Zenuo Biotech 
Traditional sperm selection methods include swim-up method and density gradient centrifugation method, which mainly ｓｅｌｅｃｔ sperm with normal vitality and morphology based on sedimentation and migration speed, which is quite different from the multiple screening mechanisms in vivo. In addition, long sorting time and excessive centrifugation operations can easily lead to sperm DNA peroxidation damage and breakage, reduce sperm quality, and affect the success rate of ART. Therefore, establishing a simple, fast, non-/low-damage new sperm selection method has become an urgent need to improve the success rate of assisted reproductive technology (ART). The application of microfluidic technology is in line with this demand, and a stable fluid environment can avoid mechanical damage to sperm to the greatest extent.
Clinically, sperm selection mainly depends on two indicators: sperm vitality and morphology. At present, the methods for evaluating and screening sperm vitality using microfluidic technology include microchannel screening, dielectrophoresis screening, and laminar flow effect screening.
Microchannel screening is to form a stable fluid so that sperm can swim freely in the fluid, without involving too much human operation, so it is very direct and reduces/avoids mechanical damage to DNA.
Foreign teams have designed a series of chips such as single straight channels, branched cascade channels, and channels with obstacles to count and measure human sperm motility. The results showed that sperm swam in one direction and could pass through single straight channels with widths of 40, 100, and 120 um. Highly motile sperm could pass through longer channels, and sperm could also pass through branched cascade curved channels. At the same time, they conducted sperm function tests, such as penetrating cervical mucus, and the effects of hyaluronic acid, spermicides, and anti-human IgG antibodies, to prove the effectiveness and versatility of microfluidic technology in evaluating sperm function. They improved the chip structure, connected the injection pool to the collection pool through 2 or 4 curved channels, and judged sperm activity by the time it took for sperm to reach the collection pool, thus achieving graded evaluation. This method has a good correlation with the results of the Makler counting plate based on the grading and evaluation of sperm forward movement. It takes 360 to 480 seconds for high-motility sperm (grade 3, grade 3+) to reach the collection pool, and 660 to 770 seconds for low-motility sperm (grade 1, grade 1+, grade 2). This experiment is mainly for sperm evaluation, not for sperm screening.
McCormack et al. designed a straight channel to use fluorescent labeling technology to screen the fertilization ability of semen samples. According to the WHO standard, the results of the screening experiment of sperm concentration and forward-moving sperm concentration showed that the Pearson correlation coefficients of the fluorescence signal of the system and the concentration of the two were 0.79 and 0.80, respectively, the sensitivity was 94% and 96%, and the specificity was 97% and 90%, respectively. However, the device is expensive and the fluorescent labeling is complex, which limits its wide application.
Seo et al. selected bull, mouse, and human sperm based on the characteristics of sperm countercurrent swimming. During the experiment, the fluids in channels A, B, and C flowed to the intersection, reservoir 2, and reservoir 3 respectively. The semen sample was introduced into reservoir 2, and the active sperm flowed back through channel B to reach the intersection, and then was transported to reservoir 3 by high-speed fluid to achieve sperm selection. In particular, this method sorted 3 bull sperm, and the sperm motility increased from an average of 18.4% to 78.8%. Based on the same principle, Chen et al. designed a portable sperm quality analyzer, which integrated Coulter technology for sperm counting and evaluated sperm motility and concentration. Experiments have shown that the number of voltage pulses 0 to 335 corresponds to 0 to 19*10^6/ml for clinical hemocytometers and 0 to 204 for sperm motility index of sperm quality analyzers. This system does not require fluorescent labeling, is low in cost, easy to operate, and does not require sample pretreatment, but the counting hole size is small (6 um wide).
Application of microfluidic technology in sperm selection
Application of microfluidic technology in sperm selection
Han et al. designed a simple structure of two microchambers connected by an array microdam to realize the selection of active sperm, and integrated a stable and reliable temperature control module. The results showed that the system was more suitable for sperm than the incubator and room temperature. At the same time, the system integrated an inverted optical microscope and could be used for sperm evaluation, embryo culture and in vitro fertilization (IVF) research.
The fastest moving sperm may not be the sperm with the strongest fertilization ability, so sperm classification capture and evaluation are beneficial to the diagnosis of male infertility and the study of sperm physiological characteristics. Qiu et al. designed different channel cross-sections to achieve different flow rates in each section, captured active sperm matching it in each flow rate section, and counted the number of sperm in each section to obtain the percentage of active sperm at each level. The authors captured active sperm with average linear velocities of (40.5±3.0), (27.2±0.6), and (18.7±0.6) μm/s, respectively.
Straight channel sperm screening is simple and convenient, and is close to the physiological environment, but there is no recognized size standard. Xie Lan used the relative number of sperm in the outlet pool and sperm motility as evaluation criteria, and verified the straight channel screening effect by fluorescently labeling sperm. Width screening channel: 7mm long, 25μm deep, 200μm, 500μm, 1mm and 1.5mm wide; length screening channel: 1mm wide, 25μm deep, 5mm, 7mm, 1cm and 1.5cm long; obtain the best channel width and length, optimize the channel depth: 25μm, 50μm, 100μm and 200μm deep; obtain the best channel width, length and depth, and optimize the screening time of 5 min, 15 min, 30 min and 60 min. When the straight channel is 1 mm wide, 7 mm long and 25μm deep, the ICR mouse sperm screened for 15 min not only has higher motility (82.6±2.9)% but also disperses from each other, which helps to improve the success rate of IVF.
