MicroRNAs Derived from Extracellular Vesicles: Keys to Understanding SARS-CoV-2 Vaccination Response in Cancer Patients?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provoked a global pandemic identified as coronavirus disease (COVID-19), with millions of deaths worldwide. However, several important questions regarding its impact on public health remain unanswered, such as the impact of vaccination on vulnerable subpopulations such as cancer patients. Cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, being manifested in most immunocompromised individuals. This strong immunosuppression can lead to a dysfunctional antiviral response to natural viral infection and compromised vaccination response. Extracellular vesicles (EVs) are membrane-bound vesicles released from cells that are involved in intercellular communication. EVs carry various molecules including microRNAs that play a crucial role in COVID-19 pathophysiology, influencing cellular responses. This review summarizes the state of the art concerning the role of EV-derived miRNAs in COVID-19 infection and their potential use as prognosis biomarkers for vaccination response in cancer patients.

MicroRNAs as Potential Tools for Predicting Cancer Patients' Susceptibility to SARS-CoV-2 Infection and Vaccination Response.

Coronavirus disease (COVID-19) is an infectious disease that is caused by a highly contagious and severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). This infection started to spread across the world in 2019 and rapidly turned into a global pandemic, causing an urgent necessity for treatment strategies development. The mRNA vaccines against SARS-CoV-2 can trigger an immune response, providing genetic information that allows the production of spike glycoproteins. MiRNAs play a crucial role in diverse key cellular processes, including antiviral defense. Several miRNAs are described as key factors in SARS-CoV-2 human infection through the regulation of ACE2 levels and by the inhibition of SARS-CoV-2 replication and spike expression. Consequently, these molecules have been considered as highly promising biomarkers. In numerous human malignancies, it has been recognized that miRNAs expression is dysregulated. Since miRNAs can target SARS-CoV-2-associated mRNAs, in cancer patients, the deregulation of these molecules can impair the immune response to the vaccines. Therefore, in this review, we propose a miRNA profile of seven SARS-CoV-2-related miRNAs, namely miR-214, miR-98-5p, miR-7-5p, miR-24-3p, miR-145-5p, miR-223-3p and miR-15b-5p, that are deregulated in a high number of cancers and have the potential to be used as prognostic biomarkers to stratify cancer patients.

Expression of LncRNAs in HPV-induced Carcinogenesis and Cancer Cachexia: A Study in K14-HPV16 Mice.

AIM: To evaluate the expression of lincRNA-p21, H19, EMX2OS, SNHG12 and MALAT1 in a mouse model of human papillomavirus 16 (HPV16)-induced carcinogenesis and cachexia. MATERIALS AND METHODS: Chest skin, ear, tongue, penis and gastrocnemius muscle samples from wild-type mice (HPV-) and K14-HPV16 male mice (HPV+) were collected to evaluate the expression of the selected lncRNAs using real-time PCR (qPCR). RESULTS: In chest skin and ear, H19, SNHG12, EMX2OS and lincRNA-p21 were down-regulated in HPV+ versus HPV- mice. In tongue and penile tissues, there was only down-regulation of lincRNA-p21 in HPV+ mice. Additionally, in penile tissue, lincRNA-p21 expression decreased in HPV-induced lesions comparing with normal tissues. In gastrocnemius muscle, MALAT1 was up-regulated and lincRNA-p21 was down-regulated in HPV+ versus HPV-mice. CONCLUSION: H19, SNHG12, EMX2OS and lincRNA-p21 may be involved in HPV-induced carcinogenesis. In addition, MALAT1 and lincRNA-p21 may play a role in muscle wasting and contribute to cancer cachexia.

Towards Drug Repurposing in Cancer Cachexia: Potential Targets and Candidates.

