Verification of Male Infertility Biomarkers in Seminal Plasma by Multiplex Selected Reaction Monitoring Assay

Andrei P Drabovich,Keith Jarvi,Eleftherios P Diamandis

doi:10.1074/mcp.m110.004127

Abstract

Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.

Highlights

From the ‡Samuel Lunenfeld Research Institute, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5T 3L9, the **Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, Canada M5G 2C4, the §Department of Surgery, Division of Urology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5T 3L9, the ¶Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada M5S 1A8, and the ‡‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
nonobstructive azoospermia (NOA) is a more complicated infertility syndrome with the azoospermia being secondary to a failure to produce sperm; NOA may be further subclassified as maturation arrest, Sertoli cell-only syndrome, and hypospermatogenesis [4]
The differential diagnosis of normal, NOA, and obstructive azoospermia (OA) (or post-vasectomy (PV)) men is required for the following reasons: (i) in infertile patients, use of markers capable of differentiating NOA and OA could eliminate the requirement for a diagnostic testicular biopsy and (ii) in healthy individuals who have undergone a vasectomy, markers capable of differentiating normal and PV seminal plasma will reveal whether the vasectomy was successful

Summary

Introduction

For most men with azoospermia, testicular biopsy is the only currently used method to definitively distinguish between OA and NOA [5, 6]. The differential diagnosis of normal, NOA, and OA (or post-vasectomy (PV)) men is required for the following reasons: (i) in infertile patients, use of markers capable of differentiating NOA and OA could eliminate the requirement for a diagnostic testicular biopsy and (ii) in healthy individuals who have undergone a vasectomy, markers capable of differentiating normal and PV seminal plasma will reveal whether the vasectomy (and later, its possible reversal) was successful. Verification of Male Infertility Biomarkers in Seminal Plasma diagnostics [9]. PTGDS protein has been recently proposed as a seminal plasma biomarker for the diagnosis of OA [10]. PTGDS could not distinguish NOA from normal and OA (PV) groups with high confidence

Results

Discussion

Conclusion