Study On Diagnosis In Preimplantation Human Embryos By Fluorescence In-situ Hybridization

Partâ… Developmental potential of in-vitro fertilized mouse embryos biopsied by laserObjective To investigate developmental potential of in-vitro fertilized mouse embryos biopsied by laser.Methods mouse oocytes obtained from superovulation were fertilized and cultured in the HTF media.Single blastomere from mouse embryos at 4-cell or 8-cell stage was removed after zona was drilling by laser.Then,the further development of the biopsied embryos was observed.Result A total of 360 oocytes were retrieved.Among them,the cleavage rate and blastcyst formation rate were 92.0%and 94.3%,respectively.There was no significant difference in the blastcyst rate(4-cell 76.6%versus78%,8-cell 70.4%versus 80.0%) and in the hatching rate(4-cell 65.6%versus 60.0%,8 -cell 59.2%versus 68.3%) between the biopsy group and the control group.And no significant difference was found in the blastcyst rate and in the hatching rate of the biopsied embryos between the 4-cell group and the 8-cell group.Conclusion Laser-assisted biopsy of embryos at 4-cell or 8-cells stage does not affect their development potential. Partâ…¡An improvement of the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridizationObjective To improve current preimplantation genetic diagnosis fixation techniques in order to find a more efficient fixation method by using polyspermy embryos.Methods At first,to remove the zona pellucida by laser.The Integrity Blastomeres were distributed into two groups:conventional hypotonic- fixationgroup and direct-fixation group.Fluorescence in situ hybridization was accomplished after fixation using chromosome X and Y probes,and then the fixation rate and the fluorescent signals were analyzed.Result1.A total of 30 Blastomeres were analyzed and 171 Blastomeres fixated.Of the 80 Blastomeres in conventional group(A group),68 got fine fixation,the successful rate was 85.0%.Among 91 Blastomeres in direct-fixation group(B group),82 got fine fixation,the successful rate was 90.1%.2.In conventional group,the hybridization rate was 77.5%(/ Blastomere) or 91.2% (/Nuclear).The rate were 83.5%and 92.7%respectively with hypotonic-fixation group and the direct-fixation group.There was no Significant difference.Conclusion:This improved fixation technique essentially eliminates the possibility of losing a cell during fixation and simplifies the process of fixation of blastomere for preimplatation genetic diagnosis. Partâ…¢The exploring on detecting sex chromosome on human embryos by dual color fluorescent in-situ hybridizationObjective To detect sex chromosome on human embryos by dual color fluorescent in-situ hybridization,and to establish the techniques of sex determination of preimplatation human embryos in order that PGD can be used clinically.Methods The Blastomeres were fixated with direct-fixation method after being biopsied by laser-assisted.FISH were stepped by degeneration,hybridization and wash of the impurity on the slides.The chromosome signals were reviewed with fluorescence microscope,and the images were collected and analyzed.Lymphocyte FISH in meiosis metaphase were designed as control group.Result1.Totally we biopsied 40 embryos.The success rate of biopsy is 90%(36/40).2.Of the 90 Blastomeres,83 got fine fixation,the successful rate was 92.2%.3.Among these fixed blastomeres,78 revealed hybridization signal,the successful hybridization rate was 94%.34 among the Biopsied 40 embryos got results which could be analyzed.Including 10 female embryos,14 male embryos and 8 mosaic embryos.4.In control group,successful hybridization rate in lymphocyte was 99%.Conclusions1.human embryos can been Biopsied by Laser-assisted method.2.FISH is an efficient and accurate technique for determination of the sex diagnosis of preimplantation human embryos.Sex chromosome mosaicism will not affect preimplantation gender diagnosis.3.We have established a series of techniques of sex determination of human preimplatation embryos.