Received: 16 January 2018 | Revised: 8 June 2018 | Accepted: 18 July 2018 DOI: 10.1111/and.13126

INVITED REVIEW

Role of oxidative stress, and inflammation in

Ashok Agarwal1 | Mohit Rana1 | Emily Qiu1 | Hashem AlBunni1 | Albert D. Bui1,2 | Ralf Henkel1,3

1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio Abstract 2Ohio University Heritage College of Oxidative stress (OS), defined as an overabundance of reactive oxygen species (ROS) Osteopathic Medicine, Athens, Ohio or a deficiency of antioxidants, has been linked to sperm damage and male infertility. 3Department of Medical There are many sources of OS and inflammation including varicocele, tobacco usage, Biosciences, University of the Western Cape, Bellville, South Africa alcohol, obesity/metabolic syndrome, leukocytospermia, sexually transmitted dis‐ ease (i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum), bacterial Correspondence Ashok Agarwal, American Center for , microorganism mutations leading to more OS, and viral (i.e., Reproductive Medicine, Cleveland Clinic, human virus, hepatitis). This review is focusing on infection and OH 44195. Email: [email protected] inflammation‐mediated OS, the inflammatory markers underlying pathology, clinical significance in male infertility, and a brief description of the recommended treatment modalities.

KEYWORDS causes of oxidative stress, male genital tract infections, oxidative stress, sexually transmitted diseases

1 | INTRODUCTION binding domains on the plasma membrane (De Lamirande & O’Flaherty, 2008; Signorelli, Diaz, & Morales, 2012; Visconti et al, Reactive oxygen species (ROS) production by spermatozoa was 1995). On the other hand, ROS can prevent tyrosine kinase inhibition initially discovered by MacLeod (1943). Subsequently, Aitken and by oxidising phosphatase, subsequently allowing tyrosine kinases to Clarkson (1987) observed that spermatozoa also produce ROS when move to the phosphotyrosine binding domains on the plasma mem‐ exposed to a calcium cascade induced by ionophore A12187. The in‐ brane (Hecht & Zick, 1992). However, the intermediate steps from flux of calcium and ROS allows capacitation to occur, which is the pro‐ tyrosine kinase activation to the end of capacitation remain to be cess by where the spermatozoa’s plasma membrane increases fluidity fully understood; yet, the end result is an efflux of cholesterol and and gains the ability to fuse with the oocyte (de Lamirande & Gagnon, greater membrane fluidity (Cross, 1998; Davis, 1981) (Figure 1). 1993). By adding catalase, a ROS scavenger, researchers found that The plasma membrane of spermatozoa contains extraordinary the amount of capacitated spermatozoa decreased by 47% (Griveau, high amounts of polyunsaturated fatty acids (PUFAs) which con‐ Renard, & Lannou, 1994). This proved that ROS play a vital role in tribute to its membrane fluidity. In turn, these PUFAs render sper‐ sperm maturation and fertilisation capability. matozoa highly susceptible to oxidative damage (Aitken, Harkiss, & Reactive oxygen species can either inhibit or activate several en‐ Buckingham, 1993). zymes to facilitate capacitation. The activation of kinases occurs ei‐ Reactive oxygen species attack of PUFAs generates a cyclic, ther directly or via a secondary messenger to facilitate the necessary propagating cycle called a radical chain reaction whereby the physiological response. These highly reactive, mainly radical‐based unconjugated double‐bond groups of the PUFAs will undergo oxygen derivatives bind directly to phosphokinase C (PKC) which, an initial electrophilic attack by ROS (Aitken, 2017), which will in turn, activates PKC and allows it to move to the phosphotyrosine eventually result in the formation of malondialdehyde (MDA),

Andrologia. 2018;50:e13126. wileyonlinelibrary.com/journal/and © 2018 Blackwell Verlag GmbH | 1 of 13 https://doi.org/10.1111/and.13126 2 of 13 | AGARWAL et al.

FIGURE 1 Role of ROS in sperm hyperactivation and capacitation

4‐hydroxynonenal (4‐HNE) and acrolein (Agarwal, Saleh, & Bedaiwy, 2003; Aitken, 2017). These reactive aldehydes are mark‐ ers for oxidative stress, as they will subsequently cause damage by reacting with positive, hydrophilic amino acids in proteins, which will not only yield further ROS production, but also cause FIGURE 2 Factors involved in generation of ROS associated mitochondrial dysregulation and ROS leakage from the inner mi‐ with male infertility tochondrial membrane. If the ROS production exceeds concen‐ trations of antioxidants in the spermatozoa, oxidative stress (OS) ensues. OS affects the plasma membrane’s integrity and induces NADPH leads to the generation of ROS by means of two path‐ premature capacitation, which renders spermatozoa less suitable ways. The first pathway includes a membrane‐bound NADPH oxi‐ for fertilisation (Velando, Torres, & Alonso‐Alvarez, 2008; Villegas dase enzyme that uses oxygen as a source to produce the superoxide et al, 2003). When the concentration of ROS exceeds the phys‐ anion, which can further produce other highly reactive molecules iological needs, spermatozoa experience OS, which results in such as hydrogen peroxide (Frederiks & Vreeling‐Sindelárová, 2001). infertility (Agarwal et al, 2003; Sanocka, Miesel, Jedrzejczak, & The second pathway consists of NADPH dehydrogenase, also known Kurpisz, 1996). as diaphorase, which is responsible for oxidation–reduction (redox) reactions in the mitochondria and the generation of ROS (Gavella & Lipovac, 1992; Siegel, Gibson, Preusch, & Ross, 1990) (Figure 3). 2 | SOURCES OF OS

