###### Abstract  
We applied femtosecond laser pulses for microsurgery of the zona pellucida (ZP) of mouse embryos in terms of assisted reproductive technologies. The embryos were subjected to a series of laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz). Optical microscopy was used to study the dependence of the ZP cut width on the pulse energy \(E\) and velocity \(v\) of the laser beam. It is shown that the same value of the cut width can be obtained for different combinations of these parameters. The boundaries of admissible values were found to be \(E\) = (19-52) nJ, \(v\) = (0.001-0.03) mm/s; recommendations on their proper choice are given. An analytical expression of the cut width of the ZP for a given combination of laser-pulse energy, beam velocity, and pulse repetition rate is proposed. This simple and easy-to-use equation allows for quick prediction of ZP cut width in embryo microsurgery procedures of laser-assisted hatching and embryo laser tagging using femtosecond laser pulses. femtosecond laser pulses; microsurgery; embryo; zona pellucida; laser-assisted hatching; assisted reproductive technologies +
Footnote †: journal: _aciences_  
study by [PERSON] and [PERSON] [9], who constructed isotherms for two types of laser sources: Zylos-tk (0.5 ms, 300 mW) [10] and Fertilase (20 ms, 47 mW) [11]. Laser absorption models and thermal conductivity of the medium [12; 13] were taken into account. The size of the area heated to 60 \({}^{\circ}\)C/80 \({}^{\circ}\)C was shown to be 16 \(\upmu\)m/13 \(\upmu\)m and 37 \(\upmu\)m/15 \(\upmu\)m for Zylos-tk and Fertilase lasers, respectively, much broader than the beam focus diameter. So, the choice was made in favor of a system with a shorter pulse duration. Since then, clinical guidelines for ZP microsurgery of the embryo using millisecond laser dissectors have been developed. The optimal parameters for laser-assisted hatching have been estimated to be 300 mW and 0.5-1 ms to reduce the thermal effect [13]. Local thinning of the ZP or opening formation, performed typically on embryo day three (postfertilization, \(E3\)), are the possible approaches implemented for LAH (see the figures in e.g., Ref. [14]). A comprehensive review of current protocols applied for embryo-assisted hatching can be found elsewhere [15], while a detailed description of all procedures performed in fertility clinics is presented in [16; 17]. Another procedure widely applied in fertility clinics is the trophectoderm (TE) biopsy. It is considered to be a sensitive and reliable tool for preimplantation genetic screening (PGS) to identify aneuploid embryos. The latter have little potential to result in a viable pregnancy and cannot be distinguished from normal embryos using standard morphological evaluations [18]. Some significant results in embryo quality estimations were, however, demonstrated with special image-processing protocols using an artificial neural network [19]. For TE biopsies, the blastocyst is held with the holding pipette, and TE cells are drawn carefully into the biopsy pipette by applying gentle suction. Subsequently, the TE cells are gently pulled away from the blastocyst, while laser pulses are applied. The detachment of the TE cells are normally achieved with less than five laser pulses [20]. Special care should be taken when selecting laser-pulse energy to avoid thermal damage of blastocyst cells and carbonization of the biopsy sample. Femtosecond lasers have been widely applied in developmental biology for several decades; their high potential in the ART has also been recently demonstrated [21]. These lasers represent a promising alternative to millisecond dissectors. The difference in the fundamental processes of laser-matter interaction makes it possible to eliminate the above disadvantages attributed to high pulse duration and temperature concerns.