Study Of The Relationship Between Human Embryonic Morphology And Mitochondrial Function

Part I. Studies on the relationship between the morphological classification of human embryo and the copy number, the diameter of the embryo, the potential of the membrane and the distribution of the mitochondriaObjective Mitochondria as a source of energy for the development of the embryo, the energy provided by the mitochondria directly affects the development of the embryo and the quality of the embryo. This study was mainly to investigate the relationship between the embryo at the different stages different morphological types and embryo mitochondrial copy number, the membrane potential, embryo diameter and their effects on the embryos development.Method We selected the frozen embryos 305 with discarded voluntarily by successful pregnancy in patients with IVF-ET infertility and the embryos with poor development after normal fertilization of fresh cycle oocytes were not suitable transferred of 152 embryos. Place them under inverted microscope to observe and take pictures after frozen thawed and cultured 2h then carry out the stage of embryo morphology and grading, divide the embryos which are at same sorce in the same period into a group. According to the different developmental stages of the embryo, divide all embryos into fresh embryo group (poor cleavage embryos, n=152), D3 frozen embryo group (cleavage stage embryos, n=144) and D5 frozen embryo group (blastocyst, n=161), then according to the grading criterion of frozen embryos, freezing cleavage stage embryos were divided into high quality embryo group (n=89) and poor embryo group (n=55), the frozen blastocysts were divided into high quality blastocyst group (n=101) and poor blastocyst group (n=60). And then use the Real-time PCR method to detect mtDNA copy number of a single embryo, JC-10 staining to detect the mitochondrial membrane potential and Tracker Green Mito staining to observe the distribution of mitochondria. Compare the differences between embryo quality and mitochondrial copy number, embryo diameter, membrane potential and distribution of each group.Result 1. The copy number of Mt DNA in D3 frozen embryo group were significantly lower than that in D5 frozen blastocyst group(P＜0.05). The copy number of Mt DNA in fresh group was significantly lower than that in D3 frozen high quality embryo group and D3 frozen non high quality embryo group (P＜0.05). The D3 high quality embryo group Mt DNA copy number compared with D3 frozen non high quality embryo group, there was no significant difference (P＞0.05). Mt DNA copy number of D5 frozen high quality blastocyst group was significantly higher than that in D5 frozen non high quality blastocyst group (P＜0.05).2. The diameter of D3 frozen embryos group was significantly lower than that of D5 frozen blastocyst group (P＜0.01). The diameter has no significant difference between the D3 high quality embryos group and D3 non high quality embryos group. The diameter of high quality embryos in D5 frozen blastocyst group was significantly higher than that in non high quality blastocyst group (P＜0.05).3. The mitochondrial membrane potential in D3 frozen embryo group were significantly lower than that in D5 frozen blastocyst group(P＜ 0.05). The mitochondrial membrane potential in fresh group was significantly lower than that in D3 frozen high quality embryo group and D3 frozen non high quality embryo group (P<0.05). The mitochondrial membrane potential in D3 frozen high quality embryo group were significantly higher than that in D3 frozen non high quality embryo group (P<0.05). Mt mitochondrial membrane potential of D5 frozen high quality blastocyst group was significantly higher than that in D5 frozen non high quality blastocyst group (P<0.05).4.Mitochondria were dispersed in the cytoplasm in the fresh group, and the mitochondria were not evenly distributed. In the high-quality embryo of D3 frozen embryo group, mitochondria are uniformly distributed at the periphery of each blastomere nuclei, uniform staining, and at the poor quality of the embryo, mitochondria not evenly distributed in the blastomeres, blastomere uneven dyeing. In the high-quality embryo of blastocyst stage, the mitochondria are mainly concentrated in a wider area around the nucleus, deeply staining and evenly distributed and in the poor quality of the blastocyst, mitochondria are mainly dispersed around the cell nucleus, lighter and uneven staining.Conclusion 1. With the continuous development of the embryos, Mt DNA copy number is gradually increased, the Mt DNA copy number of the better quality embryo is higher.2.The Mt DNA copy number