Separation of sperm based on rheotaxis mechanism using a microfluidic device
Article information
Abstract
Objective
In the application of assisted reproductive technologies (ART), selection of the optimal sperm presents a challenge. This study introduces an innovative microfluidic device that utilizes rheotaxis to efficiently sort sperm, offering superior selection of high-quality sperm compared to conventional methods.
Methods
We analyzed 30 normal samples from couples undergoing intracytoplasmic sperm injection cycles at the Infertility Center of Fatemieh Hospital in Hamadan, Iran. Each sample was divided into three groups: the initial sample, representing the control group; direct swim-up sperm selection; and sperm selection using rheotaxis. A syringe pump connected to the microfluidic device generated optimal flow conditions. Spermatozoa were evaluated regarding concentration, motility, morphology, mitochondrial membrane potential (MMP), and sperm DNA fragmentation index (DFI). Statistical significance was determined using one-way analysis of variance and the Student t-test.
Results
The concentration (7.46±2.84 million cells/mL vs. 56.67±18.27 million cells/mL, p<0.0001) and DFI (2.93±2.70 vs. 21.13±5.27, p<0.0001) were significantly lower in the sperm selected using the rheotaxis microfluidic device than in the control sperm. Progressive motility (98.10%±2.41% vs. 44.13%±7.06%, p<0.0001), normal morphology (8.36%±1.47% vs. 5.20%±1.15%, p<0.0001), and MMP (99.63%±0.71% vs. 81.13%±9.19%, p<0.0001) were significantly higher with the device than in the control group.
Conclusion
The use of a rheotaxis-based microfluidic device appeared effective in selecting high-quality sperm, demonstrating improvements in motility, morphology, and MMP and a reduction in DFI. This advancement has the potential to improve the outcomes of ART.
Introduction
Over 48 million couples (nearly 15%) experience infertility, an issue that is on the rise globally [1,2]. Assisted reproductive technology (ART), with methods such as intrauterine insemination, in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI), offers a viable option for couples facing infertility or recurring challenges in conceiving naturally [3]. Recent studies on ART usage indicate that ICSI has surpassed conventional IVF, now representing over 70% of all IVF procedures [4,5]. However, a major concern with ICSI is that it bypasses the natural selection barriers that sperm typically face in vivo, potentially allowing a ‘bad’ spermatozoon to fertilize the oocyte. This could alter embryonic development and increase the risk of genetic disorders [6]. For sperm preparation during ART, traditional techniques such as swim-up and density gradient centrifugation are frequently employed [7]. For years, these methods have been utilized to prepare spermatozoa for conventional IVF. By default, they are also used to select sperm for ICSI, despite being less than ideal for this purpose [8]. Many sperm preparation techniques are ineffective at isolating the most suitable spermatozoa, as selection is based solely on motility and morphology [3]. Furthermore, these methods involve centrifugation, which can be extremely harmful to sperm and may damage sperm DNA [9]. The capacity of human spermatozoa to produce a high-quality embryo with a strong likelihood of implantation and development is influenced by various factors, including DNA integrity [10]. Elevated levels of sperm DNA fragmentation have been found to impair embryo morphokinetic characteristics following ICSI [11]. Accordingly, the extent of DNA damage in spermatozoa is directly associated with the incidence of spontaneous pregnancy loss [12].
To improve the success rates of ART, it is crucial to develop methods that simulate the natural selection process that occurs in vivo. These techniques should enable the rapid, automated, and non-invasive selection of high-quality sperm [13]. Natural guiding systems play a key role in identifying the most suitable spermatozoa for fertilization. Research indicates that thermotaxis and chemotaxis are essential natural mechanisms used by spermatozoa to recognize the oocyte [14]. These mechanisms may help sperm overcome various challenges, such as muscle contractions, biochemical barriers, and ciliary currents [15,16]. However, these processes may be important only when sperm are extremely close to the oocyte [17]. In contrast, rheotaxis, the ability of sperm to swim upstream against the flow, is considered a key guiding mechanism over longer distances [18]. In the absence of external fluid flow, sperm tend to move in circular patterns [19]. In contrast, when subjected to shear flow, sperm reorient themselves. This is due to the increased resistive force on the sperm head compared to the tail, as well as the drag forces from the flow acting on the conical envelope of the flagellar wave [20]. Hydrodynamic principles provide a clear explanation for this process [21].
