Flagellar beating forces of human spermatozoa with different motility behaviors

Abstract

Background: One of the causes of male infertility is associated with altered spermatozoa motility. These sperm features are frequently analyzed by image-based approaches, which, despite allowing the acquisition of crucial parameters to assess sperm motility, they are unable to provide details regarding the flagellar beating forces, which have been neglected until now.

Results: In this work we exploit Fluidic Force Microscopy to investigate and quantify the forces associated with the flagellar beating frequencies of human spermatozoa. The analysis is performed on two groups divided according to the progressive motility of semen samples, as identified by standard clinical protocols. In the first group, 100% of the spermatozoa swim linearly (100% progressive motility), while, in the other, spermatozoa show both linear and circular motility (identified as 80 - 20% progressive motility). Significant differences in flagellar beating forces between spermatozoa from semen sample with different progressive motility are observed. Particularly, linear motile spermatozoa exhibit forces higher than those with a circular movement.

Conclusions: This research can increase our understanding of sperm motility and the role of mechanics in fertilization, which could help us unveil some of the causes of idiopathic male infertility.

Keywords: AFM; Flagellar beating forces; FluidFM; Human spermatozoa; Single-cell motility analysis.

Fig. 1

A sequence of frames extracted from movies acquired for sperm cells having two different motile behaviors: linear trajectory (a) and circular movement (b). Scale bar: 5 μm

Fig. 2

A sequence of frames extracted from a movie of a sperm cell being captured and released by FluidFM probe. When cantilever is far away from the sperm cell no pressure is applied (a), the sperm cell is captured by a negative pressure, which is then reduced for force measurement of the motile sperm cell (b), positive pressure is applied for sperm cell release (c). Scale bar 10 μm

Fig. 3

Representative cantilever oscillations (a, b) and Fast Fourier transform (c, d) of the signal when a spermatozoon with progressive linear motility is linked to the cantilever (a, c) or when one with circular movement (b, c) is trapped

Fig. 4

Inverse Fast Fourier transform of the main frequency ranges of the flagellar beating for a linear spermatozoon (a) and a circular one (b)

Fig. 5

A sketch of the sperm cell trapping regions, as well as representative bright field images of sperm cells trapped by the various cellular regions: tail (a), midpiece (b) head (c), and head (d). Scale bar 5 μm

Fig. 6

Semen samples with progressive motility from 80–20%: values of forces exerted by spermatozoa trapped by the tail (n = 16) and by the head/midpiece (n = 10) (a), and comparison of forces exerted by spermatozoa with linear (n = 26) and circular motility (n = 17) (b). Data are reported in box plots (+) shows the mean, while (-) indicates the median value

Fig. 7

Comparison of the forces exerted by the spermatozoa with linear motility from in semen sample with 100% progressive motility (n = 15) and from semen sample with a progressive motility lower than 80% (n = 16), in both cases, the spermatozoa are trapped by the tail (a). Forces of spermatozoa swimming linearly in samples with 100% progressive motility (n = 15) and spermatozoa with circular movement (n = 17), the circular motile sperm cells are trapped by the head, while the linear motile spermatozoa by the tail (b). Data are reported in box plots (+) shows the mean, while (-) indicates the median value. Significance level *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001