Different methods of processing epididymal and testicular sperm samples

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Shredding method

The preparation of testicular samples is more difficult if the sperm are contained within the seminiferous tubules. Many of the sperm found are immature, with a large cytoplasmic drop attached to the head, and some sperm are motile; however, most are still viable for ICSI. It is advantageous to perform the sperm retrieval procedure at least 24 hours in advance of the egg retrieval process, as motility improves with time in culture.

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A common procedure associated with the shredding method is the excision of testicular tissue by a surgeon and its subsequent examination by an embryologist. The tissue can be finely minced and teased apart with fine needles or glass slides in HEPES-buffered media. An embryologist must retrieve sperm from the field of debris, red blood cells, and Sertoli cells for ICSI. Then, the sperm suspension can be incubated at 37°C until the time of ICSI.

Squeezing method

Seminiferous tubules are teased apart and rinsed to remove blood contamination, and they are then placed into a petri dish with fresh culture media. Tubules are then cut into short lengths (1-2 cm) with fine needles. A long, thin Pasteur pipette is pulled over a flame and then bent (ideally at an angle of 45°). A second pipette (without a bend) should be heated and used to pick up the tubule contents. By holding one end of a cut tubule with the point of a needle, the bent pipette can be run along the length of the tubule while simultaneously pushing down against the base of the petri dish. This procedure squeezes the entire contents of the tubule into the medium. The contents can now be picked up with the second pulled pipette and placed in a test tube filled with clean sperm media or placed on a slide to look for sperm.

Cell strainer

A method for processing large biopsy samples that has the benefit of removing unwanted debris consists of the use of a cell strainer. First, the biopsy sample is teased apart and sliced with fine needles. These slices are rinsed in a series of petri dishes containing sperm preparation media to remove any blood contamination. The tubules are then placed in a cell strainer.

Heat-treating the end of a clean, sterile Pasteur pipette produces a sphere-shaped tip approximately 5 mm in diameter. This pipette is used as a pestle to grind and break up the seminiferous tubules against a mesh strainer.

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Tissue grinder method

Another method for processing large tissue samples is to use a mini-tissue grinder. The tissue is teased apart, sliced with fine needles and placed in a test tube with fresh culture media and a glass pestle. With the glass pestle, the sample is ground at the bottom of a glass tube. Then, the sample is concentrated via centrifugation. After centrifugation, the pellet is re-suspended with fresh culture media and placed on a Petri dish in 10-μl drops to look for sperm.

Erythrocyte lysing buffer method

Sperm recovery from epididymal fluid is usually rapid and efficient, whereas the recovery of sperm cells from testicular biopsy specimens is more difficult, especially in cases of non-obstructive azoospermia. The main difficulty is due to the abundance of red blood cells in shredded testicular biopsy specimens. The presence of a very high concentration of erythrocytes and the very infrequent occurrence of sperm cells sometimes result in a much longer examination.

The hypoosmotic swelling test (HOS test)

The hypoosmotic swelling test (HOS) is to evaluate the functional integrity of the sperm membrane. Viable sperm with normal membrane function will exhibit tail swelling and curving due to the influx of water when exposed to hypoosmotic conditions. There is a reduced success rate of fertilization for azoospermic males due to the injection of non-viable spermatozoa, which cannot be distinguished from viable spermatozoa with no motility.

Pentoxyfylline method

Pentoxyfylline is a phosphodiesterase inhibitor of the methylxantine group. It inhibits the breakdown of cyclic adenosine monophosphate (CAMP), a molecule known to play a role in sperm motility. It was found that adding pentoxyfylline to a testicular sperm sample caused sperm to be motile. This procedure is performed by adding pentoxyfylline to the sperm suspension at a 1:1 ratio so that the final

concentration of pentoxyfylline in the sample is 0.5 mg/ml. Some studies have shown that pentoxyfylline is toxic and should not be used.

Collagenase method

The enzymatic treatment of testicular biopsies with collagenase type IV was applied in clinical ICSI cases where no spermatozoa had been found after the sample was minced using two fine needles. This procedure was performed to determine whether spermatozoa can be recovered from the residual tissue pieces. This method is performed by shredding the testicular biopsy sample with fine needles. Microscopic examination of the wet preparation is carried out at 400x magnification under an inverted microscope.

Spermatozoa are directly recovered from the pellet after centrifuging the supernatant of the shredded tissue at 300 g for 5 minutes. Then, erythrocyte-lysing buffer is used to increase the probability of visualizing any spermatozoa or elongated spermatids.

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