Semen Assessment And Sperm Preparation

Prior to the oocyte retrieval procedure, the male partner provides a semen sample. After a period in the laboratory of approximately 30 minutes to allow liquefaction, the sample is thoroughly mixed and carefully assessed. By the time of oocyte retrieval, the laboratory should already be familiar with the male partner's semen profile, and can refer to features that might influence the choice of sperm preparation method used. The choice of sperm preparation method or combination of methods depends upon assessment of:

• The motile count

• Ratio between motile/immotile count

• Presence of antibodies, agglutination, pus cells or debris

Ejaculated semen is a viscous liquid composed of a mixture of testicular and epididymal secretions containing spermatozoa, mixed with prostatic secretions produced at the time of ejaculation. This seminal plasma contains substances that inhibit capacitation and prevent fertilization. The purpose of sperm preparation is to concentrate the motile spermatozoa in a fraction that is free of seminal plasma and its debris. Although sperm can be prepared by simple washing and centrifuga-tion, the method applied to early IVF practice, this method also concentrates cells, debris, and immotile sperm, the presence of which jeopardizes fertilization. Aitken and Clarkson (1987) have demonstrated that white cells and dead sperm in semen are a source of reactive oxygen species which can initiate lipid peroxidation in human sperm membranes. Peroxidation of sperm membrane unsaturated fatty acids leads to a loss of membrane fluidity, which inhibits sperm fusion events during the process of fertilization. Therefore, when preparing sperm for assisted conception motile sperm should be separated from leukocytes and dead sperm as effectively and efficiently as possible.

Layering and Swim-up

Sperm samples which show moderate to high counts (>35x10 motile sperm per millitre) with good forward progression and motility can be prepared using a basic overlay and swim-up technique: motile sperm swim up into a layer of medium, which is then washed and concentrated by centrifugation.

Discontinuous Buoyant Density Gradient Centrifugation

This is the method of choice for samples that show:

1. Low motility.

2. Poor forward progression.

3. Large amounts of debris and/or high numbers of cells.

4. Antisperm antibodies.

Original methods used Percoll, a suspension of silica particles coated with polyvinylpyrrolidone (Braude & Bolton, 1984). It was withdrawn for use in human ART programmes in 1996, and buoyant density gradient 'kits' for sperm preparation are now commercially available. These are based upon either coated silica particles, a mixture of Ficoll and iodixanol, or highly purified arabinogalactan. Individual experiences comparing the use of these products have reported no significant differences between them.

Buoyant density gradients apparently protect the sperm from the trauma of centrifugation, and a high proportion of functional sperm can be recovered from the gradients. Discontinuous two- or three-step gradients are simple to prepare and highly effective in preparing motile sperm fractions from suboptimal semen.

High-speed Centrifugation and Washing

Cryptozoospermic (or nearly cryptozoospermic) samples which must be prepared for ICSI can be either centrifuged directly (without dilution) at 1800g for five minutes, or diluted with medium and then centrifuged at 1800g for five minutes.

It is important to bear in mind that every semen specimen has different characteristics and parameters, and it is illogical to treat each specimen identically. All preparation methods are adaptable in some way. layering can be carried out in test tubes, but a wider vessel increases the area exposed to culture medium and decreases the depth of the specimen, increasing the potential return of motile sperm from oligoasthenospermic samples. Centrifugation times for buoyant density gradients may be adjusted according to the quality of the specimen to give optimum results. It is important to tailor preparation techniques to fit the parameters of the semen specimen, rather than to have fixed recipes. A trial preparation prior to oocyte retrieval may be advisable in choosing the most suitable technique for particular patients.

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