Making immotile sperm motile using high-frequency ultrasound

Sperm motility is a natural selection with a crucial role in both natural and assisted reproduction. Common methods for increasing sperm motility are by using chemicals that cause embryotoxicity, and the multistep washing requirements of these methods lead to sperm DNA damage. We propose a rapid and noninvasive mechanotherapy approach for increasing the motility of human sperm cells by using ultrasound operating at 800 mW and 40 MHz. Single-cell analysis of sperm cells, facilitated by droplet microfluidics, shows that exposure to ultrasound leads to up to 266% boost to motility parameters of relatively immotile sperm, and as a result, 72% of these immotile sperm are graded as progressive after exposure, with a swimming velocity greater than 5 micrometer per second. These promising results offer a rapid and noninvasive clinical method for improving the motility of sperm cells in the most challenging assisted reproduction cases to replace intracytoplasmic sperm injection (ICSI) with less invasive treatments and to improve assisted reproduction outcomes.

). Statistical significance was determined using unpaired t-test.

Fig. S1 .
Fig. S1.Schematic of sperm motility parameters and sperm VCL pre-and post-exposure (A) Schematic of sperm swimming trajectory along its instantaneous swimming path (continuous yellow line) and the projected average path (green dash-dot line).Circles indicate sperm head position and orange lozenges show points at which instantaneous swimming path crosses the average path.Sperm motility parameters were defined as curvilinear velocity (VCL): sperm head point-to-point velocity in successive frames, average path velocity (VAP): projected sperm head point-to-point velocity in successive frames along its average path, straight line velocity (VSL): the velocity of sperm based on net displacement between the first and last tracking points, amplitude of lateral head displacement (ALH): the average deviation of the sperm head from the average path, beat cross frequency (BCF): the rate at which the sperm head crosses the average path, linearity (LIN): the ratio of VCL to VSL. (B) Comparison of the VCL values of 50 individual sperm cells (from three independent experiments using three biologically independent human samples) before and after exposure to ultrasound.

Fig. S2 .
Fig. S2.Biocompatibility of our ultrasound sperm motility boost platform.(A) DNA fragmentation for exposed (n=621 sperm), and unexposed (control, n=437 sperm) samples, indicating no significant change in DNA integrity (P= 0.62).DNA fragmentation was evaluated using Acridine Orange staining.(B) Sperm viability for exposed (n=343 sperm) and unexposed (control, n=191 sperm) samples, indicating no significant change in viability (P= 0.93) post-exposure.Sperm from two biologically independent human samples in two independent experiments were used.Sperm viability was evaluated using LIVE/DEAD™ Sperm Viability Kit.Values are reported as mean ± s.d.(tableS7).Statistical significance was determined using unpaired t-test.

Fig. S6 .
Fig. S6.Percentage of live immotile sperm cells rendered motile after exposure for each donor.n=33 for donor 1, n=30 for donor 2, and n=48 for donor 3. Values are reported as mean ± s.d.from six independent experiments.Statistical significance was determined using one-way ANOVA with Tukey's multiple-comparison test (ns denotes non-significant).

Fig
Fig. S7.Representative sperm trajectories used for the calculation of VSL.Overlaid images showing representative sperm trajectories for quantifying VSL in (A) a medium and (B) a small size droplet.Scale bar, 20 µm.

Table S1 . Percentage of DNA fragmented and dead sperm in control and ultrasound-exposed groups.
Values are reported as mean ± s.d.

Table S3 . Averaged boost in motility parameters at the population level (representing changes in averaged values) and single-cell level (representing average of changes for each individual sperm) referring to Fig. 2 and fig. S3.
Values calculated at the population level represent the percentage of change in the averaged value of each motility parameter before and after exposure, calculated as ( Mb(i) and Ma(i) are the value of each motility parameter for i th sperm before and after exposure, respectively, and N represents the total number of analyzed sperm cells.Values reported at the single cell level were calculated