of blastocyst stage was positively correlated with the diameter of the embryo itself.3.With the continuous development of the embryos, the mitochondrial membrane potential of embryos is gradually increased, the mitochondrial membrane potential of the better quality embryos is higher.4.When mitochondria were evenly distributed in blastomeres and surrounding wider area of inner cell mass and nuclear, there is a embryo of strong biological developmental potential.Part II. Study on the relationship between human embryonic morphology and the level of Ca2+, ROS and ATP in the embryoObjective ATP is the energy guarantee of oocyte maturation, fertilization and embryo development, ROS is important redox signaling molecules within the embryo, Ca2+ is important second messenger of embryonic metabolic activity, those three are products of mitochondria and there is a close interaction between each other, which participate in the proliferation, differentiation and apoptosis of embryonic cells by acting on mitochondria. This study was mainly to investigate the relationship between the embryo at the different stages different morphological types and ATP, ROS, Ca2+ levels of embryos and their effects on the embryos development.Method We selected the frozen embryos 305 with discarded voluntarily by successful pregnancy in patients with IVF-ET infertility and the embryos with poor development after normal fertilization of fresh cycle oocytes were not suitable transferred of 152 embryos. Place them under inverted microscope to observe and take pictures after frozen thawed and cultured 2h then carry out the stage of embryo morphology and grading, divide the embryos which are at same sorce in the same period into a group. According to the different developmental stages of the embryo, divide all embryos into fresh embryo group (poor cleavage embryos, n=152), D3 frozen embryo group (cleavage stage embryos, n=144) and D5 frozen embryo group (blastocyst, n=161), then according to the grading criterion of frozen embryos, freezing cleavage stage embryos were divided into high quality embryo group (n=89) and poor embryo group (n=55), the frozen blastocysts were divided into high quality blastocyst group (n=101) and poor blastocyst group (n=60). And then use the luciferase assay to detect single embryo ATP levels, activated oxygen detection kit staining to detect the ROS level, Fluo-4AM fluorescent probe staining to detect the Ca2+ levels, and compare the differences between each embryo quality and ATP, ROS and Ca2+ levels in embryo.Result 1. The mitochondrial ATP in D3 frozen embryo group were significantly lower than that in D5 frozen blastocyst group (P＜0.05). The mitochondrial ATP in fresh group was significantly lower than that in D3 frozen high quality embryo group and D3 frozen non high quality embryo group (P＜0.05). The mitochondrial ATP in D3 frozen high quality embryo group were significantly higher than that in D3 frozen non high quality embryo group (P＜0.05). The mitochondrial ATP of D5 frozen high quality blastocyst group was significantly higher than that in D5 frozen non high quality blastocyst group (P＜0.05).2. The mitochondrial ROS in D3 frozen embryo group were significantly lower than that in D5 frozen blastocyst group (P<0.05). The mitochondrial ROS in fresh group was significantly higher than that in D3 frozen high quality embryo group and D3 frozen non high quality embryo group (P＜0.05). The mitochondrial ROS in D3 frozen high quality embryo group were significantly lower than that in D3 frozen non high quality embryo group (P＜0.05). The mitochondrial ROS of D5 frozen high quality blastocyst group was significantly lower than that in D5 frozen non high quality blastocyst group (P＜0.05).3. The mitochondrial Ca2+ in D3 frozen embryo group were significantly lower than that in D5 frozen blastocyst group (P＜0.05). The mitochondrial Ca2+ in fresh group was significantly higher than that in D3 frozen high quality embryo group and D3 frozen non high quality embryo group (P＜0.05). The mitochondrial Ca2+ in D3 frozen high quality embryo group were significantly lower than that in D3 frozen non high quality embryo group (P＜0.05). The mitochondrial Ca2+ of D5 frozen high quality blastocyst group was significantly lower than that in D5 frozen non high quality blastocyst group (p＜0.05).Conclusion 1. With the continuous development of the embryos, the mitochondrial ATP is gradually increased, the mitochondrial ATP of the better quality embryo is higher and has the better development potential.2. The higher the level of ROS and Ca2+ in embryo has the worse the embryo quality, and even cause the embryo apoptosis.