2.1 | Varicocele Although numerous factors (Figure 2) contribute to oxidative stress‐related male infertility, the majority of seminal OS is at‐ Varicocele is defined as an atypical dilatation and tortuosity of the tributed to leucocytes and immature spermatozoa. During normal Pampiniform venous plexus inside the spermatic cord (Agarwal spermatogenesis, spermatozoa will reduce the size of their cyto‐ et al., 2009; Will et al., 2011). Globally, 15% to 20% of men with plasm as Sertoli cells will phagocytise it during spermiogenesis fertility problems are diagnosed with varicocele (French, Desai, & (Gomez et al, 1996). However, an arrest in spermiogenesis will re‐ Agarwal, 2008), resulting in elevated seminal ROS levels and lower sult in excess cytoplasm around the midpiece (Hampl, Drábková, total antioxidant capacity (Pasqualotto et al., 2008). Varicocele pro‐ Kanďár, & Stěpán, 2012) with the resulting immature spermatozoa motes ischaemia in the spermatic veins and results in increasing lev‐ producing excessive amounts of ROS, which, in turn, leads to OS els of inflammatory cytokines and nitric oxide (NO; Ozbek, Turkoz, (Aitken & Baker, 1995). The retained cytoplasm allows the forma‐ Gokdeniz, Davarci, & Ozugurlu, 2000). tion of nicotinamide adenine dinucleotide phosphate (NADPH) Moderate concentrations of specific cytokines play a critical from retained glucose‐6‐phosphate content (G6PDH) via the role in maintaining physiological functions of cells inside the testes. hexose monophosphate shunt (Frederiks & Vreeling‐Sindelárová, Specifically, interleukin (IL)‐1 regulates the functions of testicular 2001). cells including Sertoli and Leydig cells (Sultana, Svechnikov, Weber, & AGARWAL et al. | 3 of 13

FIGURE 3 Production of ROS by membrane‐bound NADPH oxidase enzyme and NADPH dehydrogenase

Söder, 2000). Moreover, certain cytokines, including IL‐37 and IL‐18, sperm chromatin integrity through the induction of OS and generat‐ are upregulated in the seminal plasma of varicocele patients (Zeinali, ing oxidised DNA base adducts such as 8‐hydroxy‐2’‐deoxyguano‐ Hadian Amree, Khorramdelazad, Karami, & Abedinzadeh, 2017). sine (8‐OHdG) (Aitken, Gibb, Baker, Drevet, & Gharagozloo, 2016). These elevations lead to inflammatory response activation, leu‐ Spermatozoa only possess one enzyme, 8‐oxoguanine glycosylase cocyte recruitment and ROS production, which are detrimental to (OGG1), for the base excision repair mechanism and do not possess normal testicular functions (Allen & Gow, 2009). Consequently, ROS any downstream components of the base excision repair (BER) path‐ can disrupt the blood–testis barrier, the sperm plasma membrane way such as apurinic endonuclease 1 (APE1) and DNA repair pro‐ and DNA integrity (Ha, Park, & Park, 2011; Sabeti, Pourmasumi, teins such as X‐ray repair complementing defective repair (XRCC1); Rahiminia, Akyash, & Talebi, 2016). this unique feature makes spermatozoa susceptible to DNA damage Furthermore, endothelial nitric oxide (NO) synthase is upregu‐ (Aitken et al, 2016). lated in varicocele testes to increase blood flow into testicular tissues The consequences of tobacco usage include decrease in sperm and compensate for the hypoxia caused by venous stasis (Agarwal et concentration, motility, viability and morphological abnormalities al., 2009). In this case, high NO concentrations can be detrimental (Said, Ranga, & Agarwal, 2005). Through the processes of hydro‐ as NO can react with superoxide free radicals to form highly reac‐ genation, pyrolysis, oxidation, decarboxylation and dehydration, tive nitrogen species such as peroxynitrite and peroxynitrous acid, tobacco releases mutagens and carcinogens such as radioactive po‐ which could lead to infertility (Romeo et al., 2003). Other potential lonium, benzopyrene, dimethylbenzanthracene and polycyclic aro‐ causes of OS in varicocele patients include leptin receptors, glial cell matic hydrocarbons (Colagar, Jorsaraee, & Marzony, 2007; Richthoff, line‐derived neurotrophic factor receptor‐α1 (GDNF‐α1), HO‐iso‐ Elzanaty, Rylander, Hagmar, & Giwercman, 2008). Many of these enzyme 1 and voltage‐dependent calcium channels (Benoff et al., toxic compounds are also found to induce chromosomal aberrations 2005; Konukoglu, Serin, & Turhan, 2006; Shiraishi & Naito, 2005). in spermatozoa, leading to a reduced likelihood of success in assisted reproductive therapies (ART) (Anderson, Nisenblat, & Norman, 2010; Frey, Navarro, Kotelchuck, & Lu, 2008; Ozgur, Isikoglu, Seleker, & 2.2 | Tobacco usage Donmez, 2005; Rubes et al, 1998; Soares & Melo, 2008; Vine, 1996). Tobacco smokers exhibit higher levels of seminal ROS and OS mark‐ Smoking may also have direct and indirect effects on various ers than nonsmokers (La Maestra, Flora, & Micale, 2015). Smoking male urogenital organs by causing conditions such as , not only increases leucocyte levels by about 48% and ROS level varicocele, , reduced accessory gland function by 107%, but also decreases reactive oxygen species‐total antioxi‐ and alternations of the hypothalamic–pituitary–gonadal axis (Harlev, dant capacity (ROS‐TAC) by 10 points in the seminal plasma (Close, Agarwal, Gunes, Shetty, & Plessis, 2015). For example, smoking Roberts, & Berger, 1990; Saleh et al., 2002; Zhang et al., 2013). In could also inhibit adequate sperm maturation in epididymitis, in‐ smokers, proinflammatory leucocytes are recruited, which eventu‐ crease the likelihood of oligozoospermia for tobacco abusers and ally increases seminal ROS levels affecting spermatogenesis and varicocele patients and reduce various vesicular and prostatic pa‐ sperm quality (Zhang et al., 2013). Thus, tobacco smoking affects the rameters (Harlev et al, 2015). 4 of 13 | AGARWAL et al.

