The flagellar wave originates from the 9+2 axoneme structure at the central core of the flagellum, where nine pairs of outer microtubule doublets are mechanically linked to a central pair. [2] The sequential sliding of the nine outer microtubules via dynein arms over the neighboring doublet bends the flagellum in 3D to produce a flagellar waveform. [3] This beating behavior is self-regulatory in nature, triggered by the combined activity of dynein motors. [3a,4] Several regulation mechanisms have been suggested to control the flagellar waveform in space and time including the local curvature-controlled dynein motor activity, [4,5] selective activation/deactivation of sliding microtubules due to transverse forces, [6] and regulation of dynein motor activity because of the sliding forces. [7] The resulting motion is 3D in nature, with the flagellar wave starting from the midpiece and traveling along a conical helix toward the end of the flagellum, causing the whole sperm body to counter-rotate. [8] Consequently, the flagellar waveform and sperm swimming path display helical, [5a,8a,c,9] twisted-planar, [8e] Sperm swim through the female reproductive tract by propagating a 3D flagellar wave that is self-regulatory in nature and driven by dynein motors. Traditional microscopy methods fail to capture the full dynamics of sperm flagellar activity as they only image and analyze sperm motility in 2D. Here, an automated platform to analyze sperm swimming behavior in 3D by using thinlens approximation and high-speed dark field microscopy to reconstruct the flagellar waveform in 3D is presented. It is found that head-tethered mouse sperm exhibit a rolling beating behavior in 3D with the beating frequency of 6.2 Hz using spectral analysis. The flagellar waveform bends in 3D, particularly in the distal regions, but is only weakly nonplanar and ambidextrous in nature, with the local helicity along the flagellum fluctuating between clockwise and counterclockwise handedness. These findings suggest a nonpersistent flagellar helicity. This method provides new opportunities for the accurate measurement of the full motion of eukaryotic flagella and cilia which is essential for a biophysical understanding of their activation by dynein motors.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smtd.202101089.
sperm injection (ICSI), [3] in vitro fertilization (IVF), [4] and intrauterine insemination (IUI). [5] In ICSI, as the most invasive method, an individual sperm is selected and injected directly into an egg. In IVF or IUI, a subpopulation of pre-selected sperm is introduced close to the egg to achieve fertilization. [3][4][5] The quality of selected sperm is crucial to ART, contributing significantly to the treatment success rate, live-birth rate, and offspring health. [6] However, the current clinical sperm selection practices are highly manual, subjective, and prone to operator errors, resulting in a suboptimal ART success rate, stagnating at ≈33% per cycle over the past 30 years. [7,8] The inherent challenge is to mimic the highly parallelized and 3D selection process in vivo that enables a single-cell level sorting mechanism within a relatively short timeframe. [9] Specifically, the 3D folded structure of the female reproductive tract breaks down the initial semen volume (≈1-4 mL) into a few microliters per selection event within the folded epithelial of the fallopian tube. These subfractioning events can process hundreds of millions of sperm in just a few hours. [10] Current clinical practices, however, only provide a 2D bulk scale selection alternative.Density gradient centrifugation (DGC) [11] and swim-up [12,13] are the most commonly used methods for human sperm selection in fertility clinics. In swim-up assay (SU), highly motile sperm are selected based on their ability to swim upward from the raw semen sediment into a fresh sperm media, where they are collected after ≈60 min. This method is not applicable in the case of asthenozoospermia (poor sperm motility). [14] In DGC, human sperm are separated based on their density by being forced to cross a viscosity gradient via centrifugation force that could cause sperm DNA damage and alter sperm morphology and function. [11,15] The sperm preparation method that is used in clinics is mainly chosen based on the count and motility of sperm in the human semen sample. [9] Both SU and DGC are time consuming (30-60 min), require multiple preparation steps, depend on the clinician expertise, and differ significantly from the natural selection process in vivo. [16] Moreover, the relatively long processing time of these methods and the low percentage of recovered sperm in the swim-up method (less than 20%) have considerably restricted the clinical workflow, especially Selection of high-quality sperm is crucial to assisted reproduction. However, conventional clinical methods for sperm selection are manual and prone to operator errors. This article presents 'sperm syringe', a scalable technology that mimics the highly parallelized 3D selection process in vivo via a 3D network of 560 microchannels to select high-quality sperm. Sperm syringe retrieves more than 41% of healthy sperm from the initial sperm sample in under 15 min, providing a sufficient volume (≈500 µL) and number (1,600,000) of high-quality sperm for fertility treatments. Experiments with bull and human sper...
Sperm selection is an essential component of all assisted reproductive treatments (ARTs) and is by far the most neglected step in the ART workflow in regard to technological innovation. Conventional sperm selection methodologies typically produce a higher total number of sperm with variable motilities, morphologies, and levels of DNA integrity. Gold-standard techniques, including density gradient centrifugation (DGC) and swim-up (SU), have been shown to induce DNA fragmentation through introducing reactive oxygen species (ROS) during centrifugation. Here, we demonstrate a 3D printed, biologically inspired microfluidic sperm selection device (MSSP) that utilizes multiple methods to simulate a sperms journey toward selection. Sperm are first selected based on their motility and boundary-following behavior and then on their expression of apoptotic markers, yielding over 68% more motile sperm than that of previously reported methods with a lower incidence of DNA fragmentation and apoptosis. Sperm from the MSSP also demonstrated higher motile sperm recovery after cryopreservation than that of SU or neat semen. Experiments were conducted side-by-side against conventional SU methods using human semen (n = 33) and showed over an 85% improvement in DNA integrity with an average 90% reduction in sperm apoptosis. These results that the platform is easy-to-use for sperm selection and mimics the biological function of the female reproductive tract during conception.
Sperm Selection is an essential component of all Assisted Reproductive Treatments (ART), and is by far and large the most neglected step in the ART work ow when it comes to technological innovation. Conventional sperm selection methodologies typically produce a higher total number of sperm with variable motilities, morphologies and levels of DNA integrity; Gold-standard techniques Density Gradient Centrifugation (DGC) and Swim Up (SU) have been proven to induce DNA fragmentation through the introduction of reactive oxygen species (ROS) during centrifugation. Here, we demonstrate a 3D printed, biologically inspired micro uidic sperm selection device (MSSP) that utilizes multiple methods to simulate a sperms journey towards selection. Sperm are rst selected based on their motility and boundary following behavior, then on their expression of apoptotic markers, yielding over 68% more motile sperm than previously reported methods within a lower incidence of DNA fragmentation and apoptosis. Sperm from the MSSP also demonstrated higher motile sperm recovery after cryopreservation than SU or neat semen. Experiments were conducted side-by-side against conventional SU methods using human semen (n = 33) and showed over an 85% improvement in DNA integrity with an average 90% reduction in sperm apoptosis. These results demonstrate an easy-to-use platform for sperm selection mimicking the biological function of the female reproductive tract during conception. CapsuleMicro uidic sperm selection is capable of e ciently and consistently preparing sperm, resulting in signi cantly lower DNA fragmentation, lower apoptosis, better cryosurvival, and higher-grade motility compared with the SU method.
Study question How does viscosity influence the flagellar beating behaviour of free-swimming bull, mouse, and human sperm? Summary answer Sperm flagellar beating behaviour exhibits a transition from an irregular three-dimensional (3D) beating at 5 mPa·s to an organized two-dimensional (2D) waveform at 20 mPa·s. What is known already Sperm migrate in a complex viscoelastic environment through the female reproductive tract. The viscoelastic properties of the oviductal fluid significantly influence the progressive motility of sperm, acting as one of the key guidance mechanisms in vivo. However, the biomechanics of sperm flagellar activity in response to varying viscosity of the oviductal fluid is poorly understood. Understanding sperm flagellar behaviour in physiologically relevant environments is crucial to understanding reproduction and may help to describe unknown causes of infertility. Lack of high-speed high-resolution imaging techniques and automated image-analysis capabilities have been the main barriers to fully describe the flagellar beating behaviour. Study design, size, duration We used a custom-built high-speed high-resolution dark-field microscopy platform to resolve the flagellar dynamics of human, bull, and mouse sperm near surfaces in viscoelastic media ranging in viscosity from 1 to 250 mPa·s. The imaging system includes an automated image analysis algorithm to quantify sperm flagellar waveform and motility characteristics by extracting the flagellar centreline, reconstructing the waveform and calculating tangent-angle profiles. 20 sperm from 3 different bull, mice and humans were analyzed. Participants/materials, setting, methods Bull, mouse, and human sperm were used in this study. In each experiment, a diluted sperm sample in a buffer supplemented with methylcellulose was used and free-swimming sperm were imaged using dark-field microscopy at 200 frames per second. An automated image analysis algorithm was used to extract sperm flagellar centreline and Proper Orthogonal Decomposition (POD) was then used to study sperm flagellar waveform. Statistical analysis was performed using one-way ANOVA. Main results and the role of chance The reconstructed flagellar beating pattern was different for sperm swimming in low and high-viscosity media. Bull sperm exhibited a lower flagellar beating amplitude along the end piece when swimming in a high-viscosity media, a potential energy-efficient strategy to navigate a high viscosity fluid. The first two dominant POD modes (shape modes) describe more than 90% of the beating pattern for all species. Bull sperm exhibited a transition mode with irregular loops in 5 mPa·s buffer, but the flagellar shape cycle created an organised repetitive circular cycle in 1 mPa.s (3D beating) buffer and at viscosities higher than 5 mPa·s (2D beating). Human sperm also indicated a similar behaviour but with the transition happening at higher viscosities. Mouse sperm in high-viscosity media had a lower flagellar beating amplitude across the principal piece and higher beating amplitude across the end piece. The flagellar shape cycle in mouse sperm showed a periodic flagellar beating behaviour at high-viscosities (>20 mPa·s), but a shape cycle with distorted loops at lower viscosities. Our results showed in quantitative detail that increasing viscosity alters sperm flagellar beating pattern, and how sperm migration behaviour in low viscosity media can be distinct from their swimming behaviour in vivo. Limitations, reasons for caution A more comprehensive study of sperm motility parameters such as curvilinear velocity, average path velocity and straight line velocity with a larger sample size is required to fully characterise sperm swimming behaviour as a function of viscosity. Wider implications of the findings The increasing viscosity of the oviductal fluid regulates the sperm flagellar beating behaviour to switch from a 3D swimming behaviour with irregular shape cycles at lower viscosities to a 2D slithering mode with repetitive circular shape cycles at higher viscosities to achieve a more energy-efficient beating pattern for navigation. Trial registration number Not applicable
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