Comparison of chemiluminescence and flow cytometry in the estimation of reactive oxygen and nitrogen species in human semen.
OBJECTIVE
To determine the relative contribution of different cell types in washed sperm to the overall intracellular production of H(2)O(2) and peroxynitrite.


METHODS
Prospective study.


METHODS
University hospital.


METHODS
Thirty-one fertile volunteers and 166 men undergoing fertility assessment were included.


METHODS
Aliquots of sperm suspension in phosphate-buffered saline solution were used for the reactive oxygen species (ROS) detection by chemiluminescence and for the detection of H(2)O(2) and peroxynitrite by flow cytometry, with use of specific fluorescent probes, carboxy-2',7'-dichlorodihydrofluorescein diacetate dye for H(2)O(2) and dihydrorhodamine 123 for peroxynitrite. Gated analysis determined the relative contribution of spermatozoa, leukocytes, and "other round cells."


METHODS
Simultaneous estimates of global ROS production assessed by chemiluminescence assay compared with flow cytometric measurements.


RESULTS
The estimates of ROS with use of chemiluminescence positively correlated with the estimates of H(2)O(2) (r = 0.53) and peroxynitrite (r = 0.62) as assessed with flow cytometry. H(2)O(2) and peroxynitrite were measurable also in samples in which chemiluminescence did not detect measurable values. Increased production of H(2)O(2) by one cell type was associated with a relative increase in its peroxynitrite production.


CONCLUSIONS
The levels of ROS production measured by chemiluminescence and flow cytometry were related. Each cell type in semen contributed differently to the global intracellular levels of H(2)O(2) and peroxynitrite.
Aziz
Nabil
N
Gynaecology and Reproductive Medicine, Liverpool Women's Hospital and the University of Liverpool, Merseyside, United Kingdom. naziz@liv.ac.uk
Novotny
Jiri
J
Oborna
Ivana
I
Fingerova
Helena
H
Brezinova
Jana
J
Svobodova
Magda
M
D000328:Adult; D016022:Case-Control Studies; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D007248:Infertility, Male / Q000175:diagnosis / Q000378:metabolism; D007958:Leukocyte Count; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D030421:Peroxynitrous Acid / Q000032:analysis / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000032:analysis* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism; D012661:Semen / Q000737:chemistry* / Q000378:metabolism; D055101:Semen Analysis / Q000379:methods; D055815:Young Adult
D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D006861:Hydrogen Peroxide