| Background and ObjectiveMitochondria,which are involved in energy production,signal transduction,calcium ions homeostasis,apoptosis,are among the most abundant organelles in eukaryotic cells.Mammalian mitochondria and mitochondrial genome are maternally transmitted,and they play important roles in oocyte maturation,fertilization and early embryo development.It is believed that oocyte mitochondria are distributed to early embryonic cells,and there is no mitochondrial biogenesis until blastocyst stage.Recently,it has been confirmed that the mitochondrial DNA level of aneuploid embryos was significantly higher than that of euploid embryos.The mtDNA copy number was associated with maternal age and embryo euploidy,and they proposed to take mtDNA copy number as biomarker which could be used for chromosomal abnormality prediction.It also has been suggested that mitochondria premature replication before blastulation is a cause for suboptimal embryo development.Therefore,the increased level of mtDNA observed in abnormal or inferior embryos might be a sign of dysplasia,and this may be caused by a compensatory mechanism to internal or external environment.However,it has recently been reported that the standardized calculation method of mitochondrial DNA does not have a predictive value,and that age,ploidy as well as implantation potential are not linked to mtDNA content at all.Based on the existing results,it is not clear whether the higher level of blastocyst mtDNA is related to the chromosome abnormal ploidy states or the relatively poor embryo quality.So in this study,we analyzed the mt DNA copy number in blastocysts of different ploidies to clarify the correlation between embryo ploidy with mtDNA copy number and make it clear that whether mtDNA copy number could be served as a biomarker to judge egg/embryo quality.Materials and methodsWe made haploid embryo by parthenogenetic activation,triploid embryo by ICSI(intracytoplasmic sperm injection)and tetraploid embryo by cell electrofusion technology.We also made diabetic model by drug inducer methods to gain embryos in poor quality.Each experiment group has its certain diploid embryo counterpart by corresponding methods.Mitochondrial DNA numbers of each blastocyst from different models was tested by absolute quantitative real-time polymerase chain reaction(qPCR).Each experiment was repeated at least three times.Experimental data were presented as means ± SD.Different groups were compared by independent-sample t-test.Associations between ploidy and development speed(different rate of fast/slow develop blastocysts)as well as the various rates of embryo development were analyzed using Chi-square test.A probability level of P<0.05 was considered significant.Graphpad 7.0 was used to make relevant statistical figures.Results1 We investigated the correlation between blasctocyst ploidy and mtDNA copy numbers.The results demonstrates that mtDNA content of the two kinds of embryos were not statistically different compared with control groups(96h: haploid: P=0.4291,triploid: P=0.6001,tetraploid: P=0.5325;120h: haploid: P=0.3005,triploid: P=0.6073,tetraploid;P=0.9767).2 We divided the same chromosome ploidy blastocysts into a faster development speed group(96 h)and a slower one(120 h).According to our results,regardless of chromosome ploidy,blastocysts derived from a faster development rate do not contain a higher mtDNA levels tendency as we thought before(haploid: P=0.7791;triploid: P=0.8456;tetraploid: P=0.7113).3 Observing the development process of haploid,triploid and tetraploid embryos,we found that most of the non-diploid embryos grew slightly slower than the control group,especially the haploid and triploid ones.4 We concluded that blastocyst cell numbers had negative correlation with ploidy(haploidy vs diploidy: 60.09 ± 2.61 vs 48.88 ± 2.37;triploidy vs diploidy: 29.00 ± 2.46 vs 42.43 ± 3.04;tetraploidy vs diploidy: 21.77 ± 1.52 vs 44.46 ± 1.90).That is to say,with the increasing chromosome ploidy,the cell numbers of blastocysts obtained at the same time is reduced.5 We sampled the blastocysts from the diabetic group and examined their mtDNA copy numbers.The data obtained showed that the mtDNA copy number of diabetic mouse blastocysts was significantly elevated and there was a statistical difference(P=0.0062).In addition,we acquired in vitro aged oocytes and carried out IVF in order to make another model to certify the relationship between mtDNA content and embyo quality.According to our data,mtDNA content level of blastocysts from in vitro aged oocytes was higher than that of normal blastocysts(P=0.0092),supporting the idea that low embryo quality induced premature proliferation of mitochondria,as in Type 1 diabetic embryos.Conclusion:1 Mitochondrial DNA copy number in embryos has no direct correlation with chromosome ploidy.2 Mitochondrial DNA copy number has an increasing trend in nonoptimal embryos,thus mtDNA copy number can be served as an indicator to judge egg/embryo quality. |
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