Digital holographic microscopy in human sperm imaging

  • Igor Crha
  • Jana Zakova
  • Martin Huser
  • Pavel Ventruba
  • Eva Lousova
  • Michal Pohanka
Technical Innovations



The aim of this study was to use digital holographic microscopy (DHM) in human sperm imaging and compare quantitative phase contrast of sperm heads in normozoospermia (NZ) and oligoasthenozoospermia (OAT).


DHM spermatozoa imaging and repeated quantitative phase shift evaluation were used. Five NZ and 5 OAT samples were examined. Semen samples were examined by semen analysis and processed for DHM. Main outcome measures were maximum phase shift value of the sperm heads. Differences of the phase shift and in NZ and OAT samples were statistically tested.


In NZ samples median phase shifts were in the range 2.72–3.21 rad and 2.00–2.15 in OAT samples. Differences among individual samples were statistically significant (p < 0.001) in both groups. Median phase shift according to sperm count was 2.90 rad in NZ samples and 2.00 rad in OAT samples. This difference was statistically significant (p < 0.001).


Quantitative evaluation of the phase shift by DHM could provide new information on the exact structure and composition of the sperm head. At present, this technique is not established for clinical utility.


Digital holographic microscopy Sperm imaging Spermatozoon Male infertility Chromatin integrity 


  1. 1.
    Marquet P, Rappaz B, Magistretti PJ, Cuche E, Emery Y, Colomb T, et al. Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy. Opt Lett. 2005;30:468–70.PubMedCrossRefGoogle Scholar
  2. 2.
    Vesely P, Blase C, Matouskova E, Bereiter-Hahn J. Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment. Anticancer Res. 2006;26:967–72.PubMedGoogle Scholar
  3. 3.
    Depeursinge C, Colomb T, Emery Y, Kuhn J, Charriere F, Rappaz B, et al. Digital holographic microscopy applied to life sciences. Conf Proc IEEE Eng Med Biol Soc. 2007;2007:6244–7.PubMedGoogle Scholar
  4. 4.
    Janeckova H, Vesely P, Chmelik R. Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy. Anticancer Res. 2009;29:2339–45.PubMedGoogle Scholar
  5. 5.
    Kolman P, Chmelik R. Coherence-controlled holographic microscope. Opt Express. 2010;18:21990–2003.PubMedCrossRefGoogle Scholar
  6. 6.
    Chmelik R, Harna Z. Parallel-mode confocal microscope. Opt Eng. 1999;38:1635–9.CrossRefGoogle Scholar
  7. 7.
    Chmelik R. Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes. J Mod Opt. 2006;53:2673–89.CrossRefGoogle Scholar
  8. 8.
    Dubois F, Joannes L, Legros JC. Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence. Appl Opt. 1999;38:7085–94.PubMedCrossRefGoogle Scholar
  9. 9.
    Janeckova H, Kolman P, Vesely P, Chmelik R. Digital holographic microscope with low-spatial and temporal coherence of illumination. In: Optical and Digital Image Processing. Proc. of SPIE 2008; 7000:1–8.Google Scholar
  10. 10.
    World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed, 1999. Cambridge University Press.Google Scholar
  11. 11.
    Crha I, Kralikova M, Melounova J, Ventruba P, Zakova J, Beharka R, et al. Seminal plasma homocysteine, folate and cobalamin in men with obstructive and non-obstructive azoospermia. J Assist Reprod Genet. 2010;27:533–8.PubMedCrossRefGoogle Scholar
  12. 12.
    Oliva R. Protamines and male infertility. Hum Reprod Update. 2006;12:417–35.PubMedCrossRefGoogle Scholar
  13. 13.
    Lefièvre L, Bedu-Addo K, Conner SJ, Machado-Oliveira GS, Chen Y, Kirkman-Brown JC, et al. Counting sperm does not add up any more: time for a new equation? Reproduction. 2007;133:675–84.PubMedCrossRefGoogle Scholar
  14. 14.
    Zini A, Phillips S, Courchesne A, Boman JM, Baazeem A, Bissonnette F, et al. Sperm head morphology is related to high deoxyribonucleic acid stainability assessed by sperm chromatin structure assay. Fertil Steril. 2009;91:2495–500.PubMedCrossRefGoogle Scholar
  15. 15.
    Mudrak O, Tomilin N, Zalensky A. Chromosome architecture in the decondensing human sperm nucleus. J Cell Sci. 2005;118:4541–50.PubMedCrossRefGoogle Scholar
  16. 16.
    Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod. 2010;16:30–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Moskovtsev SI, Willis J, Azad A, Mullen JB. Sperm DNA integrity: correlation with sperm plasma membrane integrity in semen evaluated for male infertility. Arch Androl. 2005;51:33–40.PubMedCrossRefGoogle Scholar
  18. 18.
    Akgul M, Ozkinay F, Ercal D, Cogulu O, Dogan O, Altay B, et al. Cytogenetic abnormalities in 179 cases with male infertility in Western Region of Turkey: Report and review. J Assist Reprod Genet. 2009;26:119–22.PubMedCrossRefGoogle Scholar
  19. 19.
    Winkle T, Rosenbusch B, Gagsteiger F, Paiss T, Zoller N. The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center. J Assist Reprod Genet. 2009;26:41–6.PubMedCrossRefGoogle Scholar
  20. 20.
    García-Herrero S, Garrido N, Martínez-Conejero JA, Remohí J, Pellicer A, Meseguer M. Ontological evaluation of transcriptional differences between sperm of infertile males and fertile donors using microarray analysis. J Assist Reprod Genet. 2010;27:111–20.PubMedCrossRefGoogle Scholar
  21. 21.
    Gianaroli L, Magli MC, Ferraretti AP, Crippa A, Lappi M, Capitani S, et al. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertil Steril. 2010;93:807–13.PubMedCrossRefGoogle Scholar
  22. 22.
    Petersen CG, Vagnini LD, Mauri AL, Massaro FC, Cavagna M, Baruffi RL, Oliveira JB, Franco JG Jr. Relationship between DNA damage and sperm head birefringence. Reprod Biomed Online. 2011 Mar 21. [Epub ahead of print] doi:10.1016/j.rbmo.2011.03.017
  23. 23.
    Hammadeh ME, Bernardi A, Zeginiadou T, Amer A, Schmidt W. Relationship between nuclear chromatin decondensation (NCD) in vitro and other sperm parameters and their predictive value on fertilization rate in IVF program. J Assist Reprod Genet. 2005;22:301–5.PubMedCrossRefGoogle Scholar
  24. 24.
    Kolle S, Reese S, Kummer W. New aspects of gamete transport, fertilization, and embryonic development in the oviduct gained by means of live cell imaging. Theriogenology. 2010;73:786–95.PubMedCrossRefGoogle Scholar
  25. 25.
    Ierardi V, Niccolini A, Alderighi M, Gazzano A, Martelli F, Solaro R. AFM characterization of rabbit spermatozoa. Microsc Res Tech. 2008;71:529–35.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Igor Crha
    • 1
  • Jana Zakova
    • 1
  • Martin Huser
    • 1
  • Pavel Ventruba
    • 1
  • Eva Lousova
    • 1
  • Michal Pohanka
    • 2
  1. 1.Department of Gynecology and Obstetrics, Faculty of MedicineMasaryk University, and Faculty HospitalBrnoCzech Republic
  2. 2.Department of Functional Diagnostics and RehabilitationSt. Anne’s Faculty HospitalBrnoCzech Republic

Personalised recommendations