Abstract
To determine whether Raman microspectroscopy can identify different levels of oxidative sperm nDNA damage and to corroborate the findings using an established method and an alternative but complementary spectroscopic technique. Three-way comparison of Raman profiles, Fourier transform infrared spectroscopy (FTIR) spectra, and flow-cytometric assessments of sperm nDNA damage. University-based research laboratory. Thirty-eight men attending the infertility clinic at the Centre of Reproductive Medicine and Andrology. Induction of oxidative damage by Fenton's reaction on semen samples. Raman profiles, FTIR spectra, and flow-cytometric analysis of DNA fragmentation. Raman and FTIR spectra contained distinctive differences between untreated and fragmented nDNA sperm that were indicative of oxidative attack. The changes in Raman profiles were similar to those previously seen and corresponded to the DNA backbone. The peak attributions were corroborated by the FTIR spectra. Principal component analysis of the entire Raman spectra distinguished samples with varying degrees of damage. After determination of a cutoff value (0.63), estimation of the percentage of sperm with nDNA damage using the intensity ratio of Raman peaks (1,050/1,095 cm(-1)) correlated linearly to the flow-cytometric assessment. Raman microspectroscopy still requires further validation but may potentially provide a means of assessing the nDNA status of a living sperm.
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