Oxidative DNA damage in human sperm can be detected by Raman microspectroscopy

Capsule:
Raman microspectroscopy was able to identify oxidative sperm nDNA damage. The differences in spectral profiles were corroborated by Fourier transform infrared spectroscopy, and the levels of fragmentation were consistent with flow-cytometric assessment.

Authors:
Victoria Sánchez, B.Sc, Klaus Redmann, Ph.D., Joachim Wistuba, Ph.D., Frank Wübbeling, Ph.D., Martin Burger, Ph.D., Harriette Oldenhof, Ph.D., Willem F. Wolkers, Ph.D., Sabine Kliesch, M.D., Stefan Schlatt, Ph.D., Con Mallidis, Ph.D.

Volume 98, Issue 5, Pages 1124-1129.e3, November 2012

Abstract:

Objective:
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.

Design:
Three-way comparison of Raman profiles, FTIR spectra and flow cytometric assessments of sperm nDNA damage.

Setting:
University-based research laboratory.

Patients:
Thirty-eight men attending the infertility clinic at the Centre of Reproductive Medicine and Andrology.

Interventions:
Induction of oxidative damage by Fenton’s reaction on semen samples.

Main Outcome Measure(s):
Raman profiles, FTIR spectra, and flow cytometric analysis of DNA fragmentation.

Result(s):
Raman and FTIR spectra contained distinctive differences between untreated and fragmented nDNA sperm which 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. PCA of the entire Raman spectra distinguished samples with varying degrees of damage. After determination of a cut off value (0.63), estimation of the percentage of sperm with nDNA damage using the intensity ratio of Raman peaks (1050/1095 cm-118) correlated linearly to the flow cytometric assessment.

Conclusion(s):
Raman microspectroscopy still requires further validation but may potentially provide a means of assessing nDNA status of a living sperm.

  • Cristina G. Ravina

    Congratulations for such an interesting article, presenting a new noninvasively and nondestructively technology for the analysis of sperm nDNA quality. As you well mentioned, nowadays it is necessary to advance in searching an useful technique which allow us the selection in the same sample of the best spermatozoa for ICSI, and this could be a new future tool for this interest.
    However, I would appreciate a deeper explanation about the way of selecting the perfect sperm after using these techniques. I mean, in Mat&Met you mentioned that for analysis, aliquots of samples were left to air-dry overnight or at least for 15 minutes. I wonder if this is compatible with a good survival of spermatozoa.
    Finally, I want to mention the new tool for selection of apoptotic spermatozoa by inmunomagnetic removal: MACS (Magnetic cell sorting using annexin V). Certain features are characteristic of cells undergoing apoptosis, including nuclear fragmentation. MACS is a feasible and safe method that may be used to produce a high-quality sperm fraction, targeting spermatozoa with deteriorated membranes.

    • Con Mallidis

      Thank you for the kind words. Our paper is intended as the next phase of the proof of principle, consequently we dealt with showing that the technique is capable of identifying DNA damage resulting from different causes. We were in particular interested to show that oxidative damage (the proposed main physiological cause) could be found. As for the selection process, there are numerous means possible from micromanipulators to nanotraps.

      We are familiar with MACs and have used it in some of our experiments. As you
      mention Annexin V has been suggested as has Yo-Pro-1, IgG and possibly p53,
      however these work on surface molecules and hence don’t really provide a high
      yield of DNA intact sperm.

  • eykko

    Interesting article that brings an existing technology to the forefront of identifying sperm with “normal” DNA for ICSI. Current methods for evaluating oxidative sperm DNA damage render the sperm dead. Therefore, we us an aliquot as a sample of a larger group, hoping that this “sample” adequately reflects the larger community of sperm. However, there is no guarantee that the Embryologist and Andrologist picks the sperm without the defects.

    I hope this technology takes off and we can build upon this technology – specifically in the realm of evaluating living sperm that can then be utilized for ICSI. This article is succinctly summarized by Dr. M. Sigman in the preceding article: “…it is this type of innovative work that may lead to significant advances in the search for the perfect sperm.”

    A question though: IVF/ICSI is already expensive. Is this technology going to substantially increase these high costs for the couple desiring a child?

    • Con Mallidis

      Thank you for your kind appraisal. As to the cost, the main expenses would be the purchase of an instrument and the salary of the person operating it. Presently we are using a research grade instrument which is expensive (~ 150,000€) that said it has features and capabilities that far exceed those that would be needed for routine use. I’m sure, if the method fulfills its promise and enters the commercial arena, that more cost efficient and practical instruments will be developed.

  • Con Mallidis

    Views and opinions are most welcome. I’ll do my best to answer your questions.

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