Video. Micromanipulation of gametes and embryos: cryopreservation of a single human spermatozoon within an isolated zona pellucida
Authors: J Cohen
Journal: Human Reproduction Update (1997)

There are several reasons for a need to cryostore a smallnumber of spermatozoa. Men who require testicular biopsybenefit tremendously if the presence of spermatozoa isknown before the onset of follicular recruitment in thepartner. This can usually be achieved by cryopreserving thebiopsy material or suspension of cells. But post-thawsuccess depends on the presence of relatively largenumbers of spermatozoa, since the addition of cryo-protectant requires removal by centrifugation whichdestroys many viable cells. The cut-off level forconventional freezing in mini-straws is 1200 spermatozoa,but many men have even less cells. Freezing of multiplesamples of very few spermatozoa would neverthelessguarantee several attempts at intracytoplasmic sperminjection (ICSI) and avoid repeated testicular aspirations incase further treatments are required.The recent of use of testicular spermatozoa has greatlyemphasized the need for the storage of aspirated testiculartissue or of spermatozoa that are recovered from suchtissue (Salzbrun et al ., 1996). Other methods of cryostoringsmall numbers of spermatozoa retrieved from the testisinclude the use of small droplets of medium which areplaced on a dry ice surface before freezing in liquidnitrogen, to give pregnancy rates similar to those obtainedwith fresh testicular spermatozoa (Gil-Salom et al ., 1996).Though both techniques are elegant and extremely useful,application falters when levels drop below one thousand.Special methods are necessary to store spermatozoa incases of extreme oligozoospermia. This circumstancearises constantly during ICSI when epididymal ortesticular spermatozoa are very rare. The most extremecases involve finding <100 spermatozoa, and sometimeswell below this number. This video shows a method ofovercoming this problem, by cryostoring single humanspermatozoa (Cohen et al ., 1997). It involves placing themin a zona pellucida after its oocyte has been removed. Thesequences in the video include the initial preparation ofzonae pellucidae, which came from oocytes that wereimmature when aspirated from their follicles, eggs whichdisplayed abnormal fertilization, and unfertilized mouseand hamster oocytes. Fine lesions were made in the zonaeusing mechanical methods. The ooplasm was thenaspirated from the oocytes and eggs, to provide emptyzonae for storage. The example shows a hamster profile.Up to 15 spermatozoa, are then isolated and injected intothe evacuated zona pellucida, which was crypopreservedusing glycerol; this cryopreservation procedure is notshown on this video. The final sequences illustrate therecovery of the frozen/thawed spermatozoa. In practice,only one zona pellucida among 50 was not recovered aftercryopreservation, and no spermatozoa were lost throughthe narrow incisions in the zona pellucida. Fertilizationrates using the cryopreserved spermatozoa for ICSI wereabove 65%.AcknowledgementsThis video was prepared with the assistance of Mina Alika-ni, Adrienne Reing, Toni Ferrarra, Sasha Sadowy and TimSchimmel.References