AIM: The aim of this prospective study was to find possible relationship between ROS production measured by chemiluminescence and flow cytometry in human semen and sperm DNA damage estimated by Sperm Chromatin Structure Assay. METHODS: Study included 39 men from infertile couples and 23 fertile volunteers who served as a control group. Aliquot of neat semen was used for ROS detection by chemiluminescence. Aliquot of sperm suspension in phosphate buffered saline was used for the detection of ROS by flow cytometry. Another aliquot of sperm suspension was used for SCSA to measure DNA fragmentation index and High DNA stainability. RESULTS: DNA fragmentation index correlated negatively with sperm morphology and motility. High DNA stainability correlated positively with ROS production and negatively with sperm morphology and concentration. Although there were similar trends of rising DNA fragmentation index and ROS production among the three groups of men, the relationship did not reach statistical significance. CONCLUSIONS: Higher values of DNA fragmentation index and high DNA stainability may also reflect developmental and/or environmental problems and not only oxidative stress.
- MeSH
- chromatin MeSH
- dospělí MeSH
- genetické techniky MeSH
- lidé MeSH
- poškození DNA * MeSH
- prospektivní studie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- sperma metabolismus MeSH
- spermie * MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Thirty-one fertile volunteers and 166 men undergoing fertility assessment were included. INTERVENTION(S): 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." MAIN OUTCOME MEASURE(S): Simultaneous estimates of global ROS production assessed by chemiluminescence assay compared with flow cytometric measurements. RESULT(S): 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. CONCLUSION(S): 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. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
- MeSH
- analýza spermatu metody MeSH
- dospělí MeSH
- kyselina peroxydusitá analýza metabolismus MeSH
- lidé MeSH
- luminiscenční měření * metody MeSH
- mladý dospělý MeSH
- mužská infertilita diagnóza metabolismus MeSH
- peroxid vodíku analýza metabolismus MeSH
- počet leukocytů MeSH
- průtoková cytometrie * metody MeSH
- reaktivní formy dusíku * analýza metabolismus MeSH
- reaktivní formy kyslíku * analýza metabolismus MeSH
- sperma * chemie metabolismus MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
AIMS: Excessive production of reactive oxygen species (ROS) in semen has been linked to male infertility. Main sources of ROS in male genital tract are immature and/or damaged spermatozoa and a subpopulation of leukocytes known as polymorphonuclear neutrophils (PMN). METHODS: Study group included male partners of infertile couples, 67 normospermic males (group B) and 98 males with sperm abnormalities in one or more parameters (group C), 36 fertile volunteers (group A) served as controls. Sperm parameters were determined according to WHO guidelines. The ROS production was measured by chemiluminiscence in sperm suspension in phosphate buffered saline. RESULTS: All fertile volunteers in the control group had seminal PMN concentrations below 0.5x10(6)/ml. Therefore study subjects, 67 normospermic and 98 men with sperm abnormalities, were further subdivided into two subgroups of PMN concentrations: (1) < 0.5x10(6)/ml and (2) 0.5 to 1.0x10(6)/ml. The ROS production in individuals varied greatly from 1.0x10(2) to 1.7 x10(7) RLU/min per 20x10(6) spermatozoa. The ROS production in both subgroups of normospermic men and the subgroup (1) of men with sperm abnormalities was not different from the ROS production in fertile controls. The ROS production in the subgroup (2) with sperm abnormalities was significantly higher than in controls (P = 0.00004). CONCLUSIONS: Our findings suggest that the contribution of PMN to the ROS production in semen is negligible only up to a concentration of 0.5x10(6)/ml. This suggests that the current WHO Guidelines threshold of 1.0x10(6) PMN per ml of semen is too high and might be re-evaluated.