Microfluidics is the study of systems that can process and transport minute volumes of liquid through microchannels at the microscale [22]. The capacity of microfluidics to precisely regulate and manipulate fluidic microenvironments has prompted its use in the isolation of motile sperm through rheotaxis [23]. Recent advancements in microfluidics are increasingly recognized as a promising platform for reproductive clinics to optimize IVF procedures [24]. However, most of these devices are currently employed for research purposes and have limited therapeutic applications. To improve ART outcomes, the process of selecting high-quality spermatozoa must be optimized. The aim of the present research is to design a simple, cost-effective microfluidic device that does not require centrifugation. This device will employ rheotaxis to selectively sort healthy sperm, facilitating a subsequent assessment of its effectiveness compared to both swim-up and control groups.
Methods
1. Ethics statement
All ethical protocols were followed in accordance with the guidelines set forth by the Ethics Committee of the Iran University of Medical Sciences (ethics number IR.IUMS.REC.1402.1191). Written consent was acquired from every participant involved in this research.
2. Patient selection
Semen samples were collected from 30 couples undergoing ICSI cycles who were referred for semen analysis to the Andrology Unit of the Infertility Center at Fatemieh Hospital in Hamadan, Iran, between July 2023 and March 2024. The selected participants met specific inclusion criteria; namely, they had normal sperm parameters as defined by the World Health Organization (WHO) [25] and were between 20 and 40 years old [14]. Exclusion criteria for the study included severe male factor infertility, use of cryopreserved sperm samples, sperm retrieval via testicular aspiration or extraction, history of varicocele, drug abuse, or heavy smoking or alcohol consumption.
The semen sample from each of the 30 patients was divided into three equal portions for further analysis, as outlined in the experimental design section. This strategy facilitated intrapatient comparisons across the various methods.
3. Experimental design
After 3 to 5 days of abstinence, couples referred to the andrology unit of the infertility center provided ejaculated sperm samples, which were collected in sterile plastic containers and allowed to liquefy at room temperature (RT). Portions of each semen sample were then allocated to three groups for analysis (Figure 1). These included (1) a control, consisting of the initial sample; (2) sperm selection via the direct swim-up method (DSU) (Figure 2A); and (3) sperm selection using a rheotaxis microfluidic device (Figure 2B). Within the device, we employed a syringe pump to generate a controlled flow at a predetermined velocity. This allowed spermatozoa to actively swim against the flow, enabling their movement through the device. The spermatozoa that successfully navigated and penetrated the microfluidic device were then collected from the outlet for subsequent analysis.
Schematic diagram of the study design. JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide.
Schematic illustration of the procedure. (A) Rheotaxis in the microfluidic device for sperm selection. The microfluidic channel measured 200 µm wide, 150 µm deep, and 8 mm long. It connected to a syringe pump (MMT-SP-102; Mizan Micro Tech) via silicone tubing and a connector. A 20-µL sperm sample was loaded into inlet A of the microfluidic channel, which allowed spermatozoa to orient and swim against the oncoming flow from inlet B (thus demonstrating positive rheotaxis) for 45 minutes. The device also featured an outlet area for the egress of immotile and nonprogressive sperm along with the flow of the medium. After this period, spermatozoa that had reached the collection chamber were collected for further analysis. (B) Standard direct swim-up sperm selection.
To better understand the rationale behind the microfluidic device design, it is important to consider the natural conditions within the human reproductive system. Sperm navigate through microenvironments such as the cervical mucus, the uterotubal junction, and the fallopian tubes, where they encounter varying fluid flows. Our device uniquely mimics these in vivo conditions by optimizing flow velocities to closely reflect the physiological range found in the fallopian tubes, which is between 6.5 and 30 µm/sec. This range of velocities, unlike those used in other devices, was selected based on detailed studies of tubal fluid dynamics. The strategic placement of the outlet adjacent to inlet A further replicates natural conditions by allowing only motile sperm to swim against the flow. This simulates the natural selection mechanism found in vivo and increases the likelihood of selecting sperm with high motility and genetic integrity.
