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Related Experiment Videos

A new multiparameter flow cytometric method for human semen analysis.

S Perticarari1, G Ricci, M Granzotto

  • 1Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo, Italy.

Human Reproduction (Oxford, England)
|November 3, 2006
PubMed
Summary
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This study demonstrates that combining Syto 16 and 7-amino-actinomycin-D (7-AAD) effectively detects sperm apoptosis. A new multiparameter flow cytometry method enables simultaneous assessment of sperm concentration, viability, apoptosis, and leukocyte concentration.

Area of Science:

  • Reproductive biology
  • Flow cytometry applications
  • Spermatozoa analysis

Background:

  • Sperm apoptosis detection is crucial for male fertility assessment.
  • Existing methods for semen analysis can be time-consuming and lack multi-parameter capabilities.

Purpose of the Study:

  • To evaluate the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) for sperm apoptosis detection.
  • To introduce a novel multiparameter flow cytometry method for simultaneous assessment of sperm concentration, viability, apoptosis, and leukocyte concentration.

Main Methods:

  • Semen samples from 68 patients were analyzed using World Health Organization (WHO) criteria.
  • Sperm apoptosis was detected via flow cytometry using Syto 16 and 7-AAD staining.
  • A multiparameter flow cytometric analysis, including fluorescent microspheres for sperm concentration and anti-CD45 for leukocytes, was performed.

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Main Results:

  • A significant correlation was found between low Syto 16 fluorescence and caspase activation, indicating apoptosis (r=0.68, P=0.0106).
  • Betulinic acid incubation increased apoptotic cells, confirmed by both Syto 16/7-AAD and caspase activation methods.
  • Flow cytometry showed high correlation with optical microscopy for sperm (r=0.98) and leukocyte counts (r=0.64).
  • Apoptotic sperm percentage inversely correlated with sperm concentration and morphology, but not motility.

Conclusions:

  • The Syto 16/7-AAD combination offers a sensitive assay for detecting sperm apoptosis.
  • The described multiparameter flow cytometry method allows for simple, rapid, and accurate simultaneous assessment of multiple semen parameters.