Tunable motile sperm separation based on sperm persistence in migrating through shear barriers

Abstract

Rheotaxis is one of the major migratory mechanisms used in autonomous swimmers such as sperms and bacteria. Here, we present a microfluidic chip using joint rheotaxis and boundary-following behavior that selects sperms based on the motility and persistence. The proposed device consists of a channel decorated with diamond-shaped pillars that create spots of increased velocity field and shear rate. These spots are supposed as hydrodynamic barriers that impede the passage of less motile sperms through the channels, while highly motile sperms were able to overcome the generated barrier and swim through the structures. The proposed device was able to populate the chamber with sorted sperms that were fully viable and motile. The experimental results validated the separation of highly motile sperms with enhanced motility parameters compared with the initial sample. Our device was able to improve linear straight velocity, curvilinear velocity, and average path velocity of the sorted population surpassing 35%, compared with the raw semen. The processing time was also reduced to 20 min.

I. INTRODUCTION

The issue of infertility is a pervasive problem that affects countless couples all over the world. Particularly noteworthy is the fact that nearly half of all infertility cases can be attributed to male factor infertility.¹ As a result, the development of assisted reproductive technology (ART) has become a vital solution for couples struggling with infertility, often representing their last chance to achieve successful conception.² ART, serving as the prevailing technique in the field, encompasses a variety of commonly utilized procedures. These include intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI).³

To perform these methods correctly, the first step is to collect a sperm sample from the patient. Then, this sample is put through various processing techniques that aim to improve its quality. The effectiveness of treatment largely depends on some important factors related to sperm quality. Specifically, major factors include sperm motility, DNA integrity, and morphology.⁴ Density gradient centrifugation (DGC) represents a prominent conventional approach employed for enhancing the above-mentioned factors related to sperm quality. DGC involves subjecting sperm samples to centrifugal force in order to separate and isolate sperm cells with superior motility, morphology, and maturity. However, it is important to note that the benefits attained through DGC come at a cost. The process of DGC exposes sperm cells to high shear forces and oxidative stress, which can potentially lead to detrimental effects. These adverse effects include irreversible damage to the integrity of the sperm membrane and fragmentation of DNA within the cell.⁵

In the context of treating male factor infertility, ICSI stands as a promising option, but its success heavily relies on the meticulous selection criteria applied to sperm. Given that ICSI circumvents the majority of natural barriers encountered by sperm during its journey through the female reproductive tract, any abnormalities present in the selected sperm can be readily passed on to the oocyte following fertilization. This, in turn, has the potential to lead to aberrant embryonic development, an elevated risk of congenital abnormalities, and an increased likelihood of miscarriage.^6,7

The selection of sperm during ICSI is typically performed through visual inspection, a process conducted by embryologists.⁸ However, this method of selection carries inherent risks as it can lead to the inadvertent choice of sperm with undesirable traits.⁹ Moreover, the presence of inter-individual variability within the selection processes can further diminish the overall success rate of the procedure. These factors underscore the need for more precise and reliable methods of sperm selection in order to optimize treatment outcomes.

The field of microfluidics has emerged as a promising tool for various biomedical applications, offering a range of advantages that can rival conventional practices.¹⁰ This technology, characterized by its ability to manipulate fluids and cells at the microscale level, provides a unique set of qualities that contribute to its appeal. Notably, microfluidics offers the advantage of being minimally invasive, which is particularly valuable in biomedical procedures. Additionally, it enables precise control over the consumption of raw materials, minimizing wastage and reducing costs.¹¹ Furthermore, microfluidic systems can automate various processes, enhancing efficiency and accuracy in biomedical applications.¹² These distinctive qualities make microfluidics a capable and versatile tool with the potential to revolutionize various aspects of biomedical research and clinical practice.

In recent years, there has been increasing evidence regarding the feasibility and effectiveness of employing microfluidic chips for the purpose of sperm sorting.^13–15 These chips can be categorized into two main types: passive devices and active devices.¹⁶ In passive devices, the sorting mechanism primarily relies on hydrodynamic interactions between the sperms and the sidewalls of the microfluidic channels.^17,18 These interactions play a crucial role in guiding the movement and separation of the sperms within the chip.^16,19 On the other hand, active devices utilize various external stimuli, such as the application of chemoattractants, fluid flow manipulation, and temperature gradients, to facilitate the selection of desired sperms.^20,21 These stimuli are carefully designed and controlled to exert specific forces and induce directional movement of the sperms, enabling the sorting process. The utilization of microfluidic chips, whether passive or active, represents a promising approach for precise and efficient sperm sorting, offering potential advancements in ART and reproductive research. However, recent research has clearly demonstrated the superiority of rheotaxis over other approaches such as thermotaxis and chemotaxis given its ability to guide sperms over longer distances.²² In addition, unlike thermotaxis and chemotaxis, which require specific gradients of temperature and chemicals, respectively, rheotaxis merely relies on fluid flow dynamics naturally present in the reproductive tract.^23–26 This mechanism enables highly motile sperms to navigate through the uterus and reach the fallopian tube where the fertilization occurs.^22,27,28 Furthermore, the capacity of rheotaxis in a given sample has been found to be strongly associated with lower DNA fragmentation and higher fertility rate, potentially reinforcing rheotaxis position as both a reliable sorting method and an indicator of sperm fertility as well.^29,30

The microfluidic devices presented by Zeaei et al.³¹ and Heydari et al.³² leveraged rheotaxis by incorporating rheotaxis zones within the device. Upon entering these zones, sperms reorient and migrate opposite the flow, following embedded channels that lead to an isolated chamber. While these devices show promise in separating highly motile sperms, their reliance on brief, spatially confined rheotaxis zones do not perfectly evaluate sperms for persistent rheotactic behavior over extended distances and time frame. In one study, it was shown that when sperms encounter constriction while exhibiting rheotaxis, their movement is controlled by a gate-like behavior associated with constriction.³³ At a critical flow velocity, spermatozoa are trapped near the constriction opening and exhibit a characteristic butterfly-like motion. Based on this gate-like behavior, Yaghoobi et al. developed a microfluidic device with prism-like structures designed to select sperms with higher motility.³⁴ Their results demonstrated that faster sperms selected through rheotaxis contribute to improved early embryonic development. However, the requirement to load the semen via controlled flow, along with the device's susceptibility to air bubbles, may introduce potential complications that can hinder its ease of use and reliability. In our previous study, the gate-like behavior was also used to separate motile sperms into two subpopulations with various degrees of motility.³⁵ The design featured two sequential constrictions, inspired by the geometry of the uterotubal junction. While it successfully achieved sperm sorting, the inherent design limitations restricted its scalability and hindered the possibility of parallelization. As an expansion to the discussed devices, we propose a microfluidic chip that can efficiently enrich highly motile sperms by selectively trapping less motile sperms while also overcoming stated limitations. In this chip, an array of diamond-shaped pillars is designed to create areas characterized by high-velocity fields and shear rates, creating a hydrodynamic shear barrier against the progressive movement of sperms. These areas act to effectively capture and confine sperms with less motility in the vicinity of the diamond-shaped patterns. The dependence of these shear barrier strengths to the applied flow rate can be utilized to tune the sorting threshold of the proposed device. The multiple channels arising from multiple pillars in each row allow for simultaneous processing of a larger sample volume in the device. In addition, the repetition of the designed structures along the sperms migrating path guarantees the selection of persistent sperms in overcoming these barriers. Using this sorting mechanism, the proposed microfluidic chip holds promise for enhancing the enrichment of highly motile sperm populations in a controlled and efficient manner.

II. MATERIALS AND METHODS

A. Sample preparation

Fresh human semen samples were acquired from Sara Infertility Center and stored in the incubator at 37 °C. The microfluidic device was pre-heated in the incubator and was loaded with 50 μl of the semen sample. The microfluidic device was then placed under the microscope and the fluid flow was controlled using a syringe pump (TS-1B/W0109-1B, Longer Pump, China).

B. Device fabrication and preparation

The device utilizes a polydimethylsiloxane (PDMS)-based microfluidic chamber fabricated using standard soft lithography techniques. Initially, a 70 μm-thick layer of negative-tone photoresist (SU8-2050) was spun coated, patterned, and developed to form the desired master mold. Sylgard 184A silicone elastomer base and Sylgard 184B silicone elastomer curing agent were then mixed with a 10:1 weight ratio, poured onto the mold, and degassed in a vacuum desiccator for 20 min to remove air bubbles. The mixture was subsequently cured at 80 °C for 30 min. After removing the cured PDMS from the mold, inlets and outlets were punched using a reusable biopsy punch. To bond the microfluidic device to the glass substrate, both surfaces were treated with oxygen plasma for 60 s. The final device was fully submerged in de-ionized water for three days to discharge the air in the channels. During the experiment, the water inside the channels was displaced with sperm medium using a syringe pump connected to the inlet. The microfluidic device was placed on a warm plate and maintained at 37 °C during the extent of the experiment.