As a multifactorial and multiorgan syndrome, cancer cachexia is associated with decreased tolerance to antitumor treatments and increased morbidity and mortality rates. The current approaches for the treatment of this syndrome are not always effective and well established. Drug repurposing or repositioning consists of the investigation of pharmacological components that are already available or in clinical trials for certain diseases and explores if they can be used for new indications. Its advantages comparing to de novo drugs development are the reduced amount of time spent and costs. In this paper, we selected drugs already available or in clinical trials for non-cachexia indications and that are related to the pathways and molecular components involved in the different phenotypes of cancer cachexia syndrome. Thus, we introduce known drugs as possible candidates for drug repurposing in the treatment of cancer-induced cachexia.

Long non-coding RNAs regulate the hallmarks of cancer in HPV-induced malignancies.

High-risk human papillomavirus (HPV) is the most frequent sexually transmitted agent worldwide and is responsible for approximately 5% of human cancers. Identifying novel biomarkers and therapeutic targets for these malignancies requires a deeper understanding of the mechanisms involved in the progression of HPV-induced cancers. Long non-coding RNAs (lncRNAs) are crucial in the regulation of biological processes. Importantly, these molecules are key players in the progression of multiple malignancies and are able to regulate the development of the different hallmarks of cancer. This review highlights the action of lncRNAs in the regulation of cellular processes leading to the typical hallmarks of cancer. The regulation of lncRNAs by HPV oncogenes, their targets and also their mechanisms of action are also discussed, in the context of HPV-induced malignancies. Overall, accumulating data indicates that lncRNAs may have a significant potential to become useful tools for clinical practice as disease biomarkers or therapy targets.

Protein Fingerprinting of Seminal Plasma Reveals Dysregulation of Exosome-Associated Proteins in Infertile Men with Unilateral Varicocele.

PURPOSE: Aberrant expression of seminal plasma proteins are associated with altered homeostasis that may affect the fertilizing ability of spermatozoa. However, the precise roles of seminal exosomes on sperm function remain unclear. The objective of this study was to identify the differentially expressed proteins (DEPs) associated with varicocele-mediated infertility by comparing seminal plasma protein profile of unilateral varicocele patients with proven fertile donors. MATERIALS AND METHODS: Semen samples were obtained from 10 proven fertile donors with normal semen parameters and 33 infertile patients with unilateral varicocele. For proteomic analysis, 5 samples from each group were pooled and run in triplicate. Key DEPs (ANXA2, TF, CD63, KIF5B, SEMG1) associated with the exosome function were selected by bioinformatic tools and validated using Western blotting. RESULTS: A total of 47 seminal plasma proteins were differentially expressed in unilateral varicocele patients compared to fertile donors. Validation of exosome-associated DEPs in unilateral varicocele patients (n=7) and fertile donors (n=7) revealed significant upregulation of ANXA2 (p=0.0016) and downregulation of KIF5B (p=0.009). The main upstream regulators of the DEPs in seminal plasma of unilateral varicocele group were androgen receptor, YB1 and NRF2. CONCLUSIONS: This is the first report to identify DEPs in seminal plasma of unilateral varicocele patients compared to fertile donors. Based on the detection of DEPs associated with exosomal function, Western blotting was used to validate the presence of defective exosome machinery in seminal plasma of unilateral varicocele patients. KIF5B and ANXA2 can be utilized as potential biomarkers of infertility in unilateral varicocele patients.

Measurement of Reactive Oxygen Species in Semen Samples Using Chemiluminescence.

Reactive oxygen species (ROS) are continuously produced in semen and are essential for important spermatozoa functions that allow fertilization. However, an excessive amount of ROS is associated with poor sperm quality, which can compromise male fertility potential. This chemiluminescence assay is based on the production of light through the reaction between luminol and ROS. The emitted light is converted to an electrical signal by a luminometer, and the ROS levels in the sample are calculated as relative light units (RLU) per second per million spermatozoa per milliliter (RLU/s/million sperm/mL).

Distinct Proteomic Profile of Spermatozoa from Men with Seminomatous and Non-Seminomatous Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are predominant in young males (15-44 years). Seminomatous and non-seminomatous TGCTs account for about 98% of all TGCTs cases. In this study, we aimed to compare the sperm proteome of patients with seminomatous and non-seminomatous TGCTs to identify possible protein biomarkers that could help distinguish between them in a non-invasive manner. We analyzed semen samples from patients with seminomatous or non-seminomatous TGCTs (n = 15/group) that were cryopreserved before the start of cancer treatment. Quantitative proteomic analysis was conducted on pooled samples (n = 3/group) and a total of 258 differentially expressed proteins (DEPs) were identified. The overexpression of acrosin precursor (ACR) and chaperonin containing TCP1 subunit 6B (CCT6B) as well as the underexpression of S100 calcium-binding protein A9 (S100A9) in the spermatozoa of patients with non-seminomatous TGCTs were validated by western blotting conducted on individual samples (n = 6 for seminomatous group and n = 6 for non-seminomatous group). Our overall results suggest an association between the higher and faster invasiveness of non-seminomatous TGCTs and the altered protein expressions, providing important information for future studies.

Endogenous and Exogenous Antioxidants As a Tool to Ameliorate Male Infertility Induced by Reactive Oxygen Species.

Significance: Antioxidants are essential for the maintenance of cellular redox homeodynamics in the male reproductive tract, playing a key role in fertilizing potential. Reactive oxygen species (ROS), at physiological levels, are essential for sperm function and fertilization. Under pathological conditions, abnormal production of ROS may occur. Redox control is primarily regulated by the inner antioxidant system. However, these endogenous antioxidants may be present at abnormal amounts or may be insufficient. Exogenous antioxidants obtained through the diet may have an important role, particularly in specific pathological conditions. This review addresses the regulation of redox homeodynamics in the male reproductive tract by endogenous and exogenous antioxidants and the importance of their cooperation for the maintenance of fertility. Recent Advances: Many studies have shown the importance of antioxidants for the preservation of male fertility, mostly under pathological conditions. Excessive antioxidants can inhibit ROS-induced signaling pathways that are essential for the reproductive system. The challenge is to keep the balance between oxidants and antioxidants to maintain ROS-amount at physiological concentration. Critical Issues: Although antioxidant therapies are gaining popularity and showing promising results in the improvement of male fertility, there is a lack of knowledge regarding the type of exogenous antioxidant, the doses and time to be administered. Future Directions: It would be of great importance to find a way to restore redox homeostasis under stress conditions. Understanding the poorly studied mechanisms by which exogenous antioxidants cooperate with the inner cellular antioxidant system to counteract free radicals may help in the development of new fertility therapies.

New Insights on the Mechanisms Affecting Fertility in Men with Non-Seminoma Testicular Cancer before Cancer Therapy.

PURPOSE: Patients with non-seminoma testicular cancer (NSTC) cancer can be subfertile or infertile, and present reduced sperm quality, but the underlying mechanisms are unknown. The aim of this study was to compare the sperm proteome of patients with NSTC, who cryopreserved their sperm before starting cancer treatment, with that from healthy fertile men. MATERIALS AND METHODS: Semen volume, sperm motility and sperm concentration were evaluated before the cryopreservation of samples from patients with NSTC (n=15) and the control group (n=15). Sperm proteomic analysis was performed by liquid chromatography-tandem mass spectrometry and the differentially expressed proteins (DEPs) between the two groups were identified using bioinformatic tools. RESULTS: A total of 189 DEPs was identified in the dataset, from which five DEPs related to sperm function and fertilization were selected for validation by Western blot. We were able to validate the underexpression of the mitochondrial complex subunits NADH:Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1) and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2), as well as the underexpression of the testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) in the NSTC group. CONCLUSIONS: Our results indicate that sperm mitochondrial dysfunction may explain the observed decrease in sperm concentration, total sperm count and total motile count in NSTC patients. The identified DEPs may serve as potential biomarkers for the pathophysiology of subfertility/infertility in patients with NSTC. Our study also associates the reduced fertilizing ability of NSTC patients with the dysregulation of important sperm molecular mechanisms.

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

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.

A new thiocyanoacetamide (2-cyano-2-p-nitrophenyl-N-benzylthioamide) reduces doxorubicin-induced in vitro toxicity in Sertoli cells by decreasing apoptosis and autophagy.

Despite conflicting data on doxorubicin (DOX) reproductive toxicity, its chemotherapeutic potential sustains its use to treat different types of cancer. This work was designed to study the protective effect of a newly synthesized thiocyanoacetamide (TA), in comparison with selenium (Se), against doxorubicin-induced in vitro toxicity in rat Sertoli cells (SCs). DOX was administered alone or in combination with Se or TA. The possible protective role of increased concentrations of TA (0.25, 0.5 and 1 mM) or Se (12, 25 and 50 µM) on SCs was tested against 1 µM of DOX. From this screening, only the least toxic doses of TA and Se were used for further analysis. DOX cytotoxicity, as well as its impact on SCs viability, mitochondrial membrane potential (ΔΨm), oxidative stress biomarkers, apoptosis and autophagy were assessed. Our results showed that DOX exerted its cytotoxic effect through a significant increase in cell death. DOX-mediated cell death was not related to autophagy nor to an overproduction of reactive oxygen species. It was rather due to apoptosis, as shown by the increased number of apoptotic cells and increased activity of caspase-3, or due to necrosis, as shown by the increase in lactate dehydrogenase (LDH) extracellular activity. Still, Bax and Bcl-2 protein expression levels, as well as ΔΨm were not altered by the different treatments. Some individual doses of Se or TA induced a significant toxicity in SCs, however, when combined with DOX, there was a decrease in cell death, LDH extracellular activity, number of apoptotic cells and caspase-3 activity. Overall, our results indicate that DOX-mediated apoptosis in cultured SCs can possibly be averted through its association with specific doses of Se or TA. Nevertheless, TA showed a higher efficiency than Se in reducing DOX-induced toxicity in SCs by decreasing not only apoptosis, but also necrosis and autophagy.

L-Theanine promotes cultured human Sertoli cells proliferation and modulates glucose metabolism.

PURPOSE: L-Theanine is the major free amino acid present in tea (Camellia sinensis L.). The effects of several tea constituents on male reproduction have been investigated, but L-theanine has been overlooked. Sertoli cells (SCs) are essential for the physical and nutritional support of germ cells. In this study, we aimed to investigate the ability of L-theanine to modulate important mechanisms of human SCs (hSCs) metabolism, mitochondrial function and oxidative profile, which are essential to prevent or counteract spermatogenesis disruption in several health conditions. METHODS: We evaluated the effect of a dose of L-theanine attained by tea intake (5 µM) or a pharmacological dose (50 µM) on the metabolism (proton nuclear magnetic resonance and Western blot), mitochondrial functionality (protein expression of mitochondrial complexes and JC1 ratio) and oxidative profile (carbonyl levels, nitration and lipid peroxidation) of cultured hSCs. RESULTS: Exposure of hSCs to 50 µM of L-theanine increased cell proliferation and glucose consumption. In response to this metabolic adaptation, there was an increase in mitochondrial membrane potential, which may compromise the prooxidant-antioxidant balance. Still, no alterations were observed regarding the oxidative damages. CONCLUSIONS: A pharmacological dose of L-theanine (50 µM) prompts an increase in hSCs proliferation and a higher glucose metabolization to sustain the pool of Krebs cycle intermediates, which are crucial for cellular bioenergetics and biosynthesis. This study suggests an interplay between glycolysis and glutaminolysis in the regulation of hSCs metabolism.

Data on metabolic profile of insulin-degrading enzyme knockout mice.

Insulin-degrading enzyme (IDE) degrades and inactivates bioactive peptides such as insulin. As insulin is a master regulator of glucose homeostasis, lack of IDE is expected to have a profound impact on both insulin and glucose levels. This article shares data on glucose and insulin homeostasis of control, heterozygous and knockout mice for Ide after 18 weeks of a normal chow diet. This data article is related to a research article entitled "Knockout of insulin-degrading enzyme leads to mice testicular morphological changes and impaired sperm quality" (Meneses et al., 2019).

Round cells do not contaminate or mask human sperm proteome in proteomic studies using cryopreserved samples.

Semen contains leucocytes and round cells, besides spermatozoa. The objective of this study was to identify whether the proteins from round cells and leucocytes affect the proteomic analysis of spermatozoa. Cryopreserved human sperm samples were divided into four groups: (1) samples with ≥1 × 106 /ml leucocytes unprocessed; (2) samples with ≥1 × 106 /ml leucocytes processed by 65% density centrifugation; (3) samples with round cells <1 × 106 /ml unprocessed; and (4) samples with round cells <1 × 106 /ml processed by 65% density centrifugation. Samples from each group (1, 2, 3 and 4) were pooled (n = 5) for quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparative analysis revealed nine differentially expressed proteins (DEPs) groups 1 and 2. Moreover, five DEPs were identified between groups 3 and 4. We observed that cylicin-1, Atlastin-1 and vesicle transport protein SFT2B are specific to spermatozoa, and none of them were associated with leucocytes. The number of DEPs in spermatozoa of processed and unprocessed cryopreserved semen samples was negligible. Our results indicate that the presence of round cells (<1 × 106 /ml) in the seminal ejaculation does not interfere in the accurate detection of spermatozoa proteome by LC-MS/MS.

Knockout of MCT1 results in total absence of spermatozoa, sex hormones dysregulation, and morphological alterations in the testicular tissue.

Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17ß-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.

Carbonic anhydrases are involved in mitochondrial biogenesis and control the production of lactate by human Sertoli cells.

The process that allows cells to control their pH and bicarbonate levels is essential for ionic and metabolic equilibrium. Carbonic anhydrases (CAs) catalyse the conversion of CO2 to HCO3- and H+ and are thus essential for this process. Herein, we inhibited CAs with acetazolamide - ACT and SLC-0111 - to study their involvement in the metabolism, mitochondrial potential, mitochondrial biogenesis and lipid metabolism of human Sertoli cells (hSCs), obtained from biopsies from men with conserved spermatogenesis. We were able to identify three isoforms of CAs, one mitochondrial isoform (CA VB) and two cell membrane-bound isoforms (CA IX and CA XII) in hSCs. When assessing the expression of markers for mitochondrial biogenesis, we observed a decrease in HIF-1α, SIRT1, PGC1α and NRF-1 mRNAs after all CAs were inhibited, resulting in decreased mitochondrial DNA copy numbers. This was followed by an increased production of lactate and alanine in the same conditions. In addition, consumption of glucose was maintained after inhibition of all CAs in hSCs. These results indicate a reduced conversion of pyruvate to acetyl-coA, possibly due to decreased mitochondrial function, caused by CA inhibition in hSCs. Inhibition of CAs also caused alterations in lipid metabolism, since we detected an increased expression of hormone-sensitive lipase (HSL) in hSCs. Our results suggest that CAs are essential for mitochondrial biogenesis, glucose and lipid metabolism in hSCs. This is the first report showing that CAs play an essential role in hSC metabolic dynamics, being involved in mitochondrial biogenesis and controlling lactate production.

Knockout of insulin-degrading enzyme leads to mice testicular morphological changes and impaired sperm quality.

Insulin-degrading enzyme (IDE) is a zinc metalloprotease responsible for degrading and inactivating several bioactive peptides, including insulin. Individuals without this enzyme or with a loss-of-function mutation in the gene that codifies it, present hyperinsulinemia. In addition, impairment of IDE-mediated insulin clearance is associated with the development of metabolic diseases, namely prediabetes. Although insulin regulates male fertility, the role of IDE on male reproductive function remains unknown. We proposed to study the influence of IDE in the reproductive potential of males. As insulin mediates key events for the normal occurrence of spermatogenesis, we hypothesized that IDE functioning might be linked with sperm quality. We used C57BL/6N mice that were divided in three groups according to its genotype: wild type (WT), heterozygous and knockout (KO) male mice for Ide. Spermatozoa were collected from the cauda of epididymis and sperm parameters were evaluated. Testicular tissue morphology was assessed through hematoxylin and eosin stain. Mitochondrial complex protein levels and lipid peroxidation were also evaluated in the testicular tissue. Our results show that KO mice present a 50% decrease in testes weight compared to WT mice as well as a decrease in seminiferous tubules diameter. Moreover, KO mice present impaired sperm quality, namely a decrease in both sperm viability and morphology. These results provide evidence that IDE plays an important role in determining the reproductive potential of males.

Molecular Pathways Associated with Sperm Biofunction Are Not Affected by the Presence of Round Cell and Leukocyte Proteins in Human Sperm Proteome.

In human sperm proteomic experiments, leukocyte and round cell proteins may contaminate the sperm proteome and affect the bioinformatic results. The main objective of this study was to identify the possible interference of these proteins, especially from leukocytes, in identification of sperm functional pathways through proteomic and bioinformatic tools. We have evaluated the sperm proteome by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in four groups: (1) neat semen with round cells and leukocytes ≥1 × 106/mL; (2) samples with round cells and leukocytes ≥1 × 106/mL processed by 65% density gradient centrifugation; (3) neat semen with round cells <1 × 106/mL; and (4) samples with round cells <1 × 106/mL processed by 65% density gradient centrifugation. Pure leukocyte culture was used as a control group. The difference in the conserved DEPs (common to both sperm and leukocytes) between the sperm samples with leukocytes ≥1 × 106/mL and round cells <1 × 106/mL was negligible. Comparative analysis between groups 1, 2, 3, and 4 with the control group revealed that the presence of leukocyte proteins does not significantly alter the activation z-score of the identified canonical pathways or biological functions in sperm proteome. Our experimental results demonstrate that the presence of round cell and leukocyte proteins do not affect the identification of the molecular pathways associated with human spermatozoa protein function. Hence, the use of neat frozen semen samples for proteomic studies showed no significant impact on the downstream bioinformatic analysis.

Proteomic Signatures Reveal Differences in Stress Response, Antioxidant Defense and Proteasomal Activity in Fertile Men with High Seminal ROS Levels.

Elevated levels of reactive oxygen species (ROS) are a major cause of male infertility. However, some men with high seminal ROS levels are still fertile. The main objective of this study was to understand the molecular mechanism(s) responsible for the preservation of fertility in those men. Semen samples from fertile men were divided into two groups: control (n = 10, ROS < 102.2 RLU/s/106 sperm) and ROS+ (n = 10, ROS > 102.2 RLU/s/106 sperm). Proteomic analysis of seminal plasma and spermatozoa was used to identify the differentially expressed proteins (DEPs) between the experimental groups, from which some proteins were validated by Western blot (WB). A total of 44 and 371 DEPs were identified between the study groups in the seminal plasma and spermatozoa, respectively. The identified DEPs were primarily involved in oxidoreductase, endopeptidase inhibitor, and antioxidant activities. We validated by WB the underexpression of NADH:ubiquinone oxidoreductase core subunit S1 (p = 0.01), as well as the overexpression of superoxide dismutase 1 (p = 0.03) and peroxiredoxin 4 (p = 0.04) in spermatozoa of ROS+ group. Our data suggest that fertile men with high ROS levels possess an effective antioxidant defense system that protects sperm proteins, as well as an active proteasomal system for degradation of defective proteins.