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.
Molecular & cellular proteomics : MCP. 2023 Apr 20 [Epub ahead of print]
Junyan Zhang, Mirzo Kanoatov, Keith Jarvi, Andree Gauthier-Fisher, Sergey I Moskovtsev, Clifford Librach, Andrei P Drabovich
CReATe Fertility Centre, Toronto, ON, Canada., Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada., Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; Department of Surgery, Division of Urology, Mount Sinai Hospital, Toronto, ON, Canada., CReATe Fertility Centre, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada., CReATe Fertility Centre, Toronto, ON, Canada; Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada; Department of Physiology, University of Toronto, Toronto, ON, Canada; Sunnybrook Research Institute, Toronto, ON, Canada., Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada. Electronic address: .