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Annexin V Staining

The plasma membrane is one of the key structures in spermatozoa of infertile men displaying apoptotic feature. When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Annexin V is characterized by high affinity for PS and does not have the ability to pass the intact sperm membrane. Therefore, annexin V binding to spermatozoa characterizes disturbed integrity of the sperm membrane.
Colloidal super-paramagnetic microbeads (~50 nm in diameter) conjugated with annexin V separate the dead and apoptotic spermatozoa by magnetic-activated cell sorting (MACS). Cells exposing PS bound to these microbeads (annexin positive) are enriched to high extent within a column containing iron balls when placed in a very strong magnetic field. Cells with intact membranes remain unlabelled (annexin negative), and pass freely through the column. The binding of paramagnetic annexin V microbeads (ANMB) during MACS is an effective method to eliminate spermatozoa at early apoptotic stages from fresh and cryopreserved samples.
Magnetic-activated cell sorting (MACS) is used to provide a high-quality sperm fraction and as sperm preparation technique prior to assisted reproduction. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes and subjecting it to IVF or ICSI is a step further in optimizing the outcome of assisted reproduction.

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