Abstract: PD68-01
Sources of Funding: None

Introduction

Testicular sperm extraction (TESE) is successful in identifying a small number of sperm in 50% of men with non-obstructive azoospermia (NOA). Traditionally, sperm are isolated from testicular tissue using a combination of standard light microscopy, tissue digestion and time analyzing the specimen in hope to isolate rare spermatozoa. Here we discuss our results utilizing fluorescence-activated cell sorting (FACS) of testis tissue to increase the efficiency in the isolation of spermatozoa.

Methods

Testicular tissue was obtained from 10 patients: 2 cadaveric specimens with normal spermatogenesis and 8 specimens from wasted testicular tissue from microTESE. The specimens were prepared by sharp cutting followed by mechanical disaggregation with a Medimachine (BD Biosciences, USA) and passed through a 50- and 30-micron filter. The specimens were then stained with DNA-stains To-Pro-3 or SYTO 17(ThermoFischer, USA) and incubated. Sperm from a normal semen sample were stained similarly and used as controls for gating during flow cytometry . Then, cell sorting was completed using a FACSAria II (BD Biosciences, USA) to isolate spermatozoa. Each sorted specimen underwent standard light microscopy to identify spermatozoa.

Results

Using this technique, spermatozoa were successfully isolated and recovered in both cadaveric specimens. Of the 8 patients undergoing microTESE , 3 (38%) had spermatozoa recovered using standard tissue processing and 4 (50%) had spermatozoa recovered using FACS. Notably, in our cohort, both patients with maturation arrest had a negative microTESE with standard tissue processing, but had successful isolation of spermatozoa using FACS.

Conclusions

Our initial experience using fluorescence-activated cell sorting for rare spermatozoa isolation from testicular tissue proves the technical feasibility of this process. As this research continues to be refined and implemented, the clinical application of this technique has the potential to increase the rate of successful TESE to isolate spermatozoa.

Funding

None