4. Sperm selection techniques
In the DSU group, a total volume of 1.2 mL of VitaSperm (Inoclon) containing albumin was carefully layered over 1 mL of semen. The tube was then positioned at a 45° angle and incubated at 37 °C in an atmosphere containing 6% CO2 (Figure 2A). After 45 minutes, the supernatant was aspirated and transferred to an empty tube, where it was diluted with an equal volume of VitaSperm+albumin and centrifuged at 1,500 rpm for 15 minutes. Following centrifugation, the supernatant was removed, and 1 mL of VitaSperm with albumin was added to the pellet. The contents were then thoroughly mixed [25].
For the rheotaxis group, a microfluidic device was employed, designed based on natural sperm selection mechanisms [26]. The device was manufactured at the Mizan Microchip Technology Laboratory in Tehran, Iran. Using CAD software (AutoCAD; Autodesk Inc.), the three-dimensional geometry of the microfluidic device was carefully designed. A soft lithography technique was employed for its construction. The device, made from the polymer polydimethylsiloxane (PDMS), was sterilized with ethylene oxide gas and stored in a clean plastic bag after each use. PDMS was chosen for its favorable mechanical properties, elasticity, optical transparency, biocompatibility, and simple manufacturing process.
The design of the microfluidic device was optimized to minimize shear stress on sperm cells, while exposing them to a controlled and regulated flow for selection via rheotaxis. In contrast to other microfluidic devices that may not fully account for fluid dynamics, the dimensions of our channel—200 μm in width, 150 μm in depth, and 8 mm in length—were selected based on hydrodynamic modeling. This provides a more accurate simulation of sperm movement in vivo. Such attention to detail complements the non-invasive nature of our selection process, which preserves the integrity and functionality of sperm DNA compared to traditional centrifugation-based methods.
The device features a chamber with a diameter of 1,300 µm, as well as two inlets: inlet A, which introduces semen into the system, and inlet B, through which the VitaSperm flow enters. An outlet area enables the expulsion of immotile and nonprogressive sperm along with the flow of the medium. The chamber is designed to collect motile sperm that successfully navigate the device (Figure 3).
Sections of the microfluidic device.
5. Experimental setup for rheotaxis sperm selection
Inlet B of the microfluidic device was connected to a syringe pump (MMT-SP-102; Mizan Micro Tech) using silicone tubing and a connector. This setup produced a controlled flow of VitaSperm at a predetermined velocity within the channel. A 20-µL sample of normozoospermic sperm was introduced into inlet A of the microfluidic channel. This allowed the spermatozoa to align themselves against the incoming VitaSperm flow and actively swim upstream, a phenomenon known as positive rheotaxis, for 45 minutes.
Our microfluidic device operates at four distinct flow velocities: 6.5, 12, 24, and 30 µm/sec. This mirrors the range of natural flow rates observed in the female reproductive tract, particularly within the fallopian tubes, where sperm encounter varying shear forces. The physiological relevance of these flow rates differentiates our device from other microfluidic systems that utilize arbitrary or fixed flow rates, ensuring that sperm are exposed to more natural conditions during selection. Of these four flow velocities, we identified the one that facilitated the most effective sperm separation based on rheotaxis within the microfluidic device, designating it as the optimal flow velocity. This optimal velocity had the greatest impact on sperm separation, as evidenced by the higher sperm concentration (in million cells/mL) and better progressive motility of the collected sperm. Subsequently, we compared the sperm separated at this optimal flow velocity with those from the DSU and control groups. Following the experiment, the sperm that successfully navigated to the collection chamber were collected for further analysis.
6. Evaluating the cellular toxicity of the microfluidic device
The human sperm survival assay (HSSA) was employed to assess the cytotoxic effects associated with the microfluidic device. Twelve semen samples were collected from individuals with normal sperm parameters. These samples were processed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered medium, and the resulting sperm suspensions were used in the HSSA. For the experimental group, the device was aseptically inserted into a conical tube containing 1 mL of the sperm suspension. Conversely, in the control group, the conical tube contained only the 1-mL sperm suspension. The tubes were maintained at RT. At predetermined time points—0, 1, 2, 3, 4, 5, 24, 48, and 72 hours—samples of the sperm suspension were collected. Using a sterile pipette, 10 μL of the suspension was deposited onto a glass slide, covered with a coverslip, and examined microscopically. The survival index (SI) was calculated based on the percentage of experimental progressive motile spermatozoa. The SI was calculated by dividing the percentage of progressive motile spermatozoa in the experimental group by the corresponding percentage in the control group at the respective time points. An SI value below 85% was considered indicative of potential toxicity [27].
