Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development

Spermatid injection into the oocyte cytoplasm has been shown recently to yield viable human embryos developing to term after transfer to the mother. This study provides details of the laboratory techniques related to round spermatid injection (ROSI) and elongated spermatid injection (ELSI) and focuses on some special features of zygote development associated with the use of these types of sperm precursor cells for fertilization. A spermatid-enriched fraction was obtained by centrifugation of cells from azoospermic ejaculates through a discontinuous Percoll gradient column. Individual round or elongated spermatids were identified in this fraction and injected deep into oocytes. Oocyte activation was boosted by a vigorous aspiration of the ooplasm at the time of injection. The fertilization rates after ROSI and ELSI were 45 and 44% respectively. A single large syngamy nucleus was detected in 36% of the zygotes that previously showed two normal-sized pronuclei. This condition did not appear to delay the first cleavage division. These observations underscore the importance of distinguishing the syngamy nucleus of diploid zygotes from the female pronucleus of haploid, parthenogenetically activated eggs.