Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection?

Abstract

Purpose: Although, at present, the selection of sperm prior to ICSI is based on motility and morphology, undetectable anomalies, and more importantly damaged DNA are overlooked. In this regard, novel sperm selection procedures have gained much interest. For instance, sperm has been selected by Magnetic-Activated Cell Sorting (MACS) based on early apoptotic marker, the externalization of phosphatidylserine (EPS). Review of the literature has revealed that the efficiency of this technique has been mainly evaluated post Density Gradient Centrifugation (DGC). Therefore, there is a need to prove the efficiency of this technique independent of DGC. In addition, considering the fact that DGC induces EPS due to capacitation and acrosome reaction, therefore, the role of MACS before DGC(MACS-DGC) and MACS after DGC (DGC-MACS) should be assessed.

Methods: Semen samples from fifteen infertile men were divided into three separate fractions: control, DGC, and MACS. To carry out DGC-MACS, DGC samples were further divided into two fractions and MACS was carried on the second fractions. Similarly to carry out MACS-DGC, the MACS samples were further divided into two fractions and DGC was carried on the second fractions. Percentages of sperm with normal morphology, DNA fragmentation, protamine deficiency, EPS and caspase-3 activity were determined in each fraction.

Results: DGC is more efficient than MACS in separating intact sperm only in terms of normal morphology, DNA and chromatin integrity but not for active caspase. However, a combination of these procedures was more efficient than a single procedure to separate intact sperm for the aforementioned parameters. Comparison of the combined procedures showed only higher efficiency to separate active caspase in the MACS-DGC group.

Conclusion: Based on these results, we propose MACS-DGC rather than DGC-MACS to be implemented in clinical settings.

Fig. 1

Flow diagram of overall experiment design. FCM Flow Cytometry; LM Light Microscopy; FM Fluorescence Microscopy; EPS Externalization of Phosphatidylserine; CMA3 Chromomycin A3; TUNEL TdT-mediated dUTP Nick-End Labeling

Fig. 2

Comparison of the mean percentages of normal morphology (I), protamine deficiency (II), DNA damage (III) and caspase-3 activity (IV) in the control, DGC, MACS, DGC-MACS and MACS-DGC procedures. Common letters show significant difference at p < 0.05

Fig. 3

Comparison of mean percentages of annexin in the control, DGC, MACS, DGC-MACS and MACS-DGC procedures