Improving The Technqiue Of ICSI Used In Mouse And To Study The Relationship Of Deformity Incubation In Vitro And The Risk Of Blastocyst ICM Division

Objective:(1) Improving the technique of ICSI used in mouse eggs, and a significantly higher survival rate was achieved. (2) To study the relationship of deformity incubation in vitro and the risk of blastocyst inner cell mass division with mouse embryo.Methods:(1) To improve the technologies using in mouse oocyte ICSI with sharp injection pipette Firstly we modified the conventional injection by slowing oolemma sucking during oolemma puncture. Secondly, we modified the holding pipette with a trumpet-shape opening, which can prolong the depth of injection pipette inserting during injection. Then we applied it in ICSI of mouse oocyte and compared the oocytes survival, fertilization and embryonic development rates of the traditional group and modified group. (2) By promoting the ovulation in mice, we received large quantities of blastocysts which hatched in different ways and with different outcome of hatching. Divided them into four groups according hatched state:group of normal hatched,group 1 of deformity hatched ("8" form incubation), group 2 of deformity hatched (porous incubation),group of not hatched. To immunostain randomly which selected from all kinds of hatched blastocysts, watch the states of inner cell mass (ICM) signals with the confocal microscope and record the ratio of ICM of normal morphology,dispersion and division. Results:(1) Compared the operation of modified holding needle and sharp injection pipette, we found that enhanced survival rate was obtained when the speed of oolemma sucking was slowed (22% vs.48%, P<0.001).Further, a significantly higher survival rate was achieved when a modified holding pipette was used (82%, P<0.001), with a normal fertilization rate> 70% and varied blastocysts rate (0-86%) in different batches.(2)By observing the deformity incubation in vitro of Mouse blastocysts, we found the Mouse blastocyst is a good research model to watch the division state of ICM. It is also found,in the natural, and mouse blastocysts incubated in vitro has a high incidence of ICM deformity incubation ("8" form incubation 19%, porous incubation 4.6%), however, there was a significantly increased division rate of ICM of deformity incubation blastocyte ("8" form incubation group 8.9%, porous incubation group 14%, normal hatched group 2.5%, P of the former two groups is more little than 0.05 compare to the last group).Conclusion:(1) we inproved the technique of ICSI used in mouse eggs and a significantly higher survival rate was achieved. (2) By observation of deformity incubation in vitro of blastocyst and it is the verification of deformity incubation could induce the division of blastocyte inner cell mass.