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In this study, we aimed to develop a novel femtosecond laser-based technique for individual embryo tagging. We have demonstrated for the first time the possibility to create individual alphanumeric codes be means of femtosecond laser microsurgery directly on embryo’s outer shell. Femtosecond laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz, pulse energy of 20 nJ) were applied for precise alphanumeric code engraving on the zona pellucida of mouse embryos at the zygote stage. The code consisted of 4-5 characters (numerical and/or alphabetical) typically. Embryo quality assessment was performed every 24 hours post laser-assisted marking by light microscopy and compared with that of non-treated control embryos. The codes engraved could be clearly recognized until the thinning of the zona pellucida prior to hatching. No differences in morphology and developmental rates of laser-treated embryos and control embryos were found. Our results demonstrate the suitability of femtosecond laser as a novel tool for noninvasive embryo tagging, enabling embryo identification from day 0.5 post coitum to at least early blastocyst stage. The technique proposed is relatively fast, reliable and simple, and can be fully automated in the future. It can be performed in a contactless mode under sterile conditions and does not require any additional equipment (except microscope) to visualize the code and to identify the embryo. As far as femtosecond lasers offer several important advantages (such as high precision, minimal invasiveness, versatility etc.) over conventional milli/microsecond lasers we believe that further advances in ultrafast laser technology aimed at reducing complexity, size and high price of femtosecond lasers will help them gain popularity in the field of assisted reproduction.
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