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A seminal plasma microRNA combination and its application related to male reproductive dysfunction

A microRNA and reproductive function technology, applied in the biological field, can solve problems such as the semen quality that cannot directly reflect the spermatogenic function of the testis, and achieve the effects of being simple and easy to implement, improving the accuracy level, and being convenient to use.

Inactive Publication Date: 2011-12-21
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current routine seminal plasma testing items include fructose, α-glucosidase, acid phosphatase, zinc and carnitine, and some immunological indicators, including anti-sperm antibodies and anti-toxoplasma antibodies. It can not directly reflect the spermatogenic function of the testis and comprehensively evaluate the quality of semen for several kinds of male accessory gonad dysfunction and individual immunological factors with limited diagnosis [3]

Method used

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  • A seminal plasma microRNA combination and its application related to male reproductive dysfunction
  • A seminal plasma microRNA combination and its application related to male reproductive dysfunction
  • A seminal plasma microRNA combination and its application related to male reproductive dysfunction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Specific miRNA expression profile screening for reproductive dysfunction

[0054] (1) The subjects of the study are infertile men who have not taken any contraceptive measures for more than 2 years after marriage (the spouse's examination is normal, and they have no sexual dysfunction, varicocele, cardiovascular and cerebrovascular diseases, malignant tumors, abnormal liver and kidney function and other diseases. The patients were between 24-34 years old, and the disease course was 1-7 years), and age-matched males who had had children for no more than two years were used as normal controls (normal semen quality, no systemic diseases, and aged between 24-34 years old) ), semen was collected from all subjects after 3-7 days of abstinence, and the sperm quality and function were analyzed with WLJY-9000 Weili color sperm quality detection system (Beijing Weili Company). The analysis standards were all carried out according to the standards of the World Health Or...

Embodiment 2

[0064] Embodiment 2: RT-PCR experiment of miRNA in seminal plasma

[0065] The miRNAs initially screened in Table 1 were further screened and verified by quantitative PCR method, and miRNAs with significant expression differences were screened out.

[0066] First, the extracted seminal plasma total RNA was reverse-transcribed into cDNA. For each miRNA, design a gene-specific reverse primer (product of ABI Company) containing the same stem-loop structure, and use the miRNA-specific reverse primer for reverse transcription to obtain a cDNA that contains a common stem-loop structure but belongs to a specific miRNA.

[0067] The instrument used is ABI Prism 7300 fluorescent quantitative PCR instrument.

[0068] In this experiment, each miRNA mature body synthesized at different concentrations was used as a standard curve. The artificially synthesized miRNA mature stock solution was reverse-transcribed into cDNA, diluted with DEPC water to 10 fmol / L to 10 5 Concentration gradien...

Embodiment 3

[0072] Embodiment 3: Quantitative PCR method re-screens the serum miRNA screened out

[0073] There were 60 cases of male infertility, including 30 cases of non-obstructive azoospermia and 30 cases of asthenospermia, and 24 cases of age-matched males with normal fertility were used as controls. The screening criteria of patients and normal people are the same as in Example 1. The clinical information of the test subjects is shown in Table 2. The seminal plasma samples of the subjects were detected one by one with the TaqMan quantitative PCR method, and the miRNAs with significant differences in expression between the patient group and the control group were screened from the 19 miRNAs initially screened by the Solexa method (see Example 2 for specific steps). The results found that the expression levels of 7 miRNAs among the 19 miRNAs screened out in the patient group were significantly different from those in the control group (p<0.01). These seven miRNAs were miR-34c-5p, m...

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Abstract

The invention belongs to the field of biotechnology, disclosing a seminal plasma micro RNA combination relevant to male reproductive function disorder and an application thereof. The miRNA (Ribose Nucleic Acid) combination comprises the following miRNA: miR-34c-5p, miR-181a, miR-374b and miR-509-5p. The combination can be used for preparing reagent for diagnosing or monitoring male reproductive function disorder.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a combination of seminal plasma microRNA (miRNA for short) related to male reproductive dysfunction and its application. Background technique [0002] According to WHO statistics, infertile (pregnant) couples account for about 10% to 15% of couples of childbearing age, of which male factors account for about 30% to 40%. [1，2] . 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. At present, domestic clinical laboratories judge the spermatogenic function of male testes by testing the density and motility of sperm in semen, seminal plasma biochemical indicators, sex hormone levels in blood, chromosome karyotype, and Y chromosome microdeletion....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113
Inventor 张春妮张辰宇王成杨翠华姚兵
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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