Measure volume using a sterile 2 mL pipet.Transfer specimen from a plastic cup to a sterile 15 mL- conical centrifuge tube. Gently mix the specimen with equal volume of Sperm Washing Media. Centrifuge the tubes at 1500 rpm for 10 minutes.Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 50mcm of fresh washing medium, then place layer of 0.5 ml of washing medium gently on the surface. Incubate the tubes at a 45° angle for 1 hour for swim-up in vertical rack in a 37°C incubator. After the incubation period, aspirate the entire supernatant from the round bottom tube. Aliquots of the detached sperm were analyzed by halosperm assay for DNA fragmentation another aliquots of same supernatant were used for ICSI then fertilization, division, blastulation, pregnancy and implantation rates were tabulated and statistically tested.

PureSperm gradients 40 % and 80 % were used for the experiment. All procedures were conducted under sterile conditions. Using a sterile pipette, 2.0 mL of the "lower layer" (80% PureSperm gradient) was transferred into a conical centrifuge tube.

Using a new sterile pipette, 2.0 mL of the "upper layer" (40% PureSperm gradient) was gently dispensed on top of the lower layer. A liquefied semen sample was then placed on top of the upper layer and the tube was centrifuged for 20 minutes at 300g. The upper and lower layers were carefully aspirated without disturbing the pellet. Using a transfer pipette, 2-3 mL of Ham's F10 +10% HAS was added to the pellet and the resuspended pellet was centrifuged for 7 minutes at 300g. The supernatant was then removed and the pellet was suspended in a volume of 0.5 mL of Ham's F10 + FCS 10%. Aliquots of the detached sperm were dealt with like previous arm

different sperm processing method for selcting most proper sperm for ICSI

measure sperm integrity by halosperm assay

no. of oocytes fertilized /total no. of oocytes

no of embryos blastulated / total no. of embryos