| Infertility has been a severe problem in the field of reproductive medicine,and it has become the third largest disease after cancer and cardiovascular diseases;among them,male-factor infertility accounts for about 50% of the overall infertility disease.Molecular spectroscopy has a wide range of research and applications in medicine,chemistry,biology,materials and other fields,Raman spectroscopy offers a potential new method for non-destructive,rapid detection of semen,seminal plasma and sperm due to its ability to specifically obtain the "fingerprint" characteristic information of the molecular composition and structure of substances,and does not require special preparation of the test samples.It is expected to develop into a new technique for the assessment and diagnosis of male reproductive function in the field of reproductive medicine research.Therefore,given the current lack of objective,non-destructive assessment of sperm DNA damage in clinical reproductive field,the aim of this paper is to perform a label-free assessment of sperm DNA damage based on Raman spectroscopy.In the first chapter,we introduced the basic principles of Raman spectroscopy and the development of Raman spectroscopy technology,then briefly discuss the existing technical methods and shortcomings of male fertility and sperm quality assessment,and finally review the current development of male fertility assessment and research based on Raman spectroscopy.In the second chapter,we compared the Raman signal detection of sperm cells on metal substrates and glass substrates,and found that sperm cells on metal substrates had no background signal interference,and sperm cells could be successfully stained,but sperm cells were prone to fall off during the staining process,and the sperm DNA fragmentation rate calculated on the metal substrate is relatively large.In contrast,medical glass slides as the substrate can be used to achieve rapid sperm preparation for detection despite the interference of glass background signal,and sperm cells are stuck to the glass surface during the staining process.We used medical glass substrates for subsequent experiments,and the Raman spectrum of the sperm head on the glass substrate was obtained and the results of sperm DNA staining were compared one by one.The Raman spectral data of 426 sperm cells(361 with DNA non-damage and 65 with DNA damage)were obtained in the experiment;the Raman spectrum was analyzed and processed by combining principal component analysis-linear discriminant analysis(PCA-LDA)and partial least squares discriminant analysis(PLS-DA)algorithms to compare and evaluate the classification effect.Among them,the diagnostic sensitivity and specificity calculated by the PCA-LDA algorithm were 76.5% and 60.0%,respectively,with an accuracy of 74.4%;while the diagnostic sensitivity and specificity calculated by the PLS-DA algorithm were 83.1% and 75.4%,respectively,with a discrimination accuracy of 81.9%;the area under the curve(AUC)values calculated by the PCA-LDA algorithm and the PLS-DA algorithm were 0.811 and 0.837,respectively.The results show that Raman spectroscopy combined with statistical analysis methods can provide effective differentiation of sperm cells with different DNA qualities.In the third chapter,we conducted a preliminary evaluation of the Raman signals of sperm from the acrosome region,nuclear region and middle region on metal substrates and glass substrates,and found that the Raman spectra of three regions of sperm cells on the two substrates are slightly different.Then,the Raman imaging studies of sperm on metal substrates and glass substrates were carried out respectively.Our preliminary results show that Raman imaging technique can be applied to study the spatial distribution of DNA in sperm cells and the DNA damage status as well.Our experiments revealed that Raman imaging of sperm cells on glass substrates saves time and requires low costs compared with metal substrates,and the imaging based on glass substrates is compatible with clinical research and applications.Micro-Raman imaging of sperm cells provides a preliminary work and feasibility analysis for the subsequent in-depth study of sperm cells with normal and abnormal DNA in spatio-temporal resolution.In the fourth chapter,we mainly combined Raman spectroscopy and fluorescence staining to evaluate the application of hyaluronic acid in the selection of high-quality sperm cells.First,glass substrates coated with hyaluronic acid and the effect was characterized.The Raman signals of sperm head bound to hyaluronic acid(experimental group)and that not bound to hyaluronic acid were collected,and it was found that there are significant differences in the Raman spectra between them.Next,we performed nuclear protein staining and nuclear DNA staining on the sperm cells(after incubation and washing)both from experimental group and the control group.The staining results verified that the sperm cells combined with hyaluronic acid in the experimental group had more mature nuclear proteins and higher DNA double-stranded content.In summary,the application of Raman spectroscopy to the study of DNA damage in single sperm cells on glass substrates,combined with multivariate statistical analysis methods,can distinguish between DNA-damaged and DNA non-damaged sperm cells;Raman spectroscopy imaging mode can provide assessment of the spatial distribution of sperm DNA quality/damage status.In addition,Raman spectroscopy and fluorescence imaging methods have verified the feasibility of using hyaluronic acid to screen high-quality sperm cells with intact DNA.The research work in this paper is expected to provide a non-destructive and rapid method for the assessment of sperm DNA quality involved in male infertility. |
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