Protein profiling in unlocking the basis of varicocele‐associated infertility

Varicocele is one of the major causes of male infertility and has a negative impact on spermatogenesis. The conventional semen analysis does not reveal the underlying subcellular mechanisms associated with defects in spermatozoa. Proteomics and bioinformatics analysis can be used to identify the molecular aetiologies associated with poor semen quality in varicocele patients. Mitochondrial dysfunction has been identified as the main factor affecting normal physiological functions of spermatozoa. This article discusses the proteomic studies of spermatozoa and seminal plasma in varicocele patients. Proteomics can identify potential spermatozoa and seminal plasma biomarkers in varicocele‐mediated male infertility. In future, these protein biomarkers can be useful in the development of noninvasive diagnostic and therapeutic strategies for varicocele patients.


| INTRODUC TI ON
Varicocele is a clinical condition characterised by an abnormal dilation and tortuosity of the pampiniform plexus of veins draining the testicle. It is one of the most common and correctable causes of male infertility. The prevalence of varicocele is about 15% in the general population, and 25%-40% and 45%-81% among men with primary and secondary infertility, respectively (Alsaikhan, Alrabeeah, Delouya, & Zini, 2016;Shabana et al., 2015). About 78%-93% of varicocele patients are affected with unilateral left-sided lesions and 1%-7% of cases have isolated right-sided lesions. The reported incidence of bilateral varicocele is 2%-20% (Brown, 1976).
Several mechanisms have been proposed to be involved in the pathophysiology of varicocele (Naughton, Nangia, & Agarwal, 2001).
The pampiniform plexus regulates the temperature around the testicles and maintains the testicular temperature 2°C lower than that of the core body temperature, which is crucial for normal spermatogenesis. In varicocele, the countercurrent mechanism is interrupted resulting in testicular hyperthermia which causes detrimental effects on the seminiferous tubules. Furthermore, increased pressure in the pampiniform plexus and internal spermatic vein restrains the arterial flow and disrupts the osmotic regulation of metabolic products leading to deleterious effects on the testicular milieu (Naughton et al., 2001). Testicular hyperthermia, hypoxia and the associated oxidative stress induced by increased generation of reactive oxygen species (ROS) play a crucial role in the impairment of testicular functions in varicocele resulting in infertility (Cho, Esteves, & Agarwal, 2016;Kantartzi, Goulis, Goulis, & Papadimas, 2007;Naughton et al., 2001).
According to a World Health Organization (WHO) report, varicocele was twice as prevalent in men having abnormal semen than in men with normal semen parameters (WHO, 1992). Although semen analysis is considered as a fundamental test in the evaluation of male infertility, it fails to predict the reproductive outcomes (Esteves, 2014).
Advanced sperm function tests such as quantification of ROS and total antioxidant capacity in semen, evaluation of seminal oxidationreduction potential (ORP) and sperm DNA fragmentation (SDF) testing provide information on the functional integrity of spermatozoa and, thus, facilitate better selection of varicocelectomy candidates and evaluation of assisted reproductive outcomes Agarwal, Panner Selvam, Baskaran, & Cho, 2019;Majzoub, Esteves, Gosalvez, & Agarwal, 2016). However, these tests fail to provide an insight into the molecular changes at the cellular and subcellular level that are implicated in the poor fertilising potential of spermatozoa in these subjects.
Ironically, spermatozoa, the key player in fertilisation, are transcriptionally and translationally inactive and rely on translated proteins for their biological functions (Baker, Nixon, Naumovski, & Aitken, 2012). In the past two decades, the major focus of research has been on investigating the proteins associated with spermatozoa and their nutritive medium, seminal plasma, in order to pin down key proteins associated with fertilisation (Camargo, Intasqui, & Bertolla, 2018;. The analysis and characterisation of entire set of proteins of a tissue or a cell is referred to as proteomics. The advent of high-throughput proteomics tools in the field of male reproductive research has expanded our knowledge and understanding on male reproductive health. Sperm proteomics not only facilitates the identification and cataloguing of the whole proteome, but also provides insight into the cellular pathways, post-translational modifications (PTMs) and protein-protein interactions involved in the molecular mechanisms regulating spermatogenesis and crucial sperm functions (Amaral, Castillo, Ramalho-Santos, & Oliva, 2014;Brohi & Huo, 2017;Rahman, Lee, Kwon, & Pang, 2013). Furthermore, proteomics helps identifying potential diagnostic and therapeutic biomarkers for male infertility (Bieniek, Drabovich, & Lo, 2016;Panner Selvam, Agarwal, Pushparaj, Baskaran, & Bendou, 2019;Xu et al., 2012).
Several proteomics studies have been conducted in varicocele subjects and altered expression of proteins associated with key reproductive events have been reported. In fact, several biomarkers for the diagnosis and management of varicocele have been proposed Agarwal, Sharma, Samanta, Durairajanayagam, & Sabanegh, 2016;Panner Selvam & Agarwal, 2019a;Panner Selvam, Agarwal, Sharma, et al., 2019;Zylbersztejn et al., 2013). This article provides a comprehensive and up-to-date review on spermatozoa, seminal plasma and testi cular proteomics in unilateral and bilateral varicocele. Furthermore, it sheds light on the cellular pathways and potential biomarkers associated with varicocele-mediated male infertility. We also briefly discuss the role of proteomics in predicting the outcome of varicocelectomy.

| OVERVIE W OF PROTEOMI C S
The proteome of a cell can be identified using advanced proteomic tools. Computational and bioinformatic analyses of the proteomic data are required to understand the function of proteins and peptides in cellular pathways. Currently, sperm proteins are widely studied using sophisticated instruments such as matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS) integrated with conventional 2D-gel electrophoresis (du Plessis, Kashou, Benjamin, Yadav, & Agarwal, 2011). The maximum number of proteins in spermatozoa are detected due to the high sensitivity and specificity of both these techniques (Oliva et al., 2010).
Firstly, proteins are extracted from spermatozoa and seminal plasma.
Techniques such as the 1D-gel electrophoresis or 2D-gel electrophoresis are used to separate the extracted proteins. The gels are then cut into pieces, and the proteins are subjected to trypsin digestion. The protein samples are then injected into LC-MS/MS, and the generated spectral counts are used to identify and quantify the proteins. Relative expression of proteins is measured by comparing the NSAF (normalised spectral abundance factor) of each protein .
In addition to the gel-based proteomic approach, proteome profiling is also carried out using gel-free techniques. The proteins are resolved either by ion exchange or reverse-phase chromatography, 2D-liquid chromatography, peptide fractionation or off-gel electrophoresis (Abdallah, Dumas-Gaudot, Renaut, & Sergeant, 2012).
Lately, 2D-gel separation techniques are replaced by liquid chromatography (LC)-based methods. Trypsin is used to digest these extracted proteins, which are then separated using reverse-phase columns based on their hydrophobicity (Jodar, Soler-Ventura, & Oliva, 2017b). Advanced ultra-performance liquid chromatography have been used to resolve the proteins in both spermatozoa and seminal plasma fractions (Camargo et al., 2019;Intasqui et al., 2013aIntasqui et al., , 2013b

Key points
• Proteomics is a powerful tool to understand the molecular pathophysiology of sperm dysfunction.
• Molecular pathology associated with mitochondrial dysfunction is more prominent in varicocele cases.
• Mitochondrial proteins can serve as screening biomarkers for mitochondrial dysfunction-associated infertility in varicocele patients.
• There is chronic reductive stress-mediated oxidative damage to spermatozoa in patients with unilateral varicocele.

Potential areas of research
• Future studies are warranted to translate the predicted biomarkers into clinical set-up translating the predicted prognostic and diagnostic biomarkers into clinical use.
• Identification of unique biomarkers for better prognosis of varicocele-mediated infertility.
• Development of non-invasive diagnostic and therapeutic markers for varicocele patients Bioinformatic analysis provides insight into the functional and molecular mechanism based on the proteomic data (Lan, Montelione, & Gerstein, 2003). Gene ontology (GO) analysis of the proteins provides information about their localisation, distribution and biological functions. Protein-protein interactions and defective molecular pathways can be identified using sophisticated programmes such as the Ingenuity Pathway Analysis (IPA) and Metacore™. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis is commonly performed to display the link between the proteins (Agarwal, Durairajanayagam, Halabi, Peng, & Vazquez-Levin, 2014).

| Sperm proteomics
Varicocele has an adverse effect on cellular functions of spermatozoa because of its complex pathophysiology. There are only a few reports available on varicocele-related sperm proteomics in the literature (Table 1). Hosseinifar et al. were the first to profile sperm proteins in varicocele patients using 2D-gel electrophoresis. A total of 15 proteins were identified as differentially expressed proteins (DEPs) when compared to the proteome profile of normozoospermic men without varicocele. These DEPs were identified as mitochondrial proteins, cytoskeleton proteins and heat shock proteins which may compromise the fertility potential of men with varicocele (Hosseinifar et al., 2013). Furthermore, sperm proteomic studies in varicocele patients using a global proteomic approach identified the underlying molecular mechanisms associated with this pathology. Agarwal et al. used the LC-MS/MS approach and identified 369 DEPs in unilateral varicocele patients compared with fertile men . These DEPs were reported to be involved in biological processes such as signal transduction, response to stress and small molecule metabolic process . On the other hand, only 73 DEPs were identified in the case of bilateral varicocele patients . Cellular pathways associated with reproductive functions were dysregulated in both unilateral and bilateral varicocele conditions. A total of 29 proteins essential for the fertilisation process such as sperm maturation, motility, capacitation, hyperactivation and acrosome reaction were altered in unilateral varicocele patients . Furthermore, molecular pathways associated with mitochondrial function, free radical scavenging, protein ubiquitination and PTM of proteins were defective in the spermatozoa of unilateral varicocele patients.

TA B L E 1 Key differentially expressed proteins (DEPs) in spermatozoa and seminal plasma of males with varicocele and in varicocelectomy patients
In bilateral varicocele, sperm proteins APOA1, TOM22 and TGM4 associated with molecular pathology such as oxidative stress and SDF showed alteration in their expression pattern .
Lipid metabolism, small molecule biochemistry and nucleic acid metabolism functions were affected in bilateral varicocele patients . Both proteomic studies were able to predict several protein biomarkers specific to unilateral and bilateral varicocele-mediated male infertility, respectively ; . Proteins such as CABYR, AKAP, APOA1, SEMG1, ACR, SPA17, RSPH9 and DNAH17 that are crucial for the fertilisation process were proposed as candidate biomarkers in men with unilateral varicocele . On the other hand, reproductive function-related proteins (ODF2, TEKT3, TCP11, CLGN) and semen quality indicator proteins (ENKUR, SEMG1, SEMG2, SPAM1 and CABYR) were described as unique markers in cases of bilateral varicocele .

| Seminal plasma proteomics
Seminal plasma serves as a nutritive medium for spermatozoa and harbours proteins that are crucial for processes such as hyperactivation, capacitation, acrosome reaction and spermatozoa-oocyte fusion (Jodar et al., 2017;Samanta, Parida, et al., 2018). It is enriched with lipids, carbohydrates, growth factors transcriptional factors and proteins, which are secreted by the testis and accessory sex glands (Samanta, Parida, et al., 2018). The high amount of protein (33-55 g/L) in the seminal plasma makes it an ideal sample for proteomic analysis.
The first seminal plasma proteomics study was conducted in adult varicocele subjects, and the protein profile of smokers and nonsmokers were compared using 2D electrophoresis and mass spectrometry (Fariello et al., 2012). The study revealed differential expression of twenty proteins between the groups. These proteins were identified to be involved in sperm maturation, spermatozoa-oocyte fusion, inflammatory response, regulation of proteolysis and apoptosis, development and maintenance of blood-testis barrier and scavenging superoxide radicals. It was concluded that in men with varicocele, smoking results in alterations of seminal plasma protein profile contributing to poor semen quality, decreased sperm functional integrity and increased seminal oxidative stress (Fariello et al., 2012).
Seminal proteomics studies in adolescents with varicocele revealed altered expression of proteins associated with sperm motility and capacitation (Zylbersztejn et al., 2013). Furthermore, apoptosis regulating proteins (IBP-3 and SMG1) were overexpressed in adolescents with abnormal semen quality and varicocele, whereas spermatogenesis related proteins (BRE1B, pro-activator polypeptide and epididymal secretory protein E3-beta proteins) were overexpressed in adolescents with normal semen parameters and varicocele (Zylbersztejn et al., 2013). Seminal exosomes are mainly derived from the epididymis and prostate, and they exhibit a complex proteome. Spermatozoa-exosome fusion is crucial for transfer of proteins to transcriptionally and translational inert spermatozoa. These proteins are involved in the key processes associated with spermatozoa such as acquisition of fertilising ability, modulation of motility, regulation of capacitation and protection against oxidative stress (Baskaran, Panner Selvam, & Agarwal, 2020). A comprehensive proteomic analysis of exosomes derived from human seminal plasma revealed that the proteins are mainly involved in biological processes including metabolism, energy pathways, cell growth and maintenance, and transport (Yang et al., 2017).

| PROTEIN PROFILING : UNIL ATER AL VER SUS B IL ATER AL VARI CO CELE
It is important to understand the molecular pathophysiology and prognostic differences between unilateral versus bilateral varicocele. Agarwal et al. carried out a global proteomic study on spermatozoa of unilateral and bilateral varicocele patients to characterise the DEPs . They were also able to demonstrate the molecular changes responsible for the poor semen quality in bilateral varicocele patients using a comparative proteomic approach . The LC-MS/MS platform detected differences in the expression of 253 proteins between unilateral and bilateral varicocele groups. The majority of these proteins are involved in signal transduction, metabolism and apoptosis pathways. However in bilateral varicocele, the proteins GSTM3, SPANX1, CYB5R2, CALGN and PARK7 associated with sperm function (such as capacitation, hyperactivation and acrosome reaction), and the fertilisation process (such as spermatozoa-oocyte recognition and zona pellucida binding) were severely downregulated . Further in-depth analysis of the sperm proteome revealed that 135 DEPs were involved in the acetylation process

