Fluorescent probes for the detection of reactive oxygen species in human spermatozoa

Reactive oxygen species(ROS)production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state,a requirement for functional spermatozoa.However,an increase in oxidative stress,due to an abnormal production of ROS,has been shown to be related to loss of sperm function,highlighting the importance of an accurate detection of sperm ROS,given the specific nature of this cell.In this work,we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species(RNS)in human sperm,to define their specificity.Using both flow cytometry(FC)and fluorescence microscopy(FM),we confirmed that MitoSOX™Red and dihydroethidium(DHE)detect superoxide anion(as determined using antimycin A as a positive control),while DAF-2A detects reactive nitrogen species(namely,nitric oxide).For the first time,we also report that RedoxSensor™Red CC-1,CellROX®Orange Reagent,and MitoPYl seem to be mostly sensitive to hydrogen peroxide,but not superoxide.Furthermore,mean fluorescence intensity(and not percentage of labeled cells)is the main parameter that can be reproducibly monitored using this type of methodology.

Biomarkers of hypoxic-ischemic encephalopathy:a systematic review

Background Current diagnostic criteria for hypoxic–ischemic encephalopathy in the early hours lack objective measurement tools.Therefore,this systematic review aims to identify putative molecules that can be used in diagnosis in daily clinical practice(PROSPERO ID:CRD42021272610).Data sources Searches were performed in PubMed,Web of Science,and Science Direct databases until November 2020.English original papers analyzing samples from newborns>36 weeks that met at least two American College of Obstetricians and Gynecologists diagnostic criteria and/or imaging evidence of cerebral damage were included.Bias was assessed by the Newcastle–Ottawa Scale.The search and data extraction were verified by two authors separately.Results From 373 papers,30 met the inclusion criteria.Data from samples collected in the first 72 hours were extracted,and increased serum levels of neuron-specific enolase and S100-calcium-binding protein-B were associated with a worse prognosis in newborns that suffered an episode of perinatal asphyxia.In addition,the levels of glial fibrillary acidic protein,ubiquitin carboxyl terminal hydrolase isozyme-L1,glutamic pyruvic transaminase-2,lactate,and glucose were elevated in newborns diagnosed with hypoxic–ischemic encephalopathy.Moreover,pathway analysis revealed insulin-like growth factor signaling and alanine,aspartate and glutamate metabolism to be involved in the early molecular response to insult.Conclusions Neuron-specific enolase and S100-calcium-binding protein-B are potential biomarkers,since they are correlated with an unfavorable outcome of hypoxic-ischemic encephalopathy newborns.However,more studies are required to determine the sensitivity and specificity of this approach to be validated for clinical practice.