in the recruitment of macrophages in the seminal plasma (Leisegang, 2.3 | Alcohol Bouic, & Henkel, 2016; Morrison & Brannigan, 2015). However, in Alcoholic men of reproductive age have been shown to have sig‐ combination with diabetes mellitus type 2, obesity‐related infertility nificantly decreased antioxidant levels and a significant increase is exacerbated by IL‐6 and TNF‐α disruption of the hypothalamic–pi‐ in serum lipid peroxide (LPO) compared to those of nondrinkers tuitary–gonadal axis (Bhasin et al, 2010). Disruption of the axis results (Saalu, 2010). Alcohol interferes with the body’s antioxidant defence in decreased testosterone levels and, subsequently, in hypogonadism. mechanism (Agarwal & Majzoub, 2017) by impairing the metabolic pathway and generating highly reactive and toxic free radicals as 2.5 | Leukocytospermia by‐products (Das & Vasudevan, 2007) suggesting a link between OS and alcohol consumption. Studies have shown a reduction in sperm Leucocytes are the main source of ROS in semen, producing up concentration, percentage of normal sperm morphology, sperm to 1,000 times more ROS than normal spermatozoa (Plante, count, total progressive motility and testicular integrity in oligozo‐ Lamirande, & Gagnon, 1994). Globally, about 10%‐20% of infertile ospermic men (Anderson et al, 2010; Muthusami & Chinnaswamy, men have elevated seminal leucocyte concentrations caused by 2005; Vine, 1996). Alcohol usage also reduces in vitro fertilisation infections or inflammatory responses and other factors (Henkel (IVF) success and increases miscarriage rates (Klonoff‐Cohen Lam‐ et al., 2007). The most common leucocytes in the ejaculate are Kruglick, & Gonzalez, 2003; Meldrum et al, 2016; Nicolau, Miralpeix, polymorphonuclear granulocytes (50%‐60% of all leucocytes in Sola, Carreras, & Checa, 2014). ejaculate) and macrophages (20%‐30% WBCs that destroy for‐ eign material and secrete cytokines) (Wolff, 1995). Macrophages are located in the tissues. Upon activation, macrophages will re‐ 2.4 | Obesity/metabolic syndrome lease proteases, chemotactic factors and ROS that will Obesity is characterised by excessive accumulation of adipose tis‐ signal to the endothelial cells lining the blood vessels to present sues, particularly visceral adipose tissue. The hormonal basis of certain chemicals on the inner walls of the blood vessels (Mosser male infertility is adipose tissues’ formation of oestradiol (Nelson & & Edwards, 2008). Each of the WBCs produces a large amount Bulun, 2001). Adipose fibroblasts contain aromatase which converts of ROS to fight infections and eliminate pathogens by stimulating testosterone into oestradiol thereby not only decreasing serum the activity of G6PDH causing the production of high amounts testosterone levels, but by increasing serum oestradiol concentra‐ of NADPH. Furthermore, NADPH oxidase takes an electron from tions causing a decrease in inhibin B levels (Cabler, Agarwal, Flint, NADPH to convert oxygen into a superoxide anion. In turn, ex‐ & Plessis, 2010). Obesity results in male infertility by dysregulating cessive OS will activate chemokines CXCL5, CXCL8, IL‐6 and these endocrine pathways, and increased seminal ROS levels, and is IL‐8 (Comhaire, Bosmans, Ombelet, Punjabi, & Schoonjans, 1994; inducing physical manifestations such as sleep apnoea, and erectile Sandoval, Raburn, & Muasher, 2013). Consequently, an imbalance dysfunction (Kashou, Plessis, & Agarwal, 2012). between antioxidants and seminal ROS levels occurs, which leads Obesity also causes a systemic inflammatory response which to OS‐induced infertility (Agarwal et al., 2003). High concentra‐ impacts semen parameters and semen quality (Trayhurn, 2013). The tions of seminal leucocytes reduce sperm concentration/motility increased visceral abdominal fat can lead to alterations in adipokine and cause abnormal sperm morphology (Aziz, Agarwal, Lewis‐ secretion and the recruitment of proinflammatory white blood cells, Jones, Sharma, & Thomas, 2004; Saleh et al, 2002). Early detec‐ and increased NADPH oxidase activity which, in turn, generates addi‐ tion of novel biomarkers such as TLR2, TLR4, COX‐2 and Nrf‐2 can tional ROS and decreases sperm DNA integrity (Esposito et al., 2006; perhaps help facilitate better therapeutic interventions to allevi‐ Palmer, Bakos, Fullston, & Lane, 2012). In addition, those with a high‐ ate OS‐induced infertility (Hagan et al, 2015). fat diet will produce more reducing agents (NADH and FADH2) to cre‐ ate the electron gradient needed to form ATP (Tiganis, 2011). Given 2.6 | Infections the lack of ATP demand, the reducing agents will promote electron leakage and formation of superoxide radicals. Superoxide radicals are The body has three main defence mechanisms protecting against quickly removed by superoxide dismutase and generate hydrogen bacterial invasions: tight junctions between skin epithelium, the in‐ peroxide, which can be excessively produced by the skeletal muscle, nate immune response and the adaptive immune response. Potential promoting systemic ROS production (Patti et al., 2003). This systemic pathogens infect individuals through tears in membranes during ROS production has a detrimental effect on spermatogenesis and sexual intercourse. In addition, different usually have pre‐ causes DNA damage (Bachir & Jarvi, 2014; Kashou et al., 2012). dilections for infecting specific tissue types. For example, Neisseria Metabolic syndrome is a combination of three of the follow‐ species live on mucous membranes in the genital tract. ing five criteria: (a) excess waistline adiposity; (b) high fasting blood Effector B cells are the subtype of B cells that can create an‐ glucose level; (c) decreased HDL levels; (d) high triglyceride; and (e) tibodies. In order for effector B cells to be activated, MHC IIs are hypertension (Morrison & Brannigan, 2015). Men with metabolic presented on professional antigen‐presenting cells (APCs) such syndrome have increased adipokine secretion for tumour necrosis as macrophages and B cells. They signal to CD4 T‐helper cells factor‐alpha (TNF‐α) and inflammatory interleukins (ILs) which results and release cytokines to recruit more macrophages. Neutrophilic AGARWAL et al. | 5 of 13 chemotactic factors released by macrophage allow could cause harm, such as an elevated level in OS and the uninten‐ to navigate between the endothelial cells and enter the tissues. tional release of granulocytes from the phagocytes (Azenabor et Activated macrophages, neutrophils and eosinophils increase oxy‐ al., 2015). gen uptake to undergo a series of respiratory burst. tend to be highly effective in neutralising certain 2.8 | Neisseria gonorrhoeae bacteria by enhancing phagocytosis. With aggressive ad‐ ministration, oral antibiotics reach the colon in active form and thus Upon infection, the male urogenital tract reflects two types of suppress susceptible microorganisms and normal microflora; these changes—an increase in diameter and a reduction in the velocity events could lead to reduced colonisation resistance (Edlund & Nord, of seminal flow (Azenabor et al, 2015). As a result, there will be el‐ 2000). A recent murine study indicates that antibiotics may also re‐ evated levels of ROS. The causative agent of gonorrhoea, N. gon‐ duce the ability of immune cells such as macrophages to fight off orrhoeae, colonises the genital, rectal and nasopharyngeal mucosa infection as the antibiotics block off macrophages’ respiration (Yang, during the infection (Château and Seifert, 2016). Among Gram‐nega‐ Bening, & Collins, 2017). tive bacteria, N. gonorrhoeae is unique because they turn over large amounts of peptidoglycan during growth (Mavrogiorgos, Mekasha, Yang, Kelliher, & Ingalls, 2014). N. gonorrhoeae is capable of activat‐ 2.7 | Sexually transmitted disease ing TLR2, TLR4 and cytosolic receptor nucleotide‐binding oligom‐ Infections of the male genital tract with bacteria, viruses and proto‐ erisation domain (NOD)‐1 and NOD2 in regulating the immune zoa have negative impacts on male fertility. With annually 325,900 response to the bacterial antigen in the innate immune response. new cases for gonorrhoea, 500,000 for chlamydia and 70,000 for This process further activates transcription factor NF‐kB and poly‐ syphilis, these three sexually transmitted diseases (STDs) are the ubiquitination of the adaptor receptor‐interacting serine–threonine most prominent in the United States (Cates, 1999). Bacteria inter‐ kinase 2 (RIPK2) (Mavrogiorgos et al., 2014). The activation of the act with the host’s inflammatory and immune response differently NOD receptor implies that the immune response can induce not through their unique structures, special enzymes and toxins, to only cytokines (IL‐16) and chemokines (CCL17 and CCL22), but also evade the host’s response. Regardless of which specific organ has growth factors (CSF1), complement proteins and other cytosolic pat‐ the resultant inflammation, there will be an elevation of polymorpho‐ tern recognition receptors on cell surfaces, such as TLR2 and TLR4 nuclear leucocytes and granulocytes which can lead to an increase (Mavrogiorgos et al., 2014). in OS and impaired spermatogenesis (Ahmad, Plessis, & Agarwal, Furthermore, IL‐1 induces apoptosis in semen via proliferation 2017). Interestingly, the specific types of cytokines, chemokines, and differentiation of β‐cells to induce neutrophil and monocyte cell adhesion molecules and C‐reactive proteins released at the site generations with the assistance of chemoattractors such as IL‐8 of infection depend on the respective pathogen and individual host (Azenabor et al, 2015). For N. gonorrhoeae, IL‐1 and IL‐8 expression response (Newton & Dixit, 2012). affects the clinical outcome significantly (Singer & Ouburg, 2016). IL‐1 increases vascular endothelium activity and permeabil‐ Duru, Morshedi, and Oehninger (2000) showed that there is a nega‐ ity and activates lymphocytes; IL‐6 regulates temperature during tive correlation between seminal oxidative stress and sperm concen‐ inflammation; IL‐8 acts as a neutrophil chemotactic factor; IL‐10 tration, function and motility. It is also suggested that cytokines could downregulates inflammatory response by blocking NF‐kB activation be the mediator of semen quality and male infertility (Azenabor et and Th1 cell expression; TNF‐α increases vascular endothelial activ‐ al, 2015). In addition, a study indicates that increased levels of IL‐1β ity and permeability; and interferon gamma (IFN‐γ) activates macro‐ were associated with a decrease in sperm motility and an increase phages (Singer & Ouburg, 2016). Differences in cytokine expression in seminal ROS and malondialdehyde (MDA), a by‐product of lipid can alter disease progressions such as tubal pathology and related peroxidation (Koçak, Yenisey, Dündar, Okyay, & Serter, 2002). fertility problems (Singer & Ouburg, 2016; den Hartog et al, 2009; Karimi et al, 2009). 2.9 | Chlamydia trachomatis As responses to infections such as STDs, cytokine levels also increase in larger concentrations (Azenabor, Ekun, & Akinloye, Chlamydia trachomatis has the highest prevalence rate globally with 2015). There are certain types of microorganisms that can survive 4.2% for women and 2.7% for men being infected (Newman et al, for a long period of time without causing overt signs of infections 2015). Chlamydia presents men with a clinical challenge as 50% of during the latent period. For pathogens that have penetrated the men infected with chlamydia will not receive empirical treatment for structural barriers, normal inflammatory responses include pain, it (Rietmeijer, Van Bemmelen, Judson, & Douglas, 2002). The mecha‐ redness, heat and vasodilatation allowing leucocytes to exit blood nism behind Chlamydia trachomatis‐induced male infertility remains vessels and interact with vascular endothelium. The increase in controversial. Currently, there are three main theories—(a) high level vessel diameters, immunoglobulins, complements and other of WBC through cytokine stimulation; (b) interaction with CD 14 re‐ semen proteins depends on the adhesive interactions activated ceptor; and (c) development of antisperm antibodies. The first pro‐ by cytokines (Azenabor et al, 2015). Whereas the intention of the posed theory is about the interactions among high level of WBCs inflammatory response is to protect the host, the response itself through cytokine stimulations, the presence of lipopolysaccharides 6 of 13 | AGARWAL et al.

