Sperm quality influences fertility and offspring health through both genomic inheritance and epigenetic inheritance. Thus, for use in clinical-assisted reproductive technology (ART), spermatozoa must have optimal genomic and epigenetic structures. In patients with retrograde ejaculation, spermatozoa are usually recovered from urine and then cryopreserved for ART. However, the effects of urine exposure and subsequent freeze–thaw cycles on sperm quality remain unclear. This is particularly true for epigenetic changes and their underlying mechanisms. In this study, we examined how different durations of urine exposure (10 min and 40 min) followed by freeze–thaw cycles affected sperm motility, DNA integrity, and methylation levels of imprinting genes (H19-imprinted maternally expressed transcript [H19], mesoderm-specific transcript [MEST], and the transposable element Alu [Alu]). As the duration of urine exposure increased, sperm motility (median [interquartile range]) decreased from 48.0% (39.0%−52.5%) to 1.0% (1.0%−5.0%), the DNA fragmentation index (DFI; median [interquartile range]) increased from 12.0% (9.3%−19.9%) to 23.5% (13.9%−33.9%), the MEST methylation level (mean ± standard deviation [s.d.]) increased from 3.8% ± 1.5% to 11.5 ± 1.2%, and the H19 methylation level (mean ± s.d.) decreased from 86.9% ± 0.9% to 82.1% ± 0.5%. The freeze–thaw process further reduced sperm motility, while the DFI and methylation levels of MEST and H19 did not significantly change. The Alu methylation level remained stable. These findings demonstrate that urine exposure affects sperm motility, DNA integrity, and methylation levels of some imprinting genes. These effects intensify over time. In contrast, the freeze–thaw process impacts only sperm motility. In clinical practice, minimizing exposure to urine might improve sperm quality.

Keywords: DNA fragmentation; DNA methylation; retrograde ejaculation; spermatozoa