Microchannel screening is simply based on sperm motility to ｓｅｌｅｃｔ sperm, which cannot fully reflect the natural selection process of sperm under physiological conditions. Zhong Zhimin et al. simulated the interaction between sperm and cervical mucus under physiological conditions on a chip to achieve natural selection of sperm and online detection of sperm quality. This method takes a short time and does not require centrifugation. The selected sperm is superior to the pre-sorting and swim-up methods in terms of sperm motility, average trajectory speed, forward ratio, and normal morphology percentage. It also lays the foundation for simulating the fertilization process under physiological conditions on the chip.
Microchannel sperm screening is simple and easy to use, close to the physiological environment, and has little damage to sperm, so its clinical application prospects are very broad. At the same time, the activity screening channel also has the potential to integrate subsequent fertilization-related devices, laying the foundation for realizing the entire process of IVF on a chip.
Dielectrophoresis (DEP) refers to the process of cells being induced to polarize in an inhomogeneous alternating electric field. Different cells induce different dipole moments due to their different dielectric properties, conductivity, shape or size, and are therefore separated by different dielectric forces in the electric field.
Fuhr et al. achieved the capture, positioning and screening of human single sperm with different activities by applying different high-frequency electric fields. Ultramicroelectrodes are made on quartz and glass substrates. When the conductivity of the external salt solution is greater than the average conductivity of the sperm, negative dielectrophoresis is formed, and the sperm is pushed from the electrode to the minimum electric field. High-frequency alternating current is applied to a planar 4-electrode or three-dimensional 8-electrode area to form a centripetal repulsive force. Fast-swimming sperm can be captured for several seconds (when the electric field is greater than 500V/cm and the frequency is MHz, some sperm stop moving) for monitoring; using strip electrodes and interdigitated electrodes, sperm with a speed of about 40 to 70μm/s will be fixed at the breakpoint electrode for several minutes. The system can also release the captured surviving sperm to a specific area for integration of subsequent experimental operations.
When the channel scale enters the micron level and the fluid Reynolds number is low, the two parallel laminar flows close to each other can be mixed only by diffusion. After the laminar flow stabilizes, the surface tension and viscosity force cause the active sperm to swim from the stock solution through the laminar flow interface to the collection channel, while the inactive sperm and cell fragments remain in the stock solution and flow until they are discharged, thereby achieving sperm motility screening.
Cho et al. integrated a horizontal gravity-driven micropump to achieve human sperm screening based on the laminar flow effect, overcoming the defect that the flow rate decreases over time when using traditional gravity micropumps. After screening, the sperm motility of the three samples was close to 100%, the normal sperm morphology rate increased from (9.5±1.1)% to (22.4±3.3)%, and the active sperm recovery rates were 39%, 42%, and 43%, respectively. The system has high integration and high sorting efficiency, and only requires 50μl of sample volume (conventional sperm selection methods are difficult to operate). Schuster et al. have demonstrated the low damage function of laminar flow effect in sperm selection in practice, but the sperm recovery rate of the system is not high, and the screening efficiency needs to be further improved. Compared with untreated, continuous centrifugation, density gradient centrifugation, and swim-up method, Schulte et al. proved that the sperm selected by laminar flow effect had the highest motility (52.0%, 50.1%, 73.4%, 85.8%, 96.2%, respectively, P < 0.0001) and the lowest DNA damage rate (13.3%, 15.8%, 14.9%, 5.7%, 1.9%, respectively, P < 0.05). Wang Wei et al. investigated the quality of sperm sorted by laminar flow principle. Compared with the swim-up method, the sperm DNA damage rate was significantly reduced, and the motility, normal morphology rate and tail swelling rate were significantly improved. Later, the channel inlet angle and sorting channel width of the self-designed three-channel chip (sample channels on both sides) were optimized. There was no significant difference in sperm motility and normal morphology rate between the chips with three channel inlet angles (30°, 45°, 60°) and three channel widths (120, 180, 250 μm), but the sorting efficiency was the highest when the inlet angle was 30° and the sorting channel width was 250 μm.
Application of microfluidic technology in sperm optimization
Application of microfluidic technology in sperm optimization
Wu et al. used polyethylene glycol methacrylate surface modified polydimethylsiloxane (PDMS) sperm optimization chip, which has long-term (56 d) hydrophilicity and stable non-specific adsorption, effectively avoiding channel blockage, and the channel surface is easy to infiltrate and facilitate sample injection. The motility of the selected human sperm is 74%.
PDMS is not suitable for clinical application due to its lack of safety guarantee and quartz is not suitable for clinical application due to its high cost. Matsuura et al. used cycloolefin polymer, which has been approved in the clinical field, to make sperm screening chips. After screening, sperm motility increased from 53.0% to 90%, and the linear velocity increased from (21.0±1.7)μm/s in the waste liquid pool to (51.7±2.1)μm/s (n=172) and (59.5±1.6)μm/s (n=79) in the collection pool. The ART success rate was also improved, but the recovery rate of active sperm was low (0.2%～0.3%).
Tseng et al. used fluorescent dye SYBR-14/PI to label human sperm, injected the sample through a syringe pump, screened active sperm based on the laminar flow effect, observed sperm of different motility with a microscope, and identified and quantified dead/live sperm with a flow cytometer. The system has controllable and stable flow rate, and the sperm screening efficiency is improved (sperm survival rate is 94.8%).
The optimized laminar flow effect sperm motility screening chip can effectively screen high-motility sperm, which has extremely important application value for untreated semen, especially semen containing a large amount of fragments. Since this method cannot screen sperm at a specific flow rate, and the flow rate limit cannot screen a large amount of semen, and the application of this method is currently limited, there is no report on the treatment effect of moderate to severe oligoasthenozoospermia samples and the application effect in ART.
Microfluidic technology for sperm selection has the advantages of high sorting efficiency, less sperm DNA damage, simple operation and short sorting time. In ART, whether it is normal semen parameters in IVF and ICSI or sperm selection of mild oligoasthenozoospermia, it will have good application prospects.