Studying the Safety of Trophectoderm Biopsy Using Visible Femtosecond Laser Pulses

Laser-assisted trophectoderm (TE) biopsy is typically performed with millisecond diode lasers, where thermal effects remain a major safety concern. Femtosecond lasers offer a fundamentally different interaction regime, but their safety profile for TE biopsy has not been evaluated. This study aimed to assess the potential of a femtosecond laser as a tool for TE biopsy in CD1 mouse blastocysts. Biopsies were performed using single pulses at a wavelength of 514 nm with peak intensities of 5.3-16 TW/cm². The number of pulses required to separate TE samples of different isthmus widths was quantified. The study of biological effects included reactive oxygen species (ROS) and heat-shock proteins (HSP70). Laser exposure at 16 TW/cm² enabled an efficient TE biopsy within a clinically relevant number of pulses, without increasing ROS levels and with only limited HSP70 elevation, consistent with an adaptive stress response. A total of 417 embryos were transferred to pseudopregnant females to assess implantation and developmental outcomes. Implantation rates (25.8% vs. 29.6%) and the distribution of developmental outcomes (normal embryos, developmental delay and resorption) did not differ significantly between biopsied and non-exposed blastocysts in the control groups. This supports the further evaluation of femtosecond source as a tool for preimplantation genetic testing procedures.