| In mammals, only few spermatozoa succeed in arriving at the oviduct after rounds of biological selection. The population of sperm which can fertilize the egg is even smaller. It seems inconvincible that sperm can successfully find the egg just by randomly swimming, without any guidance. Chemotaxis has been proposed to be a possible mechanism during the process of sperm guiding to oocytes within the oviduct.Various research tools have been used for in vitro detection of sperm chemotaxis. However, they are typically poor in maintenance of gradient stability, not to mention their low efficiency. Microfluidic device offers a new experimental platform for better control over chemical concentration gradient than traditional ones. It is easy to establish a stable and controllable gradient in microfluidic chip. In the present study, an easy-handle diffusion-based microfluidic chip was established. This device allowed for conduction of three parallel experiments on the same chip, and improved the performance of sperm chemotaxis research. In such a chip, there were six channels surrounding a hexagonal pool. The channels are connected to the hexagon by microchannels. Firstly, fluorescein solution was used to calibrate gradient profiles in the system, demonstrating that a stable gradient could be generated in the device; secondly, the fluid flow in the system was characterized, indicating that there was no obvious flow in the area where gradient was established, posing little influence on sperm movement; thirdly, sperm behavior was observed under two concentration gradients of progesterone as a validation of the device where significant differences in chemotactic parameters were recognized between experimental and control groups (p< 0.05). Compared with control group, sperm motility was greatly enhanced in higher concentration of progesterone. In the future, the application of this device will be further developed and expanded, making it accessible for clinical work.Part ? To establish a microfluidic chip with stable concentration gradientObjective To establish a relative stable and easy-to-use concentration gradient chip which could mimics the in vivo environment where gradient is generated surrounding the oocyte-cumulus-complex, and to confirm the gradient profile in such a device.Methods The structure of the device was designed in AutoCAD software, featured by a central hexagon surrounded by six peripheral U-shape channels. Each channel was connected with one side of the hexagon through micro-channels. The master was fabricated using two-layer soft lithography. Microfluidic chip was duplicated by pouring PDMS mixture onto the master. Gradient distribution was simulated in Comsol Multiphysics and then determined using fluorescein solution.Results Simulation analysis revealed that the concentration established in the central hexagon was positively correlated with the area across the chip, from center to side. The gradient profile was observed under three different hydrostatic pressures which was correlated with the simulation analysis. Semi-quantitative analysis demonstrated that the regularity of gradient formation was better under higher hydrostatic pressures.Conclusion The microfluidic chip designed in the present study could generate stable concentration gradient, lasting a relatively long time. The area and distribution of the gradient was familiar with that in in vitro conditions, thus could be used for the study of sperm chemotaxis.Part ? To investigate the influence of fluid flow on sperm movement in the chipObjective To investigate distribution of fluid flow in the chip and to determine the possible influence on sperm movement.Methods The distribution of pressure and fluid flow was simulated in Comsol Multiphysics software. Fluorescent microspheres were used to track the fluid flow. Tracks of microspheres were analyzed in Image J to calculate speed of the flows. Then microspheres were only added in the central hexagon while other channels were doped with pure water to observe the flow in the central hexagon.Results As simulation analysis revealed, the speed was highest in the peripheral channel and was greatly reduced when liquid flowed through the interconnecting grooves. The liquid flow was very weak in the central hexagon with a speed less than 1 ?m/s. In addition, the flow speed within the device increased with the pressure difference between the inlets and outlets. Tracks of microspheres demonstrated the same results. Microspheres representing sperm were added in the central hexagon and no obvious migration was observed.Conclusion In the designed chip, there was no obvious fluid flow in the area where concentration gradient was generated. The establishment of gradient was mainly based on molecular diffusion. The flow inside the device would pose no influence on sperm movement.Part ? The effect of progesterone concentration gradient on sperm chemotactic behaviorObjective To study the effect of progesterone concentration gradient on sperm chemotactic behavior as a verification of the designed microfluidic device.Methods In experimental groups (Group A and B), progesterone solution (100 pmol/L or 1 mmol/L) was added in the peripheral channels to generate concentration gradient while sperm was added in the central hexagon. In control group (Group C), blank solution without progesterone was doped in the peripheral channels. Sperm suspension was processed using density gradient centrifugation and incubated for capacitation for 4 h.15 min after sample loading, sperm movement was recorded and tracks of 50 spermatozoa were analyzed using Image J software with MTrack J plugin. Parameters representing sperm behavior were assessed.Results Sperm motility was greatly increased after density gradient centrifugation (p <0.05). Significant differences in chemotactic parameters were recognized between experimental and control groups (p<0.05). Compared with control group, sperm motility was greatly enhanced in Group B (p<0.05), but no significant difference was found in Group A.Conclusion The microfluidic chip could be used in the study of sperm chemotaxis. Progesterone was capable of inducing chemotactic response of human sperm and possessed an motility enhancement effect on sperm at a higher concentration. |
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