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.

Viscous Loading Regulates the Flagellar Energetics of Human and Bull Sperm.

The viscoelastic properties of the female reproductive tract influence sperm swimming behavior, but the exact role of these rheological changes in regulating sperm energetics remains unknown. Using high-speed dark-field microscopy, the flagellar dynamics of free-swimming sperm across a physiologically relevant range of viscosities is resolved. A transition from 3D to 2D slither swimming under an increased viscous loading is revealed, in the absence of any geometrical or chemical stimuli. This transition is species-specific, aligning with viscosity variations within each species' reproductive tract. Despite substantial drag increase, 2D slithering sperm maintain a steady swimming speed across a wide viscosity range (20-250 and 75-1000 mPa s for bull and human sperm) by dissipating over sixfold more energy into the fluid without elevating metabolic activity, potentially by altering the mechanisms of dynein motor activity. This energy-efficient motility mode is ideally suited for the viscous environment of the female reproductive tract.

High-throughput sperm DNA analysis at the single-cell and population levels.

Clinical semen quality assessment is critical to the treatment of infertility. Sperm DNA integrity testing provides critical information that can steer treatment and influence outcomes and offspring health. Flow cytometry is the gold standard approach to assess DNA integrity, but it is not commonly applied at the clinical level. The sperm chromatin dispersion (SCD) assay provides a simpler and cheaper alternative. However, SCD is low-throughput and non-quantitative - sperm assessment is serial, manual and suffers inter- and intra-observer variations. Here, an automated SCD analysis method is presented that enables quantitative sperm DNA quality assessment at the single-cell and population levels. Levering automated optical microscopy and a chromatin diffusion-based analysis, a sample of thousands of sperm that would otherwise require 5 hours is assessed in under 10 minutes - a clinically viable workflow. The sperm DNA diffusion coefficient (DDNA) measurement correlates (R2 = 0.96) with DNA fragmentation index (DFI) from the cytometry-based sperm chromatin structure assay (SCSA). The automated measurement of population-level sperm DNA fragmentation (% sDF) prevents inter-observer variations and shows a good agreement with the SCSA % DFI (R2 = 0.98). This automated approach standardizes and accelerates SCD-based sperm DNA analysis, enabling the clinical application of sperm DNA integrity assessment.

Human sperm cooperate to transit highly viscous regions on the competitive pathway to fertilization.

Human sperm compete for fertilization. Here, we find that human sperm, unexpectedly, cooperate under conditions mimicking the viscosity contrasts in the female reproductive tract. Sperm attach at the head region to migrate as a cooperative group upon transit into and through a high viscosity medium (15-100 cP) from low viscosity seminal fluid. Sperm groups benefit from higher swimming velocity, exceeding that of individual sperm by over 50%. We find that sperm associated with a group possess high DNA integrity (7% fragmentation index) - a stark contrast to individual sperm exhibiting low DNA integrity (> 50% fragmentation index) - and feature membrane decapacitation factors that mediate sperm attachment to form the group. Cooperative behaviour becomes less prevalent upon capacitation and groups tend to disband as the surrounding viscosity reduces. When sperm from different male sources are present, related sperm preferentially form groups and achieve greater swimming velocity, while unrelated sperm are slowed by their involvement in a group. These findings reveal cooperation as a selective mode of human sperm motion - sperm with high DNA integrity cooperate to transit the highly viscous regions in the female tract and outcompete rival sperm for fertilization - and provide insight into cooperation-based sperm selection strategies for assisted reproduction.

Sperm quality metrics were improved by a biomimetic microfluidic selection platform compared to swim-up methods.

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.

High-DNA integrity sperm selection using rheotaxis and boundary following behavior in a microfluidic chip.

