From Zygote to Blastocyst: Application of Ultrashort Lasers in the Field of Assisted Reproduction and Developmental Biology

Abstract

Although the use of lasers in medical diagnosis and therapies, as well as in fundamental biomedical research is now almost routine, advanced laser sources and new laser-based methods continue to emerge. Due to the unique ability of ultrashort laser pulses to deposit energy into a microscopic volume in the bulk of a transparent material without disrupting the surrounding tissues, the ultrashort laser-based microsurgery of cells and subcellular components within structurally complex and fragile specimens such as embryos is becoming an important tool in developmental biology and reproductive medicine. In this review, we discuss the mechanisms of ultrashort laser pulse interaction with the matter, advantages of their application for oocyte and preimplantation embryo microsurgery (e.g., for oocyte/blastomere enucleation and embryonic cell fusion), as well as for nonlinear optical microscopy for studying the dynamics of embryonic development and embryo quality assessment. Moreover, we focus on ultrashort laser-based approaches and techniques that are increasingly being applied in the fundamental research and have the potential for successful translation into the IVF (in vitro fertilization) clinics, such as laser-mediated individual embryo labelling and controlled laser-assisted hatching.

Keywords: embryo; embryo biopsy; embryo labeling; femtosecond laser; in vitro fertilization; laser-assisted hatching; microsurgery; nonlinear microscopy; oocyte; ultrashort laser pulses.

Figure 3

Nonlinear microscopy modalities: (a) diagrams (from left to right) for single-photon (SPF) and two-photon excited (TPEF) fluorescence, second harmonic (SHG) and third harmonic (THG) generation, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS); (b) excited volume in case of SPF and TPEF; (c) schematic representation of the excitation and emission frequencies involved in SRS and CARS: in evidence the SRS signal in terms of gain (SRG) on the Stokes pulse or the loss (SRL) on the pump pulse. (a–c) are redrawn from an open-access (CC BY license) source; ref. [77], Authors: Parodi, Jacchetti, Osellame, Cerullo, Polli and Raimondi. Copyright © 2021, Frontiers. (d–g) An example of CARS and TPEF images of fixed mouse egg: (d) DIC image of germinal vesicle mouse egg, (e) false-colored CARS image at wavenumber 2850 cm⁻¹ and (f) TPEF xy image, accompanied by (g) false-colored overlays of germinal vesicle egg stained with BODIPY lipid stain. Scale bar: 10 µm. Fragment (d–g) is adapted with permission from an open-access (CC BY license) source; ref. [86] Copyright © 2021. Published by The Company of Biologists Ltd.

Figure 1

Ionization scheme for laser energy deposition in water.

Figure 2

Laser-based ZP microsurgery for applications in ART includes (a) in vitro fertilization, (b) polar body biopsy, (c) laser-assisted hatching; (d–i) novel techniques based on ZP microsurgery with femtosecond laser pulses (mouse embryos were used in the experiments): (d) individual laser-assisted embryo labeling by creating an alphanumerical code on the ZP’s volume and (g) example of code “07TEX” engraved on the zygote’s ZP (the dashed circles highlight the codes), (e,f) controlled laser-assisted hatching at the prescribed location by ZP microsurgery at the blastocyst stage either close to the TE (e,h) or the ICM (f,i). Scale bar 20 µm.

Figure 2

Laser-based ZP microsurgery for applications in ART includes (a) in vitro fertilization, (b) polar body biopsy, (c) laser-assisted hatching; (d–i) novel techniques based on ZP microsurgery with femtosecond laser pulses (mouse embryos were used in the experiments): (d) individual laser-assisted embryo labeling by creating an alphanumerical code on the ZP’s volume and (g) example of code “07TEX” engraved on the zygote’s ZP (the dashed circles highlight the codes), (e,f) controlled laser-assisted hatching at the prescribed location by ZP microsurgery at the blastocyst stage either close to the TE (e,h) or the ICM (f,i). Scale bar 20 µm.

Figure 4

A series of images with combined SHG and THG signals inside the in vitro cultured mouse oocyte: (a) the SHG signals reveal the spindle fibers (indicated by the yellow arrow) and the zona pellucida (indicated by the white arrow) of the oocyte, while the THG signals reveal the cell membrane, the organelles, and the polar body (indicated by the red arrow). (b,c) are images of the same mouse embryo taken at different depths. The white arrows indicate the two pronuclei. The SHG and THG signals are denoted by green and blue colors, respectively. Adapted with permission from [92] © The Optical Society.