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Germ cell-specific proteins AKAP4 and ASPX facilitate identification of rare spermatozoa in non-obstructive azoospermia.
Molecular & Cellular Proteomics : MCP 2023 April 21
Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intra-cytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis- and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+ /ASPX+ /Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multi-step gating strategy and visualization of AKAP4+ /ASPX+ /Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+ /ASPX+ /Hoechst+ assay may emerge as a non-invasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.
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