{"doi":"10.1002/cyto.a.24177","title":"Flow Cytometry of Male Reproductive Potential","abstract":"Male infertility is an increasing health problem. Studies show that in infertile couples, the malefactor contribution is 40% (1). One of the leading causes of male sterility is decreased sperm function (2). Nitrosative stress or nitrosylation of redox-sensitive thiols by reactive nitrogen species is often associated with male infertility and induces mitochondrial damage in human spermatozoa. While paternal mitochondria are generally not passed on to the next generation, they are critical for sperm swimming speed and force to propel through the female reproductive tract to meet and penetrate the egg for fertilization. Uribe and coworkers established a four-parameter functional test to evaluate sperm quality using flow cytometry (3) (Fig. 1). Propidium iodide was used as a viability dye combined with three cell-permeable tracers: boronate, monobromobimane, and tetramethylrhodamine methyl ester perchlorate. Boronate is a commonly used probe to identify cell-derived peroxynitrite which is a potent mediator of nitrosative stress (4). Pinacol boronic ester covalently bound to fluorescein allows flow cytometric quantification of the reaction between peroxynitrite and boronate. Monobromobimane is another fluorometric probe that, upon linking to non-oxidized thiols, forms a fluorescent thiol-monobromobimane adduct (excitation 380 nm; emission 478–480 nm) (5). Tetramethylrhodamine methyl ester perchlorate is a rhodamine dye that has a transmembrane potential-dependent accumulation in mitochondria and is broadly used to analyze mitochondrial health by flow cytometry (6). The authors showed that this panel of dyes offers a useful and easy tool to evaluate both fresh and frozen–thawed human semen quality. In a first approach, the “swim-up” method, a common technique in the clinic based on the self-migration of the gametes, was utilized to purify spermatocytes with the highest motility. These migratory spermatozoa exhibited high mitochondrial transmembrane potential and non-oxidized thiol levels and low amounts of peroxynitrite, compared with native semen samples. In a second approach, swim-up spermatozoa were exposed to a peroxynitrite-inducing chemical compound 3-morpholinosydnonimine or sin-1 before labeling with the dyes. The generation of peroxynitrite did not affect the viability, but lowered the mitochondrial transmembrane potential and increased thiol oxidation. This promising work further strengthened the use of flow cytometry as a valuable and quantitative tool in the repertoire of sperm evaluation techniques. Indeed flow cytometry has been used to study several aspects of cellular function in semen and potential selection of a baby's gender (7, 8). The current publication opens new venues to apply flow cytometry in andrology research. For example, by expanding the panel with additional mitochondria function probes. It would be interesting to know how the retention of the dyes utilized in this article relates to mitochondrial biomass, which can be readily accessed by flow cytometry using MitoTracker (9, 10). Quantification of nicotinamide adenine dinucleotide (NADH) fluorescence is another flow cytometric measure which provides insight into the mitochondrial complex I function (11, 12). Generally, NADH is considered as a significant contributor to cellular autofluorescence. Still, these energy-rich intermediates also serve as one of the electron sources of the mitochondrial electron transport chain that maintains the transmembrane potential. Variations in mitochondrial mass and other mitochondria functions may be additional essential parameters in the evaluation of spermatocytes (13). The newly described assay combining four probes needs further validation in a clinical trial to study its efficacy in the selection of a more functional sperm subpopulation than the classic swim-up method. Expression of the fluorescence values in absolute units rather than arbitrary mean fluorescence intensity values may also facilitate standardizati","journal":"Cytometry Part A","year":2020,"id":116775,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9579,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":280250,"name":"Kewal Asosingh","orcid":null,"position":0,"is_corresponding":true}],"reference_count":14,"raw_metadata":null,"created_at":"2026-07-18T23:13:47.803267Z","pmid":"32583489","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}