C. Imaging and video recording

The experiments were recorded using a Nikon Eclipse TS100 inverted microscope equipped with a Basler acA800-510uc camera at 60 frames per second. Open-source computer assisted sperm analysis (OpenCASA) was utilized to assess sperm motility parameters in the ImageJ software.³⁶ Linear straight velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP) were extracted from the software for both raw and sorted sample. Sperm trajectories were also tracked manually using MTrackJ module in ImageJ.

D. Vitality assay

Sperm vitality assay was conducted using Eosin–Nigrosin staining method (Sperm Vitality Assay Kit, IVF Co, Tehran, Iran) to differentiate between live and dead sperms. Sperm was mixed thoroughly with Eosin and Nigrosin in a 2:1:1 ratio and incubated at room temperature for 2 min. A smear was then prepared on a microscope slide and allowed to air-dry and was subsequently examined under a microscope. Live sperms remained intact and unstained, while dead sperms displayed a reddish-pink hue. Sample vitality was assessed by counting at least 100 sperms.

E. Statistical analysis

Each experiment was conducted in quadruplicate, using samples from four different patients, and the results were presented as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, and a P-value of less than 0.05 was deemed statistically significant.

F. Simulation

COMSOL Multiphysics (version 6.1) was used to analyze the designed microfluidic channel. Navier–Stokes [Eq. (1)] and conservation of mass [Eq. (2)] were utilized to obtain the velocity field and shear rate throughout the channel as described below,

$$\rho (v\cdot \mathrm{\nabla }v)=-\mathrm{\nabla }p+\mathrm{\nabla }\cdot \mu (\mathrm{\nabla }v+{(\mathrm{\nabla }v)}^T),$$ (1)

$$\mathrm{\nabla }\cdot v=0,$$ (2)

where ν is the velocity, p is the pressure, μ is the dynamic viscosity, and ρ indicates the fluid density. A no-slip boundary condition was assigned to the sidewalls of the microfluidic channel and a zero-gauge pressure was also applied to the outlet. The fluid flowing into the channel was also modeled as an incompressible flow with a density and dynamic viscosity of those of water.

The sperm swimming pattern inside the microfluidic channel was modeled with a two-dimensional linear motion with no acceleration as described below,

$$\begin{array}{c}{\displaystyle \frac{d\overrightarrow{r}}{dt}={\overrightarrow{v}}_{\mathrm{sperm}}+{\overrightarrow{v}}_{\mathrm{medium}}+\epsilon (0,\sigma )}\end{array},$$ (3)

where ${\overrightarrow{v}}_{\mathrm{sperm}}$ is the propulsive linear velocity of sperm, ${\overrightarrow{v}}_{\mathrm{medium}}$ is the velocity field of the fluid flow, and $\epsilon (0,\sigma )$ is the Gaussian distribution with zero mean and standard deviation of $\sigma$. The Guassian noise describes the intrinsic lateral displacement of the sperm head. The angular motion of the sperm head with respect to the fluid flow was also modeled using Eq. (4),³⁷

$$\begin{array}{c}\omega ={\displaystyle \frac{\mathrm{d}\theta }{\mathrm{dt}}=-\mathrm{\Omega }-\alpha \gamma \sin (\theta )}\end{array}.$$ (4)

In this context, θ represents the angle of the sperm head relative to the direction of the fluid flow. The γ denotes the shear rate near either the upper or lower boundary and Ω is the intrinsic angular velocity of the sperm when there is no flow present. Last, α is a dimensionless constant that is associated with the sperm geometric asymmetry. To account for sperm boundary-following behavior, sidewalls were modeled as a vector at an angle of $\delta$ with respect to the fluid flow direction. Whenever a sperm comes within a small arbitrary distance from the wall, its head angle is updated according to Eq. (5),

$$\begin{array}{c}{\theta }_{\mathrm{i}+1}=\delta -(\mathrm{\Omega }+\alpha \gamma \sin ({\theta }_{\mathrm{i}}))\mathrm{\Delta }t\end{array},$$ (5)

where $\mathrm{\Delta }t$ is the time step taken in the simulation. Sperms stay attached to the wall unless exposed to high shear rates, inducing rotations that can surpass the wall-aligned angle δ. This rotation causes sperm to distance itself enough from the wall so as to detach from the boundary. Subsequent to the detachment, sperm then reorients itself with respect to the fluid flow, governed by Eq. (4). MATLAB R2023b was used to solve the above-mentioned equations.

