Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime

Abstract

While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

Intracytoplasmic morphologically selected sperm injection outcomes: the role of sperm preparation techniques

“…The dish was placed on a microscope stage above a Uplan Apo x 100 oil/1.35 objective lens previously covered by a droplet of immersion oil. The sperm cells exhibiting normally shaped nuclei ([1] smooth, [2] symmetric, and [3] oval configuration) and [4] normal nuclear chromatin content (if it contained no more than one vacuole, which occupies <4 % of the nuclear area) were selected for injection [3].…”

“…The SUP technique relies on the ability of the motile spermatozoa to "swim up" into the culture medium, while slow and immotile sperm remain behind, along with other components in the semen pellet [2]. The DGC method separates spermatozoa according to their density and favors the isolation of motile and morphologically normal spermatozoa [24].…”

Cell handling using microstructured membranes

“…In addition, on-chip processing is gentler and less likely to affect the cells of interest by exposure to mechanical stresses during centrifugation. [28][29][30] The new cell enrichment procedure may also be useful for the isolation of cells from dissociated tissues and removal of debris, 31 or for separating cells in particles based on size, in an approach comparable to mechanical filtering of blood cells. 3 The study of cellular responses to chemical stimulation is of fundamental importance for many biology studies.…”

Application of microfluidic technologies to human assisted reproduction

“…Currently, semen processing, such as swim-up, simple washing with centrifugation, and density gradient with centrifugation, all provide efficient means of sperm isolation for IVF/CI and/or IVF/ICSI. However, centrifugation has been reported to cause sub-lethal damage to sperm (Alvarez et al, 1993). Experimental evidence suggests that sperm DNA is exposed to high levels of reactive oxygen species during centrifugation processing (Aitken and Clarkson, 1988;Hughes et al, 1998), and this oxidative stress was positively correlated with sperm DNA damage (Barroso et al, 2000).…”