Past Issue

Volume 20, Number 4, Jan-Mar(Winter) 2019, Serial Number: 80 Pages: 537-543

Induction of Sublethal Oxidative Stress on Human Sperm before Cryopreservation: A Time-Dependent Response in Post-Thawed Sperm Parameters

Maryam Hezavehei, M.Sc, 1, 2, Homa Mohseni Kouchesfahani, Ph.D, 1, *, Abdolhossein Shahverdi, Ph.D, 2, *, Mohsen Sharafi, Ph.D, 3, Ghasem Hosseini Salekdeh, Ph.D., 4, Poopak Eftekhari-Yazdi, Ph.D., 2,
Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
Department of Embryology, Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Poultry Science, College of Agriculture, Tarbiat Modarres University, Tehran, Iran
Department of Molecular Systems Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
*Corresponding Addresses: P.O.Box: 15815/3587 Department of Animal Biology Faculty of Biological Sciences Kharazmi University Tehran Iran P.O.Box: 16635-148 Department of Embryology Reproductive Biomedicine Research Centre Royan Institute for Reproductive Biomedicine ACECR Tehran Iran,



A recent innovative approach, based on induction of sublethal oxidative stress to enhance sperm cryosurvival, has been applied before sperm cryopreservation. The purpose of this study was to investigate the effects of different induction times of sublethal oxidative stress before cryopreservation on human post-thawed sperm quality.

Materials and Methods

In this experimental study, we selected semen samples (n=20) from normozoospermic men according to 2010 World Health Organization (WHO) guidelines. After processing the samples by the density gradient method, we divided each sample into 5 experimental groups: fresh, control freezing, and 3 groups exposed to 0.01 μM sodium nitroprusside (SNP) [nitric oxide (NO) donor] for 30 (T30), 60 (T60), or 90 minutes (T90) at 37˚C and 5% CO2 before cryopreservation. Motion characteristics [computer-assisted sperm analyser], viability, apoptosis [annexin V/propidium iodide (PI) assay], DNA fragmentation [sperm chromatin structure assay (SCSA)], and caspase 3 activity (FLICA Caspase Detection Kit) were assessed after thawing. The results were analysed by using one-way ANOVA and Tukey’s test. The means were significantly different at P<0.05.


Cryopreservation significantly decreased sperm viability and motility parameters, and increased the percentage of apoptosis, caspase 3 activity, and DNA fragmentation (P<0.01) compared to the fresh group. The T60 group had a higher significant percentage of total motility (TM) and progressive motility compared with other cryopreserved groups (P<0.05). We observed a significantly lower percentage of apoptotic rate and caspase 3 activity in the T60 group compared to the other cryopreserved groups (P<0.05). DNA integrity was not significantly affected by this time of sublethal stress induction (P>0.05).


Our results have demonstrated that the application of sublethal oxidative stress by using 0.01 μM NO for 60 minutes before the freezing process can be a beneficial approach to improve post-thawed human sperm quality.