Human semen is a complex mixture of cells and fluids produced by the various components of the male reproductive system. The objective of sperm preparation is to remove the vigorously swimming sperm from this mixture, leaving behind the dead, dying or otherwise poorly swimming sperm, additional cells, enzymes and other factors that comprise the seminal fluid. A sperm cell is incapable of fertilizing an oocyte until it has separated from the seminal fluid.

We use a variety of separation techniques in the laboratory that are tailored to the procedure that the sperm will be used for, and modified according to the quality and type of sperm sample we receive. The average man manufactures about 250 million sperm in a 24 hour period. From a single ejaculate, we will only use 100,000 sperm for each oocyte that we have to inseminate in an IVF cycle. But for an intrauterine insemination, we want to get as many motile sperm as possible into the female reproductive tract, so we will therefore be using a much higher overall fraction of the sperm. Alternatively, for men who have no sperm in their ejaculate and for whom we have to retrieve sperm surgically from the testicle, we want to biopsy the minimum amount of tissue that will give us one sperm for every oocyte that has to be inseminated.

There are two general methods that we employ for the vast majority of sperm processing in the laboratory. The first is a density gradient centrifugation procedure in which the sperm sample is gently spun through 1-3 columns of a viscous solution of saline coated colloidal silica particles. The layers of silica are created by delicately layering different silica particle densities on top of each other in a test tube, and then layering neat semen on top. This method takes advantage of the fact that living sperm are dense compact cells that pass easily through the columns, while dead or dying sperm that are less dense due to leaky membranes are trapped with other cells and debris in the interfaces between the layers. The second method for preparing sperm takes advantage of the sperm’s natural swimming abilities by placing neat seminal fluid in proximity to some culture medium and allowing the sperm to swim from one to the other. There are many variations in this technique including the swim-up (semen is layered under the medium), or the converse method called the swim-down, and the actual method used depends mainly on the quality of the sperm sample. The swim-up is primarily used for samples that have good numbers of highly motile sperm from which only a small fraction needs to be recovered. The swim-down technique works better when sperm are swimming weakly and need the help of gravity to separate from the seminal fluid. For an individual with vanishing numbers of sperm (say a few hundred) we may use a swim-out technique. Here, the sperm are placed in the center of a small drop of medium and an embryologist will wait with a needle at the edge of the drop, picking up the first sperm to get there. One of the big criticisms of the ICSI procedure, where individual sperm are injected into oocytes, is that the embryologist chooses the sperm. However, with the use of the swim-out procedure, there is some degree of “natural selection” as we choose the sperm that are quickest in getting to the edge of the drop. We also choose sperm that are the normal size and shape, and that are free from defects (such as a bent neck) if we have the luxury to do so. In rare cases we have to use every sperm we have, so there’s no “selection” whatsoever. In most of the cases where we’re processing samples that have normal numbers of sperm, the sperm isolated by density centrifugation or by swim-up will be “washed” once or twice before being introduced to the oocytes. This involves suspending the sperm in a volume of culture medium and then centrifuging gently so that the sperm can be concentrated and removed from the medium, while leaving behind any trace of the silica particles or seminal fluid that may have carried over from the first processing step. Although sperm can be damaged by centrifugation, these steps are necessary to ensure that the sperm are free of contaminants that could prevent fertilization.

There are many other methods used to process sperm samples but we use them so rarely that they are scarcely worth mentioning. For example, samples with a high amount of debris can be filtered through glass wool or processed by sedimentation to clean them up before they undergo any of the procedures already described. In addition, we can treat a semen sample with chemicals in certain situations, but this again only happens under somewhat desperate circumstances. If a semen sample is extremely viscous or clotted, we can digest it using the enzymes amylase or chymotrypsin. If none of the sperm are moving we can treat them with pentoxifylline or caffeine to try to stimulate movement. When performing ICSI, we need to know that sperm are alive, and movement is our primary indicator. We can try to stimulate movement using drugs, but for the sperm that are to be used to fertilize the oocytes, we prefer to go drug-free. Here, we place the sperm into a hypo-osmotic solution (regular culture medium that has more water than normal) and as water enters living sperm their tails coil. These we can then inject into oocytes.

For patients that purchase frozen sperm from a sperm bank, the bank will usually offer the option of buying the sperm processed or unprocessed†. Processed sperm, usually labeled “IUI sperm”, costs a little more since the sperm bank has already prepared it for use. Unprocessed or “ICI sperm” is essentially neat semen that has been frozen. Women who do their own inseminations at home buy this type of sperm and inject it into their vagina after it is thawed. If you buy ICI sperm with the intention of having an intrauterine insemination, we will process the sperm as above to remove the seminal fluid and dead sperm. ICI sperm cannot be placed into the uterus since semen contains many contaminants such as bacteria, but also because semen can cause painful uterine contractions.

On a given day in our laboratory, one embryologist is primarily responsible for processing sperm samples, and each embryologist is assigned to this task about once a week. Each sample has different characteristics and the individual doing the processing must make informed decisions on the best approach for recovering the sperm that we need. It is an interesting and demanding area of the laboratory, but we enjoy the challenge of maximizing the potential of each sample that we receive.

– Joe Conaghan, PhD, HCLD

† For more information on frozen sperm and the products sold by sperm banks, see the “How do I Buy Sperm?” article in the April 2005 newsletter.

Tags: ICSI, IUI, IVF - In Vitro Fertilization, Lab, Male Infertility, Treatment Options

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