Sperm survival in liquid nitrogen

Abstract

Objective: Freezing of reproductive tissue and cells has been practiced for a considerable length of time. Spermatozoa were successfully frozen since mid-50s and embryos, oocytes, and ovarian and testicular tissue in recent years. The length of survival of reproductive tissue in the frozen state while stored in liquid nitrogen is not known, but obviously very important for family-building reasons. Design: At the patients request their semen was removed from liquid nitrogen and evaluated for changes in morphology, motility, and viability after many years of cryopreservation. Materials/Methods: In the 50s, 60s, and 70s semen was frozen primarily using glycerol solution or a cryoprotectant. The glass vials, and later plastic vials to avoid spontaneous breakage of the glass ampoules, were kept in liquid nitrogen after sequential freezing protocol, which called for placing the sperm in nitrogen vapor for approximately 2 to 3 hours and plunging into the liquid nitrogen. Thawing involved removing the sample from liquid nitrogen and placing it into 37 degrees Celsius water bath for 60 seconds. After another two minutes at room temperature, an aliquot was examined under phase contrast microscopy for sperm number, morphology and motility. Post thaw viability also was assessed using 1% eosin Y in phosphate buffered saline. Data were recorded and compared with pre-freeze parameters. Results: Two semen specimens were examined, one after 19 years and 9 months of storage and the other after 21 years and 5 months of storage. The numbers as expected were not changed. The motility in both cases decreased approximately 30% after thawing. The quality of motion, i.e. linear progression, was deemed to be satisfactory in both situations (24% and 40%, respectively). The first specimen was used for in vitro fertilization and led to a successful pregnancy with delivery of twins. The second patient is considering possibility of intrauterine insemination or in vitro fertilization. Conclusions: The length of time the spermatozoa can be cryopreserved is currently not known. With suspended metabolism it could theoretically remain viable indefinitely. The presented information proves that viability, progressive motility, and even fertilizing capacity can be successfully preserved for 20 or more years. Supported by: Institute for Reproductive Medicine and Genetic Testing.