Semen samples were allowed to liquefy for 20 min at room temperature. Seminal plasma, containing exosomes, was separated from the cell fraction by centrifugation at 1000 x g for 10 min. Cell debris was removed by subsequent centrifugation at 2400 x g for 30 min followed by 0.45 μm and 0.22 μm syringe filtration (Millex HA). Exosomes were purified from the entire cell- and debris-free seminal plasma (ranging from 0.9 to 4.0 ml in our donors) by ultracentrifugation over a sucrose cushion using a method adapted from Lamparski et al. (33 (link)). Up to 2.5 ml of supernatant was added to ultracentrifuge tubes and under-layered with 300 μl of a 20 mM Tris/30% sucrose/deuterium oxide (D2O) cushion (pH 7.4) (Sigma). Samples were ultra-centrifuged at 100 000 x g for 90 min at 4°C in an SW 50 swinging bucket rotor (Beckman). The upper layer was collected and ultracentrifuged again at 100 000 x g for 14 h at 4°C over a 20 mM Tris/25% sucrose/D2O cushion (pH 7.4). The upper layer of exosome-depleted seminal plasma was stored at −80°C. The 30% and 25% sucrose cushions containing the exosome fraction were pooled and brought to 15 ml with Dulbecco's phosphate-buffered saline (PBS). The exosomes were washed by centrifuging at 2400 x g through an Amicon Ultracel 100 kDa cellulose centrifugal filter with 10 ml of PBS and concentrated to a final volume of 425 μl–3.2 ml. Exosomes were stored at −80°C.