Spectral features of nuclear DNA in human sperm assessed by Raman Microspectroscopy: Effects of UV-irradiation and hydration

Abstract

Raman Microspectroscopy represents an innovative tool for the assessment of sperm biochemical features otherwise undetectable by routine semen analysis. Previously, it was shown that induced DNA damage can be detected in smeared sperm by this technique. This novel readout may be of value for clinical settings especially if it can be transferred to living cells. Yet, starting with living sperms this study was carried-out using a variety of conditions to disclose the Raman features of sperm nuclei under different hydration conditions and UV exposure. Human sperm were immobilized and Raman spectra were obtained from individual sperm as repeated measurements. To create conditions with controlled DNA damage, sperm samples were exposed to ultraviolet light. Several media were used to evaluate their effect on Raman spectra in aqueous conditions. To substantiate differences between the experimental conditions, the spectra were analyzed by Principal Component Analysis. We observed that spectra of sperm nuclei obtained in different solutions showed a qualitatively unchanged spectral pattern showing the principal signals related to DNA. Evaluating the effect of ultraviolet light generated the finding that spectra representing DNA damage were only observed in dry conditions but not in aqueous medium. Thus, Raman microspectroscopy was successfully applied for sperm analysis in different conditions, among them in live spermatozoa in aqueous solution during the initial measurement, revealing the principle use of this technique. However, implementation of Raman spectroscopy as a technique for clinical sperm analysis and selection may be especially relevant when DNA evaluation can be established using live sperm.

Fig 1. Effect of different solutions/media.

(A) Average Raman spectrum (n = 10) of the sperm DNA in different solutions/media in which the most common peaks mentioned in the text are pointed out. (B) Principal component analysis of individual spectra. The confidence interval of the ellipses is 95% considering a normal distribution.

Fig 2. Comparison between the loading values of the PC1 in Fig 1 and a typical Raman spectrum of the sperm DNA.

The values were generated from the comparison of the different media/solutions. The black line denotes zero in the loading values.

Fig 3. Effect of hydration conditions on the DNA spectrum of individual sperm exposed to ultraviolet light (UV).

The results shown in this figure correspond to particular sperm cells that were followed through the time in all condition using a coordinate system. (A) Average Raman spectrum (n = 50) of the DNA of non-exposed (control) and UV light exposed (UV) sperm in hydrated conditions. (B) Principal component analysis of single spectra of sperm in hydrated conditions. (C) Average Raman spectrum (n = 50) of the DNA of non-exposed (control) and UV light exposed (UV) sperm in dehydrated conditions. (D) Principal component analysis of single spectra of sperm in dehydrated conditions. The confidence interval of the ellipses is 95% considering a normal distribution.

Fig 4. Comparison between the loading values of the PC1 in Fig 3B and a typical Raman spectrum of the sperm DNA.

The values were generated from the comparison of the spectra of individual sperm (control/UV exposed) evaluated in aqueous medium.

Fig 5. Comparison between the loading values of the PC1 in Fig 3D and a typical Raman spectrum of the sperm DNA.

The values were generated from the comparison of the spectra of individual sperm (control/UV exposed) evaluated in dry conditions.