DEVELOPING A METHOD FOR SPERM SELECTION TROUGH CUMULUS LIGANDS
MIRYANA GOSPODINOVA*, RUMIANA GANEVA, KRISTINA NIKOLOVA, FERIHAN SHABAN, DIMITAR PARVANOV, GEORGI STAMENOV, TODOR CHAUSHEV
Nadezhda, Women’s Health Hospital, 3 “Blaga vest” Street, Sofia, Bulgaria
*Corresponding author: mg_gospodinova@abv.bg
Keywords: immobilization, cumulus ligands, human spermatozoa
Abstract: This research focuses on the development of a direct technique for isolation and immobilisation of cumulus ligands on a plastic surface. After ovum pick up, cumulus cells samples from 199 patients, undergoing intracytoplasmic sperm injection (ICSI) were collected and stored at -20° C. All samples were thawed, mixed in a pool and stored at -20° C until their next application. Forty sperm samples from normozoospermic men were used during the process of the optimization of the implemented technique. The maximal number and percentage of the attached human spermatozoa were applied as main criteria for successful isolation and immobilization of cumulus ligands. During the optimisation process four enzymes (collagenase type I, accutase, hyaluronidase and trypsin) were tested for isolation of cumulus ligands. The immobilization of cumulus ligands on plastic surface was compared among eight concentrations and three different temperature regimes (-20°C, 4°C and 25°C). The obtained results showed that the optimal conditions were: (1) digestion of cumulus cells with collagenase type I for 20 minutes at 37°C in an incubator, (2) preparation of a pool of samples from donors and dilution in carbonate buffer to a final concentration of 0.008 cumulus samples/sample at Room Temperature (RT), (3) immobilization (placing) 1μl drops of prepared cumulus solution on the plastic surface at RT and (4) storage at 4°C until the addition of human spermatozoa. The described method for utilization of cumulus ligands on plastic surface might be very useful both for the development of new methods for diagnostics and non-invasive selection of human spermatozoa based on their functional characteristics.