IRCT201610317223N8 IRCT 2017-08-22 Royan Institute Comparison of fertilization percentage, embryo quality, implantation and pregnancy rate, between two sperm separation procedures in infertile men intra-cytoplasmic sperm injection candidate Comparison of fertilization percentage, embryo quality, pregnancy and implantation rate, between two sperm separation procedures (density gradient centrifugation with surface markers- density gradient centrifugation) in infertile men intra-cytoplasmic sperm injection candidate 2015-04-21 anticipated 113 Complete https://irct.ir/trial/7660 interventional Randomization: Randomized, Blinding: Single blinded, Placebo: Not used, Assignment: Parallel, Purpose: Health service research. N/A Male factor infertility. Intervention 1: In study group, total of oocytes were inseminated with sperm that selected with DGC following MACS processing (Separate intact spermatozoa from those that present apoptotic markers by magnetic particles conjugated with Annexin V, which has a high affinity for Phosphatidylserine)(MACS-DGC group). For MACS procedure, semen samples were washed twice with 1X buffer and diluted to ten million sperm per ml. Then, each semen sample was mixed with one hundred microliter of microbeads solution and then, this mixture was incubated for 15 min at room before being loaded to MACS column. Annexin positive sperm was retained within the column while annexin negative sperm were allowed to pass the column. Intervention 2: In control group (DGC group), oocytes were inseminated with sperm selected by Density gradient centrifugation method (based on viability and morphology). Density gradient centrifugation was prepared by laying 2 ml of 80% PureSperm into a sterile 15ml conical centrifuge tube. Then, 2 ml 40% PureSperm suspension was slowly layered over lower layer. Subsequently, semen sample were washed (5 min, 3000 rpm) with VitaSperm medium were overlayed on the 40% or upper layer. The layer was centrifuged for 20 min at 1000rpm. The supernatant was aspirated, pellet was resuspended, and washed twice with VitaSperm. After injection of sperm into the oocyte to embryo formation at day 3, events such as fertilization, cleavage and embryo formation occur that these phenomena are in vitro. After 3 days, embryos were transferred into the uterus and then assessed implantation and pregnancy in vivo condition.