Laser micromanipulation in the mouse embryo: a novel approach to zona drilling

Abstract

To introduce the use of excimer lasers for penetration of the zona pellucida for micromanipulation purposes. Cryopreserved two-cell mouse embryos were thawed and exposed to the 248-nm line of a krypton fluoride excimer laser (Lambda Physik EMG 202, Goettingen, Germany) creating a 2 to 4-micron opening in the zona pellucida. The Laser Ablation Laboratory at DuPont and the in Vitro Fertilization Laboratory at The Medical Center. The embryos were exposed in either phosphate-buffered solution (PBS) or modified human tubal fluid (HTF) with the laser power varying from 1 to 2 J/cm2 and cultured in Ham's F-10 medium (GIBCO, Grand Island, NY) with 0.4% bovine serum albumin. The outcome of each experiment was measured by blastocyst formation of laser-exposed embryos as compared with a set of unexposed control embryos handled in a similar fashion. Successful laser penetration of the zona pellucida was achieved using the 248-nm line of a krypton fluoride excimer laser. A higher blastocyst formation was found for embryos exposed in PBS. The higher optical absorption of the modified HTF partially inhibited embryo development. The blastocyst statistics increased 2.5-fold times by reducing the exposure of the embryos to ablation by-products. The use of a krypton fluoride excimer laser was introduced as a new method to open the zona pellucida of two-cell mouse embryos without interrupting blastocyst formation.