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.

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research

“…It is clear that, in order to confirm the superiority of IMSI over ICSI and to identify the infertility causes that can benefit from IMSI, other studies are mandatory. The natural sperm birefringence, detectable under polarized light, was proposed as an alternative method to evaluate sperm morphology and also viability [Collodel et al 2013a] as it may be a good indicator of ICSI's success [Gianaroli et al 2008;Vermey et al 2015].…”

Evaluation of morphological criteria of sperm quality before in vitro fertilization and intracytoplasmic sperm injection

“…Sperm cells are birefringent (optically anisotropic). When the microscope light enters an anisotropic structure, it is refracted into two different refractive rays indicating this double refraction (Gianaroli et al 2008, Petersen et al 2011, Magli et al 2012, Collodel et al 2013. Microinjection into retrieved oocytes of individually selected spermatozoa with a strictly defined morphologically normal nuclear shape and content resulted in significantly higher pregnancy rates compared to the conventional ICSI (Bartoov et al 2003, Berkovitz et al 2005, Antinori et al 2008, Cassuto et al 2009).…”

“…The ultrastructure of sperm head, neck and tail can be analyzed and quantified by scanning or transmission electron microscopy (Moretti et al 2007, Collodel et al 2009a,b, Skowronek et al 2010, Visco et al 2010, Skowronek et al 2012, Collodel et al 2013, Gatimel et al 2013.…”

Sperm phenotypes in varicocele

“…B., Bragina Е. Е., Levina А. V. Sperm phenotypes in varicocele. Andrologiya i genital'naya khirurgiya = Andrology and Genital Surgery 2019;20(4): [24][25][26][27][28][29][30][31][32][33].…”