(LPS) and the development of antisperm antibodies (Ahmad et al, 2012). The limited exposure to innate and adaptive immune re‐ 2017). More specifically, this theory indicates that when Chlamydia sponse, as a result of the lack of surface‐exposed lipoproteins, is trachomatis reaches the epithelial cell, infection often results in tis‐ not sufficient to control the bacteria; this leads to a substantial sue damage and thus stimulates IL‐1 (Arend & Dayer, 1994). IL‐1 number of spirochaetes avoiding phagocytosis (Cruz et al., 2012). (which consists of α and β subunits) stimulates polymorphonuclear Tissue lesions are one of the clinical manifestations for T. pall‐ WBCs and macrophages and then subsequently induces IL‐6, IL‐8, idum, as a result of provoking an intense cellular immune response IL‐10, TNF‐α and IFN‐γ (Lavranos, Balla, Tzortzopoulou, Syriou, & (Cruz et al., 2012). The study by Voorhis, Barrett, Nasio, Plummer, Angelopoulou, 2012; Nandipati, Pasqualotto, Thomas, & Agarwal, and Lukehart (1996) shows that expressions of Th1 cytokines, such 2005; Singer & Ouburg, 2016). The IL‐6 level in the seminal plasma as IL‐1, IL‐12 and IFN‐γ, play a role in bacterial clearance. When is higher in infertile men (Koçak et al., 2002; Naz & Kaplan, 1994). these cytokines are abnormally elevated, inflammation becomes Similarly, MDA levels were higher in the semen of infertile men, harmful because of OS‐induced sperm DNA damage and apoptosis which suggests that IL‐6 is involved in lipid peroxidation (Camejo, (Azenabor et al., 2015). Segnini, & Proverbio, 2001). This series of cascade reactions indi‐ rectly activates OS, which contributes to male infertility. 2.11 | Mycoplasma The second proposed mechanism is through the interaction be‐ tween CD14 receptor and high concentration of LPO present on There are two species of mycoplasma, which are clinically relevant the sperm membrane to induce excessive ROS (Ahmad et al, 2017), for male infertility: M. hominis and M. genitalium (Andrade‐Rocha, whereas the third proposed mechanism points to the development 2003; Gimenes et al., 2014). The prevalence of mycoplasma infec‐ of the antisperm antibodies from invasion of lymphocytes, macro‐ tions is 4.8% for M. genitalium and 9.6% for M. hominis (Gdoura et al., phages, plasma cells and eosinophils. The activation cascade of ILs 2008). 41% of men with recurrent urethritis are positive for M. geni‐ invokes more secretory IgA, IgM and IgG antibodies, in which IgA talium (Wikström & Jensen, 2006). Infections with M. hominis and and IgG are generally associated with decreased levels in semen M. genitalium decrease sperm motility and normal morphology and parameters and pregnancy outcomes (Idahl et al., 2010). A meta‐ increase DNA damage (Andrade‐Rocha, 2003; Zeighami, Peerayeh, analysis carried out on cytokine‐level variations on the progression Yazdi, & Sorouri, 2009; Zinzendorf et al., 2008). Gallegos et al. and outcome indicates that IL‐1, IL‐6, IL‐8, IL‐10, TNF‐α and IFN‐γ (2008) reported that inflammation‐induced ROS production is the variations in hosts could potentially affect the clinical outcome for source of DNA damage in mycoplasma‐mediated infections. After the patient (Singer & Ouburg, 2016). Given that there are several a course of antibiotics and antiinflammatory steroids, sperm DNA theories, early diagnostic testing and antibiotic administration (i.e., damage significantly decreased (p < 0.001; Gallegos et al., 2008). macrolides) are essential for STD eradication.

