Cryopreservation of individual spermatozoa for men with compromised spermatogenesis

Abstract

OBJECTIVE: Cryptozoospermic or NOA men are challenged by different degrees of hypospermatogenesis resulting in an inconsistent presence of sperm in their ejaculate or testicular biopsy samples. The presence of few sperm following high speed centrifugation and extensive sperm search encourage the cryopreservation of the remaining specimen in a standard fashion. Thawing of these specimens often results in the inability to identify any sibling spermatozoa.DESIGN: To test this hypothesis, we attempted to develop a cryopreservation method capable of yielding the same sperm cell after thawing.MATERIALS AND METHODS: Few motile sperm were placed in 1μl droplet of cryoprotectant on an ICSI dish under micromanipulation devoid of oil. The loaded ICSI dish was closed, Parafilm®, and placed in a cryobox containing LN2 vapor. Warming was carried out by overlaying the dish with oil and placing it on a heated stage, 37°C. Experiments on single sperm isolation and motility assessment were performed in triplicate.RESULTS: Nine samples were donated by consenting men (38.8 ± 8yrs). Five had normal semen parameters (67.5 ± 19x106/ml concentration, 52.8 ± 11% motility, and 7.0 ± 2% morph) while 4 had suboptimal sperm (35.3 ± 16x106/ml concentration, 45.2 ± 8% motility, and 2.0 ± 1% morph). An ave of 10.1 ± 1 sperm were placed in each drop and cryopreserved. During warming, each drop was examined under oil and cells identified in no more than 5min. From the 243 individual sperm cells cryopreserved, 198 (81.5%) were found. Of the sperm cells retrieved, 117 (59.1%) were motile.CONCLUSION: The cryopreservation of specimens with the extremely low number of sperm often results in disappointing chances of retrieving sibling cells. By cryopreserving individual sperm on ICSI dishes at time of initial analysis, we were able to expeditely locate, identify, and prepare for injection these same cells. In case of cryoptozoospermia and NOA, this method may represent a valuable tool to retrieve these precious gametes. OBJECTIVE: Cryptozoospermic or NOA men are challenged by different degrees of hypospermatogenesis resulting in an inconsistent presence of sperm in their ejaculate or testicular biopsy samples. The presence of few sperm following high speed centrifugation and extensive sperm search encourage the cryopreservation of the remaining specimen in a standard fashion. Thawing of these specimens often results in the inability to identify any sibling spermatozoa. DESIGN: To test this hypothesis, we attempted to develop a cryopreservation method capable of yielding the same sperm cell after thawing. MATERIALS AND METHODS: Few motile sperm were placed in 1μl droplet of cryoprotectant on an ICSI dish under micromanipulation devoid of oil. The loaded ICSI dish was closed, Parafilm®, and placed in a cryobox containing LN2 vapor. Warming was carried out by overlaying the dish with oil and placing it on a heated stage, 37°C. Experiments on single sperm isolation and motility assessment were performed in triplicate. RESULTS: Nine samples were donated by consenting men (38.8 ± 8yrs). Five had normal semen parameters (67.5 ± 19x106/ml concentration, 52.8 ± 11% motility, and 7.0 ± 2% morph) while 4 had suboptimal sperm (35.3 ± 16x106/ml concentration, 45.2 ± 8% motility, and 2.0 ± 1% morph). An ave of 10.1 ± 1 sperm were placed in each drop and cryopreserved. During warming, each drop was examined under oil and cells identified in no more than 5min. From the 243 individual sperm cells cryopreserved, 198 (81.5%) were found. Of the sperm cells retrieved, 117 (59.1%) were motile. CONCLUSION: The cryopreservation of specimens with the extremely low number of sperm often results in disappointing chances of retrieving sibling cells. By cryopreserving individual sperm on ICSI dishes at time of initial analysis, we were able to expeditely locate, identify, and prepare for injection these same cells. In case of cryoptozoospermia and NOA, this method may represent a valuable tool to retrieve these precious gametes.