Andrologia. 2018;e12960. wileyonlinelibrary.com/journal/and | 1 of 8 https://doi.org/10.1111/and.12960 © 2018 Blackwell Verlag GmbH 1 | INTRODUCTION The sperm quality is a crucial factor affecting the success rate of all in- fertility treatment options including assisted reproductive techniques (ART) (Elder & Dale, 2010), as the male factor of infertility represents 20%–30% of the infertility cases. Nevertheless, there are many factors affecting the sperm quality including the type of sperm preparation technique (Boomsma, Heineman, Cohlen, & Farquhar, 2007; Marchesi, Biederman, Ferrara, Hershlag, & Feng, 2010), the temperature during preparation (Franken, van Wyk, Stoumann, & Avari, 2011) and time of in vitro incubation of prepared semen samples (Yavas & Selub, 2004). It is well known that, in most ART protocols, the selected spermatozoa may be incubated for several hours in vitro for proper sperm capaci- tation (Liu, Liu, & Baker, 2011). In vitro sperm incubation is proved to cause oxidative stress and reactive oxygen species (ROS) production which peaks at 24 hr after incubation (Calamera, Fernandez, Buffone, Acosta, & Doncel, 2001). Prolonged exposure of spermatozoa to re- active oxygen species (ROS) causes changes in spermatozoa lipid and protein production and affects DNA integrity (Agarwal & Said, 2003; Hammadeh et al., 2006). However, there is no consensus about the optimum time for sperm incubation, before use in ART, which avoids sperm DNA fragmentation (Rougier et al., 2013). Moreover, the im- pact of sperm DNA fragmentation on the outcome of ICSI/IVF still needs more studies to be clarified (Agarwal, Cho, & Esteves, 2016). Accepted: 3 January 2018 DOI: 10.1111/and.12960 ORIGINAL ARTICLE Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate I. Ahmed 1,2 | S. Abdelateef 1 | M. Laqqan 1 | H. Amor 1 | M. A. Abdel-Lah 2 | M. E. Hammadeh 1 1 Department of Obstetrics & Gynecology, University of Saarland, Homburg, Germany 2 Department of Obstetrics & Gynecology, Sohag University, Sohag, Egypt Correspondence Islam Ahmed, Obstetrics & Gynecology Department, University of Saarland, Homburg/Saar, Germany. Email: islamabdelsalam84@gmail.com Summary The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double-strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double- strand breaks (r = −0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased num- ber of DNA strands double breaks with no influence on fertilisation rates. KEYWORDS fertilisation rate, sperm chromatin condensation, sperm DNA fragmentation, sperm preparation