2.12 | Ureaplasma 2.10 | Treponema pallidum Ureaplasma urealyticum and Ureaplasma parvum are the two clinically The primary lesion of syphilis, originating from T. pallidum, takes relevant species of ureaplasma responsible for male infertility with about three weeks to develop. Even though the infection contains a prevalence of 15.6% and 2.9% respectively (Gdoura et al., 2008). many spirochaetes and is highly contagious, the serologic test could Infection rates for ureaplasma in male infertility patients vary be‐ be negative for up to 12 weeks. Despite the administration of inex‐ tween 5% and 42% (Abusarah, Awwad, Charvalos, & Shehabi, 2013; pensive and effective antibiotics, there are still more than 10.5 mil‐ Zhou, Ma, Shi, & Liu, 2017). Whereas sperm motility and normal lion cases around the world and potential infection relapses (Schmid, sperm morphology decrease, sperm DNA damage increases. As urea‐ Rowley, Samuelson, Tun, & Guraiib, 2009; Sellati et al., 1998) with plasma does not trigger an inflammatory process which would de‐ about 30% of the patients resulting in the development of tertiary grade semen quality (Zhou et al., 2017), the rationale as to why these syphilis (Kampmeier, 1964). Yet, the circumstances of this multifac‐ semen parameters deteriorate remains unknown (Andrade‐Rocha, eted shifting balance between persistent bacteria trying to evade the 2003; Nunez‐Calonge et al., 1998; Zhou et al., 2017; Zinzendorf et host’s immune responses are not well understood (Cruz et al., 2012). al., 2008). These pathogens accumulate in the urethra and likely bind For the innate immune response, T. pallidum contains abundant directly to spermatozoa upon ejaculation (Nunez‐Calonge et al., 1998; lipoproteins capable of activating macrophages and dendritic cells Zeighami et al., 2009), a process which is thought to cause the in‐ (DC) via CD14 and Toll‐like receptor (TLR)‐1‐ and TLR2‐dependent creased DNA damage and the loss in membrane integrity by forma‐ pathways (Alexopoulou et al., 2002; Brightbill et al, 1999; Lien tion of ROS (Gallegos et al., 2008; Reichart, Kahane, & Bartoov, 2000). et al, 1999; Sellati et al., 1998; Wooten et al, 2002). Due to the special bacterial structure without surface‐exposed lipoproteins, 2.13 | pathogen‐associated molecular patterns (PAMPs) are not readily available to TLRs (Cruz et al., 2012). Afterwards, tissue‐based DC Escherichia coli is considered the most significant bacterium in infec‐ travel to lymph nodes to present the antigens to naive B and T tion‐mediated male infertility (Comhaire, Mahmoud, Depuydt, Zalata, & cells to further elicit the adaptive immune response (Cruz et al., Christophe, 1999). E. coli decreases sperm motility, decreases vitality and AGARWAL et al. | 7 of 13 increases DNA damage (Dahlberg, 1976; Diemer et al., 1996; Domes et al., proper testing for drug resistance as recent attention has also been 2012; Moretti et al., 2009; Sanocka‐Maciejewska, Ciupińska, & Kurpisz, brought upon multidrug resistance (Krupp & Madhivanan, 2015). 2005). E. coli‐mediated infertility has many modalities as to how it can cause Combination treatment is generally recommended for patients if male infertility. First, E. coli causes recruitment of leucocytes and thereby first‐line treatment is ineffective (Magri et al, 2007). Although most promotes neutrophilic production of ROS (Diemer et al., 2003; Moretti et combinations are indifferent or additive, and rifampin al., 2009). Proinflammatory cytokines, such as IL‐6, can directly rupture appear to be effective against (Oliphant & cellular membranes, which decreases sperm motility (Ramirez, Carreras, & Green, 2002) whereas fluoroquinolones chelate with cations such as Mendoza, 1992; Yamauchi‐Takihara et al., 1995). In addition, E. coli dimin‐ aluminium, magnesium, calcium, iron and zinc. This drug interaction ishes sperm membrane integrity by binding directly to their membrane and significantly lowers serum drug concentrations available to penetrate introducing porins to the spermatozoa’s membrane (Galdiero et al., 1988). target tissues (Oliphant & Green, 2002). epithelial cells are In turn, porin formation causes the release of cytoplasmic content and capable to accumulate cellular zinc that is severalfold higher than most causes a significant reduction in viability ranging from 80% to 100%. Finally, other mammalian cells (Costello, Feng, Milon, Tan, & Franklin, 2004). bound haemolytic subspecies of E. coli cause a decline in sperm vitality by Given the above explanation, the accumulation of zinc in prostate may decreasing the sperm plasma membrane potential, which mediates intra‐ prevent fluoroquinolones from reaching their optimal efficacy. cellular ROS production (Boguen, Treulen, Uribe, & Villegas, 2015).

2.15 | Microorganism mutations leading to more OS 2.14 | Prostatitis Antibiotic overuse can cause pathogens to genetically mutate, leading to Among male urogenital diseases, prostatitis remains to be the most antibiotic resistance. This is especially true with bactericidal antibiotics. in‐depth studied inflammatory diseases. In acute and chronic bac‐ The use of such antibiotics can lead to changes in cell’s metabolism to terial prostatitis, pathogens can affect spermatozoa either directly promote increased levels of reactive oxygen species (Dwyer, Kohanski, (depending on the strain of the pathogen) or indirectly by inducing & Collins, 2009). For example, fluoroquinolones bind with DNA gyrase an inflammatory response in the seminal tract via the activation of (topoisomerase II, product of gyrA and gyrB) and/or topoisomerase cytokines such as IL‐6, IL‐8 or TNF‐α (Alshahrani, McGill, & Agarwal, IV (product of parC and parE) to induce cell death (Dwyer et al, 2009). 2013; Martínez‐Prado & Camejo Bermúdez, 2010). Increased cy‐ Through the process of phagocytosis (macrophages and neutrophils en‐ tokine levels lead to OS, which not only affect the spermatozoa gulf microorganisms by a combination of ROS, RNS and enzymatic di‐ (Dobrakowski et al., 2017), but can also potentially cause a system‐ gestion), there is a sudden production of oxidative burst through both atic response by decreasing hormone levels of testosterone (Bini et macrophages and neutrophils (Vatansever et al, 2013). An elevated level al., 2015; Leisegang & Henkel, 2018). High levels of IL‐6, IL‐8 and of OS will be produced when the pathogen attempts to escape from the TNF‐α may also affect sperm transit during ejaculation if the infec‐ antibiotic treatment through mutation. To eliminate mutated bacteria, a tion spreads to the testis (Camejo et al, 2001; Nandipati et al, 2005; higher amount of antibiotic‐induced ROS will be needed to elicit the same Rajasekaran, Hellstrom, Naz, & Sikka, 1995; Martínez, Proverbio, effect (Dwyer et al, 2009), a scenario that could occur if a patient has re‐ & Camejo, 2007; Sanocka, Jędrzejczak, Szumała‐Kaękol, Frączek, ceived multiple prior administrations of antibiotics. Antibiotic resistance & Kurpisz, 2003). Increased oxidative stress also directly damages does not allow for effective elimination of the causative pathogen. sperm DNA, thus compromising the paternal genomic contribution If the pathogen is not entirely eradicated, the dosage of the an‐ to the embryo (Tremellen, 2008). tibiotic is usually increased or combined with another antibiotic to Infection and inflammation in the prostate activate leucocytes, maximise efficacy. However, multidrug resistance could also lead to which could increase ROS. For this reason, infections require im‐ oxidative stress as the microorganisms attempt to defend the body mediate attention because an elevated amount of ROS can affect against the drug; a study by Dwyer et al. (2014) showed that antibi‐ up to 35% of men consulting for infertility (Henkel, 2012). This otics induce complex redox alterations, which contribute to cellular is primarily due to the fact that untreated prostatitis can lead to damage and death. More specifically, alterations in central metab‐ oligozoospermia, or (Schuppe olism, cellular respiration and iron metabolism occur during drug et al, 2008; Weidner, Colpi, Hargreave, Papp, & Pomerol, 2002). interactions of target‐specific processes (Dwyer et al., 2014). The Untreated/undertreated infections can also lead to chronic dis‐ addition of ROS and other damaging substances such as cytokines ease, which is more difficult to treat than acute disease. In the makes it more difficult for the bacteria to be eradicated, which could example of bacterial prostatitis, four different antibiotics—fluoro‐ rather prolong acute prostatitis (Dwyer et al., 2014). quinolones, , macrolides and trimethoprim (alone or combined with sulfamethoxazole)—were introduced as an option 3 | VIRAL INFECTIONS for treatment (Lipsky, Byren, & Hoey, 2010). Antibiotic administra‐ tion remains to be the cornerstone for the treatment of bacterial 3.1 | Human