Evaluation of Laser Confocal Raman Spectroscopy as a Non-Invasive Method for Detecting Sperm DNA Contents

Abstract

Research question: Is Raman spectroscopy an efficient and accurate method to detect sperm chromosome balance state by DNA content differences?

Design: Semen samples were provided by diploid healthy men, and the analysis parameters met the current World Health Organization standards. The DNA content was assessed by analysis of the corresponding spectra obtained from a laser confocal Raman spectroscope. The sperm sex chromosome information was obtained by fluorescence in situ hybridization (FISH). Comparative analysis was performed between FISH results and Raman spectral analysis results.

Results: Different parts of the sperm head showed different spectral signal intensities, which indicated that there were different chemical components. Standard principal component analysis (PCA) can preliminarily classify sperm with different DNA contents into two groups. Further analysis showed that there were significant differences in the 785 DNA backbone peaks and 714-1,162 cm^-1 DNA skeleton regions among sperm with different DNA contents. The peak and regional peak of the DNA skeleton of X sperm were significantly higher than those of Y sperm (X vs. Y, p < 0.05). The above sperm types were confirmed by FISH. ROC curve analysis shows that there is a correlation between the Raman spectrum data and FISH results.

Conclusion: Raman spectroscopy can identify X and Y sperms by analyzing the DNA content difference. However, the accuracy of the detection still needs to be improved. Nevertheless, Raman spectroscopy has a potential application value in the field of sperm aneuploidy detection and may even be used as a non-invasive predictor of sperm aneuploid state in preimplantation genetic testing (PGT-A).

Keywords: DNA content; fluorescence in situ hybridization; human sperm; laser confocal Raman spectroscopy; preimplantation genetic testing.

Figure 1

The spectra of different regions in the sperm head. (A) Microscopic confocal picture of a human sperm cell head fixed on the glass slide. (B) The phase diagram of Raman microspectroscopy of the sperm head corresponding to (A). (C) Raman spectra of three different positions with glass (black), acrosome (red) and DNA at the sperm head. The x-axis units are displacement wavenumber (cm⁻¹), nanometer (nm), and absolute wavenumber (cm⁻¹). The y-axis unit is generally Raman strength.

Figure 2

Raman spectra of different sperm heads. (A–C) Three single sperm head Raman spectra. (D) The combined Raman spectra of the three sperms.

Figure 3

Raman light micrograph and corresponding FISH result map. (A) Analysis of sperm sex chromosome status in FISH under a fluorescence microscope. The nucleus of sperm head is stained by DAPI. The red hybridization signal on the blue sperm head represents X sperm, and green represents Y sperm (scale bar, 100 μm). (B) Planar Map of Raman Spectrum Corresponding to FISH (scale bar, 100 μm). (C) The Raman spectra of X and Y sperm. Red arrows, I785; Dashed box, Area (714–1,162).

Figure 4

PCA statistics of sperm spectra. The sperm were divided into two groups: group A included 22 sperm, and group B included 31 sperm (A: triangle B: circle).

Figure 5

Discrimination of sperm with different DNA contents by Raman spectroscopy. (A) Raman spectra of the 500–1800 cm⁻¹ area corresponding to X sperm and Y sperm. Black shows Y sperm and red shows X sperm. Raman peaks with the most significant difference for this study are highlighted by blue dotted lines. The x-axis units are displacement wavenumber (cm⁻¹), nanometer (nm). and absolute wavenumber (cm⁻¹). The y-axis unit is generally Raman strength. (B) The peak values of X and Y sperm in Area714-1,162 and I785. X, X sperm; Y, Y sperm. [n (X) = 39, n (Y) = 20, *, p < 0.05]. (C) Raman analysis data characteristics curve for sperm sex chromosomes. The area under the ROC curve indicates the prediction capacity of the model. Raman analysis data in I785 (AUC¼0.662). Raman analysis data in Area714-1,162 (AUC¼0.696).