Infertility is a significant global health concern affecting millions. Male infertility accounts for a substantial portion of cases, highlighting the need for improved ART technologies. Sperm selection is a critical step in ART, impacting fertilization rates, embryo quality, and live birth rates. Current gold-standard methods, such as density gradient centrifugation (DGC) and swim-up (SU), have limitations, including the induction of DNA fragmentation through ROS generation during centrifugation. These techniques also lack standardization and can lead to operator-dependent variability in outcomes. The inherent limitations of these methods necessitate the exploration of innovative sperm selection approaches that address these shortcomings and improve the overall success rates of ART procedures. Microfluidic devices offer a potential solution, but many existing technologies are complex and lack clinical translation. Ideal sperm selection platforms should be user-friendly, consistent, and efficiently select high-quality sperm, ideally by mimicking the natural selection process in the female reproductive tract, which utilizes multiple selection mechanisms to reduce sperm number from hundreds of millions to a few hundred.

Several studies have explored microfluidic approaches to sperm selection, primarily focusing on motility-based separation. However, these methods often lack additional selection criteria beyond motility. The use of Annexin V to remove apoptotic sperm has shown promise in improving embryo quality, but it is usually employed as an adjunct to conventional methods like DGC or SU, often in conjunction with magnetic-activated cell sorting (MACS). While MACS can improve sperm quality, it adds complexity and time to the process. The current literature underscores the need for a comprehensive sperm selection platform that combines multiple selection criteria (motility, apoptosis) in a streamlined and user-friendly manner, improving both the quality and efficiency of sperm preparation for ART.

This study utilizes a novel 3D-printed, biomimetic microfluidic sperm selection platform (MSSP). The device employs a circular array of microchannels with layered ridges to guide sperm based on their motility and boundary-following behavior. The MSSP was fabricated using Digital Light Processing (DLP) 3D printing. Human semen samples (n=33) were used, with additional diluted samples to simulate oligozoospermia. The MSSP's operation involves pre-filling the device with buffer, injecting semen, and incubating it at 37°C for 15 minutes. Selected sperm are then collected from the central outlet. A hybrid MSSP was also developed, incorporating a magnetic microbead reservoir to trap apoptotic sperm using Annexin V. The performance of MSSP was compared to a standard swim-up (SU) method. Sperm parameters assessed include concentration, motility (using OpenCASA), vitality (LIVE/DEAD staining), DNA integrity (modified sperm chromatin dispersion (SCD) test), cryosurvival, and apoptotic marker expression (Annexin V/PI binding assay and flow cytometry). Statistical analysis was performed using the Friedman test.

The MSSP demonstrated significant improvements in sperm quality compared to SU. The MSSP yielded an average 68.4% increase in motile sperm concentration compared to previously reported straight channel microfluidic devices. Specifically, MSSP showed significantly higher motility (93.5% vs. 74.1%), vitality (97.6% vs. 86.0%), and DNA integrity (1.4% vs. 7.9%) than SU. The MSSP also resulted in a substantial reduction in sperm apoptosis (5.66% vs. 26.5%). The cryosurvival rate was also significantly higher for sperm selected using MSSP (64.2% vs. 52.8%). The hybrid MSSP, incorporating apoptotic sperm removal, further enhanced sperm quality. The device operates within 15 minutes, significantly faster than the 45 minutes required for SU, making it more clinically efficient.

The findings demonstrate that the biomimetic MSSP significantly improves sperm selection efficiency and quality compared to conventional SU. The layered microchannel design and incorporation of multiple selection criteria (motility and apoptosis) enhance the selection of high-quality sperm with improved DNA integrity. The faster processing time of MSSP compared to SU enhances clinical workflow. These improvements in sperm quality have the potential to positively impact downstream ART procedures, leading to improved fertilization rates, embryo quality, and ultimately, higher chances of successful pregnancies. The device's ease of use and consistent performance make it a clinically viable alternative to existing methods.

This study presents a novel 3D-printed microfluidic sperm selection platform (MSSP) that significantly outperforms conventional swim-up methods in terms of sperm quality and processing time. The biomimetic design, incorporating multiple selection criteria, results in a higher yield of motile, viable sperm with improved DNA integrity. This technology holds great promise for improving the success rates of ART procedures. Future research could focus on large-scale clinical trials to validate the platform's effectiveness in diverse patient populations and further optimize the device design for enhanced performance.

While the study demonstrates significant improvements, further research is needed to validate the findings in a larger, more diverse patient population. The study primarily focused on comparing MSSP to SU; a direct comparison with DGC would provide a more comprehensive assessment. Long-term outcomes, such as pregnancy rates and birth outcomes, need to be evaluated in future clinical trials. The current study was conducted with specific types of reagents, a comparative analysis with different brands of Annexin V and microbeads could provide more robust information.