Table of Contents  
Year : 2020  |  Volume : 22  |  Issue : 1  |  Page : 88-93

Reduced semen quality in patients with testicular cancer seminoma is associated with alterations in the expression of sperm proteins

1 American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH 44195, USA Department of Health Sciences, Faculty of Health Sciences, University of Beira Interior, Covilhã 6201-001, Portugal Department of Microscopy and Unit for Multidisciplinary Research in Biomedicine, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto 4050-313, Portugal Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, Jeddah 21589, Saudi Arabia Division of Pathology, School of Medical Sciences, Sydney University, Lidcombe NSW 2141, Australia

Date of Submission12-Aug-2018
Date of Acceptance04-Jan-2019
Date of Web Publication16-Apr-2019

Correspondence Address:
Ashok Agarwal
American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH 44195
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/aja.aja_17_19

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Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.

Keywords: male fertility; proteomics; seminoma; sperm proteins; sperm quality; testicular cancer

How to cite this article:
Dias TR, Agarwal A, Pushparaj PN, Ahmad G, Sharma R. Reduced semen quality in patients with testicular cancer seminoma is associated with alterations in the expression of sperm proteins. Asian J Androl 2020;22:88-93

How to cite this URL:
Dias TR, Agarwal A, Pushparaj PN, Ahmad G, Sharma R. Reduced semen quality in patients with testicular cancer seminoma is associated with alterations in the expression of sperm proteins. Asian J Androl [serial online] 2020 [cited 2022 Nov 28];22:88-93. Available from:

  Introduction Top

Germ cell tumors (GCTs) represent the most common type of testicular cancer, accounting for about 90%–95% of all cases. The principal types of GCTs are nonseminomas and seminomas; the latter usually grows and spreads more slowly. In the last decades, there is a growing trend in the proportion of seminomas.[1] The survival rate of men with seminoma is very high (over 95%); thus, it is generally not seen as a threat to public health. However, its impact on male fertility represents a major concern for reproductive medicine as it frequently affects men in reproductive age (20–44 years).[2]

Men with seminoma present impaired fertilizing ability, even before diagnosis.[3] Testicular cancer seminoma affects the hypothalamic-pituitary-gonadal (HPG) axis and consequently disturbs spermatogenesis.[4] These deleterious effects are dependent on the stage and type of seminoma, resulting in poor semen quality or even azoospermia.[5] The treatment for this type of cancer, usually performed by surgery, chemotherapy, or radiotherapy, further affects semen quality [5] and hormonal function,[6] thus highly impairing male fertility. In fact, after cancer therapy, patients may become temporarily or permanently infertile.[7] For that reason, it is strongly recommended that men diagnosed with seminoma undergo sperm banking to increase the probability to father a child in the future.[8] The chances to establish a pregnancy by natural conception are 30% lower after the cancer therapy and the recovery of fertilizing ability usually takes several years.[9] Therefore, in many surviving patients with seminoma, assisted reproductive technology (ART) with cryopreserved samples is the only option for having children.[10] Still, sperm banking is not possible for many patients due to the high cost or lack of facilities, urgency to initiate the treatment, impaired spermatogenesis, and/or poor semen quality at the time of specimen collection.[11]

Proteomics studies have been recently used as a valuable tool to explore how certain health conditions affect male reproductive potential, especially by evaluating spermatozoa and seminal plasma proteome.[12],[13] Although spermatozoa are transcriptionally and translationally silent after being produced in the testis, the acquisition of sperm function occurs during maturation in the epididymis and transit through the female reproductive tract.[14] Therefore, the sperm proteome is highly susceptible to alterations according to the health status of the individual, and this impacts the quality of sperm parameters. The deleterious effects of seminoma treatment represent a challenge to understand the mechanisms behind the impairment of male fertility caused by the disease. In this study, we used semen samples from men with testicular cancer seminoma that were cryopreserved before starting cancer therapy, to investigate alterations in the sperm proteome in comparison with healthy proven fertile men.

