【摘要】 全球成年男性的精子质量呈明显下降趋势。精子质量下降与男性不育虽并非线性关系，但显然与低生育力密切相关。辅助生殖技术针对不育男性精子水平低、形态和运动活力异常等常见问题，通过评估和筛选优质精子，提供了解决男性不育问题的主要技术方案。现有临床研究局限于精子形貌和活力等特征参数的筛选，缺乏针对精子DNA损伤情况的无损评筛手段。本文首先介绍了精子无损评筛技术在辅助生殖领域男科的应用需求以及显微拉曼光谱技术的基本原理，继而对基于拉曼光谱技术的精子研究进行了综述，综合分析探讨了精子的拉曼光谱检测和光谱特征，聚焦精子DNA损伤的拉曼光谱响应问题，最后讨论和展望了精子无损评筛技术的发展前景。更多还原

【Abstract】 Significance The quality of sperm in fertile men worldwide is deteriorating. Although sperm quality is not directly related to male infertility, it is strongly linked to subfertility. Assisted reproductive technology addresses common male infertility issues, such as low sperm count, abnormal morphology, and motility, and it is the primary technological solution to male infertility through the evaluation and screening of high-quality sperm. However, current clinical research is limited to screening sperm morphology and motility, and there is a lack of non-destructive screening methods for evaluating sperm DNA damage. This review article first introduces the demand for non-destructive sperm screening technology in assisted reproduction and the basic principle of micro-Raman spectroscopy,and then summarizes current Raman-based research progress on sperm. Next, a comprehensive analysis and discussion of Raman detection and Raman spectral characteristics of sperm cells are conducted, emphasizing the Raman response to sperm DNA damage.Finally, the potential for applied research in non-destructive sperm evaluation and screening is discussed and explored.Progress Raman-based sperm analysis is still in the initial stage of development, but the unique advantages of Raman spectroscopy have opened up new routes for sperm characterization. In 2009, Huser et al. used micro-Raman spectroscopy to correlate the spectral response of chromatin differences with sperm morphology. Meister et al. achieved Raman imaging characterization of sperm nucleus,neck, and mitochondria in sperm cells. By simulating the damaging effect of UV radiation on different sperm organelles, it was verified that micro-Raman spectroscopy is expected to provide a new method for rapid assessment of sperm mitochondrial status.Subsequently, Mallidis et al. reported that the damage to sperm DNA due to UV radiation is mainly reflected in the intensity change of the PO4 skeleton peak(1042 cm-1) attributed to DNA. Additionally, the spectral response of sperm DNA damage due to UV light was only observed in dry conditions, not in solution media. A comparison of Raman results of sperm DNA and flow cytometry revealed different responses of sperm DNA damage to the characteristic peak intensities. Given the challenges of labor-intensive sperm sample processing in earlier research and the potential impact on Raman test outcomes, Huang et al. collected microscopic Raman spectrum data of normal and various abnormal morphology(pear-shaped, cone-shaped, small-headed) based on a new Raman test substrate. They achieved rapid sample preparation(tens of seconds) and effective classification using principal component analysis and linear discriminant methods(PCA-LDA) on the new substrates. Additionally, combined with the extended multiplicative signal correction(EMSC) method, Raman detection of DNA-intact and DNA-damaged sperm cells on glass slides was attempted,effectively “filtering” glass signal interference and obtaining the intrinsic Raman signal of sperm. Huang et al. also achieved rapid preparation of sperm samples, automatic segmentation of sperm head and acrosome regions, and quantification of morphology parameters using a hydrophobic substrate and image analysis. In 2016, Ferrara et al. combined holographic microscopic imaging with microscopic Raman spectroscopy not only to highlight the differences in sperm quality and morphology’s spatial distribution but also to show a novel method for sperm quality assessment in conjunction with sperm morphology assessment and Raman spectroscopy analysis.Regarding the Raman assessment of motile sperm, in 2012, Feng Liu et al. studied the interaction of sperm and the zona pellucida(ZP), the acrosome reaction induced by the zona pellucida, and the Raman spectral response of different regions of sperm.Hence, the acrosome region(800-900 and 3200-4000 cm-1) of zona-bound sperm shifted from mild hypointensity to hyperintensity compared to the Raman spectra of zona-bound sperm. In 2015, Edengeiser et al. achieved the “fixed” adsorption of motile sperm on the pretreated CaF2 substrate. Combined with the automatic measurement in the micro-Raman system, they detected a single motile sperm under physiological conditions. Raman spectroscopic detection allows the assessment of physicochemical parameters of sperminduced oxidative damage under near-physiological conditions. Conversely, in 2020, Huang et al. realized the fixed “adsorption” of motile sperm on the smooth surface of a conventional substrate. For the first time, they obtained a Raman spectrum with a high signalto-noise ratio from a single motile sperm using a water immersion objective and qualitatively compared motile and non-motile sperm.Additionally, the influencing factors of the stable “fixation” of sperm and Raman signal under different focusing conditions were discussed. Raman-based motile sperm study is still in its early stages, requiring more development and improvement in terms of experimental techniques and statistical data processing.Conclusions and Prospects Traditionally, high-quality sperm evaluation and screening have focused on the relationship between sperm DNA integrity and sperm morphology and motility. However, a lack of non-destructive and objective evaluation research directly reflects the integrity of active sperm DNA. The current research on sperm microscopic Raman detection is still in its early stages and is mainly limited to detecting and characterizing spermatozoa or immotile sperm in non-physiological environments. The combination of microfluidic devices and micro-Raman spectroscopy can achieve non-destructive “capture” and “manipulation” of motile sperm. Based on this, research on DNA damage assessment of motile sperm using micro-Raman spectroscopy is being conducted. The integration of data comprehensive analysis based on information technology, such as deep learning and Raman spectroscopy technology, can effectively extract the Raman spectral information of motile sperm DNA, establish the Raman spectral dataset of motile sperm with intact DNA, and thus provide high-quality data for clinical assisted reproduction needs. The multitechnology integration is expected to provide an objective and non-destructive new method for DNA-intact high-quality sperm screening in the field of assisted reproduction, improve the success rate of male-factor assisted reproductive technology, and promote new developments and opportunities in the study of male infertility in the field of assisted reproduction.更多还原