To enhance assisted reproductive technologies, we require sperm processing methods that imitate natural selection without compromising sperm integrity. Current techniques, which involve centrifugation, pose risks to sperm DNA and morphology. Innovating safer methods and educating about their benefits are crucial (13, 14). The objective of this study was to evaluate how sperm selection techniques, like rheotaxis, influence the characteristics and functionality of sperm. We innovatively designed and constructed an in vitro model replicating natural sperm selection based on rheotaxis. Utilizing a syringe pump connected to the microfluidic device, we ensured an ideal flow within the system. By replicating natural selection, we aim for higher success rates in assisted reproduction.
In selecting the specific flow velocities of 6, 12, 24, and 30 µm/s for the rheotaxis flow within the microfluidic device, careful consideration was given to mimic physiological conditions observed in the female reproductive tract. The natural tubal flow velocities within the fallopian tubes typically range between 6.5 and 30 µm/s (35). Our study aimed to replicate and explore this range of velocities to assess how sperm respond to various rheotaxis forces, providing insights into their movement and selection mechanisms under conditions closely resembling in vivo environments.
The choice of 6, 12, 24, and 30 µm/s flow velocities was deliberate, encompassing the lower and upper bounds of the physiological range. The lower velocities (6 and 12 µm/s) represent the lower spectrum of tubal flow, allowing us to observe sperm behavior under conditions akin to the initial stages of the fertilization journey within the female reproductive tract. The intermediate flow velocity (24 µm/s) bridge the lower and upper bounds, offering a crucial midpoint for assessing sperm response and selection as they traverse the fallopian tubes. Lastly, the highest flow velocity (30 µm/s) represents an upper physiological limit, aiding in understanding how sperm adapt and respond under faster tubal flow velocities, which might be encountered in certain regions of the female reproductive tract.
In the examining of the optimal flow velocity for separating sperm based on the rheotaxis mechanism a flow velocity of 24 µm/s was carefully chosen. This particular flow velocity was identified as optimal through a meticulous evaluation of two key parameters sperm motility and concentration.
At flow velocities below 24 µm/s, it was noted that sperm motility decreased. The slower flow dynamics allowing even less motile sperms to reach the collection chamber. This diminished mobility could potentially compromise the quality of the separated sperm sample, emphasizing the importance of an optimum flow velocity that balances mobility and separation efficiency.
on the other hand, at flow velocities higher than 24 µm/s, a significant count of sperms was observed in outlet, failing to reach the intended collection chamber. This departure resulted in a notable decrease in sperm concentration within the collection chamber. The decrease in concentration at higher flow velocities suggests that the flow dynamics became too rapid for effective retention of the sperm within the microfluidic system, significantly impacting the concentration of collected sperms.
In our investigation, we discovered a fascinating interplay between the choice of sperm isolation methods and the resulting sperm quality. Our findings demonstrated that the sperm concentration in the rheotaxis -based microfluidic device was significantly lower compared to the sperm prepared by the DSU group. This report concurs with the outcomes of earlier research carried out by Quinn M.M and colleagues, as well as Aguirregomezcorta and colleagues. (28, 36). Moreover, the total motility, progressive motility, and morphology of the sperm selected through the microfluidic method based on rheotaxis exhibited a marked increase compared to those of the sperm prepared by the DSU group. These results align with the study carried out by Huang (37).
The microfluidic method, harnessing rheotaxis, offers a gentle selection process for sperm, minimizing shear forces and potential damage to fragile sperm. This gentle approach is crucial in preserving the integrity and viability of the sperm population during isolation (38). Consequently, the microfluidic method's gentle selection process may lead to the exclusion of some less motile or compromised sperm, contributing to the observed reduction in sperm concentration. In contrast, the swim-up method, while effective, may subject sperm to more mechanical stress during the isolation process, potentially retaining a higher concentration of less motile sperm. The observed differences in sperm concentration and motility between the microfluidic and swim-up methods can thus be linked to the careful and gentle selection process inherent in the microfluidic approach (13, 39).
The morphology of sperm is pivotal in ascertaining its function and ability to move effectively. Sperm that possess a normal morphology typically exhibit improved motility, viability, and DNA integrity. Conversely, the application of microfluidic techniques can be instrumental in minimizing the occurrence of sperm with abnormal morphology, such as those with head or tail defects. By employing microfluidic technology, the presence of such anomalies can be effectively reduced, thereby enhancing the overall quality and functionality of the sperm (40, 41).
The utilization of standard sperm preparation methods in assisted reproductive technologies includes several centrifugation steps that may potentially harm the sperm (42, 43). Unlike the in vivo environment, current centrifugation-based methods for sperm selection in assisted reproductive technologies are not inspired by natural sperm selection in the female genital tract (20). As a result, semen samples obtained through these centrifugation procedures are reported to exhibit a heightened DFI and an increased level of Reactive Oxygen Species (ROS) generation (44). Our study's results demonstrate that the microfluidic group exhibited a significant decrease in sperm DNA fragmentation compared to the DSU group. Our study's findings align with those of Aguirregomezcorta and colleagues, illustrating that sperm selection using a rheotaxis-based microfluidic device significantly reduces DNA fragmentation compared to the control group (28). Ataei and colleagues also demonstrated a notable improvement in DNA fragmentation using the microfluidic method based on rheotaxis (18).
In this study, we assessed MMP using the fluorescent marker JC-1 following the protocols established by Smiley et al (33). Mitochondria serve as ATP sources in the sperm midpiece, producing and transporting energy essential for sperm motility. Our results demonstrate a significantly higher percentage of spermatozoa with favorable MMP when selected using the rheotaxis method compared 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 in the DSU group, leading to increased production of ROS, could potentially damage the mitochondrial membrane and sperm DNA, ultimately diminishing the forward progressive of spermatozoa. the forward progressive of spermatozoa. Our results align with previous reports highlighting a positive correlation between progressive motility and sperm MMP (45).