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Method for evaluating influences of smoking on quality of oocytes

An oocyte and mother cell technology, which is applied in the field of assessment of the impact of smoking on oocyte quality, can solve the problems of weakened reproductive ability, decreased blastocyst formation rate in mice, and decreased oocyte quality.

Active Publication Date: 2020-01-24
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Kendall V et al. found that when 8mM cotinine was added to the early mouse embryo culture medium, the blastocyst formation rate in mice could be significantly decreased; however, when less than 8mM cotinine was added to the mouse embryo culture medium When compared with the normal control group, there was no significant difference in the blastocyst formation rate
There are currently no studies on the effect of cotinine on oocyte quality
[0004] There is no correlation study between cotinine and oocyte quality in the prior art. Cotinine is used to directly assess the impact of smoking on oocyte quality, and further assess how much smokers and passive smokers smoke, which will cause the decline in oocyte quality and ultimately lead to weakened reproductive capacity

Method used

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  • Method for evaluating influences of smoking on quality of oocytes
  • Method for evaluating influences of smoking on quality of oocytes
  • Method for evaluating influences of smoking on quality of oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Effect of cotinine on oocyte quality

[0030] Firstly, in vitro maturation culture droplets containing 0, 100, 500, 800 and 1000 μmol / L cotinine were prepared and incubated at 37°C, 5% CO 2 Pre-equilibrate for 2 hours in the cell culture incubator. Then put 30-35 collected GV stage oocytes into each droplet and place them in an incubator for culture. After culturing for 16 hours, the oocyte maturation rate, that is, the first polar body discharge rate, was counted. The results are shown in Table 1:

[0031] Table 1 Effects of different concentrations of cotinine on oocyte development and quality in vitro

[0032]

[0033] Note: In the table, different lowercase letters indicate significant differences (p<0.05), and different uppercase letters indicate extremely significant differences (P<0.01). All groups were compared to the 0 μM group for significance.

[0034] It can be seen from the above table that when the concentration of cotinine added to the in vitro ma...

Embodiment 2

[0038] Effect of cotinine on in vitro fertilization ability of mature oocytes

[0039] In order to visually evaluate the quality of in vitro mature oocytes, the mature oocytes were first fertilized with mouse sperm in vitro to detect how many sperms the mature oocytes can combine with.

[0040] The specific method is as follows: (1) 12-13 week old male mice were killed by cervical dislocation, the tail of the epididymis was taken out with small scissors, and several slits were cut in it, the sperm were released in the in vitro fertilization solution, and then its Put in 37℃, 5% CO 2 Cell incubator culture, take 10 after 30 minutes 6 Place a sperm into a droplet containing mature oocytes at 37°C in 5% CO 2 The cells were cultured in an incubator for 1 hour, fixed, infiltrated, stained with DAPI, and the number of sperm bound to mature oocytes was counted under a fluorescent microscope.

[0041] (2) Put the mature oocytes into the parthenogenetic activation solution A (in vit...

Embodiment 3

[0044] Effects of cotinine on expression levels of different genes in mature oocytes

[0045] In order to explore the molecular mechanism of cotinine-induced decline in the quality of mature oocytes in vitro, fluorescent quantitative PCR technology was used to extract RNA from 50 mature oocytes in each group, reverse transcribe it into cDNA, and detect it with SYBR Green fluorescent dye. Relative expression of antioxidant-related genes (sod1, sod2, glrx2, and GPX1) and apoptosis-related genes (p53, BAX, and BCL-1) in each group, where GADPH was used as an internal reference gene.

[0046] The result is as Figure 4 , with the increase of the concentration of cotinine in the in vitro maturation solution, the expression of antioxidant-related genes gradually decreased. These genes were significantly and extremely significantly decreased. At the same time, with the increase of the concentration of cotinine in the in vitro maturation solution, the expression of apoptosis-related...

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Abstract

The invention discloses a method for evaluating the influences of smoking on the quality of oocytes. Specifically, oocytes are cultured to be mature in culture solutions containing different concentrations of cotinine. Finally, the influences of smoking on the quality of oocytes are evaluated by detecting some indicators, such as the maturation rate of oocytes, chromosomal aneuploidy, actin distribution, mitochondrial membrane potential, intracellular reactive oxygen species level, spindle morphology, chromosome arrangement of mature oocytes, sperm binding number after in-vitro fertilization,pronucleus formation rate after parthenogenetic activation and the like. Through a large number of objective tests, the influences of smoking on the quality of oocytes are evaluated, thus providing areference of theoretical data for the maintenance of reproductive capacity of female smokers.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for evaluating the influence of smoking on the quality of oocytes. Background technique [0002] According to "Guangming Daily" report: Among the 1.1 billion smokers in the world, my country accounts for 330 million. About 1 million people die of smoking-related diseases in my country every year, and the rest of the smokers are in a sub-health state. According to research reports, smoking can reduce female fertility. In addition, if a woman smokes during pregnancy, the fetus will face a higher risk of spontaneous abortion and perinatal death, and significantly reduce the birth weight of the fetus. However, there is still no relevant research report on whether smoking directly affects the reproductive system, especially the quality of oocytes. [0003] The main reason cigarettes are addictive is their nicotine content. Cotinine is reported to be the product of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
CPCG01N33/5014G01N33/5044C12N2503/00
Inventor 程金妹孙斐陈爱春范晓博米盼盼
Owner NANTONG UNIVERSITY
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