (Takara), 0.5 μl RNase inhibitor (Takara), 1 μl AMV (Takara) a...

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Abstract

The invention belongs to the field of gene engineering and reproductive medicines, and discloses a seminal plasma micro ribonucleic acid marker related to spermatogenesis deficiency and application thereof. The marker is selected from one or more of SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, has specificity and sensitivity to the spermatogenesis deficiency, can be used for preparing a reagent for diagnosing or monitoring the spermatogenesis deficiency, can avoid invasive diagnosis, can carry out repeated detection, and is easy to dynamically monitor the degree of the spermatogenesis deficiency.

Description

field of invention [0001] The invention belongs to the field of genetic engineering and reproductive medicine, and relates to a seminal plasma microRNA (miRNA) marker related to spermatogenesis disorder and an application 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. Specimens without sperm, after centrifugation / 15min, 3000rpm still no sperm). At present, the evaluation of the degree of spermatogenesis obsta...

Claims

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Application Information

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