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+/ACRV1+/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+/ACRV1+/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.

The authors have declared no competing interest.

Canadian Institutes of Health Research Proof of Principle Program PoP and Phase I grants; Physicians Services Incorporated (PSI) Foundation Health Research grant; Astellas Prostate Cancer Innovation Fund; Prostate Cancer Canada Movember Rising Star; and the University of Alberta.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Sinai Health System REB# 15-0149-A University of Toronto REB# 09-0156-E30252

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All data produced in the present study are available upon reasonable request to the authors