Real time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida

Raman microspectroscopy scanning of single live sperm is able to distinguish the human zona pellucida–bound sperm from unbound sperm.

Feng Liu, B.Sc., Yong Zhu Ph.D., Yufei Liu, Xiaobo Wang, Ping Ping Ph.D., Xinyuan Zhu, Ph.D., Hongliang Hu, Ph.D., Zheng Li M.D., Ph.D., Lin He, Ph.D.

Volume 99, Issue 3, Pages 684-689.e4, 1 March 2013


To determine if the Raman micro-spectroscopy (RMS) could be used to distinguish sperm bound to the human zona pellucida (ZP) from those unbound sperm.

Paired experiments in comparison Raman scanning feature of ZP bound and unbound sperm.

Public hospital-based clinical ART center.

Sperm samples from 10 fertile donors were used in this study.


Main outcomes measure(s):
Sperm-ZP binding, the ZP-induced acrosome reaction and scanning intensity of various regions of sperm.

The RMS found two slightly low intensity regions (800-900 and 3200-4000 cm-1) shifted to high intensity grade at the acrosome region of the ZP-bound sperm compared with unbound sperm. Moreover, principal component analysis (PCA) and statistical analysis showed that the RMS can distinguish the ZP-bound sperm from those unbound sperm.

RMS scanning of single live sperm could be used to distinguish ZP-bound sperm from those unbound sperm. Thus the RAM may be useful tool to detect normal functional sperm and to select sperm for ICSI.

  • fdominguez

    I agree with some of the concerns raised by previous collegues. First, it is clear that the title of the paper is quite inaccurate due to the methods and samples used in the study. But I believe that if the authors have measured such a huge number of spectra as they told (10 patients x 100 sperm x 6 localizations) in each spermatozoa they should have enough data to demonstrate that is a good tool of sperm selection. Somehow the authors should have shown at least a representative number of spectra to be confident in the results and not only one spectrum/condition. It does not help to avoid standard error in figures. I guess the authors should comment on that issue too.

  • Gail S. Prins

    I have an issue with the title of this study with regards to a “…single live cell…” in that it does not appear that this analysis was performed on a live cells (the sperm were smeared and air dried on a glass slide) nor do the data presented come from a single cells. The methods state that 100 SP-bound and 100 unbound sperm were analyzed per specimen, and there are 10 specimens examined. That would be 1000 sperm/group for the 2 types of cells. The data presented in Figure 3 with statistical analysis, on which the conclusions are based, are from 1000 sperm/group (no error bars appear on these graphs, unfortunately). So how are the findings relative to a single cell? It is not clear whether the individual selected spectra of single cells is representative, i.e. occurred on all cells tested. More importantly, how could this ever be traced back to identifying a single cell to be chosen for ICSI, which is the gist of the entire Discussion section? It appears as if the conclusions reached are premature at best.

  • This work appears to need further clarification. I share many of the same concerns of Con Mallidis. Specifically, it is unclear to me from the paper whether the sperm analyzed were live sperm as the authors imply that they were are dried and fixed prior to Raman microspectroscopy. Please clarify this point.

    I lack the background in Raman spectroscopy to address the points on Figure 1 and 2 as well as those related to the interpretation of the peaks. So would allow the authors to address these specific technical points.

    Despite these methodological issues, the paper purports to address a very important issue, selecting the optimal sperm for use in ICSI. The appeal of techniques such as FT-IR or Raman microspectroscopy lie in their ability to yield diagnostic information on live cells without damaging them. It is not clear to me that this paper performs this analysis on live sperm cells or fixed sperm. If done on fixed sperm and if the Raman spectroscopy was performed and interpreted correctly then this paper serves as a proof of principle for a new technique. However, these issues need to be clarified by the authors.

  • Con Mallidis

    The article by Liu et al contains numerous points that are
    problematic and need to be explained if the veracity of their findings
    is to be established. Specifically:

    – as the analyses were done on air dried samples, how are they “real time” and how on “live” sperm?

    how is it possible for the various segments of the cells (Figure 1) to
    have the same spectral signatures? This is particularly perplexing as
    the segments are primarily composed of differing and distinct elements
    (e.g. tail – protein; nucleus – nucleic acids etc)

    – Why do the
    spectra not show any of the distinctive peaks in the fingerprint region
    found by Huser et al, Meister et al and ourselves?

    – From Figure 2
    it is obvious that no suitable baseline correction has been performed,
    how then can differences in intensities be gauged? What is the criteria
    for such comparisons?

    – The changes listed in the text state
    that above 1441 everything increased whilst below all peaks decreased.
    How is this explained in physiological terms? Why is this not indicative
    of systematic error?

    – As the resolution of all the spectra is so low how were the peaks with differences determined?

    can an explanation be provided for the statement “The intensity of the
    nucleic acid backbone increase is due to the preacrosome’s broken
    membrane, then the nucleic-acid exposed to only the sperm membrane
    instead of two membranes, including preacrosome and cell membranes.”?
    How can the presence/absence of membranes be responsible for the signal
    emanating from nucleic acids? Why hasn’t this phenomena been seen in any
    of the other studies.

    Finally, the introduction of a new technique is dependent on the stringency
    of its application. The potential of Raman spectroscopy is as yet
    unrealised however the veracity of the technique can only be established
    by carefully conducted, controlled and analysed studies. Unfortunately
    this work does not conform to any of these requirements.

    • Yong

      Thanks for all your attention on our research.

      Firstly, as you indicated, the real time data of raman scanning usually obtained in 5-30 second in recent research, and the chemical bond state could be reflect
      immediately from the Raman spectrums. Furthermore, we have found the consistent spectrums between fresh semen and sperms air-dried for 48 hours (unpublished data).

      Secondary, it was our preliminary research and the whole shape of the Raman wave were looked less different using the original data format (Figures 2).But after the correction of the baseline and smoothing processing et al., a few peaks found distinct in control group and experimental group (Figure 4 and 5) and the
      chemical composition of these peaks have been reported by other researches in
      other cells.

      Next, the explanation of “The intensity of the nucleic acid backbone increase….
      including preacrosome and cell…..” is that the ZP-Bound sperm could be
      easily induced acrosome reaction and the depth of raman scanning is changed
      from our research.

      Finally, it is known that raman scanning is a new but an widely applied technique in other clinical diagnosis like cancer research. So we believe it has a real potential in the application of reproductive filed in 3-5 years around the world. The scientist should have the character of seeing the meaning of their research in the long run, and then the development would be systematic and positive.

      • Con Mallidis

        We welcome the response by the authors and wholeheartedly agree with the need for systematic and judicious development, that was the gist and aim of our comment. How well the answers provided address the issues raised however, we leave to the discretion of the community.

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