Flow cytometric comparison between swim-up and Percoll gradient techniques for the separation of frozen-thawed human spermatozoa.
Viable human spermatozoa were recovered from cryopreservation by either swim-up or Percoll density gradient techniques and their cell-surface oligosaccharide components were compared to those of fresh sperm. Sperm were labeled with FITC-Con A and analyzed by fluorescence microscopy and flow cytometry. By fluorescence microscopy, fresh sperm were uniformly labeled in the neck region alone. Frozen-thawed sperm, obtained by either swim-up or Percoll density gradients, showed two patterns of staining: one sperm population was stained in the neck region only, whilst another showed staining in both the neck and acrosome regions. No difference between sperm recovered by the two techniques was apparent. Flow cytometry profiles of fresh sperm revealed a single peak of fluorescence, whereas cryopreserved sperm gave two peaks of fluorescence. Samples recovered by Percoll density gradient contained significantly more sperm with the same fluorescence pattern as that observed for fresh sperm than did those recovered by swim-up. Cryopreservation alters the cell-surface oligosaccharide components of spermatozoa. Recovery of cryopreserved sperm by Percoll density gradient yields a greater proportion of sperm which resemble fresh sperm than does swim-up.
Chen
Y
Y
Department of Obstetrics and Gynaecology, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, England.
Obhrai
M S
MS
Chapman
J
J
Cuthbert
J L
JL
Williams
A
A
Tang
L
L
D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D015925:Cryopreservation; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology*