Successful Microsurgical Removal of a Pronucleus from Tripronuclear Human Zygotes

We have microsurgically extracted a pronucleus from 11 tripronuclear human zygotes under two different experimental conditions. One group of six zygotes was incubated in cytochalasin D for 30 minutes before manipulation. The remaining five zygotes were manipulated without preincubation in cytoskeletal relaxing agents. When cytoskeletal relaxants were used, all six embryos survived manipulation and cleaved. One embryo arrested spontaneously at the two-cell stage; all others cleaved to at least four cells, and one embryo reached the advanced morula stage. Of the five eggs manipulated without prior cytochalasin D and colcemid treatment, none survived for more than 30 minutes after manipulation. In none of the tripronuclear eggs was it possible to definitely identify the male pronuclei. Because development was interrupted for karyotyping, it is likely that these embryos would have otherwise continued to develop to more advanced stages. These results indicate that it is possible to microsurgically "epronucleate" tripronuclear human zygotes and obtain further development and that use of cytoskeletal relaxants facilities this manipulative procedure. However, because epronucleated embryos have significant developmental potential, chromosome studies are needed to confirm complete removal of a male pronucleus before such techniques can be applied clinically.