SUMMARY ANSWER: High-throughput flagellar waveform tracking and analysis enable measurement of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses, which are not possible by tracking the sperm head alone.
WHAT IS KNOWN ALREADY: The clinical gold standard for sperm motility analysis comprises a manual analysis by a trained professional, with existing automated sperm diagnostics [computer-aided sperm analysis (CASA)] relying on tracking the sperm head and extrapolating measures. It is not currently possible with either of these approaches to track the sperm flagellar waveform for large numbers of cells in order to unlock the potential wealth of information enclosed within.
STUDY DESIGN, SIZE, DURATION: The software tool in this manuscript has been developed to enable high-throughput, repeatable, accurate and verifiable analysis of the sperm flagellar beat.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the software tool [Flagellar Analysis and Sperm Tracking (FAST)] described in this manuscript, we have analysed 176 experimental microscopy videos and have tracked the head and flagellum of 205 progressive cells in diluted semen (DSM), 119 progressive cells in a high-viscosity medium (HVM) and 42 stuck cells in a low-viscosity medium. Unscreened donors were recruited at Birmingham Women's and Children's NHS Foundation Trust after giving informed consent.
MAIN RESULTS AND THE ROLE OF CHANCE: We describe fully automated tracking and analysis of flagellar movement for large cell numbers. The analysis is demonstrated on freely motile cells in low- and high-viscosity fluids and validated on published data of tethered cells undergoing pharmacological hyperactivation. Direct analysis of the flagellar beat reveals that the CASA measure 'beat cross frequency' does not measure beat frequency; attempting to fit a straight line between the two measures gives ${\mathrm{R}}^2$ values of 0.042 and 0.00054 for cells in DSM and HVM, respectively. A new measurement, track centroid speed, is validated as an accurate differentiator of progressive motility. Coupled with fluid mechanics codes, waveform data enable extraction of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses. We provide a powerful and accessible research tool, enabling connection of the mechanical activity of the sperm to its motility and effect on its environment.
LARGE SCALE DATA: The FAST software package and all documentation can be downloaded from www.flagellarCapture.com.
LIMITATIONS, REASONS FOR CAUTION: The FAST software package has only been tested for use with negative phase contrast microscopy. Other imaging modalities, with bright cells on a dark background, have not been tested but may work. FAST is not designed to analyse raw semen; it is specifically for precise analysis of flagellar kinematics, as that is the promising area for computer use. Flagellar capture will always require that cells are at a dilution where their paths do not frequently cross.
WIDER IMPLICATIONS OF THE FINDINGS: Combining tracked flagella with mathematical modelling has the potential to reveal new mechanistic insight. By providing the capability as a free-to-use software package, we hope that this ability to accurately quantify the flagellar waveform in large populations of motile cells will enable an abundant array of diagnostic, toxicological and therapeutic possibilities, as well as creating new opportunities for assessing and treating male subfertility.
STUDY FUNDING/COMPETING INTEREST(S): M.T.G., G.C., J.C.K-B. and D.J.S. gratefully acknowledge funding from the Engineering and Physical Sciences Research Council, Healthcare Technologies Challenge Award (Rapid Sperm Capture EP/N021096/1). J.C.K-B. is funded by a National Institute of Health Research (NIHR) and Health Education England, Senior Clinical Lectureship Grant: The role of the human sperm in healthy live birth (NIHRDH-HCS SCL-2014-05-001). This article presents independent research funded in part by the NIHR and Health Education England. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The data for experimental set (2) were funded through a Wellcome Trust-University of Birmingham Value in People Fellowship Bridging Award (E.H.O.).The authors declare no competing interests.
Methods: This was a cross-sectional study conducted among highly-trained male athletes. Only participants who showed normal knee valgus during a drop landing screening test were recruited. Twelve junior athletes performed single leg squats while maintaining a knee flexion angle of 60°. The squats were executed in three foot positions: neutral (0°), adduction (-10°), and abduction (+10°). Three-dimensional motion analysis was used to capture the lower extremity kinematics of the participants' preferred limb. The hip and knee kinematics in the sagittal, frontal, and transverse planes during squatting were compared across the three foot positions using one-way ANOVA.
Results: The participants showed a normal range of dynamic knee valgus (5.3°±1.6). No statistically significant differences were observed in hip flexion (p = 0.322), adduction (p = 0.834), or internal rotation (p = 0.967) across different foot positions. Similarly, no statistically significant differences were observed in knee flexion (p = 0.489), adduction (p = 0.822), or internal rotation (p = 0.971) across different foot positions.
Conclusion: Small changes in transverse plane foot position do not affect lower extremity kinematics during single leg squat in highly trained adolescent males with normal dynamic knee valgus. Our findings may provide guidance on safer techniques for landing, pivoting, and cutting during training and game situations.
OBJECTIVES:: The aim was to investigate their dynamic characteristics and create a relationship between these dynamic data and the prescription of foot.
STUDY DESIGN:: Experimental Assessment.
METHODS:: This article presents the modal analysis results of the full range of Össur Flex-Run™ running feet that are commercially available (1LO-9LO) using experimental modal analysis technique under a constant mass at 53 kg and boundary condition.
RESULTS:: It was shown that both the undamped natural frequency and stiffness increase linearly from the lowest to the highest stiffness category of foot which allows for a more informed prescription of foot when tuning to a matched natural frequency. The low damping characteristics determined experimentally that ranged between 1.5% and 2.0% indicates that the feet require less input energy to maintain the steady-state cyclic motion before take-off from the ground. An analysis of the mode shapes also showed a unique design feature of these feet that is hypothesised to enhance their performance.
CONCLUSION:: A better understanding of dynamic characteristics of the feet can help tune the feet to the user's requirements in promoting a better gait performance.
CLINICAL RELEVANCE: The dynamic data determined from this study are needed to better inform the amputees in predicting the natural frequency of the foot prescribed. The amputees can intuitively tune the cyclic body rhythm during walking or running to match with the natural frequency. This could eventually promote a better gait performance.