目的对精子分选用微流控芯片的微通道入口角度和分选通道宽度进行优化,探索最优分选效率的微流控芯片结构,以期获得最高的精子分选效率.方法利用自行设计和制造三种不同通道入口角度(30°、45°和60°)和三种不同分选通道宽度(120μm、180μm和250μm)的聚二甲基硅氧烷(Polydimethylsiloxane, PDMS)微流控芯片对30份健康体检者精液标本进行精子分选实验,选择出最合适的分选芯片.采用具有最佳分选效率的微流控芯片和上游法分选比较.对40例男性不育患者精液标本进行精子优选,从精子质量常规参数及DNA结构完整性两个方面评价两种精液体外处理方法对精子质量的影响.结果不同通道入口角度和不同通道宽度的微流控芯片对40份精液标本进行优选后,精子活力和正常形态精子百分率均可见显著提高,差异均具统计学意义(P<0.01).三种不同通道入口角度和三种不同通道宽度的芯片在精子活力和正常形态率的分选上比较,差异均无统计学意义(P>0.05),但分选回收效率入口角度30°的芯片显著优于45°和60°的芯片(P<0.01),通道宽度250μm的芯片显著优于120μm和180μm的芯片(P<0.01).精液经微流控芯片法和上游法处理后,在精子质量常规参数方面,微流控芯片法明显优于上游法,差异具有统计学意义(P<0.01)；在DNA链完整性方面,两种方法处理后,微流控芯片法精子的DNA损伤率明显低于上游法,差异具有统计学意义(P<0.01).结论 PDMS微流控芯片其入口角为30°、分选通道宽度为250μm时,其分选效率最高.微流控芯片优选法是一种比传统精子分选方法更高效率、DNA损伤更少、操作简便的方法,可以预见,在辅助生殖技术中特别是体外受精和卵细胞胞浆内单精子注射中具有良好的应用前景.%Objective To optimize the entrance angle and channel width of a microfluidic sperm sorter. Methods Thirty semen specimens were processed using polydimethylsiloxane (PDMS)chip based sperm sorters of different entrance angles (30°, 45°, 60°)and channel widths (120μm, 180μm, 250μm), and the sorted sperms were compared with those in raw samples in terms of motility, normal morphology ratio and hypoosmotic swelling rate. A microchip with the optimized configuration was used to process semen specimens from 40 male infertility patients, and the results were compared with those from the conventional swiming-up method. Routine sperm parameters were analyzed using the computer assisted sperm analysis (CASA), and sperm DNA integrity was determined using the sperm chromatin dispersion test (SCD). Results Microfluidic sperm sorters with different entrance angles and channel widths were all able to improve sperm activity and normal morphology ratio significantly in all the 40 semen samples (P<0.01). Microsorters with different entrance angles and widths didn ’t show significant difference in either sperm activity or normal morphology ratio. With regards to recovery efficiency, a microchannel with entrance angle of 30 ° and channel width of 250μm was superior to the other configurations (P<0.01). The microfluidic method showed advantage over the swimming-up method in terms of routine sperm parameters and sperm DNA integrity. Conclusion The highest sperm sorting efficiency was obtained using a microfludic sperm sorter with entrance angle of 30 ° and width of 250μm. Compared with the conventional sperm sorting method, the presented microfluidic approach showed advantages of high sorting efficiency, less DNA damage and simple operation. Therefore, this technology holds the potential to be used in sorting motile spermatozoa for insemination and in vitro fertilization.
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