Case report: Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection

Abstract

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.

The epididymis, cytoplasmic droplets and male fertility

“…It is more likely, however, that spermatozoa have inherently acquired some motility, independent of the epididymis, as spermatozoa trapped in epididymal spermatocoeles, which contain no epididymal secretions, 3 are poorly motile, although a few can fertilize eggs in vitro. 4 Observations made in in vitro fertilization clinics indicate that increased numbers of progressively motile spermatozoa can be recovered from testicular explants by their mere incubation for up to 3 days in vitro (Figure 1), [5][6][7][8] in the complete absence of epididymal secretions. Clinical advantage of this is routinely taken to increase the number of spermatozoa available for intracytoplasmic sperm injection.…”

Recent advances in sperm maturation in the human epididymis

“…Initiation of motility may be a time-dependent phenomenon, rather than a result of epididymal secretions, since sperm recovered from the testis and caput are only motile after ductal occlusion [30,38] and sperm obtained by testicular biopsy become motile upon incubation [13].…”

Comment identifier le spermatozoïde vivant?

“…L'incubation des prEl~vements testiculaires h 37~ avec 5% de CO2, durant quelques heures dans un milieu de culture utilis~ pour la fficondation in vitro [23], voire la culture in vitro des spermatozo'fdes pendant 24 h 72 heures, permet d'initier cette mobilitE sur des spermatozo'fdes initialemerit immobiles [9]. De mEme, la culture des prEl~vements avant leur congElation augmenterait la resistance des spermatozofdes h la congElation [10].…”