1 RBMO VOLUME 00 ISSUE 0 2018 1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Tehran, Iran 2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR Tehran, Iran 3 Department of Animal Science, College of Agriculture, Tarbiat Modarres University Tehran, Iran 4 Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Tehran, Iran © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. *Corresponding authors. E-mail addresses: kochesfehani@khu.ac.ir (H M Kouchesfahani), Shahverdi@royaninstitute.org (A Shahverdi), https://doi.org/10.1016/j.rbmo.2018.11.029 1472-6483/© 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Declaration: The authors report no fnancial or commercial conficts of interest. Q1 KEYWORDS Apoptosis Freezing Motility Nitric oxide Sublethal stress ARTICLE Preconditioning of sperm with sublethal nitrosative stress: a novel approach to improve frozen–thawed sperm function BIOGRAPHY Abdolhossein Shahverdi is Professor of Embryology and Scientifc Director of the Sperm Biology group at Royan Institute. His main research interests are fertility preservation, reproductive epigenetic and germ cell biology. He has over 20 years of experience in reproductive biology and has published more than 120 international scientifc papers. Maryam Hezavehei 1,2 , Homa Mohseni Kouchesfahani 1, *, Abdolhossein Shahverdi 2, *, Mohsen Sharaf 3 , Ghasem Hosseini Salekdeh 4 , Poopak Eftekhari-Yazdi 2 KEY MESSAGE Stress preconditioning of human spermatozoa with nitric oxide before freezing has led to enhanced cryosurvival and better maintenance of fertilizing ability. This strategy could be benefcial in sperm preservation protocols in assisted reproductive technologies. ABSTRACT Research question: Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post–thaw human spermatozoa? Design: Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 μM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO–0.00), 0.01 μM nitric oxide (NO–0.01), 0.1 μM nitric oxide (NO–0.1), 1 μM nitric oxide (NO–1), 10 μM nitric oxide (NO–10), 100 μM nitric oxide (NO–100). Frozen–thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes. Results: In fresh spermatozoa, nitric oxide did not induce a negative effect, except a signifcant reduction in motility and viability at 200 µM and 400 µM (P < 0.05). Cryopreservation signifcantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P < 0.05). NO–0.01 signifcantly increased total and progressive motility versus the other groups (P < 0.05). The lowest percentage of caspase 3 activity was in the NO–0.01 and NO–0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P < 0.05) in NO–0.01 (69%) versus controls (42%). Conclusions: Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.