Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model.
Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.
Said
Tamer
T
Center for Advanced Research in Human Reproduction, Infertility and Sexual Function, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Agarwal
Ashok
A
Grunewald
Sonja
S
Rasch
Manja
M
Baumann
Thomas
T
Kriegel
Christian
C
Li
Liang
L
Glander
Hans-Juergen
HJ
Thomas
Anthony J
AJ
Paasch
Uwe
U
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D006224:Cricetinae; D053938:DNA Fragmentation; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D066298:In Vitro Techniques; D007248:Infertility, Male / Q000473:pathology* / Q000628:therapy*; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009865:Oocytes / Q000166:cytology; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism
D017304:Annexin A5; C505407:CASP3 protein, human; D053148:Caspase 3; D020782:Matrix Metalloproteinases