title: Light, polarizing, and transmission electron microscopy: three methods for the evaluation of sperm quality.
abstract: Semen from 33 patients were evaluated by light microscopy (LM) obtaining sperm concentration, percent motility, percentage of sperm with normal morphology (PAP staining), and percentage of dead sperm (Eosin Y stained). The samples were observed by polarizing microscopy (PM), that evaluates sperm morphology and the viability by birefringence of organelles, and it provides a PM index (percentage of birefringent, viable, motile sperm) and a percentage of dead, non-birefringent sperm. Sperm were processed for transmission electron microscopy (TEM) and TEM data were elaborated with a mathematical formula able to provide a fertility index (FI, number of sperm free of structural defects) and percentages of sperm immaturity and necrosis (dead sperm). To test the reliability of these techniques, the values of normal acrosome, nucleus, midpiece, and tail and the presence of cytoplasmic residues obtained with the three methods were compared. With the exception of cytoplasmic residues (P = 0.40), significant differences in the evaluation of each organelle were observed and TEM analysis resulted as the most stringent screening. In addition, relationships among relevant sperm variables were investigated. Motility showed positive correlations with the percentage of normal tail, midpiece, and PM index (P < 0.01), but it exhibited negative correlations with indices of sperm death (non-birefringent sperm: P < 0.05; percentage of eosin Y stained sperm: P < 0.05; necrosis: P < 0.01), which were positively correlated with each other (P < 0.01). Positive correlations were found between indices expressing normal sperm morphology: FI with PM index (P < 0.01) and with the percentage of normal sperm (PAP staining) (P < 0.01), which in turn were correlated with the PM index (P < 0.001). Sperm immaturity showed positive correlations (P < 0.01) with the presence of cytoplasmic residues detected with the three methods. In conclusion, LM, PM, and TEM are reliable techniques in evaluating sperm quality. PM appears to offer several advantages 'midway' between LM and TEM and it should be considered in sperm analysis.
authors.affiliation: Department of Biomedical Sciences, Applied Biology Section, Interdepartmental Centre for Research and Therapy of Male Infertility, University of Siena, Siena, Italy. giulia.collodel@unisi.it
authors.forename: Giulia
authors.initials: G
authors.lastname: Collodel
authors.forename: Francesca
authors.initials: F
authors.lastname: Iacoponi
authors.forename: Lucia
authors.initials: L
authors.lastname: Mazzi
authors.forename: Gaia
authors.initials: G
authors.lastname: Terzuoli
authors.forename: Nicola Antonio
authors.initials: NA
authors.lastname: Pascarelli
authors.forename: Elena
authors.initials: E
authors.lastname: Moretti
mesh_terms: D000177:Acrosome; D016923:Cell Death; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis*; D008297:Male; D008853:Microscopy; D046529:Microscopy, Electron, Transmission; D008859:Microscopy, Polarization; D055101:Semen Analysis; D013076:Sperm Count; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology*