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C e 3 9 IN PATHOPHYSIOLOGY OF VARICOCELE ASSOCIATED MALE INFERTILITY n 9 tr 1 um t. E Es xcellentiae Ashok Agarwal, PhD1, Damayanthi Durairajanayagam, PhD1,2, Rakesh Sharma, PhD1, Luna Samanta, PhD1,3, Rola F. Turki, MD4, and Edmund S. Sabanegh, MD5 1American Center For Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 2Physiology, Universiti Teknologi MARA, Sungai Buloh, Malaysia, 3Redox Biology Laboratory, School of Life Science, Ravenshaw University, Orissa, India, 4Center of Excellence in Genomic Medicine, King AbdulAziz University, Jeddah, Saudi Arabia, 5Department of Urology, Cleveland Clinic, Cleveland, OH

ABSTRACT RESULTS

OBJECTIVE: Varicocele appears to a ect later stages of spermatogenesis. It causes scrotal hyperthermia, hypoxia, hormonal imbalances, and re-ow of metabolites from renal and/or adrenal glands Dierentially Expressed Proteins in fertile and varicocele group leading to oxidative stress. The objective was to study the major di erences in the distribution of spermatozoal proteins in infertile men diagnosed with varicocele compared to fertile men. 1. Of the 1250 proteins identied by global proteomics, 99 were di erentially expressed proteins (DEP) (Figure 1A). DESIGN: Prospective proteomic study. 2. Majority of the DEP (87.5%, 77/88) were underexpressed in patients with varicocele. Only 12.5 % of the DEP were overexpressed. Of these, 88 were common to both fertile and the varicocele group as shown in the Venn diagram (Figure 1B). MATERIALS AND METHODS: Proteins were extracted from infertile men with unilateral and bilateral varicocele (n=5) and men with proven fertility (n=5). 1-D gel electrophoresis followed by LC/MS-MS 3. Heat map generated for the DEP that were overexpressed or underexpressed is shown in Figure 2. (LTQ-Orbitrap Elite hybrid mass spectrometer) was used for identication. Mascot (Matrix Science, London, UK), SEQUEST (Thermo Fisher Scientic, San Jose, CA, USA) and X! Tandem (TheGPM, thegpm.org) were set up to search the human reference with database assuming trypsin as the digestion . Functional annotations of proteins were obtained using bioinformatics tools and Classi cation of the Dierentially Expressed proteins (Figures 3 and 4) pathway databases. 1. Both the underexpressed and overexpressed proteins are located in the mitochondria, cytosol, organelle membranes, nuclear pore complex and the cAMP-dependent protein complex. RESULTS: Of the 99 proteins that were di erentially expressed (DEP) in the varicocele group, 9 were uniquely expressed in the fertile group compared to 2 proteins that were unique to the varicocele 2. Underexpressed proteins were shown to be involved in spermatogenesis, sperm motility, mitochondrial dysfunction, metabolism of nucleotides, and fatty acid metabolism. groups. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in varicocele group. Key protein functions a ected in the varicocele group were 3. Overexpressed proteins were involved in formation of cellular protrusions, cellular compromise and loss of phosphatidyl-ionositol-4-5 bisphosphate in the varicocele group. spermatogenesis, sperm motility (ACRBP, SPA17, AKA7), and mitochondrial dysfunction (NDUFS1, UQCRC2). 4. Nine proteins were unique to the fertile group. This included aspartate - rich protein 1 (DRICH1), nucleoporin p58/p45 (NUPL1), uncharacterized protein C9orf135 (C9orf135), coiled-coil domain-containing protein CONCLUSIONS: We have identied proteins that are underexpressed in varicocele group. These proteins may be key players involved in the pathology of varicocele and in the onset of infertility. 42A (CCDC42), HD domain containing protein 2 (HDDC2), protein DPCD (DPCD), V-proton ATPase subunit B brain isoform (ATP6V1B2), heterogeneous nuclear ribonucleoprotein M (HNRNP M) and syntaxin-12 (STX12). 5. Two proteins unique to the varicocele group were integrin alpha-M (ITGAM), and integrin beta-2 (ITGB2). INTRODUCTION Proteins involved in major networks Varicocele is diagnosed in about 15-20% of the adult male population and is implicated as a factor in about 40% of infertile men. 1. First network: 17 focus molecules. 15 proteins underexpressed in the varicocele group and participated in the nucleic acid metabolism, small molecule biochemistry and molecular transport and were compared to the The recommendation of the American Society of Reproductive Medicine is to treat a varicocele when it is palpable and present with at least fertile group. 2 proteins were overexpressed (NDRG1 and ODF2) (Figure 5A). one abnormal semen parameter in couples presenting with infertility or when the female partner is normal. Numerous factors that have been proposed 2. Second network. 11 proteins; 8 were underexpressed and 3 overexpressed. Involved in energy production, metabolism, lipid metabolism and small molecule biochemistry (Figure 5B). to explain the occurrence of this multifactorial disease include venous stasis, heat stress, testicular hypoxia, apoptosis, heavy metal toxicity, increased 3. Protein-protein interaction network shows involvement in reproductive functions such as spermatogenesis, sperm motility and mitochondrial dysfunction (Figures 6A and B). oxidative stress and increased DNA damage. However the underlying molecular mechanism of varicocele associated testicular dysfunction and infertility remains unclear. Unilateral varicoceles present on the left side are more common (35-40%) compared to the bilateral varicoceles (10-15%). While surgical repair eliminates varicocele in a majority of the cases, its impact on infertility remains unclear.