Rheotaxis, as one of the main natural guidance mechanisms in vivo, has been used in microfluidics to separate motile sperm. However, the lack of DNA integrity assessment and the inability to separate the cells in a specific reservoir have been the main limitations for the practical application of most of the devices using rheotaxis for sperm separation. Here, we present a microfluidic chip that can separate highly motile sperm using their inherent rheotaxis and boundary-following behavior in a network of boomerang-shaped microchannels. The device design is informed by our FEM simulation results to predict sperm trajectories. Experimental results demonstrate the device's performance to separate over 16 000 motile sperm in under 20 min, sufficient for droplet-based IVF. Separated cells are classified into two motility groups, highly motile (swimming speed > 120 µm s-1) and motile (swimming speed < 120 µm s-1). The device selects sperm with over 45%, 20%, and 80% improvement in motility, the number of highly motile sperm, and DNA integrity, respectively, suggesting promising potential for applications in assisted reproduction.

High-throughput isolation of cancer cells in spiral microchannel by changing the direction, magnitude and location of the maximum velocity.

Circulating tumor cells (CTCs) are scarce cancer cells that rarely spread from primary or metastatic tumors inside the patient's bloodstream. Determining the genetic characteristics of these paranormal cells provides significant data to guide cancer staging and treatment. Cell focusing using microfluidic chips has been implemented as an effective method for enriching CTCs. The distinct equilibrium positions of particles with different diameters across the microchannel width in the simulation showed that it was possible to isolate and concentrate breast cancer cells (BCCs) from WBCs at a moderate Reynolds number. Therefore we demonstrate high throughput isolation of BCCs using a passive, size-based, label-free microfluidic method based on hydrodynamic forces by an unconventional (combination of long loops and U-turn) spiral microfluidic device for isolating both CTCs and WBCs with high efficiency and purity (more than 90%) at a flow rate about 1.7 mL/min, which has a high throughput compared to similar ones. At this golden flow rate, up to 92% of CTCs were separated from the cell suspension. Its rapid processing time, simplicity, and potential ability to collect CTCs from large volumes of patient blood allow the practical use of this method in many applications.

A novel microfluidic device with parallel channels for sperm separation using spermatozoa intrinsic behaviors.

Isolating high-quality motile sperm cells is considered to be the main prerequisite for a successful artificial pregnancy. Microfluidics has emerged as a promising platform capable of mimicking in-vivo environments to separate motile sperm cells and bypassing the need for the current invasive clinical sperm separation methods. In this study, the proposed microfluidic device exploits the parallelization concept through symmetry to increase both the processed sample volume and the injected flow rate compared with the previous conventional devices, which used rheotaxis as their primary method of sperm separation. Using the finite element method (FEM) and flow simulations, the trajectories of sperm cells exhibiting rheotaxis behavior were predicted inside the proposed device. Different flow rates, including 0, 0.5, 1.5, 3, 4.5 and 6 µl/min, were experimentally injected into the device, and the effect of flow rate on the size of the hypothetical rheotaxis zone and the number of isolated sperm cells was investigated. Furthermore, it was illustrated that 100% of the isolated motile sperm cells are motile, and by manipulating the injected flow rate into the device, different classes of sperm cells in terms of motility parameters can be separated and utilized for further uses.

Surface acoustic wave-driven pumpless flow for sperm rheotaxis analysis.

Sperm rheotaxis, the phenomenon where sperm cells swim against the direction of fluid flow, is one of the major guiding mechanisms for long-distance sperm migration within the female reproductive tract. However, current approaches to study this pose challenges in dealing with rare samples by continuously introducing extra buffer. Here, we developed a device utilising acoustic streaming, the steady flow driven by an acoustic perturbation, to drive a tuneable, well-regulated continuous flow with velocities ranging from 40 µm s-1 to 128 µm s-1 (corresponding to maximum shear rates of 5.6 s-1 to 24.1 s-1) in channels of interest - a range suitable for probing sperm rheotaxis behaviour. Using this device, we studied sperm rheotaxis in microchannels of distinct geometries representing the geometrical characteristics of the inner-surfaces of fallopian tubes, identified sperm dynamics with the presence of flow in channels of various widths. We found a 28% higher lateral head displacement (ALH) in sufficiently motile rheotactic sperm in a 50 µm channel in the presence of acoustically-generated flow as well as a change in migration direction and a 52% increase in curvilinear velocity (VCL) of sufficiently motile sperm in a 225 µm channel by increasing the average flow velocity from 40 µm s-1 to 130 µm s-1. These results provided insights for understanding sperm navigation strategy in the female reproductive tract, where rheotactic sperm swim near the boundaries to overcome the flow in the female reproductive tract and reach the fertilization site. This surface acoustic wave device presents a simple, pumpless alternative for studying microswimmers within in vitro models, enabling the discovery of new insights into microswimmers' migration strategies, while potentially offering opportunities for rheotaxis-based sperm selection and other flow-essential applications.

