Sperm preparation for Assisted Reproduction techniques

Sperm preparation for Assisted Reproduction techniques

There are different techniques for preparation of the sperm for reproductive purposes. The objective of these techniques is the enrichment of the sample with the greater possible number of mobile and functional sperm. It is considered a good capacitated sample (either for artificial insemination or IVF)if it contains a total amount greater than 1 x 106 sperm with more than 85% of mobile progressive sperm forms (final concentration of 20 x 106 spermatozoa/ml in 50 microliters).

In addition, during this capacitating process, non-mobile sperm and seminal plasma (contains toxic and bioactive substances that can damage sperm) will be eliminated using oxidative stress. Prostaglandins found in small concentrations in semen will be also removed since they could cause uterine pain in women.

Simple washing

This consists in the centrifugation, at 400 g, of the seminal sample to remove the seminal plasma and concentrate the sperm in a small volume. This method provides with concentrate the sperm, but it does not imply any selection method. However, this type of capacitating process is not usually done on its own, only in cases of serious oligozoospermias, criptozoospermias or testicular biopsy specimens.

Swim-up

Specific culture medium is added to the sperm sample, and then it is centrifuged at 400 g for 10 minutes. In this way all cells contained in the sperm, including spermatozoa, will go to the bottom of the tube forming a precipitate or pellet. Afterwards the content can be separated from pellet by decantation processes of the tube sample. The culture medium is then carefully added (0.5 to 1 ml), not removing the pellet, and staying in an incubator at 37 ° C with 5% CO2 for 45 minutes. During this time, best mobility sperm will move up swimming from the pellet towards the surface of the media. The supernatant surface will be collected after 45 minutes, and will consist in a rich media of high mobility sperm.

This technique is widely used in laboratories of fertilization in vitro, especially in cases of normozoospermia since concentrations greater than 90% of progressive motile sperm can be achieve and it is an easy-to-perform method.

Density gradient

This method consists on the centrifugation of the sperm sample to make it pass through a colloid inform of continuous or discontinuous density gradient (45%, 60% and 90% of density). Percoll can be used for its realization, which is silica particles surrounded by polyvinylpyrrolidone. However, nowadays other colloids as PureSperm or SpermGradare being used since, unlike the Percoll, they have been purified and are free from endotoxins.

Once the gradient has been established in the tube, the sperm sample can be deposited on the top of the gradient and perform the centrifugation. All cells go down because the centrifugation, going through the various layers of density, but with greater mobility and better morphology sperm will be able to get to the bottom of the tube more quickly, while stationary or slow sperm will be retained in surface layers. It is important to note that in this technique the fraction of interest will be at the bottom of the tube, the one with higher density, since sperm with better mobility and morphology will concentrate there.

This method is used mainly in cases of teratospermia, asthenozoospermia, and samples with abundant cells and detritus. It offers many advantages, since it can accumulate large amount of sperm with very good mobility and normal morphology.

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