Freeze-drying (FD) has been widely used for preserving viruses, bacteria, and fungi, because FD materials are easy to store and transport without cryopreservation in liquid nitrogen. In addition, sperm DNA is easy to be damaged by FD stress. EGTA (ethylene glycol tetraacetic acid) was reported to be a good chelator in FD buffer for sperm DNA protection. Recently, trehalose was also reported to have notable ability in stabilizing protein structure and bio-membrane in dry state. In this study, we examined effect of combination of EGTA and different concentrations of trehalose (0 M, 0.015 M, 0.03 M, 0.06 M and 0.09 M) in FD medium on sperm DNA integrity and in vitro development after ICSI using FD sperm.
Firstly, FD sperm in the groups were rehydrated and analyzed with Halomax kit to detect DNA lesion. DNA damage in 0.015 M trehalose group significantly decreased compared with the control group (P<0.001). However, higher concentrations of trehalose had no protective effects on DNA integrity. Secondary, we used FD sperm in trehalose 0 M and 0.015 M groups for ICSI. Sham injection and parthenogenetic activation were used as control groups. There were no significant differences in rates of normal fertilization and blastocyst formation (examined at 10 h and 6 days after insemination, respectively) using FD sperm between 0 M and .015M groups. The quality of blastocysts (measured by the total numbers) was not different between the groups. These parameters were also not different from those in the control groups. The results suggest that trehalose has protective effect against sperm DNA damage. Further experiments will be necessary to examine the DNA damage affect full-term development of ICSI-oocytes.