Germ Cell–Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia

Abstract

•45 germ cell proteins were measured in human spermatozoa and seminal plasma.•Novel markers of obstructive and non-obstructive azoospermia were verified.•ASPX and AKAP4 proteins were exclusively localized to sperm acrosomes and tails.•Imaging flow cytometry identified rare intact AKAP4+/ASPX+/Hoechst+ sperm in NOA. Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific 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 multistep 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 noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility. Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific 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 multistep 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 noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility. Infertility affects 15% of couples globally, with a male factor contributing to infertility in approximately 50% of the cases (1Jarvi K. Lo K. Grober E. Mak V. Fischer A. Grantmyre J. et al.The workup and management of azoospermic males.Can Urol. Assoc. J. 2015; 9: 229-235Crossref PubMed Scopus (29) Google Scholar). The routine diagnostics of male factor infertility relies on semen analysis for the spermatozoa count, motility, and morphology and on infertility in the couple (2Campbell M.J. Lotti F. Baldi E. Schlatt S. Festin M.P.R. Bjorndahl L. et al.Distribution of semen examination results 2020 - a follow up of data collated for the WHO semen analysis manual 2010.Andrology. 2021; 9: 817-822Crossref PubMed Scopus (36) Google Scholar). Common categories of male infertility based on abnormal sperm parameters include oligozoospermia, asthenozoospermia, teratozoospermia, azoospermia, and a combination of morphological abnormalities with low sperm count and diminished motility diagnosed as oligoasthenoteratozoospermia. Azoospermia is further classified as obstructive azoospermia (OA) with no sperm in ejaculate due to obstruction despite normal spermatogenesis and non-obstructive azoospermia (NOA) with no sperm in ejaculate due to testicular failure (2Campbell M.J. Lotti F. Baldi E. Schlatt S. Festin M.P.R. Bjorndahl L. et al.Distribution of semen examination results 2020 - a follow up of data collated for the WHO semen analysis manual 2010.Andrology. 2021; 9: 817-822Crossref PubMed Scopus (36) Google Scholar). Men with elevated follicle-stimulating hormone (FSH) in blood serum and smaller testicles are more likely to have testicular failure resulting in NOA. However, men with normal FSH levels could have either NOA or OA (3von Eckardstein S. Simoni M. Bergmann M. Weinbauer G.F. Gassner P. Schepers A.G. et al.Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples.J. Clin. Endocrinol. Metab. 1999; 84: 2496-2501Crossref PubMed Scopus (206) Google Scholar), and a testicular biopsy is typically required to determine the presence and degree of spermatogenesis and distinguish between OA and NOA. Breakthroughs in assisted reproductive technologies allowed fertility clinics to offer successful treatments to couples with male factor infertility, including its severe forms (1Jarvi K. Lo K. Grober E. Mak V. Fischer A. Grantmyre J. et al.The workup and management of azoospermic males.Can Urol. Assoc. J. 2015; 9: 229-235Crossref PubMed Scopus (29) Google Scholar). Patients with mild oligozoospermia or idiopathic male infertility have sufficient numbers of spermatozoa in semen to be offered either intrauterine insemination, conventional in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI). Patients with moderate oligoasthenoteratozoospermia could be managed with IVF or ICSI depending on sperm count and spermatozoa quality. NOA, the most severe form of male infertility, could only be treated with ICSI, providing spermatozoa were retrieved through microdissection testicular sperm extraction (mTESE). The mTESE procedure has a ∼50% success rate and can identify spermatozoa even in those cases when an initial diagnostic testicular biopsy revealed no spermatozoa (4Schlegel P.N. Palermo G.D. Goldstein M. Menendez S. Zaninovic N. Veeck L.L. et al.Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia.Urology. 1997; 49: 435-440Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 5Schlegel P.N. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision.Hum. Reprod. 1999; 14: 131-135Crossref PubMed Scopus (660) Google Scholar). It should be noted that testicular needle biopsies, partly due to the random sampling, fail to identify spermatozoa in ∼30% of NOA patients who had spermatozoa following mTESE (6Ramasamy R. Schlegel P.N. Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval.J. Urol. 2007; 177: 1447-1449Crossref PubMed Scopus (89) Google Scholar). Thus, extensive microsurgical dissection of seminiferous tubules and their examination with an operating microscope is the most widely used technique to identify pockets of spermatozoa. Seminiferous tubules with spermatogenesis are excised, and the mTESE spermatozoa are used for ICSI (7Aboukhshaba A. Punjani N. Doukakis S. Zaninovic N. Palermo G. Schlegel P.N. Testicular sperm characteristics in men with nonobstructive azoospermia and their impact on intracytoplasmic sperm injection outcome.Fertil. Steril. 