基于精浆SERS光谱技术的男性无精子症诊断方法研究

Azoospermia is the most serious case in male infertility, limitations of clinical diagnosis are time-consuming, cumbersome operation as well as potential risk of unpredicted damage to other organs during a testicular biopsy. Seminal plasma serves not only as the carrier to sperm, but also the "soil" and energy source for spermatozoa. Seminal plasma is rich in biochemical components and the corresponding changes can directly imply the early pathological state, which can be considered as a most important entry for detection. Based on our previous work, this project develops a new method that employs capillary tube SERS substrate to interrogate seminal plasma samples from patients suffering varying degrees of azoospermia. Through band assignment and extraction of SERS characteristic peaks, qualitative analysis of biochemical substances in seminal plasma was conducted. In addition, multivariate statistical analysis such as partial least squares (PLS) was carried out to quantitatively determine the levels of vital components (such as amino acids, glucosidase, and fructose) in seminal plasma. With the combination of qualitative and quantitative results, SERS spectroscopy based diagnostic model for diagnosis and differentiation of male azoospermia was built. Therefore, the project is expected to address the current shortcomings in clinical diagnosis of azoospermia such as high cost, time-consuming and potentially testicular biopsy damage, thus providing a rapid, objective and non-destructive method for differential diagnosis and classifications of azoospermia in the future to assist the objective diagnosis of male infertility, which will definitely have great significance.

无精子症是男性不育的最严重情形，其临床诊断存在耗时、操作繁琐、睾丸活检有损等问题。精浆不仅是精子的载体，更是其能量来源和生存“土壤”；精浆生化成分丰富，相关的生化指标改变可直接、灵敏的反映出男性生殖器官疾病的早期病理状态，也是生化检测最重要的切入点。本项目在前期工作的基础上设计新型毛细管SERS检测基底，获得无精子症患者不同病理的精浆SERS光谱数据；并通过谱峰归属、特征谱峰提取，定性分析比较精浆中生化物质的差异；此外，结合以偏最小二乘法（PLS）为主的多变量统计分析方法，开展精浆中重要生化成分（氨基酸、葡萄糖苷酶和果糖等）含量的定量检测；综合定性定量数据，构建检测、诊断区分无精子症的拉曼光谱诊断模型。因此，该项目研究有望针对目前男性无精子症诊断高成本、耗时、潜在睾丸活检损伤的不足，提供无精子症诊断和类型区分的快速、客观和无损新方法，进而有助于协助男性不育的客观诊断，具有重要的意义。

本项目针对男性无精子症问题，开展基于精浆SERS光谱技术的诊断研究。首先，成功制备了拉曼增强性能优异、重复性好的SERS毛细管基底，开展正常和无精子症不育男性精浆的SERS光谱；通过对光谱数据的谱峰归属、特征谱峰的提取，分析比较了生化物质的差异；其次，通过色谱柱方法获得精浆外泌体的分离和富集，随后开展精浆外泌体的SERS光谱检测，获得了精浆外泌体的特异性生化信息；而基于神经网络模型分析对比体外正常和无精子症患者精浆外泌体的SERS光谱数据，初步实现对正常和无精子症患者的诊断评估。最后，结合显微拉曼光谱技术获得精子头部DNA的拉曼光谱响应刻画；以及基于精子DNA完整性的特征性光谱数据，初步实现了活性精子质量的无损显微拉曼检测评估。项目的进一步深入研究有望为正常男性和无精子症不育男性的鉴别诊断提供客观、无损新方法，以及为精液中优质精子的筛选提供新思路。