Proteomics is a rapidly emerging technology that allows the simultaneous detection of thousands of proteins. A few studies have looked at altered Figure 1 Figure 2 Figure 3 Figure 4 protein proles in varicocele patients before and after varicocele repair. However, irrespective of the type (unilateral or bilateral) and/or grade of A B Heat map showing varicocele, a cohort of patients remains infertile. the DEP proteins that were overexpressed or underexpressed in the In this study, we aim at comparing the protein prole of spermatozoa from infertile men with varicocele with that of fertile men. We also examined proteins varicocele group. involved in key sperm functions related to spermatogenesis, sperm motility, and mitochondrial dysfunction.

MATERIALS and METHODS

This study was approved by the Institutional Review Board. Semen samples were collected from 50 infertile varicocele patients and 10 proven fertile men without a varicocele. Semen analysis was conducted and 5 samples from varicocele and 5 from the fertile group were pooled and Venn diagram showing A: Distribution of global proteins in fertile (control), unilateral and bilateral varicocele group and B: Di erentially expressed proteins in prepared for proteomic analysis. fertile and unilateral + bilateral varicocele group.

Liquid Chromatography Mass Spectrometer analysis (LC-MS) 5 samples from each group were normalized for protein concentration and pooled. Equal amounts of proteins were diluted in the SDS-PAGE sample bu er and fractionated using 1D SDS-PAGE. The gel bands were reduced and alkylated followed by trypsinization. The peptide extracts (~30 μL) were prepared for LC-MS analysis. Figure 5

