Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations

Abstract

The zonae pellucidae of mouse oocytes were photoablated by an ultraviolet laser (337.1 nm) to create one or two 10 μm holes. The pulse energy used was ~ 3 μJ/s, with a frequency of 10 pulses/s. These laser zona-drilled (LZD) oocytes with one hole (LZD1) or two holes (LZD2) and the zona-intact controls were inseminated with spermatozoa at standard concentrations of 5 x 104, 5 x 105, 1 x 106 and 2 x 106/ml. Fertilization was significantly improved at all sperm concentrations in LZD1 and LZD2 oocytes as compared to the controls. However, there was no significant difference in fertilization rates between LZD1 and LZD2, LZD2 produced significantly higher numbers of blastocysts at day 5. Hatching was significantly enhanced in the presence of either one or two holes in the zona. Polyploidy was generally absent, except in LZD2 oocytes (1%) inseminated at higher sperm concentrations. Differential cell counts of expanded LZD blastocysts were similar to those of the controls. Significantly fewer LZD2 blastocysts implanted and produced viable fetuses than LZD1 and control blastocysts. Morphological abnormalities of the fetuses were absent in all three groups. Laser zona-drilling using the ultraviolet laser was shown to be fast, efficient and safe.