Practices in sperm selection for artificial reproductive techniques: Are we doing right?

Sperm selection is one of the crucial steps in ARTs; the selection of quality spermatozoa leads to better embryo development and further devalues the failure rates of the associated reproductive process. The ejaculated semen sample comprises seminal plasma, reactive oxygen species (ROS), and mature and immature spermatozoa. The viable sperm cells need to be isolated from other components promptly (~30 minutes) (Agarwal et al., 2014). If not, the motility of sperm cells diminishes, which is not ideal for the ARTs executions.

Methods for quality spermatozoa separation: Numerous methods, including simple wash, density gradient centrifugation, swim-up, and swim down, are being practiced in reproductive clinics. In the abovementioned methods, swim-up and density centrifugation are the most exploited techniques for sperm separation.

SIMPLE WASH

Post liquefication of semen sample (~15–20 minutes), simple wash comprises the dilution of semen sample with culture medium. Subsequently, the sample is centrifuged twice to separate seminal plasma. The selected centrifugation steps and force need to be small and weaker respectively; else the strong centrifugal force causes the formation of ROS, which damages the DNA integrity and motility of spermatozoa (Agarwal et al., 2014; Balasuriya et al., 2014; Henkel and Schill, 2003; R.R. and W.-B., 2003).

DENSITY CENTRIFUGATION

As we are all familiar with density gradient centrifugation encompasses the separation of the liquid phases and particles based on their densities. The mature sperm cells have a density higher density (~1100 kg/m3) than immature and no-spermatozoa cells (1060 -1090 kg/m3) (Agarwal et al., 2014). The centrifugation tube prepared with double density gradients ̶ top layer 45% (v/v) density gradient medium (colloidal suspension of silica particle-stabilized with hydrophilic silane in HEPES) to HTF (human tubal fluid) supplement with HAS (human serum albumin) and lower layer 90% (v/v) density gradient medium to HTF again supplement with HSA ̶. 2ml of different concentration sample placed into the centrifugal tube and subsequently, 3ml of the liquefied semen sample added on the top and centrifugation of the sample conducted for 20min at 1600 rpm. Higher density sperm cells settle in the bottom and are isolated through the pipette.

SPERM SWIM-UP

Sperm swim-up is the most practiced technique in reproductive clinics. The liquefied semen sample is mixed with sperm wash medium (HTF) in a 1:1 ratio and subsequently kept for centrifugation at 1600 rpm for 10 minutes (Agarwal et al., 2014). The sedimented sample is gently removed and placed in a 3ml fresh sperm medium. Subsequently, the sample is again centrifuged at 500 rpm for 5 min and incubate at 45O for an hour approximately (Agarwal et al., 2014; R.R. and W.-B., 2003). Viable sperm cells swim upward. The top layer is carefully aspirated and collected samples are assessed for their motility.

The swim-up method has been customized for oligozoospermic patients. The adapted method is renamed as the direct swim-up method, where the centrifugation of the semen sample is omitted, and the sample is directly placed for incubation. Check the figure here.

SWIM-DOWN

A serum solution is prepared to be placed at the bottom of the tube. Subsequently, the centrifuged semen sample is kept on top of the serum and incubate at 37OC for an hour. The motile sperm moves towards the serum gradient (Agarwal et al., 2014; Gonzales and Pella, 1993).

Conclusively the current practices involve centrifugation and it is evident that strong centrifugal force causes ROS generation and DNA fragmentation. Conventional separation of the sperm cells is one of the chief factors in ARTs failure as bad sperm cell also are able to fertilize the egg but leads to defective embryo development. What can we learn from mother nature? Find out in our next post of the week.

REFERENCES

Agarwal, A., Sharma, R., Beydola, T., 2014. Sperm Preparation and Selection Techniques, in: Medical and Surgical Management of Male Infertility. https://doi.org/10.5005/jp/books/11840_29

Balasuriya, A., Serhal, P., Doshi, A., Harper, J.C., 2014. Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation. Andrologia 46. https://doi.org/10.1111/and.12052

Gonzales, G.F., Pella, R.E., 1993. Swim-down: A rapid and easy method to select motile spermatozoa. Syst. Biol. Reprod. Med. 30. https://doi.org/10.3109/01485019308988365

Henkel, R.R., Schill, W.B., 2003. Sperm preparation for ART. Reprod. Biol. Endocrinol. https://doi.org/10.1186/1477-7827-1-108

R.R., H., W.-B., S., 2003. Sperm preparation for ART. Reprod. Biol. Endocrinol. 1. https://doi.org/10.1186/1477-7827-1-108