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利用Transwell膜孔筛选高质量精子及其性能评估

Screening of High-quality Spermatozoa Using Transwell Membrane Pores and Assessment of Their Performance

    摘要
  • 摘要:
    目的 基于Transwell膜孔的分子筛作用，探讨利用Transwell优选人类高质量精子的可行性。
    方法 收集2023年11月–2024年5月广州中医药大学第一附属医院男科新鲜精液标本162份。分析经5 μm和8 μm孔径Transwell精子优选的差异并经透射电子显微镜（transmission electron microscope, TEM）观察微观结构差异，比较Transwell优选精子30 min、60 min、80 min后精液参数的变化，比较Transwell与密度梯度离心法（density gradient centrifugation, DGC）和上游法（swim-up methods, SU）对不同质量精子优化后精液参数的差异。本研究将精子前向运动（progressive motility, PR）百分率设定为主要结局指标，精子活力、正常精子形态率（normal sperm morphology rate, NSMR）、DNA碎片指数（DNA fragmentation index, DFI）、精子浓度及回收率设定为次要结局指标。
    结果 当孔径为5 μm、优选时间为60 min时，Transwell优选精子效果更佳。在正常精液组中，与DGC法相比，Transwell优选后的精子PR百分率中位数从77.50%升至92.05%（P<0.05），NSMR中位数从9.00%升至13.25%（P<0.01），DFI中位数从2.16%降至0.25%（P<0.01）；与SU法相比，Transwell优选后的精子浓度中位数从4.70×106 mL－1升至14.15×106 mL－1（P<0.01），DFI中位数从0.47%降至0.25%（P<0.01），回收率中位数从2.50%升至6.48%（P<0.01），但精子活力、PR百分率和NSMR差异无统计学意义。在异常精液组中，与DGC法相比，Transwell优选后的精子PR百分率中位数由37.20%升至88.75%（P<0.01），精子活力中位数由53.00%升至95.00%，DFI中位数由12.49%降至0.92%（P<0.01）；与SU法相比，Transwell优选后的精子PR百分率中位数由86.30%升至88.75%（P<0.05），DFI中位数由1.63%降至0.92%（P<0.01）。
    结论 Transwell优选方法在获得足够数量、活力高、形态良好以及DNA完整性较佳的精子方面表现出较好性能。这种特性使其有望成为辅助生殖技术中精选高质量精子的简便实用方法。

     

    Abstract:
    Objective The feasibility of using Transwell, based on the molecular sieve effect of its membrane pores, to select high-quality human sperm was explored.
    Methods  A total of 162 fresh semen samples were collected from the Department of Andrology at the First Affiliated Hospital of Guangzhou University of Chinese Medicine between November 2023 and May 2024. Differences in sperm selection using 5 μm and 8 μm pore Transwell filters were analyzed, and microscopic structural differences were observed by transmission electron microscopy (TEM). Changes in semen parameters after 30, 60, and 80 minutes of Transwell sperm selection were compared. Differences in semen parameters after optimization of sperm of varying qualities by Transwell, density gradient centrifugation (DGC), and swim-up (SU) methods were also compared. In this study, the percentage of progressive motility (PR) of sperm was set as the primary outcome measure, while sperm motility, normal sperm morphology rate (NSMR), DNA fragmentation index (DFI), sperm concentration, and recovery rate were set as secondary outcome measures.
    Results  When the aperture is 5 μm and the preferred time is 60 minutes, the Transwell method yields better results for sperm selection. In the normal semen group, compared with the DGC method, the median percentage of PR in sperm after Transwell selection increased from 77.50% to 92.05% (P < 0.05), the median NSMR increased from 9.00% to 13.25% (P < 0.01), and the median DFI decreased from 2.16% to 0.25% (P < 0.01). Compared with the SU method, the median sperm concentration after Transwell selection increased from 4.70 × 106 mL－1 to 14.15 × 106 mL－1 (P < 0.01), the median DFI decreased from 0.47% to 0.25% (P < 0.01), and the median recovery rate increased from 2.50% to 6.48% (P < 0.01). but there were no statistically significant differences in sperm motility, PR percentage, or NSMR. In the abnormal semen group, compared with the DGC method, the median PR percentage of sperm after Transwell selection increased from 37.20% to 88.75% (P < 0.01), the median sperm motility increased from 53.00% to 95.00%, and the median DFI decreased from 12.49% to 0.92% (P < 0.01). Compared with the SU method, the median PR percentage of sperm after Transwell selection increased from 86.30% to 88.75% (P < 0.05), and the median DFI decreased from 1.63% to 0.92% (P < 0.05).
    Conclusion The Transwell preferred method demonstrates excellent performance in obtaining sufficient sperm with high viability, good morphology, and good DNA integrity. This makes it a promising, simple, and practical method for selecting high-quality sperm in assisted reproductive technologies.

     

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