Purpose

This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring.

Methods

The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR.

Results

The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta.

Conclusion

These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring.

中文翻译：

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激光辅助孵化对小鼠囊胚及子代印迹基因IGF2/H19甲基化及表达模式的影响

目的

本研究旨在确定激光辅助孵化的钻孔和变薄处理对胚胎和后代中印记基因IGF2/H19的表达和甲基化的影响。

方法

将经过钻孔或变薄处理的孵化前囊胚或对照孵化前囊胚移植到代孕子宫中。使用亚硫酸氢盐转化介导的测序和实时 PCR 分别评估 IGF2/H19 印记控制区 (ICR) 的 DNA 甲基化以及 IGF2 和 H19 的表达。

结果

与对照组和细化组相比，钻孔组的孵化囊胚发育率显着增加。细化组孵化的囊胚IGF2/H19 ICR DNA甲基化水平为27.33%，而对照组和钻孔组为38.67%和36%。与对照组和钻孔组相比，细化处理增加了胎盘中 IGF2/H19 ICR 的 DNA 甲基化水平。钻孔和间苗处理降低了预孵化和孵化的囊胚以及胎盘中H19 mRNA的表达水平，而在间苗组中观察到后代H19 mRNA的表达水平显着增加。

结论

这些观察结果表明，用于孵化体外培养的小鼠囊胚的钻孔可能会提高后代的产量。