Cryopreservation of testicular and epididymal samples

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Successful sperm cryopreservation allows patients to have multiple ICSI cycles without the need for additional sperm harvesting procedures. Because ICSI enables severely impaired viable sperm to fertilize an oocyte, cryopreserved sperm can achieve rates of fertilization and pregnancy similar to those achieved using fresh sperm.

Studies have also found that the cryopreservation of testicular sperm is more problematic than the cryopreservation of epididymal sperm because testicular tissue yields a much lower number of spermatozoa with limited motility.

Nevertheless, reports have indicated that it is possible to cryopreserve testicular biopsy tissue samples and subsequently extract spermatozoa after thawing, with at least isolated pregnancies being achieved.

Although the use of cryopreserved testicular sperm for ICSI has several advantages, the data concerning the outcomes of IVF-ICSI procedures using frozen-thawed testicular sperm are still controversial. Some investigators claim that they have demonstrated, in obstructive and non- obstructive azoospermic men, that cryopreserved sperm can function as well as fresh sperm. Additionally, some labs have shown that the use of frozen-thawed testicular sperm for ICSI results in higher abortion rates and lower live birth rates compared with fresh testicular sperm.

Without cryopreservation, testicular tissue and testicular sperm can only be used for one ICSI cycle. Lacking cryopreserved sperm, each cycle of ICSI in these couples requires repeated surgeries. Repeated testicular surgeries can cause permanent testicular damage, irreversible atrophy, deterioration of spermatogenic development, and even loss of endocrine function. The cryopreservation of samples allows for multiple ICSI cycles and minimizes the number of invasive testicular surgeries.

Surgically recovered sperm from PESA can be cryopreserved in a manner similar to the cryopreservation of fresh ejaculates, although the post-thaw survival rates are usually very poor.

Due to extreme variability in post-thaw survival rates, it is important to freeze a small aliquot for a post-thaw survival test. This test requires thawing of the sample the day before the egg retrieval. In cases of virtually zero post-thaw survival, having results from the test sample allows time for the patient to consider what precautions to take if no viable sperm are identified on the day of their partner’s egg retrieval. Because there is never an absolute guarantee that cryopreserved sperm will thaw with appropriate viability, counseling patients regarding the use of donor sperm as a back-up should be considered, as well as options of repeat surgery or oocyte cryopreservation.

The biopsy specimen is placed in a petri dish with HEPES buffer supplemented with protein at 37°C. Under a dissecting microscope, the seminiferous tubules are teased apart using fine needles, and the contents are squeezed into the surrounding media. The tubules are transferred to a 15-ml conical tube containing 1 ml of fresh media. The cell suspension is then transferred to a separate centrifuge tube. Both tubes are incubated at 37°C for 15-30 minutes, and the supernatant (containing

tubules) is combined within the cell suspension in the second tube. The suspension is centrifuged at 500 g for 5 minutes, and the pellet is resuspended in 1 ml of fresh media with protein. A cell count is performed, and the suspension is diluted or concentrated to 0.5-1.0 million sperm/ml. Before freezing, an aliquot is removed to assess sperm quality. At the time of cryopreservation, multiple aliquots of sperm are frozen whenever possible along with a test cryovial. The cell suspension is slowly mixed in a 1:1 ratio with test yolk buffer and 12% glycerol so that the final concentration of glycerol is 6%. The samples are slowly cooled at a rate of 0.5°C/min to 4°C and then packaged in 1-ml cryovials. The vials are frozen at a rate of 10°C/min to -90°C and are stored in liquid nitrogen at -196°C.

A study was conducted using this protocol for 29 patients with obstructive and non-obstructive azoospermia undergoing testicular sperm extraction for a total of 46 IVF-ICSI cycles (12 fresh, 34 frozen). No statistically significant differences were noted between the fresh and frozen-thawed testicular spermatozoa (Habermann, et.al, 2000). The method where cryopreservation and recovery of

spermatozoa can be performed even in patients who have fewer than 100 spermatozoa present in the final testicular tissue homogenate. A porous capsule, such as an emptied zona pellucida, is used as a vessel to contain individual spermatozoa. Empty zona pellucida is prepared by microscopic dissection; specifically, small incisions are made in the zona using a microsuction device. The emptied zona pellucida is then maintained in Hepes-buffered media supplemented with protein. A sperm suspension in 10% PVP is then injected into the zona, placed in an 8% glycerol solution and cryopreserved using a standard freeze protocol with plastic straws. The use of an empty zona pellucida is very advantageous for samples with very low numbers of spermatozoa and also reduces the loss of motility associated with post-thaw dilution and sperm washing, which is observed when thawing frozen donor sperm.

In cases where freezing intact testicular tissue is required, testicular tubules are rinsed in HEPES-buffered medium to eliminate or reduce red blood cell contamination. The tubules are sectioned with a scalpel blade into pieces of approximately 2-3 mm and placed into a 35-mm dish that contains HEPES-buffered medium and cryoprotectant medium at a 1:1 v/v ratio at room temperature. Then, after 30 minutes, the samples are loaded into as many cryostraws as required. The 30-minute room- temperature equilibration period is ongoing during the loading process. The straws are sealed and labeled with the patient’s name. At the end of the 30-minute room-temperature equilibration period, the straws are loaded into the goblets and cryocanes and placed horizontally in the freezer for 30 minutes. At the end of the 30-minute cooling period, the specimens are hung over liquid nitrogen, well below the frost line of the tank, for 30 minutes. Then, the specimens are plunged into liquid nitrogen for continued storage. It is recommended to cryopreserve a test straw and then thaw it to determine the success of the cryopreservation procedure. To thaw these samples, the straws must be taken out of the liquid nitrogen tank and placed in a room- temperature water bath for 10 minutes. The contents are then expelled into room- temperature HEPES-buffered medium, rinsed with fresh HEPES-buffered medium and allowed to stand for 10 minutes before the sample is processed. The tissue is placed in a sterile Petri dish with a small volume of HEPES-buffered medium, sufficient to cover the tissue, and this sample is then homogenized with fine needles. Using a pulled pipette, a small volume of the homogenate is moved to the ICSI dish, and motile sperm are captured using an assisted hatching pipette. These sperm are then placed in a drop of PVP. Note that overnight incubation of the cryopreserved, thawed, and processed homogenate may increase the effective yield of the preparation (Schiewe et.al, 2011).

References

• Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril. 2000;73(5):955–60.

• Schiewe M. Fertility Conundrums, Testicular Sperm and ICSI. 2011 Available from: http://ivfconundrums.com/node/137