Germ cell-specific proteins ACRV1 and AKAP4 facilitate identification of rare spermatozoa in semen of non-obstructive azoospermia patients

Non-obstructive azoospermia (NOA), the most severe form of male infertility due to testicular failure, could be treated with intra-cytoplasmic sperm injection (ICSI), providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). Here, we hypothesized that some 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 and extensive verification by targeted proteomic assays and immunofluorescent microscopy revealed a panel of testis-specific proteins expressed at the continuum of germ cell differentiation, including the late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) with the exclusive expression in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay revealed low numbers of the morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa in semen pellet of NOA patients. While the previously suggested soluble markers for spermatozoa retrieval suffered from low diagnostic specificity, our multi-step gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells bearing elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. Pending further validation, our assay may emerge as a non-invasive test to predict the retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of the severe forms of male infertility.