The Use Of Immunofluorescence In Microdissection Testicular Sperm Extraction

Microdissection testicular sperm extraction is a procedure used to extract sperm from patients with irreversible non-obstructive azoospermia. Sperm extracted are then used for in vitro fertilization (IVF). While this technique has improved the sperm retrieval rate compared to other biopsy techniques, sperm are still often not retrieved. The purpose of our research was to develop a technique for identifying small foci of sperm for retrieval for use in IVF.

After injecting the seminiferous tubules of fertile mice testes with a mouse antibody specific for the human acrosome, we could identify sperm in the majority of animals using confocal microscopy on excised testes. Performance of the same procedure on sterile mice did not reveal any fluorescent signal.

Though pleased with the results of our research, there are several limiting factors that must be addressed prior to practical clinical use. First, injection of human seminiferous tubules and achieving adequate antibody distribution might prove more difficult as the seminiferous tubules are longer, and it will likely require increased hydrostatic pressure to disperse the antibody. This pressure may prove problematic and lead to rupture of the tubules.

Second, while the antibody did bind to the sperm, it was not as selective as anticipated. There was binding of the antibody to both the head and tail of spermatozoa, not just the acrosome. This could have been due to the fact that a mouse-derived antibody was used in a mouse model, which could possibly be associated with the occurrence of a significant amount of non-specific binding of the sperm. There was also background staining within the tubules, making identification of the sperm more difficult. Also, it is still unknown the extent to which the antibody will prove to be detrimental to the sperm, rendering it unusable for IVF.

Lastly, in our animal model the testes were excised from the mice prior to microscopic analysis since the specimen being observed under the confocal microscope required water submersion. Obviously, a different method of analyzing human testicular tissue in situ is required. Intraoperative performance of the confocal microscopy could address this problem, but providing the necessary equipment for this procedure in the operating room may prove challenging.

Despite the obstacles facing practical use of immunofluorescence in microdissection testicular sperm extraction, we are encouraged by the results of our study. More research is certainly needed to evaluate the feasibility of this technique in humans.

Written by Jason R. Greenhalgh, MD, Thomas S. Griffith, PhD, and Moshe Wald, MD as part of Beyond the Abstract on UroToday.com.