Androcare Fertility Laboratories and Cryobank

Sperm Preparation

The Artificial Insemination (AI) is the first option treatment for infertile couples with cervical factor subfertility, mild-moderate male subfertility and unexplained infertility. With the exception of cases in which the use of in vitro fertilization (IVF or ICSI) is strictly due as a consequence of a severe male or female factor, the artificial insemination must be part of a gradual approach to the techniques of artificial insemination. This is particularly the case since the AI is a valid low-cost method, minimally invasive and easily acceptable for the female’s hormone treatment. Some different techniques are used to prepare the spermatozoa for the AI, but the choice strongly depend on the quality of the semen, that is on the concentration, motility and morphology, in order to obtain the higher number of good spermatozoa, even from the poorest semens.

Sperm washing 

For the best quality samples (number and motility of sperms) the sperm washing is often performed (Boomsma et al., 2004) for the AI. The procedure simply consists in the washing of the semen with a sterile medium added with human albumin. After the fluidification of the sample, the entire volume is divided in fractions of not more than 2 ml into centrifuge tubes. The sterile medium of the equal volume (e.g. for the volume of the sample of 2 ml the medium added is 2 ml) is added in each tube and gently mix with a sterile pipette. After that, the samples are centrifuged at 300g (the rpm must be calculated for the centrifuge in each laboratory) for 10 min and than the supernatant is very carefully removed with a sterile pipette. The pellet is resuspended in 1 ml of the medium, gently mixed and centrifuged again for 5 min at 300g. The supernatant is removed again and the final pellet is resuspended in sterile medium for the AI. It is very important to determine the count and the motility of the final preparation before the insemination. In spite of the simplicity and velocity of the method, it must be reminded that the repeated centrifugations without the separation of the good sperms from leukocytes and dead sperms can produce many oxidative species and the damage of the sperms function.

Swim up method 

The swim up is the most common technique used especially if the semen sample has a normal number of good sperms (normozoospermia). By this technique, the sperms are selected on their motility and the capability to swim out of the seminal plasma. If the “direct swim up” is performed, after the fluidification of the sample, the entire volume (well mixed) is divided in fractions of 1 ml into centrifuge tubes (round bottom is preferred). 1,3 ml of culture medium is placed over the semen with extreme attention in each tube. The tubes must be put in the incubator, inclined at an angle around 45° and incubated at 37°C for 30-60 min. By inclining the tubes at 45°, we increase the surface between the medium and the semen and we improve the capability of the sperms to swim out of the semen and to reach the medium. After that, the tube must be returned in the vertical position and 1 ml of the supernatant of each tube can be gently removed, aspirating the sperms from the upper meniscus downwards with a sterile pipette .In alternative, the culture medium can be placed in each tubes and the semen can be stratified under the medium, in order to obtain a much cleaner surface between the semen and the medium. In addition, the recovery of the sperms can be optimized by increasing the number of the tubes and decreasing the volume of the semen in each tube. 2 ml of medium are added to the supernatant of each tubes and than centrifugated at 300g for 10 minutes. The supernatant is removed again and the pellet is resuspended in the sterile medium for the AI. 
The “not direct” swim up from pellet is performed with the centrifugation of the semen followed by the stratification of the medium over the resuspended pellet. The liquefied semen is divided in fractions of 1 ml into each tubes, the medium is added (1:1) and after the centrifugation the supernatant is gently removed. Over the resuspended pellet, 1,3 ml of medium is replaced with caution and the tubes is put into the incubator from 30 to 60 min at 37°C (inclined at 45°); after the migration of the sperms, the volume of the semen for the AI is removed and the sperm count and motility are assessed.

Density Gradient Centrifugation

This  technique is used to select the greater number of motile spermatozoa in cases of severe oligozoospermia, teratozoospermia or asthenozoospermia. In this method, good quality sperms can be separated from dead sperms, leukocytes and the other components of the seminal plasma by a density discontinuous gradient. Cells with different density and motility can be selected during the centrifugation by the colloidal silica coated with silane of the gradient; the sperms with high motility and good morphology are at the bottom of the tube, finally free from dead spermatozoa, leukocytes, bacteria and debris. This is also the method we used in washing HIV contaminated sperm.The most applied discontinuous density-gradient is a two layers density-gradient, formed by a top layer of 40% (v/v) and a lower layer of 80% (v/v). Density gradient media are available in commerce ready to use or ready to make the different density layers; the top layer phase (40%) is prepared by adding 4 ml of density gradient medium to 6 ml isotonic sterile medium (BWW, Earle, Ham F-10 or HTF) supplemented with HAS (Human Serum Albumin); the lower layer phase (80%) is prepared by adding 8 ml of density gradient medium to 2 ml of isotonic sterile medium. The density gradient is prepared by layering 1 ml of 40% medium over the 80% medium, or by layering the 80% medium under the 40% medium in a conical centrifuge tube (not the round bottom tube!). The number of the tubes depends on the volume of the semen sample, but the total volume could be divided in not more of 1 ml of semen per tube. 

After the fluidification, 1 ml of the semen is layered over the upper layer (40%) and centrifuged at 300g for 15 minutes. If the volume of each layer is reduced (<1ml) the spermatozoa have to migrate for a less distance between the layers and so the greater number of motile spermatozoa can be recovered. The centrifugation time and force can be varied depending on the quality of the sample: for example, the centrifugation time can be increased for specimens with high viscosity. After the centrifugation, most of the supernatant must be gently removed and the pellet is placed into a new, clean tube; here, the pellet is well resuspended in 5 ml of medium to remove the density gradient medium. It is centrifuged at 200g for 10 minutes. At the end of the centrifugation, the supernatant is removed and 5 ml of new medium are added. The centrifugation is repeated again and the final pellet is resuspended in the sterile medium for the AI. The concentration and the motility after the preparation can be determined. We observed  sterile conditions when we perform this  technique for therapeutic applications.