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

Preclinical Evaluation of TEX101 Protein as a Male Infertility Biomarker and Identification of its Functional Interactome

TEX101 is a testis-specific cell surface protein expressed exclusively in the male germ cells, and it was previously suggested as a biomarker for the differential diagnosis of male infertility. Molecular function of TEX101 is not known, but previous studies demonstrated that murine TEX101 was a cell membrane chaperone, essential for fertilization. In this work, we employed quantitative proteomic strategies to investigate the clinical and the functional aspects of human TEX101 protein. To facilitate translation of TEX101 into clinic, we first developed and optimized an immunoassay for TEX101 measurement in biological fluids. We then evaluated the performance of TEX101 ELISA in 805 seminal plasma samples of fertile, sub-fertile, and infertile men. We demonstrated the clinical utility of TEX101 to evaluate vasectomy success, to stratify azoospermia forms and subtypes, and to predict the success of sperm retrieval in patients diagnosed with non-obstructive azoospermia. To gain insights into human TEX101 function, we employed mass spectrometry approaches to investigate the physical and the transient TEX101 interactome. Co-immunoprecipitation-mass spectrometry revealed a testis-specific complex TEX101-DPEP3 which was validated by hybrid immunoassay in testicular tissues and spermatozoa. In addition, we discovered a TEX101 rs35033974 homozygous knockdown model, which carried a missense variation leading to near-complete protein degradation. We then performed global proteomic analysis of TEX101 knockdown spermatozoa, and we identified the transient interactome of TEX101. New knowledge on TEX101 will provide insights into reproductive biology, facilitate better diagnostics of male infertility, contribute to rational selection of assisted reproduction treatments, and offer new protein targets to develop non-hormonal male contraceptives.Ph.D

Characteristics of Patients with Obstructive Sleep Apnea at High Risk for Cardiovascular Disease

Background and Objectives: To evaluate the influence of obstructive sleep apnea (OSA)-related symptoms on prevalent cardiovascular disease (CVD) in a large clinical population of patients. Materials and Methods: A total of 2127 patients (mean age 55 years, 24% women) underwent diagnostic polysomnography and were evaluated using the Epworth sleepiness scale (ESS), the Athens Insomnia Scale (AIS), and the Beck Depression Inventory (BDI). We investigated the predictive value of OSA-associated symptoms for prevalent cardiovascular disease, after adjustment for relevant confounding factors including age, obesity, and co-morbidities. Results: Patients with OSA and CVD were older and had a higher Body Mass Index (BMI); the percentage of obese patients was also higher (83% vs. 70%, p p = 0.64], insomnia symptoms (AIS ≥ 6) [odds ratio (95% CI) 0.748 (0.473–1.184), p = 0.21], frequent awakenings [odds ratio (95% CI) 1.599 (1.019–2.508), p = 0.06], and nocturia [odds ratio (95% CI) 1.359 (0.919–2.009), p = 0.124] were not associated with CVD after adjustment for the previous confounders. On the other hand, depressive symptoms (BDI ≥ 10) independently predicted prevalent CVD [odds ratio (95% CI) 1.476 (1.154–1.887), p = 0.002]. Further analysis in subgroups stratified by age, BMI, and gender demonstrated that depressive symptoms predicted prevalent CVD but only in the subgroup of younger (age group p = 0.006) OSA patients. Conclusions: OSA patients with CVD were more likely to complain of less typical OSA symptoms and depressive symptoms compared to patients without CVD in this large clinical patient cohort, supportingthecomplexity and heterogeneityof OSA

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

Abstract 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