Reproduction Abstracts

Introduction: During spermiogenesis, which is the last stage in spermatozoa maturation, a spermatozoa plasma membrane remodelling stage occurs which promotes the up-regulation of membrane receptors to aid the zona pellucida binding e.g. hyaluronic acid (HA) receptors.

Researches have demonstrated that spermatozoa that are able to bind to HA in vitro are, in fact, more mature spermatozoa that have completed specific maturation processes and are ultimately more likely to reach the oocyte and fertilise it. These maturation processes include plasma membrane remodelling, cytoplasmic extrusion and finally, nuclear maturation of the spermatozoa. It has been noted that spermatozoa selected using HA as a selector exhibit characteristics such as minimal DNA fragmentation, normal morphology, and lower frequency of chromosomal aneuploidies, ultimately supporting the use of the HA binding assay as a sperm selection method for ARTs.

In the current project the ability of good quality (recovered from 90% density gradient or pelleted) and bad quality spermatozoa (recovered from 45% density gradient or interphase) in binding to HA was tested. We also compared the levels of DNA damage and chromatin maturity in pelleted and interphase and also in HA-bound and unbound spermatozoa.

Methods: The HBA® slides (Origio) were used to check the ability of binding pelleted and interphase spermatozoa to HA.

After separation of human spermatozoa using 45–90% density gradient centrifugation (DGC), spermatozoal suspension (pelleted and interphase spermatozoa) was placed onto the assay chamber and the chamber was incubated at RT. As a result, spermatozoa which have HA receptors are able to bind to the HA-coated slide with an active beating tail.

The mean percentage (S.D.) of HA-bound spermatozoa was calculated (for both the interphase and pelleted spermatozoa) for 15 different human samples.

The levels of DNA damage and chromatin maturity was checked using acridine orange (AO) and aniline blue (AB) staining in pelleted and interphase and also in HA-bound and unbound spermatozoa.

Results and discussion: The results showed that there is a significant difference (P<0.0001) between the mean percentage (S.D.) of HA-bound spermatozoa in pelleted compared to the interphase spermatozoa.

Our results also confirmed that pelleted spermatozoa had significantly lower levels of DNA damage (P<0.0001) and higher levels of chromatin maturity (P<0.05) compared to interphase spermatozoa.

In addition we illustrated higher levels of chromatin maturity and lower levels of DNA damage in HA-bound spermatozoa (separated using a HA-coated surface) compared to HA-unbound spermatozoa (P<0.0001).

In conclusion our results confirmed that HA binding assay is a good method for selection of mature spermatozoa.