In situ visualization of damaged DNA in human sperm by Raman microspectroscopy

Abstract

Background: Beyond determining the percentage of damaged sperm, current Methods: of DNA assessment are of limited clinical utility as they render the sample unusable. We evaluated Raman microspectroscopy, a laser-based non-invasive technique that provides detailed chemical 'fingerprints of cells and which potentially could be used for nuclear DNA-based sperm selection. Methods: Eight healthy donors provided ejaculates. After system optimization, a minimum of 200 air-dried sperm/sample/donor, prior to/and after UVB irradiation, were assessed by two observers. Spectra were analysed by Principal Component, Spectral Angle and Wavelet Analyses. Results: Spectra provided a chemical map delineating each sperm head region. Principal Component Analysis showed clear separation between spectra from UV-irradiated and untreated samples whilst averaged data identified two regions of interest (1040 and 1400 cm -1). Local spectral analysis around the DNA PO4 backbone peak (1042 cm-1), showed that changes in this region were indicative of DNA damage. Wavelet decomposition confirmed both the 1042 cm-1 shift and a second UVB susceptible region (14001600 cm-1) corresponding to proteinDNA interactions. No difference was found between observer measurements. Conclusions: Raman microspectroscopy can provide accurate and reproducible assessment of sperm DNA structure and the sites and location of damage. © 2011 The Author.