Magnetic-activated Cell Sorting before Cryopreservation Preserves Mitochondrial Integrity in Human Spermatozoa

Abstract

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation-thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved-thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation-thawing (45.4+/-8.6%) compared to sperm that were not separated (15.8+/-4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation-thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.

REVIEW ARTICLE: Clinical Relevance of Oxidative Stress in Male Factor Infertility: An Update

“…MACS has been shown to yield motile, viable, morphologically normal spermatozoa that displays significant cryopreservation tolerance and higher fertilization potential. [91][92][93][94][95][96][97] It allows separation of apoptotic spermatozoa, which may be one of the causes of ART failure even in patients with otherwise normal sperm parameters.…”

Effect of sperm storage and selection techniques on sperm parameters

“…The sperm sample separated by MACS before cryopreservation also contained a larger number of sperm with an intact mitochondrial membrane. The mitochondrial survival rate (MSI), which is the ratio of the percentage of sperm with intact mitochondrial membrane intergrity (MMP) after cryopreservation to that in the neat sample, increased by 36.1% ± 18.9% when MACS was used before cryopreservation [Grunewald et al 2006]. Hence, sperm selection using MACS before cryopreservation can improve motility and cryosurvival of a sperm sample.…”

Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa

“…These increases (either activated caspases or YO-PRO-1C/PIK) only affected part of the spermatozoa, just as we detected in the H 2 O 2 treatment. The confirmation of this kind of subpopulations would represent a breakthrough finding, since many separation and evaluation methods for spermatozoa rely on the detection of apoptotic markers (such as phosphatidylserine externalization; Grunewald et al 2006). Alternatively, if these pathways were involved in sperm DNA damage (by the activation of endogenous endonucleases; Gorczyca et al 1993, Lamond et al 2003, caspase inhibitors (Eley et al 2005, Marti et al 2008 or mitochondrial protectors (Lamond et al 2003, Fernandez-Gomez et al 2005, Hardeland 2005) might improve artificial reproductive techniques.…”