Lab on a chip devices for fertility: from proof-of-concept to clinical impact.

Microfluidics offers tremendous opportunities to understand the underlying biology of fertilization at the single-cell level and improve infertility management, however, its true clinical impact is yet to be realized. Lab-on-a-chip devices have generally failed to diffuse into clinical practice due to issues associated with their translation or their practicality and performance in clinical settings. In this perspective, I reflect on how the full potential of microfluidic technologies for fertility can be realized by considering regulatory and manufacturing considerations at the development stage and by redefining our developmental goals to directly target the ultimate clinical needs. I also challenge the common rationale around developing technologies for infertility treatment based on reducing cost and complexity in operation as the ultimate outcome is invaluable, human life.

High-Frequency Ultrasound Boosts Bull and Human Sperm Motility.

Sperm motility is a significant predictor of male fertility potential and is directly linked to fertilization success in both natural and some forms of assisted reproduction. Sperm motility can be impaired by both genetic and environmental factors, with asthenozoospermia being a common clinical presentation. Moreover, in the setting of assisted reproductive technology clinics, there is a distinct absence of effective and noninvasive technology to increase sperm motility without detriment to the sperm cells. Here, a new method is presented to boost sperm motility by increasing the intracellular rate of metabolic activity using high frequency ultrasound. An increase of 34% in curvilinear velocity (VCL), 10% in linearity, and 32% in the number of motile sperm cells is shown by rendering immotile sperm motile, after just 20 s exposure. A similar effect with an increase of 15% in VCL treating human sperm with the same setting is also identified. This cell level mechanotherapy approach causes no significant change in cell viability or DNA fragmentation index, and, as such, has the potential to be applied to encourage natural fertilization or less invasive treatment choices such as in vitro fertilization rather than intracytoplasmic injection.

Unraveling the Kinematics of Sperm Motion by Reconstructing the Flagellar Wave Motion in 3D.

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 thin-lens 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.

Rheotaxis-based sperm separation using a biomimicry microfluidic device.

Sperm selection is crucial to assisted reproduction, influencing the success rate of the treatment cycle and offspring health. However, in the current clinical sperm selection practices, bypassing almost all the natural selection barriers is a major concern. Here, we present a biomimicry microfluidic method, inspired by the anatomy of the female reproductive tract, that separates motile sperm based on their rheotaxis behavior to swim against the flow into low shear rate regions. The device includes micropocket geometries that recall the oval-shaped microstructures of the female fallopian tube to create shear protected zones for sperm separation. Clinical tests with human samples indicate that the device is capable of isolating viable and highly motile sperm based on their rheotaxis responses, resulting in a separation efficiency of 100%. The device presents an automated alternative for the current sperm selection practices in assisted reproduction.

CRISPs Function to Boost Sperm Power Output and Motility.

