Analysis of Seminal Plasma from Patients with Non-obstructive Azoospermia and Identification of Candidate Biomarkers of Male Infertility

Abstract

Infertility affects approximately 15% of couples with equivalent male and female contribution. Absence of sperm in semen, referred to as azoospermia, accounts for 5-20% of male infertility cases and can result from pretesticular azoospermia, non-obstructive azoospermia (NOA), and obstructive azoospermia (OA). The current clinical methods of differentiating NOA cases from OA ones are indeterminate and often require surgical intervention for a conclusive diagnosis. We catalogued 2048 proteins in seminal plasma from men presented with NOA. Using spectral-counting, we compared the NOA proteome to our previously published proteomes of fertile control men and postvasectomy (PV) men and identified proteins at differential abundance levels among these clinical groups. To verify spectral counting ratios for candidate proteins, extracted ion current (XIC) intensities were also used to calculate abundance ratios. The Pearson correlation coefficient between spectral counting and XIC ratios for the Control-NOA and NOA-PV data sets is 0.83 and 0.80, respectively. Proteins that showed inconsistent spectral counting and XIC ratios were removed from analysis. There are 34 proteins elevated in Control relative to NOA, 18 decreased in Control relative to NOA, 59 elevated in NOA relative to PV, and 16 decreased in NOA relative to PV. Many of these proteins have expression in the testis and the epididymis and are linked to fertility. Some of these proteins may be useful as noninvasive biomarkers in discriminating NOA cases from OA.

The Use of Proteomics in Assisted Reproduction

“…Batruch et al utilized MudPIT and high resolution MS to proteomically characterize the seminal plasma of both normal donors and vasectomized azoospermic men and subsequently compared the derived profiles to those of men with non-obstructive azoospermia, to identify 18 and 59 proteins that were overexpressed, as well as 34 and 16 proteins that were underexpressed in the NOA group compared to controls and PV, respectively, allowing, thus, possible discrimination between OA and NOA patients. Two of these proteins, SPAG11B and TEX101, were considered highly important as infertility biomarkers (73,74). In a more recent study by Drabovich et al immunohistochemistry and an immunoenrichment MSbased assays were utilized in SP samples of azoospermic and normal men and succeeded in recognizing epididymisexpressed ECM1 and testis-expressed TEX101 as highly specific and sensitive markers differentiating obstructive and non-obstructive azoospermia (75).…”

Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

“…To facilitate our diagnostic strategy, we considered as candidates only secreted and membrane-bound proteins which were previously identified in our SP proteome of more than 3,000 proteins [20][21][22] . We assumed that some candidates selected with large-scale -omics approaches might be false-positives due to various pre-analytical, analytical and data analysis biases.…”

“…Nearly a quarter of molecular composition of SP is secreted by prostate 18,19 , with the rest produced by seminal vesicles, epididymis, testis and periurethral glands. We previously completed several years of work on proteome of SP and identified more than 3,000 proteins in SP of healthy men and patients with infertility, prostatitis and prostate cancer [20][21][22] .…”

Proteomics of human seminal plasma: Identification of biomarker candidates for fertility and infertility and the evolution of technology

“…The flight time of each ionized fragment is proportional to the square root of the m/z ratio. As the charge (z) of the ionized proteins is often þ1, the m/z value is often equal to the mass (2003) MALDI-TOF Impaired spermatogenesis Utleg et al (2003) LCMS/MS Prostasome Fung et al (2004) MALDI-TOF; LC-MS/MS Healthy fertile men Pilch and Mann (2006) MALDI-TOF; LTQ-FT Healthy fertile man Yang et al (2007) SELDI-TOF Biomarkers of oligospermia Yamakawa et al (2007) LCMS/MS Infertile versus fertile men Bai et al (2008) SELDI-TOF Severe oligospermia versus healthy fertile men Kumar et al (2008) MALDI-TOF Heparin binding proteins in human seminal plasma Kumar et al (2009) MALDI-TOF Heparin binding proteins in human seminal plasma Wang et al (2009) LCMS/MS Asthenozoospermia Drake et al (2010) LTQ-Orbitrap XL Prostatic secretions Batruch et al (2011) LTQ-Orbitrap XL Post-vasectomy versus healthy fertile men Milardi et al (2012b) LTQ-Orbitrap XL Healthy fertile men Batruch et al (2012) LTQ-Orbitrap XL Non-obstructive azoospermia Kagedan et al (2012) LTQ-Orbitrap XL Prostatitis Molecular Reproduction & Development value. The spectral output produced by MALDI-TOF is represented by a number of protein peaks, which are described by a m/z value on the horizontal axis and by a peak-intensity value on the vertical axis (Schuchardt and Sickmann, 2007).…”