Separation Of Human Spermatozoa Research Articles

Separation of X- and Y-bearing human spermatozoa by sperm isolation medium gradients evaluated by FISH

An eight-layer discontinuous sperm isolation medium (PureSperm gradient) was evaluated in separation of human spermatozoa according to sex chromosomes by fluorescence in-situ hybridization (FISH). This study was carried out on spare samples from normozoospermic and oligozoospermic patients referred to the Royan Institute for infertility treatment. Semen analysis was assessed according to World Health Organization criteria. X- and Y-bearing spermatozoa were simultaneously identified in the neat semen (control) and sperm isolation medium fractions from the same samples using FISH and chromosome specific DNA probes. At least 1000 spermatozoa were scored for each sample. The proportions of X- and Y-bearing spermatozoa were determined by presence of red or green fluorescent signals. Before separation, there was no significant difference in the percentage of spermatozoa with the specific signals of X and Y chromosomes. After separation, in both normal and oligozoospermic patients, the percentage of X-bearing spermatozoa in the bottom layer slightly exceeded that in the top layer (P = 0.001). In both the normal and oligozoospermic groups, the difference between the frequencies of Y-bearing spermatozoa in the top and bottom layers was significant (P = 0.001). It seemed that eight-step discontinuous gradient was not a reliable method for the separation X- and Y-bearing spermatozoa for clinical purposes.

Separation of human spermatozoa with physiological integrity of plasma membrane.

Objective: To evaluate the physiological integrity of sperm membrane after preparation of semen simultaneously using 4 different manipulation methods: Washing-Centrifugation (WC), Swim Up (SU), Two-Layer Density Column (LC) and Real Time Micro-separation System (RT) without the supplementation of antibiotics in cell culture media.

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select) and Percoll, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll gradient. After translocation of separated spermatozoa from the Percoll solution to a culture medium, serum, Percoll or hyaluronic acid (Sperm Select) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of 45Ca2+ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of 45Ca2+ and the acrosome reaction, whilst Percoll inhibited both of these actions. Hyaluronic acid (Sperm Select) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll inhibited, influx of 45Ca2+ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select) and Percoll affect the acrosome reaction and the prerequisite for Ca2+ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.

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.

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.

The separation of human spermatozoa into X or Y chromosome rich fractions and Quinacrine staining for advanced light microscopy

The separation of human spermatozoa into X or Y chromosome rich fractions and Quinacrine staining for advanced light microscopy