Fertilization requires sperm to travel long distances through the complex environment of the female reproductive tract. Despite the strong association between poor motility and infertility, the kinetics of sperm tail movement and the role individual proteins play in this process is poorly understood. Here, we use a high spatiotemporal sperm imaging system and an analysis protocol to define the role of CRISPs in the mechanobiology of sperm function. Each of CRISP1, CRISP2, and CRISP4 is required to optimize sperm flagellum waveform. Each plays an autonomous role in defining beat frequency, flexibility, and power dissipation. We thus posit that the expansion of the CRISP family from one member in basal vertebrates, to three in most mammals, and four in numerous rodents, represents an example of neofunctionalization wherein proteins with a common core function, boosting power output, have evolved to optimize different aspects of sperm tail performance.

Curvature in the reproductive tract alters sperm-surface interactions.

The fallopian tube is lined with a highly complex folded epithelium surrounding a lumen that progressively narrows. To study the influence of this labyrinthine complexity on sperm behavior, we use droplet microfluidics to create soft curved interfaces over a range of curvatures corresponding to the in vivo environment. We reveal a dynamic response mechanism in sperm, switching from a progressive surface-aligned motility mode at low curvatures (larger droplets), to an aggressive surface-attacking mode at high curvatures (smaller droplets of <50 µm-radius). We show that sperm in the attacking mode swim ~33% slower, spend 1.66-fold longer at the interface and have a 66% lower beating amplitude than in the progressive mode. These findings demonstrate that surface curvature within the fallopian tube alters sperm motion from a faster surface aligned locomotion in distal regions to a prolonged physical contact with the epithelium near the site of fertilization, the latter being known to promote capacitation and fertilization competence.

Selection of high-quality sperm with thousands of parallel channels.

Sperm selection is essential for successful fertilization and embryo development. Current clinical sperm selection methods are labor-intensive and lack the selectivity required to isolate high-quality sperm. Microfluidic sperm selection approaches have shown promise but present a trade-off between the quality and quantity of selected sperm - clinicians demand both. The structure of the female reproductive tract helps to isolate a sufficient quantity of high-quality sperm for fertilization with densely folded epithelium that provides a multitude of longitudinally oriented pathways that guide sperm toward the fertilization site. Here, a three-dimensionally structured sperm selection device is presented that levers this highly parallelized in vivo mechanism for in vitro sperm selection. The device is inserted in a test tube atop 1 mL of raw semen and provides 6500 channels that isolate ∼100 000 high-DNA-integrity sperm for assisted reproduction. In side-by-side clinical testing, the developed approach outperforms the best current clinical methods by improving the DNA integrity of the selected sperm subpopulation up to 95%. Also, the device streamlines clinical workflow, reducing the time required for sperm preparation 3-fold. This single-tube, single-step sperm preparation approach promises to improve both the economics and outcomes of assisted reproduction practices, especially in cases with significant male-factors.

Machine learning for sperm selection.

Infertility rates and the number of couples seeking fertility care have increased worldwide over the past few decades. Over 2.5 million cycles of assisted reproductive technologies are being performed globally every year, but the success rate has remained at ~33%. Machine learning, an automated method of data analysis based on patterns and inference, is increasingly being deployed within the health-care sector to improve diagnostics and therapeutics. This technique is already aiding embryo selection in some fertility clinics, and has also been applied in research laboratories to improve sperm analysis and selection. Tremendous opportunities exist for machine learning to advance male fertility treatments. The fundamental challenge of sperm selection - selecting the most promising candidate from 108 gametes - presents a challenge that is uniquely well-suited to the high-throughput capabilities of machine learning algorithms paired with modern data processing capabilities.

Flagellar energetics from high-resolution imaging of beating patterns in tethered mouse sperm.

We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered mouse sperm at high resolution. Beating waveforms are reconstructed using proper orthogonal decomposition of the centerline tangent-angle profiles. Energy conservation is employed to obtain the mechanical power exerted by the dynein motors from the observed kinematics. A large proportion of the mechanical power exerted by the dynein motors is dissipated internally by the motors themselves. There could also be significant dissipation within the passive structures of the flagellum. The total internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in wildtype samples, indicating that ion-channel regulation by cysteine-rich secretory proteins controls energy flows powering the axoneme.

A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions.

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.