| Redox regulation and mitochondrial dysfunction in varicocele-associated male infertility
Oxidative stress and mitochondrial dysfunction are more pro- Underexpression of cAMP-dependent type-I protein kinase is considered a crucial factor for dysregulation of mitochondrial structural and functional proteins  whereas, downregulation of Cytochrome bc I complex subunit in spermatozoa is indicative of a hypoxia-induced oxidative stress pathology in varicocele cases (Agarwal, Sharma, Samanta, et al., 2016). Using cluster analysis, 3-di-

| Proteomic profiling as a predictor of varicocelectomy outcome
In general, varicocele repair or varicocelectomy is the most common method to correct varicocele. Several studies have reported an improvement in semen quality (Madgar, Weissenberg, Lunenfeld, Karasik, & Goldwasser, 1995;Nieschlag, Hertle, Fischedick, Abshagen, & Behre, 1998), sperm DNA damage (Lacerda et al., 2011) and reproductive outcomes (Esteves, Oliveira, & Bertolla, 2010;Schlegel, 1997) et al., 2014) were the first to demonstrate the differential expression of sperm proteins in varicocele patients before and after surgery. The proteins ATP5 associated with mitochondrial function, SOD1 involved in antioxidant mechanism and HSPA5 related to stress response mechanism were upregulated in spermatozoa after varicocele repair (Hosseinifar et al., 2014). Another mass spectrometry-based quantification using dimethyl labelling of sperm proteins reported that expression of 27 proteins increased after varicocele repair. These proteins were associated with sperm production such as B2GM and A2MG, and mitochondrial functions such as ATPB, QCR1, ATPA, MDHM, DX39A, ECH1, TSN16 and KAP2 (Camargo et al., 2017).
Camargo et al. compared the protein profile and proteomic enriched pathways of seminal plasma using an unbiased free-label quantitative proteomic approach (2D nano UPLC-ESI-MS E ) in men before and after microsurgical varicocelectomy (Camargo et al., 2013). Of the 316 proteins identified, 68 were reported to be differentially expressed among the groups. Enriched functional analysis revealed an enhanced nitric oxide metabolism and tetratricopeptide repeat domain-binding functions in the pre-varicocelectomy group, while response to ROS, gluconeogenesis, nicotinamide adenine dinucleotide (NAD)-binding and protein stabilisation were enriched in post-varicocelectomy patients (Camargo et al., 2013). Overall, the analysis demonstrated a shift back to homeostasis following varicocelectomy, which indicated that varicocele repair facilitates the return of semen quality to its physiological state (Camargo et al., 2013). A similar shift towards homeostasis in semen was demonstrated following surgical intervention in adolescent varicocele . A recent prospective study analysed seminal plasma proteomic profile of F I G U R E 1 Mitochondrial dysfunction due to hypoxia and reductive stress-induced oxidative stress is a mediator of sperm dysfunction in unilateral varicocele patients adult varicocele subjects before and 12 months after varicocelectomy as well as based on the improvement of semen quality following varicocelectomy (positive vs. negative outcome) (Camargo et al., 2019). Tripeptidyl peptidase-1 (TPP-1), a seminal protein, was reported to be three-fold higher in men with a positive outcome following varicocelectomy when compared to men with a negative outcome. Furthermore, the study reported TTP-1 as an outcome predictor for varicocelectomy in adults with an area under the receiver operating characteristics (ROC) curve of 84.5% (Camargo et al., 2019).

| Current challenges and future perspectives
A tremendous increase in spermatozoa and seminal plasma proteomic studies over the last decade provides better understanding of molecular pathology in male infertility. Proteomic and bioinformatic tools were successfully used to identify the candidate biomarker(s) for specific male infertility conditions. However, proteomic studies involving spermatozoa and seminal plasma are not without their limitations and challenges. Due to the complex nature of semen, experiments related to PTM require enrichment of sperm proteins prior to MS analysis . In spermatozoa, the abundance of PTMs such as phosphorylation, acetylation and methylation is low, and hence, a target-based proteomic approach is the most suitable technique. Apart from that, analysis of proteomic data is also very challenging and requires expertise in the area of bioinformatics. Computational tools such as IPA, Metacore, Cytoscape and Reactome can help with the interpretation of proteomic data to identify the pathways and cellular processes affected in specific conditions. Moreover, powerful meta-analysis is required to validate the biomarkers. Future clinically oriented proteomic research should involve the validation of biomarkers implicated in varicocele-associated male infertility. Strategies for the transfer of validated biomarkers from bench to bedside should be developed in order to improve the quality and better management of male infertility issues.