Semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity

Abstract

The objective of this study was to determine the effects of density gradient centrifugation on sperm cell DNA integrity and to correlate any detected DNA damage with semen analysis parameter. A total of 40 semen samples were collected from nonazoospermic men presenting for infertility evaluation at our department. Individual samples were divided into two parts: one part of the semen was washed and the remainder was prepared using the PureSperm density gradient centrifugation. Sperm DNA fragmentation as evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay, was monitored in the initially washed sample and in the different layers of the density gradient centrifugation. No significant correlations were observed between sperm DNA fragmentation, age of patient, concentration and motility. However, a significant correlation existed with strict spermatic morphology. Following density gradient centrifugation, the proportion of spermatozoa with DNA fragmentation decreased significantly when compared with whole semen. In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of DNA damage when compared with those remaining in the 70% and 50% layers. These results demonstrate that semen processing by the PureSperm gradient is useful in selecting sperm with higher double-strand DNA integrity.

Proteomic analysis of mature and immature ejaculated spermatozoa from fertile men

“…It is well-known that immature spermatozoa especially those with the presence of excessive residual cytoplasm demonstrate higher ROS level than mature spermatozoa 612303149. The present study also found that many proteins were decreased along with the decrease in ROS level during the process of spermatozoa maturation.…”

“…In our previous study, we demonstrated that by using different density gradients, spermatozoa in the human seminal ejaculate can be separated into four fractions of sperm undergoing varying levels of maturation 6. As expected, with increases in motility and normal morphology in the four fractions, a decrease in the ROS levels and DNA fragmentation was observed,63031 demonstrating that spermatozoa in the whole ejaculate can be separated into four stages of maturation according to their density.…”

“…However, the role of 26S protease regulatory proteins in spermatozoa maturation, or in sperm function is not reported. Since oxidative stress and DNA fragmentation are higher in immature spermatozoa than in mature spermatozoa,630 and the 26S proteasome complex takes part in multiple cellular events including apoptosis, immune response, and DNA damage response,45 we deduce that besides protein ubiquitination and cell cycle arrest, these 26S proteosomal proteins might also take part in oxidative stress response and apoptosis of the immature spermatozoa. However, the exact function of 26S protease regulatory proteins in spermatozoa maturation needs further exploration.…”

“…This is because of the fact that antibody availability for each stage of spermatozoa maturation is not available, and more importantly after sorting, cell numbers in each fraction would be too small for extraction of the proteins. Many of the studies, including our two previous studies612 have demonstrated that density gradient can separate spermatozoa at different maturation stages 612173031. Obtaining mature, morphologically normal, motile sperm with minimum DNA damage by sperm preparation on a density gradient is the most common accepted procedure in assisted reproductive laboratories for intrauterine insemination and in vitro fertilization.…”

Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid

“…These findings are supported by other studies showing that spermatozoa from the 90% fractions of DDGC processed samples have a high percentage of normal morphology and chromatin maturity and generally lower levels of DNA fragmentation (Le Lannou and Blanchard, 1988;Sakkas et al 2000;Tomlinson et al, 2001;Brahem et al 2011a).…”