O-209 Separating spermatozoa from debris, non-germ and red blood cells during differential centrifugation as a new effective approach of sperm recovery from surgically-retrieved specimens

Abstract

Abstract Study question What is a more efficient method to separate spermatozoa from debris, non-germ and red blood cells in surgically-retrieved testicular and epididymal specimens? Summary answer The newly method based on differential centrifugation is able to recover a pure fraction of spermatozoa from the supernatant; no sperm are contaminated with sediment. What is known already Conventional methods of processing testicular and epididymal specimens are based on simple washing so that spermatozoa are contaminated with non-germ cells, large numbers of red blood cells and debris and difficult to identify and recover from the sediment. Thus, even if the sample contains spermatozoa, it is not always possible to recover and use them for ICSI. The newly method is based on differential centrifugation, which has never been used for sperm 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. Study design, size, duration A total of 223 testicular open biopsies with microdissection (micro-TESE) was performed in patients with NOA. The recovered spermatozoa were used to fertilize the fresh spouse's oocytes (synchronous IVF cycle) and/or cryopreserved ones for delayed transfer in the IVF/ICSI program (non-synchronous IVF cycle). Surplus embryos obtained in IVF programs were cryopreserved for delayed transfer in the FET (frozen-thawed embryo transfer) programs. Participants/materials, setting, methods The biopsy material was first processed mechanically. In all cases, the further processing of cell suspensions was carried out by differential centrifugation, with parameters of force and time set individually (on average 200-350 g for 1-2 min, 8-12 times). Main results and the role of chance The microscopic examination of cell suspensions, mechanically processed, revealed at least one spermatozoon in all fields of view in 89 patients (39.9%). The further processing of cell suspensions by differential centrifugation was 100% efficient: spermatozoa for ICSI were recovered from the testicular sample in all the 89 cases. Pregnancy occurred in 47 out of 89 patients, in 147 ART cycles (52.8% per patient, 31.9% per ART cycle). Limitations, reasons for caution Not applicable Wider implications of the findings The newly proposed method of processing cell suspensions for spermatozoa recovery is based on differential centrifugation and 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 are inefficient. Trial registration number not applicable