A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

Abstract

Introduction. Conventional methods of processing testicular and epididymal specimens are based on simple washing, so that spermatozoa are contaminated with other cells and difficult to recover from the sediment. The newly method is based on differential centrifugation, which has never been used for male germ cells before. Cell particles are separated according to their sedimentation rate so that spermatozoa remain in the supernatant, whereas the sediment with extra cells and debris is removed. A pure fraction of spermatozoa from supernatant is retrieved even in severe cases. Methods. A total of 436 specimens surgically obtained from epididymis/testis in 398 patients were treated using method of differential centrifugation. To evaluate the effectiveness of the new approach, each biological sample was assigned to a specific quality category based on sperm content during intraoperative examination. The recovered spermatozoa were used to fertilize the fresh spouse’s oocytes and/or cryopreserved. A retrospective data analysis was performed. Results. The microscopic examination surgically obtained specimens revealed at least one spermatozoon in all fields of view in 237 patients (59,5%). The further processing by differential centrifugation was 99,6% efficient (spermatozoa for fertilization were recovered in 236 cases, including cryopreservation in 214 cases). In 199 patients no spermatozoa were found during intraoperative examination. After treatment by differential centrifugation, single spermatozoa were isolated and used for oocyte fertilization in 40 cases (20%). Conclusion. The newly proposed method of processing cell suspensions for spermatozoa recovery allows obtaining male germ cells from biopsy samples and using them for fertilization, especially if they are critically low in number and conventional methods for sperm recovery do no work or inefficient.