7. Measurement and analysis of sperm parameters
Sperm parameters were evaluated according to 2010 WHO guidelines.
1) Evaluation of average sperm concentration
Spermatozoa were observed and counted using an improved Neubauer hemocytometer under a microscope. Average sperm concentrations were determined in accordance with the prescribed methodologies outlined in the WHO laboratory manual [25].
2) Assessment of sperm morphology
To evaluate the impact of sperm selection on morphology, Diff-Quik staining (MICROPTIC S.L. Co.) was performed [28]. The procedure was conducted in accordance with the manufacturer’s instructions. Approximately 10 µL of the sperm sample was spread on a clean slide and allowed to air dry for a minimum of 10 minutes. Following the handbook guidelines, the slides were then stained and examined using a bright-field microscope. A total of 200 spermatozoa from each sample were evaluated and classified according to their morphology, including normal or abnormal head, midpiece, and tail structures. The proportion of abnormal spermatozoa was reported as a percentage [29].
3) Determination of sperm motility
Sperm motility was evaluated by ascertaining the proportions of spermatozoa exhibiting progressive and nonprogressive motility. Progressive motility refers to spermatozoa that move actively in a straight line or in large circles, regardless of speed. Nonprogressive motility encompasses all other patterns of movement that do not result in forward progression [14]. To assess sperm motility, semen samples were warmed to a temperature of 37 °C. Motility was then measured using computer-assisted sperm analysis. A 10-μL aliquot of semen was loaded into a pre-warmed sperm counting chamber equipped with a cover slide, exhibiting a chamber height of 20 μm. At least 200 spermatozoa were evaluated across a minimum of five fields. The results were reported as percentages, representing the total and progressive motility [30].
8. DNA fragmentation assay
The DNA fragmentation index (DFI) was assessed using an sperm DNA fragmentation assay (SDFA) kit on sperm samples (Halo kit; Idehvarzan Farda Company). A 50-μL aliquot of sperm suspension was combined with low-melting agarose. Then, 20 µL of this mixture was placed on a pre-coated glass slide. The droplet was covered with a coverslip and maintained at 4 °C for 5 minutes. After the coverslip was gently removed, the slide was treated with a denaturing solution, referred to as solution A, for 7 minutes at RT in the dark. This was followed by the application of lysing solution (termed solution B) for 15 minutes at RT. The slide was then rinsed with distilled water for 5 minutes and sequentially dehydrated in ethanol solutions of 70%, 90%, and 100% for 2 minutes each before being allowed to air dry at RT. Staining was performed using solution C for 75 seconds, solution D for 3 minutes, and solution E for 2 minutes. After a final rinse with distilled water, at least 200 sperm were evaluated under bright-field microscopy. The extent of DNA fragmentation was determined by the size of the halo around the sperm head. Spermatozoa with large or medium halos were classified as having intact DNA, whereas those with small or no halos were considered to have fragmented DNA [31]. A DFI value of >30% was employed as a cutoff [32].
9. Evaluation of sperm mitochondrial membrane integrity using JC-1 dye
The mitochondrial membrane potential (MMP) was assessed using the lipophilic cationic dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) [33]. For JC-1 staining, stock solution (Cayman Chemical Company; cat. No. 110009172) was diluted at a 1:10 ratio in the culture medium according to the manufacturer’s instructions. To conduct the assay, 1 μL of the JC-1 working solution was combined with 9 μL of sperm suspension and incubated in a humidified chamber at 37 °C with 5% CO2 for 30 minutes. Mitochondrial activity was then analyzed using a fluorescence microscope (Olympus) at a magnification of ×1,000, with the samples placed under a coverslip in immersion oil. The MMP status was determined based on the fluorescence pattern of 200 spermatozoa across five different microscopic fields per slide. Orange-red fluorescence was indicative of high MMP, corresponding to active mitochondria [34]. The results were expressed as the percentage of sperm with high MMP.
10. ICSI, fertilization rate, and embryo quality assessment
Ovarian stimulation was conducted using the antagonist protocol. Metaphase II oocytes from each couple were randomly allocated into two groups. One group underwent ICSI with sperm prepared using the DSU method, while the other group received ICSI with sperm selected using the rheotaxis-based microfluidic device. Fertilization rates were evaluated 16 to 18 hours following ICSI, and embryo quality was assessed 44 to 48 hours after ICSI.
