A microscopic approach of sperm preparation for intracytoplasmic sperm injection

Objective: To develop a microscopic sperm preparation technique that is specially suited for ICSI. Design: Microscopic sperm separation column is designed to be constructed on a 60 mm petri dish that is usually used in ICSI so an ideal number of capacitated and hyperactivated sperm are available at the time of lCSI procedure. Materials and Methods: A 15 uL volume microgradient column was constructed in the shape of a square with an open corner on a petri dish. One open end of the square contained semen, while the three arms contained 3 variable concentrations of albumin used to maintain the gradient. The microgradient column, covered with mineral oil, was observed under an inverted microscope. When sufficient number of sperm reached the harvest zone (column-end), it was disconnected from the rest of the column by mild scratching at the specific location with the piper tip. Twelve semen samples with different male factor etiologies were processed with the column and the quality of the sperm yield was evaluated. Results: Sperm preparation was successful from 12/12 (100%) semen samples. The motility of the prepared sperm in all samples were 100±0% with grade 'a' quality (WHO criteria). 96±6% sperm in the yield exhibited hypo-osmotic swelling. The percentage of spermatozoa with normal morphology (Kruger's strict criteria) was 53±7%. The number of sperm reaching the harvest zone correlated with the time in all samples. However, one hour incubation was found sufficient to harvest more than file number of sperm required to perform ICSI. Conclusion: Microscopic sperm preparation does not induce any gross and ultrastructural physical damage to the sperm since the procedure does not require any pretreatment such as centrifugation of the semen. Furthermore, since the technique is based on self migration of motile sperm in horizontal column, it always yields morphologically superior sperm with 100% motility from semen irrespective of its male factor quality. Quality sperm obtained in microscopic sperm preparation technique thus might enhance ICSI fertilization rate and embryo quality.