| Approximately7to10%couples face the fertility problem in china, among which the male factor infertility accounts for nearly a half. In clinical, routine semen analysis is a basic evaluation for male reproductive ability. However, test results may differ greatly due to different testing conditions and different operators. That is, semen quality may change over time, and on the other hand, routine semen analysis performance is still affected by subjective factors. Currently, there is a lack of objective and effective methods for assessing male fertility. Concerning this issue, in this study, preliminary study of semen quality based on Raman spectroscopy (including conventional Raman and SERS) were performed, and the major results are as follows.At first, fundamental theroy of Raman spectroscopy was introduced and a brief review of Raman applications in human body fluid was summarized.Then, the biochemical differences in normal and abnormal semen (seminal plasma) were analyzed and compared based on conventional Raman spectroscopy, Raman spectra obtained from normal and abnormal semen samples (seminal plasma) combined with principal component analysis (PCA) and partial least square (PLS) were performed to give good discriminant results. Moreover, SERS-based investigation of seminal plasma which obtained from fertile and infertile male was conducted. By using PLS-DA method, a diagnostic sensitivity of76%and specificity of83%were obtained, respectively (samples from33infertile male and59fertile male were analyzed). Our results demonstrated that some specific differences of biochemical concentrations were found between normal and abnormal semen samples (also semen samples obtained from fertile and infertile male), and Raman spectroscopy is expected to provide a new method for objective and comprehensive evaluation of male fertility, thus has a great potential of being developed as an non-destructive and important method for clinical assistant diagnosis of male fertility.Also, ultrafiltrate of seminal plasma with proteins removed were prepared and conventional Raman and DCDR (drop coating deposition Raman) methods were separately employed to perform the label-free quantitative analysis of citric acid and fructose, two important substances of semen, which reflect the functions of prostate and seminal vesicle. Our results showed that compared with tedious sample preparation needed in routine quantitative assay kit methods, Raman-based quantitative analysis demonstrates the advantages of relatively high accuracy, convenient and rapid, and is expected to be developed as a novel quantitative application for biochemical test in seminal plasma.In the end, spermatozoa with normal and abnormal morphologies were compared based on micro-Raman spectroscopy, and the characteristic Raman spectra of "normal" spermatozoa, selected by using the pre-treatment of density gradient centrifugation plus swim-up method were obtained. More importantly, a protocol of stabilizing the live sperm with the sperm head attached onto a metal substrate has been successfully developed for the first time, and the preliminary Raman measurement was carried out. Our results showed that with further development and optimization of this new strategy, it has the potential to provide a new method to identify and select the real high-quality spermatozoa for clinical assisted reproductive application, and overcome the potential risks involved in current manipulation which solely rely on evaluation of sperm morphology and motility.In conclusion, Raman spectroscopy has great application prospect in characterization and diagnosis of normal and abnormal semen, diagnosis of fertile and infertile male based on seminal plasma SERS spectra, as well as the label-free quantitative analysis of contents of seminal plasma. Even More important, Raman spectroscopy is expected to provide a new objective and non-destructive method for evaluation and selection of high-quality spermatozoa in ICSI (intracytoplasmic sperm injection) treatment. |
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