The embryo grading system was defined as follows: top-quality embryos were characterized by regular blastomere symmetry, less than 10% fragmentation, and no evidence of multinucleation; fair-quality embryos had uniform blastomeres, less than 25% fragmentation, and unclear multinucleation; and poor-quality embryos exhibited uneven blastomeres, greater than 25% fragmentation, and clear signs of multinucleation [35].
11. Statistical analysis
To assess the normality of the data distribution, the D'Agostino-Pearson test was employed. One-way analysis of variance was then performed, followed by the Student t-test and Fisher exact test, to determine the statistical significance of the observed differences. p-values of less than 0.05 were considered to indicate statistical significance. All statistical analyses were conducted using GraphPad Prism ver. 8.4.2 (GraphPad Software Inc.). The results were presented as box-and-whisker plots, with the box representing the 25th and 75th percentiles, the line within the box indicating the median value, and the whiskers showing the 10th and 90th percentiles.
Results
1. Determination of optimal flow velocity within the microfluidic chip
To identify the optimal flow conditions within the microfluidic chip, we considered two parameters: the average sperm concentration (in million cells/mL) and the progressive motility of sperm collected by the system. We evaluated four flow velocities: 30, 24, 12, and 6.5 µm/sec. At a flow velocity of 6.5 µm/sec, the progressive sperm motility rate was 80.40%±8.35%, with an average sperm concentration of 2.074±9.40 million cells/mL. Increasing the flow velocity to 12 µm/sec resulted in a higher progressive motility rate of 87.80%±6.90%, although the average sperm concentration decreased to 1.581±7.00 million cells/mL. At 24 µm/sec, the progressive motility rate further improved to 93.00%±4.12%, and the average sperm concentration was 2.34±6.00 million cells/mL. Finally, at the highest tested flow velocity of 30 µm/sec, the progressive motility rate peaked at 95.20%±3.56%, and the average sperm concentration was 1.22±3.00 million cells/mL (Figure 4). Thus, regarding the optimal flow velocity for sperm separation based on the rheotaxis mechanism, a flow velocity of 24 µm/sec was carefully selected.
Examination of varying flow velocities within the chip in relation to sperm concentration (Conc.) and progressive motility (Motil.).
2. Evaluation of sperm parameters
The effects of sperm selection using either a rheotaxis microfluidic device or DSU on average sperm concentration, morphology, and motility are depicted in Figure 5. Analysis of average sperm concentration across the tested methods indicated a significantly lower average sperm concentration following preparation with both DSU and microfluidic methods compared to the initial sample (p<0.0001). Furthermore, the microfluidic method resulted in a more pronounced decrease in average sperm concentration than the DSU method (p<0.0001) (Figure 5A).
Evaluation of (A) average sperm concentration, (B) progressive motility, and (C) morphology in the studied groups. a)p<0.0001.
Analysis of the initial sample revealed a progressive sperm motility rate of 44.13%±7.06%. In comparison, this rate was 85.03%±9.64% with the DSU method and 98.10%±2.41% with the microfluidic device. This represented a significant improvement in the average progressive motility of sperm for both the swim-up and microfluidic preparation techniques relative to the initial sample (p<0.0001). Furthermore, compared to the DSU preparation method, the microfluidic approach was associated with a significantly higher average progressive sperm motility (p<0.0001) (Figure 5B).
The assessment of sperm morphology revealed that the initial sample contained 5.20%±1.15% normally shaped sperm. In contrast, the DSU method yielded 6.33%±1.51% normal morphology, while the microfluidic method showed a substantial improvement, with 8.36%±1.47% normal morphology. The comparison of sperm morphology indicated significant increases in the proportion of normal sperm for both the DSU and microfluidic methods compared to the initial sample (p<0.0001) (Figure 5C).
3. Evaluation of sperm DFI in the studied groups
The analysis of sperm DFI following sperm selection using the rheotaxis microfluidic device and DSU is presented in Figure 6A. The initial sample exhibited a sperm DFI of 21.13%±5.27% (Figure 7A). In comparison, the DSU method yielded a DFI of 5.54%±11.03% (Figure 7B), while the microfluidic method resulted in a substantially lower DFI of 2.70%±2.93% (Figure 7C). Notably, the sperm DFI rate was significantly lower following preparation via the microfluidic method compared to DSU (p<0.0001).