2022; 117: 522-527Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The mTESE procedure is laborious, and patients often spend more than 3 hours under general anesthesia. The heterogeneous etiology of male infertility, the low diagnostic sensitivity of FSH, the inaccuracy of the initial testicular biopsy diagnostics, and the laborious surgical retrieval of spermatozoa may result in different and unpredictable mTESE outcomes at different fertility clinics. A noninvasive diagnostic test to predict mTESE outcome constitutes a recognized clinical need. Likewise, a viable approach for the rapid identification of rare mature spermatozoa within dissected seminiferous tubules of NOA patients, or within semen cell debris of patients with severe oligoasthenoteratozoospermia, would be highly beneficial to IVF programs. Numerous studies provided potential biomarkers of male infertility, including genomic (8Yatsenko A.N. Georgiadis A.P. Ropke A. Berman A.J. Jaffe T. Olszewska M. et al.X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.N. Engl. J. Med. 2015; 372: 2097-2107Crossref PubMed Scopus (238) Google Scholar, 9Shen F. F. et in male infertility with morphological abnormalities of the sperm PubMed Scopus Google Scholar, A. P. et in and male infertility and in and 9: PubMed Scopus Google and markers K. S. M. R. S. et of in sperm 2015; PubMed Scopus Google Scholar), M. E. R. S. et of sperm with idiopathic male Med. 2015; PubMed Scopus Google Scholar), J. L. et analysis a in infertile PubMed Scopus Google Scholar), A. et cell by in male Clin. PubMed Scopus Google Scholar), and proteins L. et in is a marker and potential of idiopathic male Full Text Full Text PDF PubMed Scopus Google Scholar, R. et human male infertility with sperm motility and Med. PubMed Scopus Google Scholar, A.P. Lo biomarkers for the of male J. PubMed Scopus (89) Google Scholar, A.P. P. K. as a diagnostic for male reproductive Urol. PubMed Scopus Google Scholar, K. A.P. of as a male infertility Google Scholar). Numerous including serum FSH, inhibin and seminal were to predict mTESE sperm retrieval outcome A. K. T. K. M. et of successful outcome of microdissection testicular sperm extraction in men with idiopathic nonobstructive Urol. PubMed Scopus Google Scholar, V. J. G. A. P. inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive Reprod. PubMed Scopus Google Scholar). Likewise, a noninvasive test to between NOA and OA A.P. A. P. A. et of azoospermia with biomarkers and in seminal Med. PubMed Scopus Google Scholar, A.P. K. of male infertility biomarkers in seminal by multiplex Full Text Full Text PDF PubMed Scopus Google Scholar). While those revealed diagnostic sensitivity of to suffered from low levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are testicular sperm Steril. Full Text Full Text PDF PubMed Scopus Google Scholar, A. et of a test for the of male Med. PubMed Scopus Google Scholar, Schlegel P. A. S. A. et sperm retrieval success for men with testicular 2021; Scholar). low diagnostic for mTESE retrieval of spermatozoa even for highly testis-specific likely due to their presence at the continuum of germ cell and at the and of spermatogenesis A. et of a test for the of male Med. PubMed Scopus Google Scholar). hypothesized that testis-specific proteins further as germ cell–specific proteins and measured in seminal could facilitate noninvasive and more of mTESE suggested that germ and proteins would noninvasive and more identification of rare morphologically intact spermatozoa in the NOA semen cell pellets multiplex imaging flow The morphology of spermatozoa including acrosome-capped nuclei and elongated tails could be with the of germ and markers and could unambiguous identification of spermatozoa with no and diagnostic with biomarkers in to identify the most markers on data for the and germ cell–specific identification and of the spermatozoa extensive of markers by the targeted and of markers could be with the multiplex immunofluorescent microscopy and imaging flow cytometry We hypothesized that and germ cell–specific proteins measured in or semen could facilitate noninvasive and more of mTESE as a to and germ cell–specific proteins as biomarkers of NOA. of the most and germ cell–specific of targeted of of proteins by in of extensive of with as and flow and of a multiplex imaging flow cytometry assay for noninvasive identification of rare spermatozoa in semen of NOA patients. to to highly testis-specific as A.P. A. P. A. et of azoospermia with biomarkers and in seminal Med. PubMed Scopus Google Scholar), could be with or or NOA levels and from A.P. A. P. A. et of azoospermia with biomarkers and in seminal Med. PubMed Scopus Google be to and of with panel of testis-specific proteins in of for studies on male infertility NOA with and NOA with the mTESE A and and were by for were by the of of testis-specific proteins in and NOA as to A.P. A. P. A. et of azoospermia with biomarkers and in seminal Med. PubMed Scopus Google and of and as previously for a panel of proteins in by with the and the A.P. P. M. 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We the localization of ASPX_HUMAN within acrosomes and ASPX_HUMAN within tails of the spermatozoa as as spermatozoa in the testicular microscopy and flow cytometry and imaging flow cytometry for ASPX_HUMAN and AKAP4_HUMAN We that the of spermatozoa with flow cytometry cytometry and imaging flow cytometry and revealed the presence of levels of ASPX_HUMAN and AKAP4_HUMAN proteins in of flow cytometry analysis of AKAP4+/ASPX+/Hoechst+ spermatozoa. of spermatozoa with were imaging of spermatozoa with AKAP4 and and for AKAP4 and in spermatozoa of and semen by imaging flow cytometry facilitated the of a multistep gating strategy based on spermatozoa and and AKAP4 and rapid of the AKAP4+/ASPX+/Hoechst+ The approach facilitated the analysis of of and rapid identification of the rare morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa as in even within the debris-laden NOA semen pellets pellets of patients were to the imaging flow cytometry analysis for AKAP4+/ASPX+/Hoechst+ cells of elongated a AKAP4+/ASPX+/Hoechst+ spermatozoa were identified in semen pellets of and identified AKAP4+/ASPX+/Hoechst+ morphologically intact spermatozoa in semen pellets of NOA patients with no spermatozoa identified at the routine semen analysis and of patients with the mTESE outcomes provided diagnostic sensitivity to intact spermatozoa in NOA semen the of imaging flow cytometry with an of azoospermia with mTESE a identified morphologically intact spermatozoa in the semen pellets of patients results revealed that multistep gating strategy and imaging flow cytometry visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei could fast and unambiguous identification of rare morphologically intact spermatozoa in NOA semen flow cytometry identification of morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa in NOA semen in Schlegel P. A. S. A. et sperm retrieval success for men with testicular 2021; NOA NOA or OA provided sensitivity to spermatozoa in NOA NOA or OA in a The provided sensitivity to spermatozoa in NOA patients. The that of human were with at in to human tissue M. L. P. A. et of the human 2015; PubMed Scopus Google Scholar). The and highly of testis-specific proteins in as of the spermatozoa cell sperm and were M. N. M. a to and with the Clin. PubMed Scopus Google Scholar). A. et of the in between sperm and the for S. A. 1999; PubMed Scopus Google Scholar), the cell A. A. V. E. F. et on the for S. A. PubMed Scopus Google Scholar), and the N. M. A. M. The is required for sperm to with PubMed Scopus Google Scholar). The of the cell of and the provided and E. is the and is for PubMed Scopus Google Scholar, A. S. A. of the human sperm and fertilization PubMed Scopus Google Scholar). hypothesized that and germ cell–specific proteins with microscopy could emerge as markers for the noninvasive and highly identification of rare spermatozoa within the debris-laden semen pellets of NOA patients. studies on the identification of rare intact spermatozoa in NOA semen pellets were often due to with the of intact spermatozoa from debris (2Campbell M.J. Lotti F. Baldi E. Schlatt S. Festin M.P.R. Bjorndahl L. et al.Distribution of semen examination results 2020 - a follow up of data collated for the WHO semen analysis manual 2010.Andrology. 2021; 9: 817-822Crossref PubMed Scopus (36) Google Scholar). approach to the of the most germ and proteins on of previously identified spermatozoa K. A.P. of proteins through of human spermatozoa for the Full Text Full Text PDF PubMed Scopus Google and A.P. P. M. A. et elevated levels of in seminal of Full Text Full Text PDF PubMed Scopus Google Scholar), and on of the are for and A.P. E. an for 2015; PubMed Scopus Google Scholar, A.P. - and technologies for and to Scopus Google Scholar, A.P. E. 3 - J. for Scopus Google Scholar), suggested that targeted due to their rapid and could be to proteins and the clinical of proteins or We previously that targeted provided of proteins in human cell A.P. of and of in Full Text Full Text PDF PubMed Scopus Google Scholar, A.P. A. analysis of in cells by Full Text Full Text PDF PubMed Scopus Google Scholar), cells A. J. A. A.P. F. S. et of an in human cells by of in cell Full Text Full Text PDF PubMed Scopus Google Scholar, A.P. E. S. A. et analysis of in PubMed Scopus Google Scholar), A.P. and of the by 2021; Full Text Full Text PDF PubMed Google Scholar), A.P. of proteins in the by and of the PubMed Scopus Google Scholar, L. V. et for of proteins and their clinical PubMed Scopus Google Scholar, E. A.P. A. of a multiplex assay for of markers of in samples.J. PubMed Scopus Google Scholar, A.P. of a approach for of in multiplex PubMed Scopus Google Scholar, A. T. A. S. et of proteins in of patients with and by PubMed Scopus Google Scholar), and serum E. A.P.

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