MSACL 2018 US Abstract

Topic: Proteomics

Diagnosis of Male Infertility with Proteomic and Proteogenomic Biomarkers Measured in Seminal Plasma

Andrei Drabovich (Presenter)
University of Toronto

Bio: I received my PhD in bioanalytical chemistry from York University, Canada in 2008. After graduation, I completed my post-doctoral fellowship in clinical proteomics at Mount Sinai Hospital and the University of Toronto. Since 2014, I am appointed as a Scientist at the University Health Network, Toronto and an Assistant Professor at the Department of Laboratory Medicine and Pathobiology at the University of Toronto. My current interests include clinical proteomics, biomarker discovery, prostate cancer and male infertility.

Authorship: Mirzo Kanoatov (1), Christina Schiza (2), Keith Jarvi (1,3), Eleftherios P. Diamandis (1,2,4,5), Andrei P. Drabovich (2,4,5)
(1) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; (2) Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada (3) Department of Surgery, Division of Urology, Mount Sinai Hospital, Toronto, Canada (4) Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada (5) Department of Clinical Biochemistry, University Health Network, Toronto, Canada

Short Abstract

Non-invasive differential diagnosis of male infertility is a recognized unmet need in urology. In our search for biomarkers, we focused on testis-specific proteins secreted into seminal plasma, a proximal fluid suitable for non-invasive diagnostics. Using mass spectrometry, we previously discovered and validated TEX101 protein as a biomarker of azoospermia. In this work, we discovered and quantified by targeted proteomic assays a polymorphic TEX101 protein variant associated with idiopathic male infertility. We also identified and measured additional 45 testis-specific proteins expressed in male germ cells at different stages of spermatogenesis. Pending further validation, a comprehensive panel of these markers will facilitate diagnosis of idiopathic male infertility and prediction of sperm retrieval outcomes, thus increasing the reliability of assisted reproduction techniques.

Long Abstract

Introduction

Diagnostic testicular biopsy is currently the only definitive method to distinguish between obstructive (OA) and non-obstructive azoospermia (NOA), to identify the NOA subtypes of Sertoli Cell-Only syndrome, maturation arrest and hypospermatogenesis, and to predict the outcome of testicular sperm extraction (TESE). Non-invasive differential diagnosis of male infertility is thus a recognized unmet need in urology. In our search for biomarkers, we focused on the proteome of seminal plasma (SP), a proximal fluid suitable for discovery of novel markers and development of non-invasive diagnostics [1]. We have previously identified two SP protein biomarkers, TEX101 and ECM1, which could differentiate between OA and NOA with 81% sensitivity at 100% specificity [2]. TEX101 assay, however, showed only moderate effectiveness in predicting the success of TESE [3]. Our work on TEX101 provided us with unique criteria to identify male germ cell-specific proteins suitable for the development of non-invasive diagnostics in SP. In this presentation, we will introduce our biomarker development pipeline and discuss in detail identification, verification and validation of proteomic and proteogenomic biomarkers of male infertility.

Methods

Our platform integrates proteomic profiling of SP by shotgun mass spectrometry, mining of the Human Protein Atlas for germ cell-specific proteins and verification and validation of biomarker candidates by quantitative targeted proteomic assays, immunoprecipitation-mass spectrometry assays and in-house developed ELISA [4]. Identification of proteogenomic markers includes mining of the Genome Aggregation Database for damaging polymorphisms in testis-specific genes, DNA genotyping and measurement of polymorphic protein variants by GluC-mediated targeted proteomics assays.

Results

Mining of the Human Protein Atlas identified nearly 1,500 proteins with highly specific or enhanced expression in testis. This list was then narrowed down to 129 extracellular or secreted germ cell-specific proteins previously identified in our spermatozoa and SP proteomes and, thus, sufficiently abundant to be quantified by mass spectrometry. Following this, we selected 45 proteins specific for different stages of spermatogenesis. These included germ cell-specific proteins expressed exclusively by spermatogonia (n=1), spermatocytes (n=6), spermatids (n=10) and spermatozoa (n=3). Following that, we developed targeted proteomic assays and quantified a panel of 48 proteins in SP samples of patients with confirmed NOA subtypes and TESE outcomes. In addition, a cohort of 437 men was genotyped for a TEX101 polymorphism, and the G99V TEX101 variant protein was measured in spermatozoa of wild-type, hetero- and homozygous men by a GluC-mediated PRM assay. Measurements revealed a near-complete degradation (>97%) of the G99V variant protein, with homozygous men being previously diagnosed with idiopathic male infertility.

Conclusions & Discussion

SP is a promising fluid to discover novel markers of male infertility and develop non-invasive diagnostics. Pending further validation, a comprehensive panel of germ cell-specific proteins and polymorphic protein variants may pinpoint stage-specific termination of spermatogenesis leading to idiopathic male infertility and facilitate development of a non-invasive test to better predict TESE outcomes, thus increasing the reliability of assisted reproduction techniques.


References & Acknowledgements:

[1] Drabovich, A.P.; Saraon, P.; Jarvi, K.; Diamandis, E.P. Seminal Plasma as a Diagnostic Fluid for Disorders of Male Reproductive System. Nature Reviews Urology, 2014, 11, 278-88.

[2] Drabovich, A.P.; Dimitromanolakis, A.; Saraon, P.; Soosaipillai, A.; Batruch, I.; Mullen, B.J.; Jarvi, K.A.; Diamandis, E.P. Differential Diagnosis of Azoospermia with Proteomic Biomarkers ECM1 and TEX101 Quantified in Seminal Plasma. Science Translational Medicine, 2013, 5, 212ra160.

[3] Korbakis, D.; Schiza, C.; Brinc, D.; Soosaipillai, A; Karakosta, T.D.; Legare, C.; Sullivan, R.; Mullen, B.; Jarvi, K; Diamandis, E.P.; Drabovich, A.P.* Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility. 2017, BMC Medicine, 15, 60.

[4] Korbakis, D.; Brinc, D.; Schiza, C.; Soosaipillai, A; Jarvi, K; Drabovich, A.P.*; Diamandis, E.P. Immunocapture-selected reaction monitoring screening facilitates the development of ELISA for the measurement of native TEX101 in biological fluids. Molecular & Cellular Proteomics, 2015, 14, 1517-26


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