AKAP3 The digest was analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide ACRBP A B ACBD3 RAB2A ACBD3 PRKAR1A* Cytokine/ AKR7A2 LBR PKIA Growth Factor molecular weights and product ion spectra (MS/MS) to determine sequence in successive instrument scans. SETX LRBA C2orf88 Enzyme SUMO2 HSPA2 SPA17 PRKACG AKAP7 MGLL IDH3B NDUFS1* FN1* Ion channel PGAM2 ACO2 IQGAP1 CACNA2D2 OGDH* PRKACA APOA1 Database Searching and Protein Identi cation PFKM GFPT1 SMPDL3A GAA Ligand-dependent HSPA4L PLA2G16 Nuclear Receptor SLC12A1 A4GALT MLYCD Peptidase Tandem mass spectra were extracted by Proteome Discoverer version 1.4.1.288. All MS/MS samples were analyzed using Mascot, Sequest and X! Tandem. DLST ODF2 GABRB3 UQCRC2 AKIP1 CPT2 UBC ECE2 NFS1 AK7* TNF To validate MS/MS-based peptide and protein identications, Sca old was used. Proteins were annotated with ontology (GO) terms from NCBI. ALDOB RELA Transcription TCIRG1 PPARD Regulator NDRG1 Reactome depiction of A: Overall B: underexpressed C: overexpressed Reactome depiction of A: Overall B: underexpressed C: overexpressed CCT6B NME5 DEDD Transporter ACSL6 proteins involved in Cellular functions and D: Overall E: proteins involved in Key Pathways and D: Overall E: underexpressed F: ATP6V1E1 ATP6V1G2 LIPG Other ATP1A4* ATP6V1C1 IGHMBP2 RUVBL1 GPX4 SLK Direct Interaction underexpressed F: overexpressed proteins involved in Biological overexpressed proteins involved in Molecular Quantitative Proteomics CNDP2 Indirect Interaction ATP6V0A1 processes. functions. To account for the sample-to-sample variation seen during the replicate analysis of the samples, normalized spectral abundance factor (NSAF) approach was ATP6V1G1 ACADS ECH1 applied prior to quantication of the relative amount of protein present. The abundance of the proteins was classied according to the average spectral Interaction network showing A: Nucleic acid metabolism, small molecule biochemistry and molecular transport and B: Energy production, lipid metabolism and small molecule biochemistry. count, di erent constraints for biological and statistical variance (p values) as well as the cuto for the fold change (NSAF ratio) as: CONCLUSIONS

1. Very Low abundance: Spectral count range 1.7-7; p ≤ 0.001 and (NSAF ratio ≥ 2.5 for overexpressed, ≤ 0.4 for underexpressed proteins) Figure 6 1. Infertile men with varicocele have a large number of spermatozoa proteins that are underexpressed compared to fertile men. A B SPA17 ACRBP Neighborhood 2. Majority of the underexpressed proteins are involved in major energy metabolism pathways, transport, protein folding and proton pumps. 2. Low abundance: Spectral count range 8-19; p≤ 0.01 and (NSAF ratio ≥2.5 for overexpressed, ≤ 0.4 for underexpressed proteins) NDUFS1 OGDH APOA1 FN1 Gene Fusion 3. Medium abundance: Spectral count range between 20-79; p≤ 0.05 and (NSAF ratio ≥ 2.0 for overexpressed, ≤ 0.5 for underexpressed proteins) Cooccurrence 3. Many of these proteins are involved in spermatogenesis, sperm motility and mitochondrial dysfunction. PRKAR1A IQGAP1 Coexpression ACO2 Experiments 4. Fertile men exhibit unique proteins that are completely missing in infertile men with varicocele. 4. High abundance: spectral counts >80; p≤ 0.05 and (NSAF ratio ≥ 1.5 for overexpressed, ≤ 0.67 for underexpressed proteins) NME5 AK7 Databases Textmining 5. The underexpression of these proteins in varicocele men are indicative of development of varicocele and sperm dysfunction that ultimately CACNA2D2 PRKACA Bioinformatics analysis [Homology] results in male infertility.

Functional annotation and enrichment analysis were performed using publicly available bioinformatics annotation tools and databases (GO Term Finder, GO Term UQCRC2 IDH3B 6. Validation of some of these DEP identied in this study will narrow down the key proteins involved in the development Mapper, UniProt, STRAP), Database for Annotation, Visualization and Integrated Discovery (DAVID) and proprietary software packages such as IPA of varicocele. This may help the clinicians identify patients who are more likely to benet from varicocelectomy and have improvement (Ingenuity Pathway Analysis) and Search Tool for theRetrieval of interacting / Proteins (STRING). STRING database showing evidence of functional link between protein-protein interactions amongst A: in sperm quality, and consequently, an increased likelihood of a successful pregnancy. Spermatogenesis, sperm motility and B: mitochondrial dysfunction.