Microfluidics: The future of sperm selection in assisted reproduction.

BACKGROUND: Obtaining functional sperm cells is the first step to treat infertility. With the ever-increasing trend in male infertility, clinicians require access to effective solutions that are able to single out the most viable spermatozoa, which would max out the chance for a successful pregnancy. The new generation techniques for sperm selection involve microfluidics, which offers laminar flow and low Reynolds number within the platforms can provide unprecedented opportunities for sperm selection. Previous studies showed that microfluidic platforms can provide a novel approach to this challenge and since then researchers across the globe have attacked this problem from multiple angles. OBJECTIVE: In this review, we seek to provide a much-needed bridge between the technical and medical aspects of microfluidic sperm selection. Here, we provide an up-to-date list on microfluidic sperm selection procedures and its application in assisted reproductive technology laboratories. SEARCH METHOD: A literature search was performed in Web of Science, PubMed, and Scopus to select papers reporting microfluidic sperm selection using the keywords: microfluidic sperm selection, self-motility, non-motile sperm selection, boundary following, rheotaxis, chemotaxis, and thermotaxis. Papers published before March 31, 2023 were selected. OUTCOMES: Our results show that most studies have used motility-based properties for sperm selection. However, microfluidic platforms are ripe for making use of other properties such as chemotaxis and especially rheotaxis. We have identified that low throughput is one of the major hurdles to current microfluidic sperm selection chips, which can be solved via parallelization. CONCLUSION: Future work needs to be performed on numerical simulation of the microfluidics chip prior to fabrication as well as relevant clinical assessment after the selection procedure. This would require a close collaboration and understanding among engineers, biologists, and medical professionals. It is interesting that in spite of two decades of microfluidics sperm selection, numerical simulation and clinical studies are lagging behind. It is expected that microfluidic sperm selection platforms will play a major role in the development of fully integrated start-to-finish assisted reproductive technology systems.

Development of a novel cervix-inspired tortuous microfluidic system for efficient, high-quality sperm selection.

Microfluidic systems have been extensively studied in recent years as potential alternatives for problematic conventional methods of sperm selection. However, despite the widespread use of simple straight channels in these systems, the impact of channel geometry on selected sperm quality has not been thoroughly investigated. To explore this further, we designed and fabricated serpentine microchannels with different radii of curvature, inspired by the tortuous structure of the cervix. Our results showed that in the presence of gentle backflow, microfluidic channels with a 150 µm radius of curvature significantly enhanced the quality of selected sperms when compared to straight channels. Specifically, we observed significant improvements of 7% and 9% in total motility and progressive motility, respectively, as well as 13%, 18%, and 19% improvements in VCL, VAP, and VSL, respectively. Through careful observation of the process, we discovered a unique near-wall sperm migration pattern named boundary detachment-reattachment (BDR), that was observed exclusively in curved microchannels. This pattern, which is a direct consequence of the special serpentine geometry and sperm boundary-following characteristic, contributed to the superior selection performance when combined with a fluid backflow. After determining the best channel design, we fabricated a parallelized chip consisting of 85 microchannels capable of processing 0.5 ml of raw semen within 20 minutes. This chip outperformed conventional methods of swim-up and density gradient centrifugation (DGC) in terms of motility (9% and 25% improvements, respectively), reactive oxygen species (18% and 15% improvements, respectively), and DNA fragmentation index (14% improvement to DGC). Outstanding performance and advantages such as user-friendliness, rapid selection, and independence from centrifugation make our microfluidic system a prospective sperm selection tool in clinical applications.

Effect of Bovine Serum Albumin Supplementation in Tris-Soybean Lecithin-Based Extender on Quality of Chilled Ram Epididymal Spermatozoa.

Objective: This study was designed to assess the effects of using tris-soybean lecithin (TSL)-based extender supplemented with bovine serum albumin (BSA) on the quality of ram epididymal spermatozoa during refrigerated storage. Method: Epididymal sperm were collected from 22 Zandi rams, diluted in TSL-based extender at different concentrations (0%, 2.5%, 5%, 7.5%, and 10%) of BSA, and stored for 5 days at 4°C. Sperm parameters including motility, viability, plasma membrane integrity, chromatin protamination, and malondialdehyde (MDA) content were evaluated at 0, 24, 72, and 120 hours of refrigeration. Results: The addition of 10% BSA to the extender significantly improved sperm viability at 24 and 120 hours of refrigerated liquid storage (p < 0.05). An enhancement in plasma membrane integrity was observed along with a decrease in MDA level by increasing the concentration of BSA from 0% to 10% (p > 0.05). Sperm motion characteristics were higher in the BSA-free group at 120 hours of preservation (p < 0.05). No statistical difference was found for nuclear protamination between experimental groups (p > 0.05). Conclusion: BSA supplementation in TSL-based extender can preserve the viability of epididymal ram spermatozoa during liquid storage at 4°C.

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa.

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability.

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C-zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ-related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid-binding ability, and PLCζ level along with its distribution were evaluated using computer-assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid-binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.

Clinical outcomes of magnetic activated sperm sorting in infertile men candidate for ICSI.

Magnetic activated cell sorting (MACS) with annexin V microbeads deselected apoptotic sperm with externalized phosphatidylserine (PS) residues on their surface and decrease chance of such sperm to be inseminated. Therefore, the aim of this study was to evaluate efficiency of MACS procedure in a prospective randomized trial. Sixty-two semen samples were allocated into two groups and processed according to: (i) a combination of MACS with density gradient centrifugation (MACS-DGC) as study group (N = 29); and (ii) DGC alone as a control group (N = 33). Fertilization, embryo quality, pregnancy and implantation rates were compared between the two groups. Although, no significant difference was observed in fertilization rates between the two groups, the percentage of high-quality embryos, pregnancy and implantation rates was significantly higher in the MACS-DGC group compared to DGC alone. Therefore, MACS may help to select the most fertile sperm and improve clinical outcomes of intra-cytoplasmic sperm injection (ICSI).

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant.

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.