P-042 Enhancing human sperm motility using high-frequency ultrasound
Authors: Vafaie A , Raveshi M  R , Devendran C , Nosrati R , Neild A 
Published: July 2024

Abstract

Study question
Is high-frequency ultrasound effective in the treatment of low-motility sperm for use in assisted reproduction?

Summary answer
Our study reveals that immotile and poorly motile sperm respond to ultrasound treatment and show up to 266% boost to motility parameters upon ultrasound exposure

What is known already
Sperm motility is central to both natural and assisted reproduction. This crucial importance has led to the utilization of chemical drugs such as pentoxifylline, theophylline, and other phosphodiesterase inhibitors to improve motility. However, these drugs are embryotoxic with a potentially harmful effect on sperm longevity. In a recent study, we showed that sperm cells at the population level showed a 32% boost to motility after 15 seconds of exposure to ultrasound. However, to translate these findings into clinical applications, it is crucial to identify which subset of sperm within a population is most affected by the treatment.

Study design, size, duration
We conducted single-cell study aimed at comprehensively assessing how different sperm motility grades are affected by ultrasound exposure. We utilized a droplet acoustofluidic system using which sperm cells were isolated in droplets and their responses to ultrasound were monitored. A total of 239 individual sperm from three biologically independent human samples were included in this study and parameters such as sperm motility, viability, DNA integrity, and mitochondrial membrane potential (MMP) upon ultrasound exposure were analysed.

Participants/materials, setting, methods
Fresh human semen samples were collected from healthy donors with the approval of the Monash University Human Research Ethics Committee. Three human donors contributed to a total of sixteen independent experiments performed in this work. The manual tracking plugin in ImageJ was used to measure sperm motility. The LIVE/DEAD™ Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability and JC-1 (Invitrogen, USA) was used to monitor the MMP.

Main results and the role of chance
We classified the sperm cells based on their pre-exposure straight-line velocity (VSL) into three categories of grade C (non-progressive, VSL &lt; 5 µm s-1), grade B (slow progressive, 5 ≤ VSL&lt; 25 µm s-1), and grade A (rapid progressive, 25 µm s-1 ≤ VSL) cells. Our single-cell analysis results show that 20 seconds of ultrasound exposure boosts the motility of grade C sperm up to 266% and as a result, 90% of total sperm are graded as progressively motile after exposure. Through ANOVA tests, we showed that non-progressive sperm cells show a boost to all of the motility parameters more than the other grades. In addition, ultrasound exposure renders 34% of live immotile sperm motile with 24% of the cells graded as progressive and the remaining 10% showed a twitching motility after exposure. We also showed that ultrasound exposure induces flagellum deformation to 16% of total live immotile sperm cells, providing opportunities to detect live sperm in testicular samples using this approach. Finally, we explored the non-invasiveness of the ultrasound platform and showed that there is no detrimental effect on sperm DNA integrity and viability post exposure.

Limitations, reasons for caution
A larger sample size could provide a higher statistical confidence to our study. Treatment of patient samples (testicular or asthenozoospermic) and evaluation of embryo safety must be included in future studies to evaluate the effectiveness of this method for clinical use.

Wider implications of the findings
Our acoustic mechanotherapy method could enhance sperm motility to potentially benefit assisted reproduction outcomes. It also enables new opportunities for detecting live sperm in testicular samples.

Trial registration number
not applicable