(A) Evaluation of DNA fragmentation index in the studied groups. (B) Assessment of mitochondrial membrane potential status in the studied groups. a)p<0.0001; b)p<0.01.
Representative images of sperm DNA fragmentation assessment under different conditions using the sperm DNA fragmentation assay with the Halo kit (Idehvarzan Farda Company), stained according to the manufacturer’s protocol, at ×400 magnification. (A) initial semen sample, (B) sperm separated using the direct swim-up method, and (C) sperm selected with the microfluidic method. The images reveal variations in DNA fragmentation patterns among the samples, highlighting differences between the methods. The figure illustrates the comparatively low level of DNA fragmentation following sperm selection via the microfluidic approach, with sperm in 'a' displaying large halos, indicative of intact DNA, and sperm in 'b' exhibiting small or absent halos, signifying fragmented DNA.
4. Evaluation of the MMP of motile sperms in the studied groups
The MMP status of motile sperm following sperm selection using the rheotaxis microfluidic device and DSU is depicted in Figure 6B. The MMP of motile sperm in the initial sample was measured at 81.13%±9.19% (Figure 8A). Following sperm selection, the MMP increased significantly to 97.60%±3.10% with the DSU method (Figure 8B) and was even higher, at 99.63%±0.71%, using the microfluidic method (p<0.0001) (Figure 8C). Moreover, the difference in MMP between the microfluidic method and the DSU approach was statistically significant (p<0.01).
Representative images of mitochondrial membrane potential (MMP) status assessment of motile sperm under different conditions with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) staining, at ×400 magnification. (A) initial semen sample, (B) sperm separated using the direct swim-up method, and (C) sperm selected with the microfluidic method. The images illustrate variations in MMP patterns among the samples, featuring 'a' sperm with low mitochondrial activity (green fluorescence) and 'b' sperm with high mitochondrial activity (orange-red fluorescence).
5. Fertilization rate and embryo quality
The impact of sperm preparation technique on ICSI outcomes was assessed by examining fertilization rates and embryo quality after ICSI. Table 1 compares these parameters between sperm prepared using the DSU method and those selected with the microfluidic device. The results demonstrate that sperm selection via the microfluidic device led to a significantly higher fertilization rate in ICSI compared to the DSU method (p=0.03). Additionally, as detailed in Table 1, the microfluidic technique markedly improved embryo quality, yielding a greater number of top-quality embryos relative to the DSU method (p=0.03).
Results of fertilization rate and embryo quality
Discussion
The optimization of ARTs necessitates sperm processing methods that mimic natural selection without compromising sperm integrity. Current techniques, which involve centrifugation, pose risks to sperm DNA and morphology. Thus, developing safer methods and educating the medical community about their benefits is essential [13,14]. The aim of this study was to assess the impact of sperm selection techniques, such as rheotaxis, on sperm characteristics and functionality. We designed and constructed an innovative in vitro model that simulates natural sperm selection based on rheotaxis. By connecting a syringe pump to the microfluidic device, we established optimal flow within the system. Our goal is to replicate natural selection processes to achieve higher success rates in assisted reproduction.
In selecting flow velocities of 6, 12, 24, and 30 µm/sec for the rheotaxis within the microfluidic device, we aimed to closely mimic the physiological conditions of the female reproductive tract. The natural flow velocities within the fallopian tubes typically range from 6.5 to 30 µm/sec [36]. Our study sought to replicate and examine this range of velocities to understand how sperm respond to various rheotaxis forces. This approach provides insights into their movement and selection mechanisms under conditions that closely resemble in vivo environments.
Additionally, toxicity assessment revealed that the microfluidic device is non-toxic and safe for use, as evidenced by sperm survival rates consistently exceeding 85% across various time intervals. This suggests that the device preserves sperm viability and motility without causing harmful effects. These findings confirm that the microfluidic system offers a gentle, non-invasive environment for sperm selection, further supporting its potential for use in assisted reproduction without compromising sperm integrity.
The choice of flow velocities of 6, 12, 24, and 30 µm/sec was deliberate, covering the lower and upper limits of the physiological range. The lower velocities, 6 and 12 µm/sec, correspond to the slower end of tubal flow, enabling the observation of sperm behavior in conditions resembling the early stages of their journey through the female reproductive tract. The intermediate velocity of 24 µm/sec serves as a bridge between the lower and upper extremes, providing a critical point for evaluating sperm response and selection as they navigate the fallopian tubes. The highest velocity of 30 µm/sec represents the upper physiological threshold, helping to elucidate how sperm adapt and react to the faster flow velocities that may be present in certain areas of the female reproductive tract.
The selection of 24 µm/sec as the optimal flow velocity for sperm separation based on rheotaxis reflects a balance between sperm motility and concentration. This velocity was determined through careful experimental evaluation to optimize both parameters, thus yielding the highest-quality sperm sample for ICSI procedures. At flow velocities below 24 µm/sec, sperm motility decreased, as the slower flow dynamics permitted even less motile sperm to reach the collection chamber. This reduced motility could compromise the quality of the separated sperm sample, underscoring the importance of choosing a flow velocity that balances motility with separation efficiency. In contrast, at velocities exceeding 24 µm/sec, the data reveal a more complex relationship between flow velocity, motility, and sperm concentration. Contrary to the expectation that higher flow velocities would result in a lower average sperm concentration due to more rapid flushing, our findings did not exhibit a strict negative correlation. A possible explanation is that at elevated flow velocities, such as 24 and 30 µm/sec, the microfluidic system preferentially captures highly motile sperm, which are more capable of withstanding the increased flow forces and are thus retained in the collection chamber. This selective retention could explain why sperm concentration remained relatively stable, even as progressive motility increased. Furthermore, at higher flow velocities, sperm exhibiting greater motility are more likely to be retained within the system, while less motile sperm are expelled more swiftly. This creates a dynamic interplay that may not lead to a straightforward reduction in concentration. The average sperm concentration in the collected samples reflects a balance between these effects, which may not consistently demonstrate a simple inverse correlation with flow velocity. Additionally, the introduction of shear stress at higher flow velocities can improve motility but may also disrupt sperm aggregation, contributing to variability in the measured concentration.
In our investigation, we uncovered an intriguing relationship between the methods used for sperm isolation and the resulting sperm quality. Our findings demonstrated that the average sperm concentration obtained with the rheotaxis-based microfluidic device was significantly lower than that of the DSU group. These findings align with previous research conducted by Quinn et al. [37], as well as Romero-Aguirregomezcorta et al. [28]. Furthermore, sperm selected via the microfluidic technique demonstrated notably higher total motility, higher progressive motility, and improved morphology compared to sperm isolated using DSU. These results are consistent with the findings of Huang et al. [38].
The microfluidic method utilizes rheotaxis to provide a gentle selection process for sperm, minimizing shear forces and potential damage to these fragile cells. This approach is essential for preserving the integrity and viability of the sperm population during isolation [39]. The gentle selection occurring in the microfluidic method may exclude some less motile or compromised sperm, contributing to the observed reduction in average sperm concentration. In contrast, the swim-up method, although effective, may expose sperm to greater mechanical stress during isolation, potentially resulting in higher retention of less motile sperm. The differences in average sperm concentration and motility observed between the microfluidic and swim-up methods can therefore be attributed to the careful selection process that is characteristic of the microfluidic approach [13,40].
The morphology of sperm is pivotal in determining its function and ability to move effectively. Sperm with normal morphology generally exhibit better motility, viability, and DNA integrity. The application of microfluidic techniques can be instrumental in minimizing the proportion of sperm with abnormal morphology, such as those with head or tail defects. Utilizing microfluidic technology can effectively reduce the presence of these anomalies, thus improving the overall quality and functionality of the sperm [41,42].
The standard sperm preparation techniques used in ARTs involve multiple centrifugation steps that may be detrimental to sperm integrity [43,44]. Current centrifugation-based methods for sperm selection in ARTs are not inspired by natural sperm selection in the female genital tract, thus differing from in vivo conditions [20]. Consequently, semen samples processed through these centrifugation techniques have been shown to exhibit an elevated DFI and an increase in reactive oxygen species (ROS) production [45]. In our results, sperm processed using the microfluidic approach had significantly lower sperm DNA fragmentation compared to the DSU group. This aligns with the findings of Romero-Aguirregomezcorta et al. [28], who reported that sperm selection via a rheotaxis-based microfluidic device significantly reduced DNA fragmentation in comparison to a control group. Similarly, Ataei et al. [18] observed a marked improvement in DNA fragmentation with the use of a rheotaxis-based microfluidic method.
