Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility

BACKGROUND: TEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men. METHODS: Mass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples. RESULTS: We demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 μg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia. CONCLUSIONS: We demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-017-0817-5) contains supplementary material, which is available to authorized users

Seminal biomarkers for the evaluation of male infertility

For men struggling to conceive with their partners, diagnostic tools are limited and often consist of only a standard semen analysis. This baseline test serves as a crude estimation of male fertility, leaving patients and clinicians in need of additional diagnostic biomarkers. Seminal fluid contains the highest concentration of molecules from the male reproductive glands, therefore, this review focuses on current and novel seminal biomarkers in certain male infertility scenarios, including natural fertility, differentiating azoospermia etiologies, and predicting assisted reproductive technique success. Currently available tests include antisperm antibody assays, DNA fragmentation index, sperm fluorescence in situ hybridization, and other historical sperm functional tests. The poor diagnostic ability of current assays has led to continued efforts to find more predictive biomarkers. Emerging research in the fields of genomics, epigenetics, proteomics, transcriptomics, and metabolomics holds promise for the development of novel male infertility biomarkers. Seminal protein-based assays of TEX101, ECM1, and ACRV1 are already available or under final development for clinical use. Additional panels of DNA, RNA, proteins, or metabolites are being explored as we attempt to understand the pathophysiologic processes of male infertility. Future ventures will need to continue data integration and validation for the development of clinically useful infertility biomarkers to aid in male infertility diagnosis, treatment, and counseling

Dynamic Kinetic Capillary Isoelectric Focusing: A Powerful Tool for Studying Protein-DNA Interactions

A new method called dynamic kinetic capillary isoelectric focusing (DK-CIEF) is presented for the study of protein-DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein-DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants

Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas

Abstract Background Cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. The main purpose of this study was to generate an extensive CSF proteome and define brain-related proteins identified in CSF, suitable for development of diagnostic assays. Methods Six non-pathological CSF samples from three female and three male individuals were selected for CSF analysis. Samples were first subjected to strong cation exchange chromatography, followed by LC-MS/MS analysis. Secreted and membrane-bound proteins enriched in the brain tissues were retrieved from the Human Protein Atlas. Results In total, 2615 proteins were identified in the CSF. The number of proteins identified per individual sample ranged from 1109 to 1421, with inter-individual variability between six samples of 21 %. Based on the Human Protein Atlas, 78 brain-specific proteins found in CSF samples were proposed as a signature of brain-enriched proteins in CSF. Conclusion A combination of Human Protein Atlas database and experimental search of proteins in specific body fluid can be applied as an initial step in search for disease biomarkers specific for a particular tissue. This signature may be of significant interest for development of novel diagnostics of CNS diseases and identification of drug targets

Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

Abstract Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21