  Participants and Methods Top

Semen analysis and cryopreservation

This study was conducted after approval by the Institutional Review Board (IRB) of Cleveland Clinic, Cleveland, OH, USA. Semen samples were obtained from healthy volunteers with proven fertility (control, n = 15) and patients with seminoma (n = 15). All the participants signed informed written consent to allow the use of their samples in this study. The inclusion criteria were as follows: (1) control group, healthy fertile men who had fathered a child in the last 2 years; (2) seminoma group, patients diagnosed with seminoma and undergoing sperm banking before starting cancer therapy. Following 2–3 days of abstinence, semen samples were collected at the Andrology Center, Cleveland Clinic. Samples were liquefied for 20–30 min in an incubator (Panasonic, Newark, NJ, USA) at 37°C, and a routine semen analysis was conducted according to the World Health Organization (WHO) 2010 guidelines.[15] Semen volume, sperm motility, and sperm concentration were recorded. Total sperm count and total motile count were also calculated and the results were expressed as mean ± standard error of the mean (s.e.m.). Whole ejaculate samples were immediately cryopreserved in TEST-yolk buffer (TYB; Irvine Scientific, Santa Ana, CA, USA) in a ratio of 1:1 as previously described [16] and finally labeled and stored in liquid nitrogen at −196°C.

Protein extraction and estimation

Samples were thawed on ice and centrifuged at 4000g for 10 min (Eppendorf, Hauppauge, NY, USA). To remove the freezing medium (TYB) as much as possible, the sperm pellet was washed four times in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 4000g for 10 min at 4°C. Total sperm protein was extracted overnight at 4°C with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). Subsequently, samples were centrifuged at 10 000g for 30 min at 4°C, to recover the protein fraction (supernatant). Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to estimate the protein concentration, according to the manufacturer's instructions.

Quantitative proteomic analysis

Three samples from the control or seminoma group were randomly selected for the proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were pooled (n = 3) using the same amount of protein from each sample. Each pool was then evaluated as an individual sample in the proteomic analysis. The system used was a Finnigan LTQ-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific) using the previously described conditions and software.[17] Scaffold (version, Proteome Software Inc., Portland, OR, USA) was used for the identification of differentially expressed proteins (DEPs) between the control and seminoma groups. The spectral counts were used to determine the abundance of each protein (very low, low, medium, or high). The identified DEPs were categorized as underexpressed, overexpressed, or unique to one of the groups, based on the normalized spectral abundance factor (NSAF) ratio according to previously reported criteria.[17]

Bioinformatic analysis

Bioinformatic analysis of DEPs identified by LC-MS/MS was carried out using the Ingenuity Pathway Analysis software (IPA; Qiagen, Hilden, Germany). IPA was used to evaluate the canonical pathways, top diseases and bio-functions, and upstream regulators related to the identified DEPs. Proteins were selected for validation by western blot considering the following criteria: (1) proteins involved in reproductive system development and function; (2) proteins involved in the top canonical pathways; (3) proteins with a higher difference of abundance between the experimental groups; and (4) proteins with a well-described function in the literature. Only proteins that met all the above-mentioned criteria were subjected to western blot.

Western blot

Western blot was performed using individual samples from the control and seminoma groups (n = 6 per group). A total of 25 μg protein per sample was mixed with 4 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) in a ratio of 1:3 and made up to 25 μl with PBS. Samples were boiled at 95°C for 10 min and immediately loaded into a 4%–15% (w/v) polyacrylamide gel (Bio-Rad). Electrophoresis was performed with constant voltage (90 V) for 2 h. Precision Plus Protein™ Dual Xtra Standard (Thermo Fisher Scientific) was used as the molecular weight marker. The resolved proteins were transferred (20 V for 30 min) to methanol-activated polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Marlborough, MA, USA) and blocked for 90 min at room temperature, with a 5% (w/v) nonfat milk (Bio-Rad) solution prepared in tris-buffered saline with tween-20 (TBST; Sigma-Aldrich). Membranes were incubated overnight (4°C) with specific primary antibodies followed by the respective secondary antibodies at room temperature, for 90 min [Supplementary Table 1 [Additional file 1]]. Membranes were incubated with enhanced chemiluminescence (ECL) reagent (GE Healthcare) for 5 min, and the chemiluminescence signals were read in the ChemiDoc™ MP Imaging System (Bio-Rad). Densities from each band were quantified with Image Lab™ Software (version 6.0.1, Bio-Rad) and divided by the corresponding total protein lane density. Total protein density was obtained by incubation of the membranes with total colloidal gold protein stain (BioRad). The results were expressed as fold variation relative to the control group.

