M. Bungum; N. Forsell; A. Giwercman  

Author Affiliations – Skane University Hospital, Reproductive Medicine Centre, Malmö, Sweden 



Prolonged in vitro incubation of spermatozoa has been shown to have adverse effects on sperm motility, vitality as well as on DNA integrity. Knowledge regarding how shorter incubation periods prior to the IVF/ICSI procedure affect semen quality is however limited. The aim of the present study was to examine if sperm DNA integrity was affected during incubation in three different conditions for 2 hours after sperm preparation prior to the IVF/ICSI procedure.

Materials and Methods:

Density gradient centrifuged samples from two hundred men undergoing infertility work-up were included in the study. Following gradient centrifugation one reference sample was frozen immediately. Thereafter samples were divided into three aliquots and incubated for two hours in either1) room temperature (23-24 °C); 2) in a 37°C humidified incubator with 6%CO2 and 5%O2 or 3) in a 37°C humidified incubator with atmospheric air. The Sperm Chromatin Structure Assay (SCSA) was used to assess the extent of sperm DNA damage. Sperm DNA fragmentation was expressed as DNA fragmentation index (DFI).


A statistically significant increase in DFI was seen in density gradient prepared samples incubated for 2 hours at 37°C, 6%CO2 and 5%O2 compared to the reference sample taken immediately after preparation. This was the case also for samples incubated at 37°C in atmospheric air. Moreover, statistically significant lower DFI levels were seen in the group incubated at room temperature compared to those incubated at 37°C, 6%CO2 and 5%O2 or at 37°C in atmospheric air.


In order to prevent against further sperm DNA damage after density gradient preparation prior to the IVF/ICSI procedure, spermatozoa should be stored at room temperature.