Sperm Morphology by High Magnification in Fertility Men

Overview

A new concept for observing the fine morphology of spermatozoa at high magnification (x6000) with an inverted microscope, a numeric camera using differential interference contrast has been developed (1). This technique called Motile Sperm Organellar Morphology Examination allows to see some abnormalities, mainly vacuoles on the head of spermatozoa. These vacuoles appear to be related to sperm DNA damage and to affect embryo developmental potential (2, 3, 4). The application of Motile Sperm Organellar Morphology Examination may represent an improvement in the evaluation of semen quality, with some potential clinical repercussions at the diagnostic/prognostic level. First of all, the investigators need data on fertile men in order to define " normality " of sperm morphology at high magnification. The aim of this study is therefore to better characterize these vacuoles (number, surface, position) in a population of men fertile in order to establish normality criteria.

The population studied consisted in 50 men aged 18 to 45 years with proven spontaneous fertility. All subjects gave their informed consent to participate in the study. After questioning on full medical and andrological history, semen samples were collected by masturbation after 2 to 5 days of sexual abstinence and were processed for analysis after liquefaction for 20 min at 37°C. We carried out a sperm count, motility, vitality and conventional morphology analysis as well as a detailed morphometric analysis of the vacuoles at high magnification using an image analysis software. For the analysis at high magnification, fifty microliters of fresh sperm was washed in 2.5 ml of washing solution by centrifugation for 5 minutes at 400g. The pellet was resuspended in 100 µl of washing solution and the spermatozoa were fixed by addition of 100 µl Phosphate Buffer Saline-formaldehyde 3.7%. Two microliters of this suspension was placed in a glass-bottomed dish and examined by Nomarski interference contrast microscopy with a camera mounted on a microscope with an immersion objective lens x100. For each sample, sperm head vacuoles were analyzed on 100 spermatozoa that were randomly photographed and separately analyzed using digital imaging system software. Measurements using the software were carried out by a single operator. The Interactive Measurement module allows measurement of sperm head areas and vacuole areas by manually depicting their outline. The area and position of each vacuole were recorded.