Evaluation of Embryo Quality After Enrichment of Non-apoptotic Spermatozoa Using Magnetic-activated Cell Sorting (MACS): Study on Sibling Oocytes.

Studies of infertile couples have demonstrated that male factor plays a role in infertility. Spermatozoa can present damages at molecular levels that have an effect on fertilization and embryo development. One of these molecular damages is represented by DeoxyriboNucleic Acid (DNA) fragmentation whose negative impact on embryological outcome appears during genome activation after fertilization and is also correlated with increased miscarriage rate.

DNA fragmentation represents one of the late manifestations of apoptosis. Identification of spermatozoa showing DNA fragmentation (apoptotic spermatozoa) requires fixation and staining. Therefore this method cannot be applied in clinical practice to select for non-apoptotic spermatozoa for ICSI. However, this selection is possible by using magnetic-activated cell sorting (MACS). The procedure is based on the identification and depletion of spermatozoa showing the early marker of apoptosis, namely the externalization of phosphatidylserine (PS), by using the ability of Annexin V to recognize this antigen in the plasma membrane of apoptotic sperm cells. After MACS, the fraction that is enriched in non-apoptotic cells (Annexin V-negative cells) can be further used for clinical application.

The MACS system makes use of a separation column placed in a magnetic field, and of Annexin-V reagent labeled with paramagnetic beads.

Initially, the semen sample will be subject to Density Gradient Centrifugation (DGC). The sample obtained will be divided in 2 fractions: one fraction will be subject to magnetic sorting (fraction 1) and the other fraction will represent the control (no sorting; fraction 2).

The cells from fraction 1 will be incubated with Annexin-V and then passed through the separation column located in a magnetic field. The apoptotic cells (Annexin V-positive cells) will be selectively retained in the column. The non-apoptotic cells, not being labeled by Annexin V, will pass through the column and will be collected for further use.