Long-term effects of widespread pharmaceutical pollution on trade-offs between behavioural, life-history and reproductive traits in fish.

In our rapidly changing world, understanding how species respond to shifting conditions is of paramount importance. Pharmaceutical pollutants are widespread in aquatic ecosystems globally, yet their impacts on animal behaviour, life-history and reproductive allocation remain poorly understood, especially in the context of intraspecific variation in ecologically important traits that facilitate species' adaptive capacities. We test whether a widespread pharmaceutical pollutant, fluoxetine (Prozac), disrupts the trade-off between individual-level (co)variation in behavioural, life-history and reproductive traits of freshwater fish. We exposed the progeny of wild-caught guppies (Poecilia reticulata) to three field-relevant levels of fluoxetine (mean measured concentrations: 0, 31.5 and 316 ng/L) for 5 years, across multiple generations. We used 12 independent laboratory populations and repeatedly quantified activity and risk-taking behaviour of male guppies, capturing both mean behaviours and variation within and between individuals across exposure treatments. We also measured key life-history traits (body condition, coloration and gonopodium size) and assessed post-copulatory sperm traits (sperm vitality, number and velocity) that are known to be under strong sexual selection in polyandrous species. Intraspecific (co)variation of these traits was analysed using a comprehensive, multivariate statistical approach. Fluoxetine had a dose-specific (mean) effect on the life-history and sperm trait of guppies: low pollutant exposure altered male body condition and increased gonopodium size, but reduced sperm velocity. At the individual level, fluoxetine reduced the behavioural plasticity of guppies by eroding their within-individual variation in both activity and risk-taking behaviour. Fluoxetine also altered between-individual correlations in pace-of-life syndrome traits: it triggered the emergence of correlations between behavioural and life-history traits (e.g. activity and body condition) and between life-history and sperm traits (e.g. gonopodium size and sperm vitality), but collapsed other between-individual correlations (e.g. activity and gonopodium size). Our results reveal that chronic exposure to global pollutants can affect phenotypic traits at both population and individual levels, and even alter individual-level correlations among such traits in a dose-specific manner. We discuss the need to integrate individual-level analyses and test behaviour in association with life-history and reproductive traits to fully understand how animals respond to human-induced environmental change.

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.

Paper-Based Acoustofluidics for Separating Particles and Cells.

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 µm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Magnetotaxis Enables Magnetotactic Bacteria to Navigate in Flow.

Magnetotactic bacteria (MTB) play an important role in Earth's biogeochemical cycles by transporting minerals in aquatic ecosystems, and have shown promise for controlled transport of microscale objects in flow conditions. However, how MTB traverse complex flow environments is not clear. Here, using microfluidics and high-speed imaging, it is revealed that magnetotaxis enables directed motion of Magnetospirillum magneticum over long distances in flow velocities ranging from 2 to 1260 µm s-1 , corresponding to shear rates ranging from 0.2 to 142 s-1 -a range relevant to both aquatic environments and biomedical applications. The ability of MTB to overcome a current is influenced by the flow, the magnetic field, and their relative orientation. MTB can overcome 2.3-fold higher flow velocities when directed to swim perpendicular to the flow as compared to upstream, as the latter orientation induces higher drag. The results indicate a threshold drag of 9.5 pN, corresponding to a flow velocity of 550 µm s-1 , where magnetotaxis enables MTB to overcome counterdirectional flow. These findings bring new insights into the interactions of MTB with complex flow environments relevant to aquatic ecosystems, while suggesting opportunities for in vivo applications of MTB in microbiorobotics and targeted drug delivery.

Paper-Based Quantification of Male Fertility Potential.

BACKGROUND: More than 70 million couples worldwide are affected by infertility, with male-factor infertility accounting for about half of the cases. Semen analysis is critical for determining male fertility potential, but conventional testing is costly and complex. Here, we demonstrate a paper-based microfluidic approach to quantify male fertility potential, simultaneously measuring 3 critical semen parameters in 10 min: live and motile sperm concentrations and sperm motility. METHODS: The device measures the colorimetric change of yellow tetrazolium dye to purple formazan by the diaphorase flavoprotein enzyme present in metabolically active human sperm to quantify live and motile sperm concentration. Sperm motility was determined as the ratio of motile to live sperm. We assessed the performance of the device by use of clinical semen samples, in parallel with standard clinical approaches. RESULTS: Detection limits of 8.46 and 15.18 million/mL were achieved for live and motile sperm concentrations, respectively. The live and motile sperm concentrations and motility values from our device correlated with those of the standard clinical approaches (R(2) ≥ 0.84). In all cases, our device provided 100% agreement in terms of clinical outcome. The device was also robust and could tolerate conditions of high absolute humidity (22.8 g/m(3)) up to 16 weeks when packaged with desiccant. CONCLUSIONS: Our device outperforms existing commercial paper-based assays by quantitatively measuring live and motile sperm concentrations and motility, in only 10 min. This approach is applicable to current clinical practices as well as self-diagnostic applications.

Direct DNA Analysis with Paper-Based Ion Concentration Polarization.

DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

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.

Fallopian tube rheology regulates epithelial cell differentiation and function to enhance cilia formation and coordination.

The rheological properties of the extracellular fluid in the female reproductive tract vary spatiotemporally, however, the effect on the behaviour of epithelial cells that line the tract is unexplored. Here, we reveal that epithelial cells respond to the elevated viscosity of culture media by modulating their development and functionality to enhance cilia formation and coordination. Specifically, ciliation increases by 4-fold and cilia beating frequency decreases by 30% when cells are cultured at 100 mPa·s. Further, cilia manifest a coordinated beating pattern that can facilitate the formation of metachronal waves. At the cellular level, viscous loading activates the TRPV4 channel in the epithelial cells to increase intracellular Ca2+, subsequently decreasing the mitochondrial membrane potential level for ATP production to maintain cell viability and function. Our findings provide additional insights into the role of elevated tubal fluid viscosity in promoting ciliation and coordinating their beating-a potential mechanism to facilitate the transport of egg and embryo, suggesting possible therapeutic opportunities for infertility treatment.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.