Laser Immobilization of Sperm for ICSI
Costa-Borges et al., Fertility and Sterility, 2024
Objective
Laser immobilization of spermatozoa has been previously proposed as an alternative to standard mechanical breakage of the tail to streamline the ICSI procedure. However, its use in IVF routine has been limited due to varying laser parameters and the uncertainty on the possible biological consequence of hitting the sperm head. Our study aimed to determine the optimal laser intensity for sperm immobilization and assess its biological impact in mice.
Materials and Methods
A 1.48 micron wavelength diode laser (Navilase,Octax,Vitrolife) was used in all experiments. Mouse B6CBAF1 sperm collected from cauda epididymis were initially used to establish the minimum laser intensity required for permanent immobilization. Laser intensities ranged from 0.3 to 10 mS and shots were applied with accuracy by carefully positioning the sperm head or the middle region of the tail outside a micropipette. Subsequently, sperm were evaluated for permanent immobilization, and then injected into fresh mouse oocytes using a piezo-driven unit. Embryo development was monitored up to the blastocyst stage, followed by vitrification and transfer to pseudo-pregnant recipients. Resulting mice were followed to the next generation. In another set of experiments, human donor semen samples were processed with gradients, and sperm used to validate laser settings using the mouse oocyte activation test (MOAT). A laser intensity of 1.2 mS was tested with either a single or sequential two laser shots applied to the sperm tail. Subsequently, sperm were injected into mouse oocytes and cultured in a time-lapse incubator to assess second polar body extrusion and pronuclei formation. Chi-square test was employed for group comparison.
Results
A linear correlation was observed between laser energy and permanent sperm immobilization. A single laser shot of 1.2 mS (equivalent to 0.2 mJ) near the middle sperm tail permanently immobilized 100% of mouse sperm. This setting showed no impact on blastocyst formation rates (n=183,71%), similar (p=0.116) to mechanically immobilized sperm (n=143,78.9%). Direct head application also immobilized sperm, but significantly compromised blastocyst formation (n=67,11.9%), even at 0.3 mS intensity (n=384,40.4%). However, surviving blastocysts from head-shot sperm (n=90) developed into live pups (48.6%), similar to tail-shot (n=102, 40.4%, p=0.818) or control (n=92,47.8%, p=0.170). All mice showed normal health, behavior, and fertility for two generations. In human sperm, two consecutive 1.2 mS laser shots immobilized 100% of sperm, with oocyte activation efficiency (n=60, 87%) matching controls (n=37, 93.7%).
Conclusions
The careful adjustment of laser settings enables the efficient immobilization of spermatozoa with minimal impact on mouse embryo development. However, direct laser application to the sperm head compromises blastocyst formation, even at low intensities. Further research is needed to assess clinical safety of this approach in human sperm.
Impact Statement
By optimizing intensity parameters and avoiding direct sperm head targeting, laser sperm immobilization holds promise to simplify the ICSI procedure, potentially enhancing its efficiency and success rates.