Effect of Laser Optoperforation of the Zona Pellucida on Mouse Embryo Development in vitro

Laser operations on cells and embryos are an important field of current photobiology and biophotonics. The high power density of tightly focused laser irradiation provides an efficient impact on matter of cells or embryos. Precise focusing of the laser spot allows strictly controlled perforation of the membrane. The present work was devoted to studying the influence of optoperforation of mammalian embryonic zona pellucida with a tightly focused laser beam with 1.48-µm wavelength on further development of the embryo. Such a laser operation was proposed for application in in vitro fertilization (IVF) practice and intracytoplasmic sperm injection into the oocyte (ICSI). For cultured in vitro oocytes and embryos, the process of natural exiting from the zona pellucida ("hatching") is often impaired, which decreases probability of implantation and pregnancy The goals of the present work were to determine the influence of different manipulations on development of embryos in vitro until blastocyst formation and on the ISSN 0006-2979, Biochemistry (Moscow), 2015, Vol. 80, No. 6, pp. 769-775. © Pleiades Publishing, Ltd., 2015. Original Russian Text © E. O. Zakharchenko, A. D. Zalessky, A. A. Osychenko, A. S. Krivokharchenko, A. K. Shakhbazyan, A. V. Ryabova, V. A. Nadtochenko, 2015, published in Biokhimiya, 2015 769 * To whom correspondence should be addressed. Abstract-The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida. Effect of Laser Optoperforation of the Zon