Study On The First Polar Body Of Human Oocytes With Aneuploidy By Biopsy And Fluorescence In Situ Hybridization

PartⅠStudy on Kunming Mouse First Polar Body BiopsyObjective Utilizing cytochalasins B(CB) pretreatment of KM mouse oocytes to set an animal model of intracytoplasmic sperm injection(ICSI) and first polar body(1PB) biopsy in the mouse.The aim is to achieve theoretical and technical preparation for 1PB biopsy in human oocytes by researching on the effect of the mouse oocyte's fertilization and embryo development after biopsy.Methods Eugamic Kunming white mice were recruited to this experiment.According to whether performing biopsy or not,the mice were divided into biopsy group and control group.In addition,these mice were also allocated to CB group and CB-free group by CB pretreatment.ICSI were manipulated on oocytes that were got after ovarian hypersitimulation of the mice.Laser-assisted microdissection of the zona pellucida were performed in the biopsy group after ICSI,then embryos were cultured to blastocyst stage by sequential media.The control-group of the culture system was same as biopsy group..The rate of fertilization,cleavage,morula and blastocyst formation were observed and compared between the two groups.Besides,The same items in the CB group with CB-free group were contrasted.Results The achievement ratio of laser-assisted biopsy in KM mice 1PB was 90.0%. Without CB pretreatment,the mortality of oocytes was higher.Among the survival,no significant difference was found in the rate of fertilization,cleavage,morula and blastocyst formation between biopsy group and the control group(P＞0.05).In the survived oocytes of CB pretreatment groups,there were also no significant differences between the two groups with the rate of fertilization,cleavage,morula and blastocyst formation(P＞0.05 ).The survival rate was higher after CB pretreatment(P＜0.01 ).Simultaneously,better fertilization rate appeared in the CB group(P＜0.05 ).Conclusions①Establishing the method of 1PB biopsy on KM mouse with CB pretreatment which facilitated the set of the animal model.②Laser-assisted biopsy of 1PB on the mouse is safe and effective.③lPB biopsy has no effect on the fertilization rate and embryo's development in the mouse.PartⅡStudy on the effects about the first polar body biopsy of human oocyteObjective To set an approach of first polar body biopsy on human oocyte and evaluate the effect of the process on the embryo's development. Methods 85 first polar bodies(1PB) were aspirated by laser-assisted microdissection of the zona pellucida after ICSI.Embryos were cultured to blastocyst stage by sequential media.The rate of 2PN formation,cleavage and blastocyst formation of successful biopsied samples were compared with that of the control group consists of 186 oocytes.Homogeneous groups in terms of 1PB morphorlogy were analysed with regard to embryo's development.Results There were 81 oocytes with successful biopsy and the achievement ratio was 95.3%.There were no significant difference between the rate of 2PN formation, cleavage and blastocyst formation(P＞0.05 ),and no significant differences were found in the rate of 2PN formation and cleavage(P＞0.05),but the significant difference appeared in the blastocyst formation with different morphology grade of 1PB(P＜0.05). Different morphological 1PB biopsy didn't affect the formation of the blastocyst by the contraction of homologous 1PB morphological grade in the two groups(P＞0.05 ).Conclusions①Laser -assisted biopsy of 1PB is safe and effective in human oocyte.②The process isn't harmful to the embryo's development.③Biopsy can be carried out in different morphological 1PB without affecting the outcome.PartⅢAneuploidy Diagnosis of Human Oocytes by Fluorescence in Situ Hybridization on First Polar BodyObjective To perform fluorescence in situ hybridization(FISH) on first polar bodies of fresh human oocytes,and analyze the aneuploidy formation of second oocyte.Methods After treatment in the hyposolution,the first polar body were fixed by methanol/acetic acid(Carnoy solution).FISH were stepped by degeneration, hybridization and washed the impurity on the slides.We reviewed the chromosome signals with fluorescence microscope,collected the image and analyzed them. Lymphocyte FISH in meiosis metaphase were designed as the control group.Results Of 74 first polar bodies,69 got fine fixation,the successful rate was 93.2%. Among these fixed polar bodies,54 revealed hybridization signal which could be analyzed.The successful hybridization rate was 78.3%.A chromatid loss was found in 4 polar bodies and a polar body gained extra chromosome.11 polar bodies showed sister chromosome balanced separation.In the control group,successful hybridization rate in lymphocyte was 97.3%.Conclusions①We successfully established the approach of FISH on first polar body for analyzing the aneuploidy formation of human oocytes;②Among aneuploidies from chromosome21,the occurrence of premature unbalanced separation of sister chromatid (PSSC) was higher than non disjunction(ND).Sister chromosome balanced separation was present in fresh oocytes,the mechanism should be researched further.