The selection of normal spermatozoa during intracytoplasmic sperm injection does not enable the detection of nuclear defects. Sperm head nuclear abnormalities have previously been identified as vacuoles by motile-sperm organelle-morphology examination. In terms of the link between the presence of vacuoles and embryo development, it has been shown that the injection of morphometrically normal spermatozoa with no vacuoles is associated with significantly higher blastocyst rate, a smaller proportion of arrested embryos, and higher pregnancy rates.1 According to previously obtained data in assisted reproduction, it is of importance to reliably select vacuole-free spermatozoa.2
This was a prospective and blinded observational study. Hyaluronic acid (HA)-bound, standard morphologically (SM)-selected (200x), and unselected sperm (control) were collected from different persons. The evaluation of vacuoles was performed by Nomarski high-power differential interference contrast optics (600x–7,200x). The number of vacuoles in each sperm head was determined and the spermatozoa were placed into 4 groups: absence of vacuoles, the presence of one vacuole, the presence of two vacuoles, or the presence of >two vacuoles. Fifteen human semen samples were prepared by 80% density gradient. From each sample, a minimum of 20 spermatozoa per method (HA, SM selection) were collected in separated polyvinylpyrrolidone droplets. Additionally, 20 unselected sperm were collected from each sample and designated as controls. All samples were observed blind by the same person. Statistical significance was defined as p<0.05. One-way analysis of variance and post-hoc Tukey-Test were performed. All statistical evaluations were carried out using SigmaStat Version 3.5.
After completing statistical analysis, the number of spermatozoa without vacuoles found in HA-selected (p<0.001) and SM-selected (p<0.01) spermatozoa were significantly higher compared to the unselected. The number of sperms with one (p<0.05), two (p<0.01), and >two vacuoles (p<0.001) was significantly higher in the unselected spermatozoa. Furthermore, in HA-selected spermatozoa, the appearance of two and >two vacuoles was significantly lower compared to the SM-selected spermatozoa (p<0.050). Although the time necessary for the isolation of individual spermatozoa from each sample was one of the limiting factors for this study, both selection methods provided spermatozoa which contained a smaller number of vacuoles compared to the unselected samples, especially in the group with >two vacuoles. Therefore, HA selection may be an effective method to identify spermatozoa with a higher fertilisation potential to improve results in assisted reproductive technology procedures.