Rapid sperm capture: High-throughput flagellar waveform analysis

Abstract

The FAST software package and all documentation can be downloaded from www.flagellarCapture.com. Summary 1 Flagella are critical across all eukaryotic life, and the human sperm 2 flagellum is crucial to natural fertility. Existing automated sperm di-3 agnostics (CASA) rely on tracking the sperm head and extrapolating 4 measures. We describe fully-automated tracking and analysis of flag-5 ellar movement for large cell numbers. The analysis is demonstrated 6 on freely-motile cells in low and high viscosity fluids, and validated on 7 published data of tethered cells undergoing pharmacological hyperac-8 tivation. Direct analysis of the flagellar beat reveals that the CASA 9 measure 'beat cross frequency', does not measure beat frequency. A 10 new measurement, track centroid speed, is validated as an accurate 11 differentiator of progressive motility. Coupled with fluid mechanics 12 codes, waveform data enables extraction of experimentally intractable 13 quantities such as energy dissipation, disturbance of the surrounding 14 medium and viscous stresses. We provide a powerful and accessible 15 research tool, enabling connection of the cell's mechanical activity to 16 its motility and effect on its environment. 17 Introduction 18About 100 million men worldwide are suggested to be subfertile (Inhorn and 19 Patrizio, 2015), but for many of them an accurate diagnostic cause remains 20 elusive. The ability of sperm to successfully migrate through the female re-21 productive tract is key to natural fertilisation, however current techniques for 22 assessing sperm motility lack diagnostic power and mechanistic insight. The 23 heterogeneity of human sperm requires tools that can acquire and analyse 24 2 (2018) and associated special issue). Such systems are widely used for vet-25 erinary work and in domestic animal breeding, conservation, and toxicology 26 (Amann and Waberski, 2014), but have not yet made the breakthrough into 27 routine clinical usage. The reasons for this have been discussed elsewhere 28 (Gallagher et al., 2018b). 29 Standard CASA systems produce a motility assessment from the track of 30 the head movement, however this does not enable causative or mechanistic 31 insight because it lacks detail on the movement of the flagellum. Knowledge 32 of this beat could provide hitherto untapped information for the estimation 33 of non-visible attributes such as the contribution of different metabolic path-34 ways, and modulation in response to the physical and biochemical environ-35 ments (Ooi et al., 2014). The capability to capture flagellar movements and 36 associated mechanistic insights has broader applicability in the life sciences 37 including the role of cilia in embryonic development (Smith et al., 2019), 38 swimming of multiflagellate microorganisms (Wan and Goldstein, 2018), the 39 use of high-speed holographic microscopy to image the flagellar waveforms 40 of malaria parasites (Wilson et al., 2013), and even the design of hybrid 41 bio-robots for biomedical applications (Xu et al., 2017).42 The use of comp...

Doing more with less: The flagellar end piece enhances the propulsive effectiveness of human spermatozoa

“…Furthermore, because the end piece has been largely considered to not have a role in propelling the cell, it has received relatively little attention. However, analysis of experimental data [15] indicates that the sperm flagellar waveform has a significant impact on propulsive effectiveness, and moreover changes to the waveform have an important role in enabling cells to penetrate the highly viscous cervical mucus encountered in internal fertilization [16]. This leads us to ask: does the presence of a mechanically inactive region at the end of the flagellum help or hinder the cell's progressive motion?…”

The human sperm beats anisotropically and asymmetrically in 3D

“…1(c,d). The rolling-ofasymmetric-bend mechanism induces a sperm kinematic illusion in which the flagellar beating appears to be symmetric for cells swimming in a straight trajectory if observed with traditional 2D microscopy [24,25,27,28,58], rendering this intrinsic flagellar asymmetry invisible thus far. Similar rolling-of-asymmetric-bend mechanism have also been reported for hamster spermatozoa in which the sperm flagellum induces a rapid side-to-side 180 • degrees switch, rather than the gradual 360 • rolling for human sperm, to achieve straight-line symmetric swimming despite the asymmetric bending [3].…”

“…This is further augmented by external sources of asymmetry, such as the hydrodynamic influence of solid surfaces during for sperm boundary accumulation near to the coverslip [21][22][23]. This raises a fundamental question: given this multilayered hierarchy of distinct asymmetries that are intrinsically present, how does the human sperm flagellum achieve symmetric beating and straight-line swimming trajectories typically observed under the microscope [10,[24][25][26][27][28]?…”

“…Indeed, our understanding of the human sperm flagellum beating is centred on the dogma of symmetric beating [29][30][31]. This have been postulated based on the wealth of observations thus far showing straightline swimming trajectories for sperm beating symmetrically, as well as symmetric travelling waves of curvature of the waveform [10,24,[26][27][28]32]. As such, beating symmetry is the cornerstone of any comparative study on flagellar undulation [24,29,32], especially so while assessing symmetry-breaking events, such as in sperm capacitation, hyperactivation and buckling phenomena [18,19,[32][33][34].…”

“…How to even reconcile symmetric swimming observations with the umbrella of asymmetries that are present at the molecular level in the human spermatozoa, is still open to debate. This scenario is aggravated by the fact that the literature to date has been predominately limited to planar projections of the flagella beat [10,[24][25][26][27][28][29][30][31], albeit the human sperm flagellum beats intrinsically in threedimensions. Despite numerous efforts to capture the flagellar beat in 3D [34,[40][41][42][43][44][45][46][47][48][49][50][51][52], the importance of threedimensional component of the beat is yet to be fully recognised at the human fertility realm [53].…”