Standardization of pretreatment and staining technique to perform automatic human sperm morphology with ASMA (assisted sperm morphometry analyses)

Clara Ramírez, José Ramón Alonso, Pedro Jiménez, Rocío Reyes, Jordi Ramis, Josep Maria Gris, Carlos Aulesa

Unidad de Andrología, Laboratorios Clínicos, Hospital Universitario Vall d’Hebron, Barcelona, Spain; Servicio de Reproducción Asistida, Hospital Universitario Vall d’Hebron, Barcelona, Spain

Abstract

Objective: Standardization ofpretreatment and staining technique to realizemorphological semen evaluation with computer-assisted sperm morphometry analysis system, with semen system analyzer SCA 5.4 (Sperm Class Analyzer, Microptic).

Material and methods: Morphological analysis was performed with semen system analyzer SCA 5.4 (Microptic SL, Barcelona, Spain). The staining method was a modification of Hemacolor kit (Merck). Between 60 and 125 semen samples were chosen randomly from our laboratory.

Results: Pretreatment of semen samples was a centrifugation for 5 minutes at 300 g, seminal plasma was rejected and the pellet was homogenized with .2 mL of the seminal plasma itself. Not change in sperm morphology have been found with this pretreatment. Staining was performed with Hemacolor kit (Merck) but with some modifications. Fixer has been buffered with phosphate buffer pH 7.2 at 10%, time recommended by the manufacturer has been reduced. Fixation for 5 seconds, 30 seconds with Eosin staining and 2 seconds with staining Azur. Finally it was washed for 30 seconds with pH 7.2 phosphate buffer, as indicated by the manufacturer. After the pretreatment and staining we have got reference values for our methodology.

Conclusions: An automation methodology to perform sperm morphology reduces coefficient variations of determination thereby we can increase its technical reliability and remove the subjectivity of the manual analysis. This standardization can be the first step to study the diagnostic value of advanced morphology in infertility and urological diseases.

DOI: 10.1016/j.androl.2016.05.002

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