Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting.
OBJECTIVE
To determine the applicability of flow cytometry to assess human sperm acrosome reaction.


METHODS
Prospective evaluation of semen samples incubated overnight for the development of spontaneous acrosome reaction or exposed to the calcium ionophore A23187 (5 microM) for 3 hours for induction of the acrosome reaction.


METHODS
University-affiliated tertiary care center.


METHODS
Normal healthy volunteers.


METHODS
The spermatozoa were stained with fluorescein isothiocyanate (FITC)-labeled pea agglutinin. The labeled samples were assessed visually and also subjected to analytic flow cytometry and fluorescence-activated cell sorting.


METHODS
Acrosome reaction assessed visually and by flow cytometry.


RESULTS
Flow cytometric analysis showed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after overnight incubation or exposure to A23187, suggesting loss of fluorescence, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between the methods was small (2.49%), the difference between the two methods for each individual sample can vary between -24% to +29%. When the sperm cells were subjected to cell sorting based on green fluorescence intensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%).


CONCLUSIONS
These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytometry is more objective and less time consuming when many samples are assessed at the same time, the visual method remains a useful and practical procedure in the clinical andrology laboratory.
Uhler
M L
ML
Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California.
Leung
A
A
Chan
S Y
SY
Schmid
I
I
Wang
C
C
D000177:Acrosome / Q000187:drug effects / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D002469:Cell Separation; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D037102:Lectins; D008297:Male; D037121:Plant Lectins; D011446:Prospective Studies; D013094:Spermatozoa / Q000166:cytology
D005456:Fluorescent Dyes; D037102:Lectins; D037121:Plant Lectins; C036292:pea lectin; D000001:Calcimycin; D016650:Fluorescein-5-isothiocyanate