Statistical analyses

After testing normal distribution by the Kolmogorov–Smirnov test, semen parameters and western blot results were analyzed by Mann–Whitney U test for independent samples, using the MedCalc Software (version 17.8; MedCalc Software, Ostend, Belgium). All data are presented as mean ± s.e.m., and differences with P < 0.05 were considered statistically significant.

  Results Top

Semen quality in patients with testicular cancer seminoma

The average volume of the ejaculates was very similar between the control and seminoma groups [Table 1]. However, there was a decrease in sperm motility (P = 0.019), sperm concentration (P = 0.003), total sperm count (P = 0.001), and total motile count (P = 0.001) in patients with seminoma relative to control [Table 1]. Nevertheless, all the samples were considered normozoospermic according to the WHO 2010 criteria.[15]
Table 1: Semen parameters of fertile men (control) and patients with testicular cancer seminoma

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Differentially expressed proteins

Proteomic analysis identified 1149 proteins in the control group and 911 in the seminoma group. After comparative analysis between the experimental groups, a total of 1192 proteins were quantified and 393 were found to be differentially expressed [Supplementary Table 2 [Additional file 2]]. More than half (52.7%) of the DEPs were underexpressed, while 20.1% were overexpressed in spermatozoa of patients with seminoma. Furthermore, 4.1% of the DEPs were unique to the seminoma group and 23.1% unique to the control group [Figure 1].
Figure 1: Number of proteins identified by proteomic analysis of spermatozoa samples obtained from fertile men (control) and men with testicular cancer seminoma, and expression profile of the DEPs identified after comparative analysis between the experimental groups. DEPs: differentially expressed proteins.

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Selection of proteins for validation

According to the IPA analysis, among the top diseases and bio-functions related to “physiological system development and function,” the category with the highest P value was “reproductive system development and function.” Within this category, we selected seven proteins involved in specific reproductive processes [Table 2]: angiotensin-converting enzyme (ACE), acrosin precursor (ACR), T-complex protein 1 subunit gamma (CCT3), sperm surface protein Sp17 (SPA17), sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4), heat shock-related 70 kDa protein 2 (HSPA2), and proteasome activator complex subunit 4 (PSME4). Some of these proteins were also involved in the top canonical pathways identified in this dataset. While HSPA2 participates in the “protein ubiquitination pathway” and “unfolded protein response,” ACE is related to “phagosome maturation.” Other top five canonical pathways included “mitochondrial dysfunction” and “oxidative phosphorylation.” Among the proteins involved in those pathways were NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1), cytochrome b-c1 complex subunit 2 (UQCRC2), and ATP synthase subunit alpha (ATP5A), which are subunits of the mitochondrial complexes I, III, and V, respectively. These three proteins were also selected for analysis by western blot. The abundance and expression pattern of the ten selected proteins obtained by the proteomic analysis is presented in [Table 3].
Table 2: Specific functions of the differentially expressed proteins related to reproductive system development and function identified by the bioinformatic analysis when comparing the sperm proteome of patients with testicular cancer seminoma with fertile men

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Table 3: Proteomic data of the differentially expressed proteins identified in the spermatozoa samples of fertile men (control) and men with testicular cancer seminoma before cancer therapy, which were selected for validation by western blot

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Prediction of the upstream regulators

The IPA analysis predicted the activation or inhibition of several proteins, which could be responsible for the altered expression in the sperm proteome of men with seminoma. The rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) was predicted to be activated, thus leading to the underexpression of NDUFS1, UQCRC2, ATP5A1, and PSME4. Moreover, it was predicted that the underexpression of ATP5A1 and ATP1A4 may involve the activation of the amyloid-beta A4 protein (APP). On the other hand, the inhibition of the heat shock factor protein 2 (HSF2) was predicted to regulate the underexpression of CCT3, as well as six other chaperonins of the T-complex protein-1 (TCP-1) family (CCT2, CCT4, CCT5, CCT6A, CCT7, and CCT8).

Western blot analysis

All proteins selected for western blot analysis were identified. There was an increase in the protein expression of ACE (P = 0.005) and ACR (P = 0.009) in the seminoma group (2.61 ± 0.38 and 2.02 ± 0.26-fold variation to control, respectively) in comparison with the control (1.00 ± 0.25 and 1.00 ± 0.19, respectively) [Figure 2]. On the other hand, there was a decrease in the protein levels of ATP1A4 (P = 0.016) and HSPA2 (P = 0.041) in men with seminoma (0.53 ± 0.03 and 0.32 ± 0.11-fold variation to control, respectively) when compared with the control group (1.00 ± 0.25 and 1.00 ± 0.22, respectively) [Figure 2]. The protein levels of CCT3, SPA17, and PSME4 were similar between the study groups. There was also a decrease (P = 0.026) in the protein expression levels of UQCRC2 (0.34 ± 0.14-fold variation to control) in the seminoma group relative to the control (1.00 ± 0.14) [Figure 3]. No differences were found in the protein levels of NDUFS1 or ATP5A1 between the experimental groups.
Figure 2: Graphical representation of the expression levels of proteins involved in reproductive functions (ACE, ACR, ATP1A4, CCT3, HSPA2, PSME4, and SPA17) in spermatozoa samples obtained from fertile men (control) and men with testicular cancer seminoma. Results are presented as fold variation to control and expressed as mean±standard error of the mean (n = 15 per group). Statistical significance is indicated as: *P < 0.05, **P < 0.01, seminoma versus control. Representative blots for each protein are also presented. ACE: angiotensin-converting enzyme; ACR: acrosin precursor; ATP1A4: sodium/potassium-transporting ATPase subunit alpha-4; CCT3: T-complex protein 1 subunit gamma; HSPA2: heat shock-related 70 kDa protein 2; PSME4: proteasome activator complex subunit 4; SPA17: sperm surface protein Sp17.

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Figure 3: Graphical representation of the protein expression levels of mitochondrial complex subunits NDUFS1, UQCRC2, and ATP5A1 in spermatozoa samples obtained from fertile men (control) and men with testicular cancer seminoma. Results are presented as fold variation to control and expressed as mean ± standard error of the mean (n = 15 per group). Statistical significance is indicated as: *P < 0.05, seminoma versus control. Representative blots for each protein are also presented. NDUFS1: NADH-ubiquinone oxidoreductase 75 kDa subunit; UQCRC2: cytochrome b-c1 complex subunit 2; ATP5A: ATP synthase subunit alpha.

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  Discussion Top

The present study is the first attempt to identify alterations in spermatozoa proteome of patients with seminoma before initiating cancer therapy, using fertile donors as control group. Our goal was to evaluate the expression levels of proteins involved in reproductive function from spermatogenesis to sperm function and fertilization. This may provide new insights on the underlying mechanisms responsible for the reduced sperm quality in men with seminoma.

Spermatogenesis consists of a complex process of spermatozoa production that involves several steps of germ cell differentiation. The bioinformatic analysis identified an underexpression of PSME4 in spermatozoa of patients with seminoma. PSME4 plays a role in the morphology of male germ cells; it is particularly important for histone replacement during chromatin remodeling and DNA double-strand break repair.[18] It has been reported that mice lacking this protein present impaired spermatogenesis and reduced fertility.[19] Thus, the downregulation of this protein may contribute to reduced fertility in men with seminoma. Although we were not able to confirm the underexpression of PSME4 by western blot in our dataset, we observed the underexpression of the molecular chaperone HSPA2 by both proteomics and western blot analysis. Molecular chaperones are essential for normal sperm production and functional transformation. HSPA2 acts as a protein quality control system as it ensures the correct folding/refolding of proteins and activates the degradation of misfolded proteins.[20] It has been described that HSPA2 participates in the stability of the microtubules during the meiotic process of germ cell differentiation.[21] In fact, animal studies show that knockout mice for Hspa2 exhibit an enormous number of apoptotic germ cells, resulting in infertility.[22] Men with abnormal spermatogenesis frequently present a reduced hspA2 mRNA expression.[23] Thus, the downregulation of HSPA2 protein in men with seminoma may contribute to the decreased production of normal spermatozoa during spermatogenesis, which is in accordance with the observed reduction in sperm concentration and total sperm count in seminoma group.

