Effect Of Laser-assisted Hatching Technique On DNA Damage Of Embryo

Objective: To assase the effect of laser assisted hatching on DNA double-strand breaks of mouse embryo.Methods:The mouse embryo were collected from 2cells ? 8cells and blastocyst stage.All of samples were fixed with 4% paraformaldehyde.Immunofluorescence technique,Phospho-Histone H2 A.X(Ser139)Rabbit m Ab as primary antibody ?Cy3-labled Goat Anti-Rabbit Ig G as secondary antibody and H2 A.X foci as marker,was applied to observe the the phenomenon of various stages of DNA damage in the process of normal development of mouse embryo.Subsequently,the mouse embryo were collected in each group of 20 or so,after laser-assisted hatching0.5h,1h,24 h,respectively.Same technique was utilized to observe the occurrence of DNA double-strand breaks.The 8 cells mouse embryo were collected and divided into two groups: the assisted hatching group and non-assisted hatching group.After two groups were cultured on 2 days,all of these embryo were stained with Hoechst 33258 to compare the blastocyst formation rate and blastocyst rate,the number of blastocyst also counted.Results: Compared with 2cells group,8 cells group and blastocyst group had significant difference(P<0.05)in the number of H2 AX.foci.There was an increasing trend of mouse embryo of the number of H2 AX.foci after assisted hatcing from 0.5 hour to 24 hours.Compared with the non-assisted hatching group,significance difference in the rate of hatching(P<0.05).No significance difference in the rate of blastocyst formation and the number of blastocysts between assisted hatching group and non-assisted hatching group.Conclusion:DNA double strand breaks occurred in the process of normal development of murine embryo.Laser assisted hatching increased DSBs of mouse embryo,and the technique contributed to the hatching of embryo.Objective: To assase the effect of laser assisted hatching on DNA double-strand breaks of human tripronuclear embryo.Methods: Collected the tripronuclear embryo from abnormal fertilazation zygotes,tripronuclear embryo were treated with laser assisted hatching,15?m inner layer and 50?m outer layer,respectively.Subsequently,embryos were cultured in incubator(37?,5% CO2)for 0.5h and 1h,respectively.At the same time,negative control and positive control were setted.All of samples were treated with 4% paraformaldehyde.Immunofluorescence technique was applied like first part to observe the the DNA double-strand damage breaks.H2 A.X foci was used as biomarker.Results:no significance difference between assisted hatching 15?m inner layer and assisted hatching 50 ?m outer layer after incubation 0.5 hour or 1hour.Conclusion:Laser assisted hatching did not increased DSBs of tripronuclear embryo.Objective:To assase the effect of different laser assisted hatching method on human embryo.Methods:A total of 121 patients with Frozen-thawed embryo transfer were selected and divided into two groups,assisted hatching 15 ?m inner layer group and assisted hatching 50 ?m outer layer group,were studied.The study indexes were as follows:implantation rate,pregnancy rate,miscarriage rate,live birth rate.The effects of two methods of laser assisted hatching on the outcome of FET were compared.Admission Criteria:Subject has experienced failure of fresh cycle embryo transfer or FET.Embryos were selected with frozen-thaw of high quality(ie,the number of cells is 6 or more,fragments below 20%,grade ?and above)exclusion criteria:Subject has demonstrated with endometrial <6mm or endometrail adhesions and other injuries,uterine malformations such as double horn uterus,uterine mediastinum,endometritis or adenomyosis,hydrosalpinx,chromosomal abnormalities and so on.Results: no significant difference between the assisted hatching 15 ?m inner layer group and assisted hatching 50 ?m outer layer in the implantation rate,pregnancy rate,miscarriage rate and live birth rate.Conclusion: no significance difference between assisted hatching 15?m inner layer and assisted hatching 50 ?m outer laye was found.