Immunodeficiency Virus prostatitis, and numerous studies have indicated that antibiotics can significantly improve semen parameters and pregnancy rates. Human immunodeficiency virus (HIV) causes a decrease in semen Yet, antibiotic treatment needs to be performed carefully and after motility, vitality and forward progression when CD4 cell counts are 8 of 13 | AGARWAL et al. below 350 cells/microlitre (Wang et al., 2014). Otherwise, HIV‐se‐ in the spermatozoa thus causing further ROS propagation and OS ropositive men who are asymptomatic do not show a decrease in (La Vignera et al, 2012, Machida et al, 2006, Lorusso et al, 2010). fertility (Garolla et al., 2013; Lorusso et al., 2010; Wang et al, 2014). Hepatitis C‐induced OS causes a decrease in sperm motility but In men with symptomatic HIV infections, recruitment of monocytes, unchanged ejaculatory volume, apoptosis and increased DNA macrophages and leucocytes is found in semen (Garolla et al., 2013). damage (Machida et al, 2006; La Vignera et al, 2012, Hofny et al With current highly active antiretroviral therapy (HAART) reg‐ 2011; La Vignera et al, 2012; Lorusso et al, 2010). imens increasing life expectancy for HIV‐infected men, investiga‐ tions on how HAART affects male fertility show a negative impact on semen quality (Frapsauce et al., 2015; Garolla et al., 2013; Kehl 4 | CONCLUSIONS et al., 2011; Wang et al., 2014). The use of nonnucleoside reverse transcriptase inhibitors may induce oxidative stress as they are mi‐ There are many specific inflammatory/infectious processes that tochondrially toxic (Kehl et al, 2011; Pavili et al., 2010) and disrupt cause male infertility. Therefore, it is important to correctly iden‐ the mitochondrial membrane potential (Frapsauce et al, 2015). The tify the causative agent as each possesses a unique mechanism disruption in mitochondria results in decreased motility and forward of damage as well as specific susceptibilities to antibiotics when velocity for men on nonnucleoside reverse transcriptase inhibitors, considering treatment. Many factors contribute to the decision to particularly efavirenz (Frapsauce et al, 2015). treat OS in male infertility. Modifiable lifestyle changes such as tobacco/alcohol cessation, healthy diet or antioxidant supplemen‐ tation with different chances of success are recommended. On the 3.2 | Hepatitis other hand, antibiotic treatment remains to be the cornerstone of Hepatitis B and hepatitis C are both viruses found to cause male treating bacterial‐mediated infections where certain antibiotics infertility. Hepatitis B is considered to worsen fertility parameters can also cause OS‐related damages. Yet, an increasing risk of in‐ given its ability to pass through the blood–testis barrier (Garolla fections with multidrug‐resistant bacterial strains has to be taken et al, 2013; Vicari et al, 2006). Given the hepatitis B virus is able into consideration. Whereas the treatment of choice for viral in‐ to pass through the blood–testis barrier, it can directly trans‐ fection is antiretrovirals, nonnucleoside reverse transcriptase in‐ fer its genome in spermatozoa which results in defective sper‐ hibitors pose a potential decrease in fertility. Thus, more efforts miogenesis and decreased fertilisation rates (Zhou et al., 2017). need to be made to better understand the causes and effects of Spermatozoa exposed to hepatitis B were found with increased oxidative stress on male fertility, so that better and more sustain‐ phosphatidylserine externalisation and lipid peroxidation meas‐ able treatment regimens can be developed. ured by MDA formation (Kang et al., 2012; Qian, Li, & Li, 2016). Men with chronic hepatitis B had high seminal concentrations of ORCID IL‐18, which causes natural killer cells to secret proinflammatory cytokine INF‐ɣ (Qian et al, 2016). These authors found a positive Ashok Agarwal http://orcid.org/0000-0003-0585-1026 correlation between inner MDA formation and IL‐18 concentra‐ Ralf Henkel http://orcid.org/0000-0003-1128-2982 tion, suggesting MDA formation is caused by an inflammatory process. 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