The protein ATP1A4 was identified as downregulated in seminoma group by the proteomic analysis, and this result was confirmed by the western blot technique. IPA analysis revealed that ATP1A4 participates in several reproductive processes, including spermatogenesis, function of sperm, cell movement of sperm, hyperactivation, and fertilization. ATP1A4 is the catalytic subunit of the Na +/K +-ATPase membrane protein, which controls the exchange of sodium and potassium ions across the plasma membrane in an ATP-dependent reaction.[24] The regulation of ions in spermatozoa is essential for the acquisition of motility and fertilizing ability. ATP1A4 plays a key role in maintaining human sperm motility.[25] It has been shown that male mice lacking this subunit are completely sterile and their spermatozoa present not only reduced motility but also impaired hyperactivation and inability to fertilize in vitro.[26] These studies highlight the importance of ATP1A4 for male fertility, and the underexpression of this protein in spermatozoa of men with seminoma may explain the decrease in sperm motility and total motile count relative to proven fertile men (control group). The downregulation of ATP1A4 was related to the activation of APP. In fact, this protein has been identified in human spermatozoa and suggested to play an important role in sperm function, especially in signaling events involved in sperm motility.[27]

Another important process crucial for sperm function is mitochondrial function. It is required for energy production necessary for spermatozoa movement and production of reactive oxygen species (ROS) in physiological amounts to trigger capacitation and regulate hyperactivation and acrosome reaction.[28] Mitochondrial function relies on the expression of the mitochondrial complexes I–IV for oxidative phosphorylation (OXPHOS) and complex V for ATP production.[29] Our proteomic data showed a downregulation of NDUFS1, UQCRC2, and ATP5A1 in the seminoma group, which are subunits of complex I, III, and V, respectively. The downregulation of these three proteins was predicted to be induced by the activation of RICTOR, which plays a key role in spermatogenesis and sperm maturation signaling pathways.[28] The mitochondrial subunits are essential for the proper assembly of the complexes; thus, alterations in their protein expression in spermatozoa are indicative of mitochondrial dysfunction, as reported by the IPA canonical pathways.[30] Although the western blot analysis demonstrated a tendency of reduced expression of the three mitochondrial subunits, only the UQCRC2 was decreased in patients with seminoma. Downregulation of UQCRC2 was associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately compromises sperm binding and fertilization.[31] In fact, an underexpression of UQCRC2 was observed in infertile men with varicocele.[32]

The acquisition of sperm fertilizing ability involves a timed triggering of events in the female reproductive system, culminating in sperm–oocyte binding. SPA17 and CCT3 are two sperm proteins involved in this function, which were identified as downregulated in the seminoma group by the proteomic analysis. SPA17 is a mannose-binding protein that binds to zona pellucida carbohydrates during fertilization.[33] It also plays an important role in germ cell differentiation during spermatogenesis, as its expression increases from early to late stages.[34] CCT3 is one of the subunits of the TCP-1 complex. Although we selected to evaluate the expression levels of this subunit, six other subunits of this complex (CCT2, CCT4, CCT5, CCT6A, CCT7, and CCT8) were also downregulated in men with seminoma. These subunits mediate capacitation-dependent binding of spermatozoa to the zona pellucida.[35] Thus, the downregulation of this system may compromise sperm fertilization.[36] The downregulation of TCP-1 complex subunits was predicted to be due to HSF2 inhibition. In fact, disruption of hsf2 in mice affected testicular size [37] and induced spermatogenic defects.[38] When active, HSF2 is likely to induce the upregulation of HSPA2.[39] Thus, the predicted inhibition of HSF2 in men with seminoma is in accordance with the downregulation of HSPA2. Although the underexpression of SPA17 and CCT3 was not confirmed by the western blot, the downregulation of HSPA2 in men with seminoma may contribute to the loss of sperm function. In fact, this protein is known to regulate the formation of zona pellucida-binding sites in spermatozoa during spermatogenesis.[40] In addition, it regulates fertilization by mediating the function of sperm surface receptors, such as sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), during sperm-egg recognition.[41] Previous proteomic studies have shown low expression levels of HSPA2 in men with asthenozoospermia [42] and primary or secondary infertility.[43] Another study also reported a downregulation of HSPA2, ATP1A4, and SPA17 in infertile varicocele patients.[32] Our results suggest that the altered expression levels of these proteins in men with seminoma may contribute to the impairment of male fertility.

The proteomic analysis also identified ACE as overexpressed in the seminoma group, and this result was confirmed by western blot. This protein is a zinc metallopeptidase responsible for the conversion of angiotensin I to angiotensin II.[44] The role of ACE in male reproductive function is not completely understood. Studies with ACE-deficient mice reported that these animals produce a normal number of spermatozoa and present normal motility and morphology. However, the spermatozoa were unable to bind and fertilize the egg.[45],[46] A negative correlation between sperm-bound ACE activity and sperm motility has also been observed.[47] The testis-specific isoform of this protein (tACE) is believed to be released from functional spermatozoa during capacitation and acrosome reaction to increase the fertilizing ability.[48] In fact, a lower tACE activity was detected in spermatozoa from normozoospermic men relative to those with oligoasthenozoospermia.[47] Thus, the overexpression of this protein in spermatozoa from men with seminoma may be responsible for the decrease in sperm motility observed in this group, and possibly explains why some men with seminoma are not able to have children even before the treatment.

Finally, the protein ACR, in its precursor form (proacrosin), was identified as underexpressed in the seminoma group by the proteomic analysis. This protein is activated and converted to its active form during acrosome reaction, playing a role in sperm–oocyte binding.[49] In contrast, using western blot, we found a high overexpression of this protein in men with seminoma. Although we cannot clearly infer about the molecular mechanisms, any of the scenarios (underexpression/overexpression) could lead to a defective acrosome reaction and impaired fertilization. The difference on these results may be due to the sensitivity of each technique and to the sample size. Further studies to assess acrosin activity in men with seminoma are needed to clarify the impact of this condition in acrosome reaction.

Overall, our study points toward important alterations in sperm proteins with a key role in male fertility in men with seminoma. As of today, no specific sperm markers have been identified for the clinical diagnosis and monitoring of testicular cancer seminoma development. The expression levels of HSPA2, ATP1A4, UQCRC2, and ACE can be helpful sperm biomarkers when evaluating the fertility status of a man, which may allow the early diagnosis of seminomas in a noninvasive approach. Although there is still a lot to explore in the pathophysiology of male subfertility/infertility in men with seminoma, our results represent a step forward in understanding the molecular mechanisms behind the reduced sperm quality in these patients. Future advances in mass spectrometry and bioinformatics will improve our understanding on human sperm function in healthy and disease conditions.

  Author Contributions Top

AA and RS were responsible for the conception and design of the study. TRD was responsible for the acquisition and interpretation of data, as well as writing the first draft. GA helped in samples processing and PNP performed the bioinformatic analysis. All authors read and approved the final manuscript.

  Competing Interests Top

All authors declared no competing interests.

  Acknowledgments Top

The authors would like to thank Dr. Belinda Willard (Director, Proteomics Core Laboratory, Lerner Research Institute, Cleveland Clinic) for her support in the proteomic analysis and Dr. Ralf Henkel and Dr. Saradha Baskaran for their help in reviewing the manuscript. Financial support for this study was provided by the American Center for Reproductive Medicine, Cleveland Clinic, OH, USA. Tania R Dias was supported by the Portuguese Foundation for Science and Technology (FCT, SFRH/BD/109284/2015) and Fulbright Program (E0585639). Sponsors were not involved in the experiments or writing/submitting the paper.

Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.

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  [Table 1], [Table 2], [Table 3]

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