Measuring apoptosis in human spermatozoa: a biological assay for semen quality?
OBJECTIVE
[1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures expression of phosphatidylserine on the outer leaflet of the cell membrane, or the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxy-uridine triphosphate] nick end labeling) assay, which measures occurrence of DNA strand breaks and [2] to correlate the outcome with routine semen variables and the hypoosmotic swelling (HOS) test.


METHODS
Pilot study and clinical trial.


METHODS
Large teaching hospital and fertility center.


METHODS
Men whose semen was studied for various reasons.


METHODS
Percentage of apoptotic spermatozoa by two different assays, percentage of necrotic spermatozoa, concentration and motility of spermatozoa, and outcome of the HOS test.


RESULTS
Apoptosis can be measured in spermatozoa by flow cytometry using the Annexin V assay and the TUNEL assay. Twenty percent of spermatozoa were apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm concentration (r = -0.389; P<.05) and motility (r = -0.289; P<.05). A highly significant inverse correlation was seen between DNA double-strand breaks (TUNEL assay) and sperm concentration (r = -0.629; P<.0001).


CONCLUSIONS
Flow cytometry can easily and reliably detect phosphatidylserine expression on the outer leaflet of the cell membrane and DNA strand breaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spermatozoa are apoptotic, and the concentration of spermatozoa is lower in men with more apoptotic spermatozoa.
Department of Obstetrics and Gynecology, Medisch Spectrum Twente Hospital Group, Enschade, The Netherlands.
G J
GJ
Oosterhuis
A B
AB
Mulder
E
E
Kalsbeek-Batenburg
C B
CB
Lambalk
J
J
Schoemaker
I
I
Vermes
D017304:Annexin A5 / Q000032:analysis / Q000378:metabolism; D017209:Apoptosis; D053938:DNA Fragmentation; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D007248:Infertility, Male / Q000473:pathology*; D008297:Male; D010718:Phosphatidylserines / Q000032:analysis / Q000378:metabolism; D010865:Pilot Projects; D011419:Propidium; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*
D017304:Annexin A5; D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate