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An amount of 850 µl untreated semen sample will be loaded into the inlet chamber with a help of a sterile insulin-type syringe. A volume of 750 µl of Sage Quinn's AdvantageTM Medium with Hepes + 5% HSA-solution medium will be placed on top of the membrane (outlet chamber). After 30 min of incubation in a humidified incubator (37°C), 500 µl of medium, containing the selected spermatozoa, will be retrieved from the upper side of the membrane (retrieval chamber). An amount of 400 µl will be necessary for the IUI procedure.

The gradient columns will be prepared by placing 1 ml 80% gradient media in a centrifuge tube and an additional 1 ml of 40% gradient layered on top. The raw semen sample will be placed on top of the gradient (1ml semen/gradient tube) and centrifuged at 300xg for 20 minutes. The sperm pellet will be collected and washed 2 times for 5 min at 800xg in Sage Quinn's AdvantageTM Medium with Hepes + 5% HSA-solution. The resulted pellet will be resuspended in the washing solution in a volume that will provide at least 1x106 forward motile spermatozoa in 400 µl, as this amount is necessary for IUI procedure.