A noninvasive, motility independent, sperm sorting method and technology to identify and retrieve individual viable nonmotile sperm for intracytoplasmic sperm injection.
OBJECTIVE
For intracytoplasmic sperm injection in the absence of sperm motility it can be virtually impossible to distinguish viable from nonviable sperm. A reliable means to identify viable nonmotile sperm is needed and would likely improve the intracytoplasmic sperm injection success rate. Optoelectronic tweezers are a new technology that uses light induced dielectrophoresis fields to distinguish individual live cells from dead cells. We assessed the ability of optoelectronic tweezers to distinguish viable from nonviable individual nonmotile human sperm.


METHODS
Fresh semen specimens from 6 healthy men were suspended in an isotonic sucrose/dextrose solution and incubated with 0.4% trypan blue dye (Sigma-Aldrich®). Within 15 minutes we randomly selected 5 motile and 50 nonmotile sperm, including 25 trypan negative, followed by 25 trypan positive sperm, under 200× magnification for optoelectronic tweezers assay. We recorded the individual sperm response (attraction or repulsion) to the optoelectronic tweezer field and trypan staining status.


RESULTS
From each subject 55 unwashed sperm were individually assayed for a total of 330. All motile sperm were attracted to optoelectronic tweezers. Of 150 trypan negative (viable) sperm 132 (88%) were attracted to the optoelectronic tweezer field with 0.88 sensitivity (95% CI 0.82-0.93) vs that of the trypan blue assay. All 150 trypan positive (nonviable) sperm were repulsed by or neutral to the optoelectronic tweezer field with 1.0 specificity (95% CI 0.98-1.00) vs that of the trypan blue assay. Type I error equaled 0 and overall assay agreement was 94%.


CONCLUSIONS
The optoelectronic tweezer assay can distinguish viable from nonviable nonmotile viable sperm with sensitivity comparable to that of the trypan blue assay and equal specificity. Optoelectronic tweezers are a promising means of selecting sperm for intracytoplasmic sperm injection.
Department of Urology, University of California-San Francisco, San Francisco, California 94143, USA. mgarcia@urology.ucsf.edu
Maurice M
MM
Garcia
Aaron T
AT
Ohta
Thomas J
TJ
Walsh
Eric
E
Vittinghof
Guiting
G
Lin
Ming C
MC
Wu
Tom F
TF
Lue
D006801:Humans; D008297:Male; D052898:Optical Tweezers; D010865:Pilot Projects; D020554:Sperm Injections, Intracytoplasmic; D013081:Sperm Motility; D053625:Sperm Retrieval; D020858:Tissue and Organ Harvesting / Q000379:methods