## Materials and Methods  
### Experimental Setup  
Embreyo microsurgery was performed using the custom-built \"Femtosecond laser scalpel\" system shown in Figure 1. A femtosecond ytterbium laser (TETA, Avesta LLC, Moscow, Russia) generated ultrashort pulses with the following parameters: duration \(\tau=280\) fs, energy \(E=300\)\(\upmu\)J, radiation wavelength \(\lambda=1028\) nm, pulse repetition rate \(f\leq 2.5\) kHz. Femtosecond laser pulses were further frequency-doubled in a DKDP crystal (radiation wavelength \(\lambda=514\) nm). Laser radiation was then directed to the right-side port of an Olympus IX-71 inverted microscope and was focused using a 20\(\times\) UPlanFL microlens (Olympus, Japan) with a numerical aperture \(\text{NA}=0.5\) into a spot \(\sim\)2 \(\upmu\)m in diameter. An attenuation unit and a telescope installed along the beam pathway between the laser and the microscope were used to adjust the energy of the pulses and to match the beam diameter with the microlens aperture. A small portion of the laser beam was reflected from a thin glass plate (Fresnel reflection of about 8%) and directed to a photodiode (DET36A2, Thorlabs Inc., Newton, NJ, USA) to control the energy of the laser pulses. The signal of the photodiode was calibrated through linear regression of its amplitude and the energy of the laser pulses at the output of the microlens registered by laser sensor (S120 VC, Thorlabs Inc., Newton, NJ, USA). A special Petri dish with a glass bottom was used for embryo microsurgery. The dish was mounted on the motorized stage (Marzhauser Wetzlar, Wetzlar, Germany) of the microscope to move the embryo relative to the stationary laser beam. Embryo images were recorded with a CMOS camera (DFK 72 AUC02, The Imaging Source, Bremen, Germany). The image magnification factor \(K=0.1082\)\(\upmu\)m/pixel was preliminarily estimated using a standard Ronchi slide (100 lp/mm, #57-888, Edmund Optics, Barrington, NJ, USA) installed on the microscope stage. Figure 1: Schematic setup of the system for femtosecond laser-assisted ZP microsurgery: 1—femtosecond ytterbium laser; 2—beam attenuator; 3—second-harmonic generator; 4—thin glass plate; 5—photodiode; 6—telescope; 7—electromechanical shutter; 8—dichroic mirrors; 9—microlens; 10—X–Y motorized stage; 11—Petri dish with embryo; 12—condenser lens; 13—microscope lamp; 14—noch filter; 15—tube lens; 16—CMOS camera; 17—inverted microscope. Schematic presentation of glass-bottom Petri dish containing a blastocyst (in a fresh drop of embryo manipulation medium) during the process of laser-assisted ZP cut. To automate the microsurgery procedure, a custom-built software was used. It allowed the operator to visualize the embryo; to control laser parameters such as the energy, the pulse repetition rate, and the laser beam divergence; as well as to set the laser beam's trajectory. To do this, a primitive element (a line or a curve) was drawn right above the live embryo image from the camera to set the form of a cut. Each drawn element was converted to a sequence of commands to the motorized stage. Position of the motorized stage during ZP cutting and synchronous laser shutter opening/closing were controlled automatically. When focusing, the laser beam's diameter decreases along the beam axis, resulting in growth of the laser intensity. The latter reaches its maximum in the beam waist. Assuming a Gaussian shape of femtosecond laser pulse in space and time and a relation for peak power \(P_{0}=2\sqrt{\frac{\ln 2}{\pi}}E/\tau=0.94E/\tau\), pulse peak intensity is determined using equation [26]:  
\[I_{0}=\frac{2\sqrt{\ln 2}E}{\sqrt{\pi\tau\omega_{0}^{2}\tau}}=\frac{0.94E}{\pi \omega_{0}^{2}\tau} \tag{1}\]  
where \(\omega_{0}=1.12\) um is the radius of the focused laser beam at \(1/e\) level, and \(\tau=280\) fs is the pulse duration full width at half maximum. Laser-assisted ZP cutting occurs as soon as the incoming laser intensity in the beam waist exceeds the threshold.