III. RESULTS AND DISCUSSION

A. Device and operation

In this study, we developed a microfluidic chip consisting of the diamond-shaped pillars in the microchannel to create the desired flow pattern. By adjusting the spacing between these diamond-shaped structures and selecting the optimum flow rate, a series of hydrodynamic barriers was generated, impeding sperm swimming ability. The generated hydrodynamic barriers allowed faster moving sperms to swim through the structures, while slower moving sperms trapped within the pillars. In addition, the designed microfluidic device consists of a reservoir for loading the raw human sample that is followed by a series of parallel channels, guiding motile sperms to the main channel, where the diamond-shaped pillars are located. Figure 1 provides an overview of the device and its schematic. Figure 1(b) depicts the device schematic consisting of a raw semen reservoir, guidance and sorting channels, sorted sperm chamber, and fluid inlet. Figures 1(c) and 1(d) illustrate guidance channels and diamond-shaped pillars within the device. Figure 1(e) highlights the zoomed version of the pillars, where the created confinement generates a shear barrier. The white arrows indicate the incoming fluid flow is restricted in the spacing between two adjacent pillars, elevating the associated velocity field and shear rate. Sperms based on their motility can overcome the barrier, or entrap in a recurring butterfly-like motion as indicated by the pink arrows, or be washed away by the fluid.³⁸

FIG. 1.

(a) The microfluidic device presented in this study for separating motile sperms. (b) Schematic of the device. (c) Schematic of the guidance channels, whereby motile sperms are guided to the sorting channels. (d) Sorting channels consisting of diamond pillars. (e) The confinement between two adjacent pillars generates the shear barrier in the path of sperms.

Sperm movement in this scenario is influenced by two key factors: the force resulting from the fluid flow in the microchannel, and the sperm rheotaxis behavior. Previous investigations have reported that fluid velocities ranging from 22 to 102 μm/s can induce rheotaxis behavior.³⁹ The primary contributor to the rheotaxis behavior exhibited by sperm is the anatomical arrangement of their head and flagella, which are not situated on a flat plane. In addition to the above-mentioned behavioral pattern, healthy sperms also exhibit a tendency to adhere closely to rigid boundaries, aligning their heads as closely as possible to the walls while allowing their tails to oscillate at a certain distance below or above the heads.²⁷ Given the parabolic flow profile in the laminar flow regime inside the microfluidic channel, the asymmetry in the experienced forces by sperm head and tail would result in asymmetric torque being generated across the sperm body. The torque will result in the reorientation of the sperm tail around its head, causing changes in its trajectories. Using these facts, the diamond-shaped pillars are designed to create constrictions in the path of the fluid flow, creating shear rate and velocity field hotspots. The sudden increase in these variables would result in periodic sperm detachment–attachment of surrounding boundaries, resembling butterfly-like motion. However, sperms possessing a higher degree of motility can surpass the hydrodynamic barrier and swim through the pillars. By carefully selecting the optimum spacing of the pillars and flow rate, one can achieve a successful sorting of highly motile sperms. In order to reduce the degrees of the freedom and associated complexity, the spacing between the diamond-shaped pillars was chosen as a fixed arbitrary value, and the fluid flow rate was changed as the variable. Figures 2(a) and 2(b) demonstrate fluid velocity field and shear rate for the designed geometry. The surface plots highlight the presence of mentioned hotspots between neighboring pillars. Figures 2(c) and 2(d) illustrate the velocity field magnitude and shear rate profiles between two arbitrary pillars. As can be seen, the profiles obey a parabolic trend that agrees with the established profile in the laminar flow regime. The center of the gap between the pillars experiences elevated shear rate and velocity field magnitude that create the desired hydrodynamic barrier. Unlike the velocity field that reaches zero near the pillar boundaries, the shear rate reaches a non-zero minimum that ensures there is enough shear stress to cause sperm detachment and reorientation in the vicinity of the barrier.

FIG. 2.

Simulation of (a) the velocity field and (b) the shear rate throughout the channel for a flow rate of 50 nl/min. The parabolic diagrams also illustrate the values of (c) velocity magnitude and (d) shear rate between two nearby pillars in the channel. As can be seen, both parameters are maximum in the center of the gap between two pillars, creating the parabolic flow profile associated with the laminar flow.

In Sec. III B, we present numerical analysis results for the selection of fluid flow range that leads to a desirable separation outcome. The sperm trajectories resulting from sperm modeling are also included to give the reader a clearer view of sperm behavior in the device. Subsequently, experimental results are presented. The device is loaded with sperm culture medium and connected to a syringe pump to provide a steady fluid flow. The raw human semen sample is loaded into the device using a micropipette via the semen reservoir. Upon the introduction, sperms swiftly follow the guidance channels to reach the sorting channel, where they face the hydrodynamic barriers. Sperms endowed with high motility and persistency had surmounted the shear barriers and eventually reached the sorted sperm chamber, where they were analyzed for their vitality and motility parameters.

B. Simulation results

In order to determine the optimum working range of the flow rate, Monte Carlo simulation was conducted with a population of 300 sperms, with their velocities obeying a normal distribution with a mean of 65 μm/s and a standard deviation of 10 μm/s. Each sperm was assigned a velocity drawn from this distribution and tracked as it interacted with the pillars. Figure 3 illustrates the trajectories of 30 sperms from the population, analyzed within a section of the sorting channels as part of the Monte Carlo simulation. The simulation primarily focused on sperm movement within the sorting channels, where hydrodynamic barriers are located. While the guiding channels serve to direct motile sperms toward this region, the sorting process itself takes place within the sorting channels.

FIG. 3.

Sperm trajectories through the microfluidic sorting channels at different flow rates: 120, 150, and 180 nl/min. The colored lines represent the movement paths of individual sperm as they interact with the hydrodynamic barriers formed by the diamond-shaped pillars. At 120 nl/min, most sperm successfully navigate through the sorting channel, while at 180 nl/min, only a few sperm with high motility manage to overcome the shear barriers. As the flow rate increases, the strength of the hydrodynamic barriers also increases, resulting in fewer sperm being able to traverse the channel. The blue arrow represents fluid flow direction in the simulation.

The provided trajectories clearly demonstrate the rheotaxis and boundary-following behaviors of sperms as they navigate through the sorting channels. When near boundaries, sperms tend to follow these edges until they encounter the hydrodynamic barriers. Upon reaching these barriers, sperms exhibit a characteristic butterfly motion as they interact with the shear forces between the pillars. At lower flow rates, more sperms are able to surpass the barriers and reach the other side. However, as the flow rate increases, the strength of the shear barriers rises, resulting in fewer sperms possessing sufficient motility to overcome them. This is particularly evident at a flow rate of 180 nl/min, where only a handful of sperms managed to pass through the first row of pillars. The trajectories also confirm that low-motility sperms, once detached from the pillar edges, can be swept away by the fluid flow, as seen at 180 nl/min. The pointed ends of the diamond-shaped pillars facilitate the detachment process, allowing sperms to reorient themselves with respect to the flow and swim toward the next barrier. This mechanism effectively ensures the separation of sperms with high motility and consistency.

Figure 4 demonstrates the histograms of the sorted sperms against the initial population for three different flow rates. As can be seen, by increasing the flow rate, the sorting threshold was shifted upward toward higher velocities. The final sorted concentration is also inversely correlated with the incoming fluid flow rate. As the flow rate increases, the more stringent and selective the barriers become, resulting in fewer sperms to pass through the structures. The trade-off between concentration and velocity/motility profile is well balanced at the flow rate of 150 nl/min, showing a desired output. As a result, the fluid flow rate was swept in the vicinity of this value during the experiment to find the optimal sorting process. Furthermore, the tunability of the device with changing the flow rate can empower this device to tailor its output for various ART procedures. In the actual design, the length scale of the channel, where the diamond pillars are located, was designed to be rather long to also put the sperm persistence to the test. As a result, the sorted sperms were those that would be able to consistently overcome the hydrodynamic barriers, generated between the diamond pillars.

FIG. 4.

The histograms depict the distribution of sperm velocity in initial and sorted population. By adjusting the flow rate to a higher value, the velocity of the sorted population tends to center around higher velocities, as less motile sperms were not able to overcome the shear barriers in migrating through the pillars. The dotted line highlights the sorting threshold for each flow rate, where the number of unsorted sperms outnumbers the sorted ones in each velocity bin.

C. Experimental results

Upon depositing the semen sample into the semen reservoir, the viable and motile sperms were promptly guided by the parallel channels to the main channel in less than 5 min. The flow rate was fine-tuned near the above-mentioned 150 nl/min, and sperm trajectories in the sorting channels were analyzed under the microscope. Figure 5 illustrates the trajectories of sperms interacting with the diamond pillars. Sperms can interact with the shear barriers differently based on their motility, as sperms with a higher degree of motility can easily overpower the barrier and pass through the structures [Fig. 5(a), Video S1 in the supplementary material]. In contrast, sperms with a lower degree of motility were trapped nearby the pillars and performed a periodic detachment–attachment motion as denoted in earlier research [Fig. 5(b), Video S2 in the supplementary material]. Sperms with degraded motility, however, have no means to counteract the incoming fluid flow and would be washed away by the fluid [Fig. 5(c), Video S3 in the supplementary material]. This, in turn, ensures the passage of highly motile sperms through the pillars, while sperms with lower degrees of motilities would be barred from traversing the structures.

FIG. 5.

Trajectories of sperms interacting with the shear barrier. (a) Highly motile sperms were able to overcome the shear barrier and passed through. (b) Less motile sperms were trapped in the vicinity to the shear barrier, performing a periodic detachment–attachment motion known as butterfly-like motion. (c) Sperms with degraded motility were pushed back by the incoming flow and were washed away. Scale bar indicates 50 μm.

Figure 6 demonstrates sperm motility parameters extracted by the OpenCASA. The graphs clearly show considerable improvements of sperm motility parameters in the sorted population compared with the initial sample. While both VSL and VCL improved significantly in the sorted sperms, the value of VSL experienced a more dramatic increase compared with the other two, indicating the selection of sperms with a higher degree of linearity. This is consistent with the initial assumption of sorting sperms based on their motility and persistence, as linear sperms are expected to have a more persistent performance in swimming upstream and overcoming the hydrodynamic barriers.

FIG. 6.

Sperm motility parameters, including straight-line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP), obtained from OpenCASA are reported as mean ± standard deviation (n = 4), *P < 0.05, **P < 0.01. The sorted population experiences a significant boost in every parameter compared with the raw semen.

Figure 7 also highlights the percentage of sperm viability and motility in two populations. As expected, the sorted population was both fully motile and viable, as the lengthy channel of diamond pillars did not allow the migration of immotile sperms or those that were somewhat defective.

FIG. 7.

Results of viability and motility of sorted and raw sperm samples. The values are reported as mean ± standard deviation (n = 4), *P < 0.05, **P < 0.01. While viability and motility of the raw sample were nearly 50% and 40%, respectively, the sorted population exhibited 100% viability and motility.

IV. CONCLUSION

We demonstrated a novel high-throughput microfluidic device for the isolation of highly motile sperms without manual intervention and initial sample processing based on rheotaxis and boundary-following behavior. The proposed device consists of parallel diamond-shaped geometries, creating hydrodynamic barriers in the path of sperms. Highly motile sperms are more likely to overcome the generated barriers, whereas less motile sperms would be trapped in a periodic swimming pattern between the pillars.

The numerical simulation demonstrated the hypothesis behind the sorting mechanism, which was further validated in the experiments. The diamond pillars were able to isolate highly motile sperms with a high degree of swimming persistency in the sorting chamber. The efficacy of the method was corroborated with analyzing sperm motility parameters, which illustrated more than 35% improvement in sperm motility parameters. The sorted population was isolated in a time-frame short of 20 min, which ensures the perseveration of sperm integrity during the process.

SUPPLEMENTARY MATERIAL

See the supplementary material for videos showing sperm motion patterns within the microfluidic channel near the shear barriers.

AUTHOR DECLARATIONS

Conflict of Interest

Authors have no conflicts to declare.

Ethics Approval

The experiments were conducted in compliance with ethical guidelines, with informed consent obtained from all patients. The study was approved by the ethical review board of Iran University of Medical Sciences with the approval number of IR.IUMS.REC.1403.625.

Author Contributions

Mohammadjavad Bouloorchi Tabalvandani: Conceptualization (lead); Investigation (lead); Methodology (lead); Supervision (equal); Writing – original draft (equal); Writing – review & editing (equal). Zahra Saeidpour: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Validation (equal); Writing – original draft (equal); Writing – review & editing (equal). Zahra Habibi: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Software (equal); Validation (equal); Visualization (equal). Saeed Javadizadeh: Data curation (equal); Methodology (equal); Software (equal). Majid Badieirostami: Project administration (lead); Resources (lead); Supervision (lead); Validation (equal); Writing – review & editing (equal).

DATA AVAILABILITY

The data that support the findings of this study are available within the article and its supplementary material.