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Study on Diagnosis in Preimplantation Human Embryos by Fluorescence In-situ Hybridization

Author: ZhangMinMin
Tutor: CaoYunXia
School: Anhui Medical University,
Course: Obstetrics and Gynaecology
Keywords: Mice Fertilization In vitro Embryo biopsy Laser method Development Fixed Preimplantation genetic diagnosis Fluorescence in situ hybridization Blastomeres Sex chromosomes Chimeric
CLC: R714.8
Type: Master's thesis
Year: 2008
Downloads: 59
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Abstract

In the first part of the laser method biopsy mice IVF embryos late development purposes to Kunming (KM) mice in vitro fertilization (IVF) embryos for research to try to apply the the laser method biopsy mouse embryo, and to explore the impact of biopsy technique for early embryonic development . Method of mouse oocyte ovulation for in vitro fertilization and culture, and then using the laser method biopsy collected 4 cell and 8 cell embryos, and finally to observe the development of embryos after biopsy. 360 1. Shared the oocytes, fertilization rate and cleavage rate of 92.0% and 94.3%, respectively. Blastocyst formation rate of the 2.4-cell embryo biopsy group and hatched blastocyst rates were 76.6% and 65.6%, blastocyst formation rate of 8 - cell embryo biopsy group and hatched blastocyst rate of 70.4% and 59.3%, respectively. compared to the control group with no biopsy, the difference was not significant (P> 0.05); 4 - cells in the biopsy group and 8 - cell biopsy group blastocyst formation rate and hatching blastocyst rate compared, no significant difference ( P> 0.05). Single-blastomere biopsy the conclusions laser mice 4 cell or 8-cell embryos, does not affect the late development of the embryo, is an effective embryo blastomere biopsy method. The second part of the human embryonic single blastomere fixed improved method for the purpose of human polyspermy embryonic blastomeres improved the traditional the hypotonic fixed method, to explore a more simple and efficient method of fixing. Methods to collect 30 human the polyspermy embryos laser drilling to remove the zona pellucida, will receive a complete blastomere a traditional hypotonic fixed law and direct fixation fixed underwent X and Y chromosome fluorescence in situ hybridization, comparative fixed-rate and hybrid rate. Results 1. Fixed blastomeres of 171. Hypotonic fixation (A group) fixed 80 success 68 success rate was 85.0%. Direct fixation (B) fixed set of 91, the success of 82, the success rate was 90.1%. Both groups had a significant difference. 2.A group hybridization was 77.5% (/ blastomere), 91.2% (/ auditing), group B hybridization rate 83.5% 92.7% (/ number of blastomeres) (/ auditing) . There was no significant difference. Conclusions directly fixed to simplify the fixed steps to reduce cell loss rate for the the ideal single blastomere interval fixed nuclear technology. The third part of the research purposes of human embryos for gender diagnosis by FISH dual-color fluorescence in situ hybridization analysis of the pre-implantation embryo sex chromosome, carried out to establish gender diagnostic technology for implantation genetic diagnosis before clinical. Laser biopsy method blastomere biopsy method with fixed, fixed directly and the gender diagnosis then fluorescence in situ hybridization. FISH step comprises the denaturation, hybridization, elution and fluorescence microscopy results were analyzed. Using lymphocytes as a comparative analysis. 1 on 40 human cleavage stage embryos, a biopsy, successful biopsy of 36, and the destruction of embryos 4 biopsy success rate was 90% (36/40). 90 blastomeres fixed, of which 83 were successfully fixed, fixed success rate of 92.2%. 3 fixed successful the 83 blastomere cell line two-color FISH gender identification, 78 cells to see the fluorescent signal, hybridization was 94.0%. 34 40 embryo biopsy can be analyzed FISH results, 85% of the total biopsy embryos (34/40), accounting for 94.4% of the successful biopsy embryos (34/36). 34 FISH results of early human embryos, including 10 normal female embryos, 14 normal male embryos, eight sex chromosome chimeric embryos. 4 Total number of nuclei or interim phase the nucleus 200 198 nucleus see the hybridization signal. Conclusion 1 laser method can be used for human embryo biopsy. Fluorescence in situ hybridization can be fast and efficient 'human preimplantation embryo sex diagnosis does not affect the accuracy in the diagnosis of gender sex chromosome mosaicism. 3 FISH technology platform to establish the diagnosis of human preimplantation embryos single blastomere gender.

Study on Diagnosis in Preimplantation Human Embryos by Fluorescence In-situ Hybridization

Abstract

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