Molecular glass wool filtration as a new tool for sperm preparation.
BACKGROUND
Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter.


METHODS
Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody.


RESULTS
Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS.


CONCLUSIONS
The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.
Grunewald
Sonja
S
Department of Dermatology/Andrology Unit, University of Leipzig, Leipzig, Germany.
Miska
Werner
W
Miska
Gisela
G
Rasch
Manja
M
Reinhardt
Martin
M
Glander
Hans-Juergen
HJ
Paasch
Uwe
U
D017304:Annexin A5 / Q000737:chemistry*; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D015925:Cryopreservation; D003911:Dextrans; D005374:Filtration / Q000379:methods; D006801:Humans; D008280:Magnetics; D008297:Male; D053078:Membrane Potential, Mitochondrial; D010718:Phosphatidylserines / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism
D017304:Annexin A5; D003911:Dextrans; D010718:Phosphatidylserines; C025614:sephadex; D053148:Caspase 3