In this study, we assessed MMP using the fluorescent marker JC-1, adhering to the protocols established by Smiley et al. [33]. Mitochondria in the sperm midpiece serve as sources of adenosine triphosphate generating and transporting the energy crucial for sperm motility. Our findings reveal a significantly higher percentage of spermatozoa with favorable MMP when selected via rheotaxis, as opposed to those in the DSU group and the initial sample.
Furthermore, the DSU group exhibited lower sperm motility and a significantly higher DFI than the microfluidic group. This suggests that the high-speed centrifugation applied in the DSU method, which leads to increased production of ROS, could potentially damage the mitochondrial membrane and sperm DNA. This would ultimately diminish the forward progression of the spermatozoa. Our results align with previous reports that highlight a positive correlation between progressive motility and sperm MMP [46].
Our study reinforces the established correlation between MMP and sperm functionality, while exploring the potential value of MMP as an additional indicator in sperm selection. By selecting spermatozoa with higher MMP, the rheotaxis-based microfluidic device may provide a more sophisticated approach to promoting fertilization potential and embryo development. Incorporating mitochondrial functionality into the sperm selection process introduces an additional layer that could improve outcomes in ART. While MMP has been examined in various contexts, its application in this setting provides further insights into the potential impact of sperm energy status on reproductive success.
Upon assessing the clinical outcomes, we found that samples processed with the rheotaxis-based microfluidic device demonstrated significantly higher fertilization rates and improved embryo quality compared to those prepared with the DSU technique. These functional outcomes strongly support the clinical relevance of the microfluidic device, given that its capacity to improve both fertilization rates and embryo quality has direct implications for the success rates of ART.
Our findings align with those of Romero-Aguirregomezcorta et al. [28], who reported improved fertilization rates and early embryo development with the use of a microfluidic device for rheotaxis-based sperm selection. Notably, however, this prior study was conducted using mouse samples, while our research utilized human samples. This distinction highlights the potential translational impact of our findings, as demonstrating improvements in human samples offers more direct clinical relevance for ART applications.
Our work is distinguished by the detailed reporting of fertilization rates and embryo quality, functional outcomes that have not often been emphasized in studies of similar microfluidic devices. This highlights the potential of rheotaxis-based sperm selection to improve ART outcomes in humans, a potentially underexplored area in previous research on microfluidic devices.
This study introduces a microfluidic device designed for sperm selection based on the rheotaxis mechanism, highlighting several advantages as well as limitations. A key benefit of this approach is that it represents a gentle and non-invasive technique for sperm selection, potentially reducing the likelihood of DNA damage compared to traditional methods such as swim-up and density gradient centrifugation. The spermatozoa selected using this in vitro model were evaluated in various experiments, demonstrating the potential applicability of the technique for patients undergoing ICSI treatment.
However, some limitations of this study must be considered. As the sample size of 30 participants was relatively small, larger studies are needed to confirm our findings and assess their reproducibility across more diverse populations. Furthermore, the data were sourced from a single infertility center; thus, conducting similar studies with a multicenter design would provide a more thorough understanding of the device’s effectiveness. Another limitation is the exclusive use of normozoospermic samples, which does not address the device’s performance with samples from patients experiencing severe male factor infertility.
This study demonstrates the potential of rheotaxis-based microfluidics in sperm selection, revealing improvements in motility, morphology, and MMP along with a reduction in DNA fragmentation. Although further research is required, this approach has the potential to improve the effectiveness of ART, offering a promising avenue for future studies in the field of reproductive science.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The authors wish to express their sincere gratitude to the Council for Development of Regenerative Medicine and Stem Cell Technologies for its invaluable support and collaboration. The Council’s unwavering dedication to advancing research in regenerative medicine and associated technologies played a pivotal role in facilitating this study. We are also deeply thankful to all the participants who contributed to this research. Their involvement was crucial for collecting the data and insights detailed in this article.
Author contributions
Conceptualization: MM. Methodology: HT, SP, RD. Formal analysis: SP, RM. Data curation: HT, RS, LT, RD. Funding acquisition: MM. Project administration: MM. Visualization: RS, MM. Writing-original draft: HT, LT. Writing-review & editing: RS, MM, RD. Approval of final manuscript: HT, SP, RS, RM, LT, MM, RD, MM.