Efficacy of Density Gradient Centrifugation Technique (DGC) in Enhancing Sperm Cell DNA Quality for Assisted Reproductive Technique

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Abstract Objective: Density-gradient centrifugation (DGC) is commonly used for sperm preparation before Assisted Reproductive Technology (ART) procedures. This technique separates superior motile spermatozoa with normal morphology from the total sperm population. However, there is still controversy as to the effects of this sperm separation technique on sperm cell DNA integrity which is a determining element in the process of fertilization and embryonic development.The objective of this study was to determine the effects of DGC on sperm cell DNA integrity as assessed by a novel association between two cytogenetic tests.Study Designs: Semen samples were collected from 30 fertile donors and 40 patients being candidates for ART treatmemt. Each sample was divided into two parts: the first portion was subjected to selection by two layers of DGC (45% and 90%) and the second fraction was rinsed with phosphate buffered saline solution and centrifuged without density gradient.Abnormal sperm chromatin structure as evaluated by sperm chromatin dispersion (SCD) test and DNA denaturation as assessed by acridine orange (AO) test were monitored in the initially washed sample and in the different layers of the density gradient centrifugation.Results: DGC significantly improved the proportion of sperm progressive motility, total motility, and sperm morphology. Moreover, following density gradient centrifugation, the proportion of spermatozoa with denaturated DNA significantly decreased when compared with whole semen (p< 0.001). In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of sperm chromatin decondensation when compared with those remaining in the 45% layer and unprocessed semen (p<0.001). Conclusions: Using double cytogenetic tests, our study shows that semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity and recommended to be used in sperm preparation for assisted reproduction.

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