Proteomics in seminal plasma as predictor of successful sperm retrieval in infertile azoospermic men
Savadi Shiraz, Elham
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Men with non-obstructive azoospermia (NOA) have zero sperm in the ejaculate. Testicular sperm suitable for assisted reproductive technology (ART) could be present in NOA patients. Sperm is collected by micro-testicular sperm extraction (M-TESE). However, no diagnostic test exists to predict the presence of sperm in the testis biopsy before surgery. About 50% of azoospermic men are unable to have sperm retrieved at M-TESE and therefore have surgery unnecessarily. The ability to predict the presence of sperm in the testis prior to TESE would improve sperm retrieval rates and avoid unnecessary surgery. Identification of specific proteins related to spermatogenesis in seminal plasma (SP) could provide a novel proteomics assay for sperm presence prior to biopsy. SP is a rich, easily-accessible and promising source of spermatogenesis biomarkers. This study was designed to compare the SP proteomes of two different groups of NOA. 1) Men with mixed-testicular atrophy (MA) with a positive sperm retrieval at M-TESE. 2) Sertoli cell-only syndrome (SCO) patients with a negative sperm retrieval at M-TESE. Results also compared with the control-group. In a part of study, SP was collected from MA and SCO patients. All samples were compared by label-free Liquid chromatography-mass spectrometry/MS proteomics. Differentially expressed proteins were defined as those with a fold-change >2.0 and a significant difference between the groups (p<0.05). In another part of the study, a comparative proteome analysis of seminal plasma was performed in men requesting vasectomy. Sampling was done before vasectomy and six weeks after vasectomy. 2D electrophoresis was performed separately with 100 µl seminal plasma for each sample. Gel images were acquired with a typhoon 9200 laser scanner and analysed with PdQuest software. Comparison between groups (before vs. after) revealed 84 significant different spots. Protein identification revealed 27 different proteins. Among the significant proteins identified by mass spectrometry SOD1, SOD3 and LGALS3BP were selected for further validation by 3 different methods: Western blotting, immunohistochemistry and ELISA (n=15/group). Also, to compare with seminal plasma, the protein concentrations of SOD3 and SOD1 were measured in the blood-serum of participants. SOD3 immunoreactivity (ELISA) in SP from SCO men was 2.3 fold lower than controls (p<0.05), and SP from MA men was 4.6 fold lower than controls (p<0.0001). The SOD3 MA/SCO protein ratio was 0.49 (p<0.05) by ELISA and 0.16 (p<0.01) by mass spectrometry. SOD3 gave the expected band of 30 kDa by Western blot of SP samples, but no significant difference between MA and SCO groups was observed. In normal human testis, SOD3 staining was predominantly observed in Sertoli cells, while in the testis of azoospermic patients staining was more variable. SOD1 immunoreactivity (ELISA) in SP from SCO and MA men was decreased significantly compared with controls (p<0.05). The MA/SCO ratio of SOD1 was significantly different by mass spectrometry but not by ELISA or quantitative Western blot. In normal human testis, SOD1 was predominantly localized in spermatogonia, while in the testis of azoospermic patients variable SOD1 staining was noted. There were no significant differences in the levels of SOD1 and SOD3 in the blood-serum of MA compared to SCO patients. Two different antibodies were used for quantitation of protein (in both cases of SOD1 and SOD3) by Western-blotting compared to ELISA which may explain the variation in results. Moreover, the selected antibodies may not target the same sequence region that was detected by the mass spectrometry. SOD3 was significantly different in SP from MA versus SCO azoospermic patients by at least 2 different proteomics-methods (mass spectrometry and ELISA), and thus has the potential to predict the presence of sperm in the testis prior to biopsy. LGALS3BP in SP from SCO and MA men was significantly lower than controls (p<0.001) by Western blot. Also, a significant difference for the LGALS3BP MA/SCO protein ratio was observed (p<0.05). Among the significant proteins identified by 2D electrophoresis, an epididymis specific candidates, namely, CRISP1 (Cystein-Rich Secretory Protein 1) were selected for further validation by Western blotting and using two different antibodies, which had affinity for two different epitopes of protein. The CRISP1 MA/SCO protein ratio was significantly different (p<0.05) by applying the both antibodies. SOD3 (Superoxide dismutase 3), LGALS3BP and CRISP1 are proteins in seminal plasma which have potential to predict the success of micro-dissection sperm retrieval in non-obstructive azoospermia men (NOA).