**1. Introduction**

The mammalian spermatozoon is a cell with a high demand for energy to perform its function. Spermatozoa obtain their energy by two main metabolic pathways: glycolysis that occurs in the principal piece of the flagellum and oxidative phosphorylation (OXPHOS) that takes place on mitochondria located at the midpiece of the flagellum [1]. Spermatozoa contain between 50 and 75

mitochondria [2] and as with any other kind of cell that performs aerobic metabolism, is associated with the production of free radicals named reactive oxygen species (ROS) that include the hydroxyl radicals (•OH), superoxide anion (•O2 −), hydrogen peroxide (H2O2), and nitric oxide (NO). These ROS are highly reactive molecules due to the presence of an unpaired electron in their outer shell. In addition, they have a very short half-life in the range of nanoseconds to milliseconds. ROS are produced as a consequence of natural cell machinery and participate in the normal function of a cell. However, when ROS production overcomes cellular antioxidant defenses surpassing a physiological range, they cause deleterious e ffects due to oxidative stress (OS) that results in oxidation of lipids, proteins, carbohydrates, and nucleotides [3].

Male subfertility and infertility have been associated with OS. Moreover, since infertile men have lower seminal plasma antioxidant capacity in comparison with fertile men, when higher levels of ROS occur, they led to an increase of lipid peroxidation (LPO) [4]. It is well described that when ROS overproduction occurs, it induces sperm DNA damage, although they have the potential to fertilize embryo development and fertility might be disturbed [5,6]. It is unclear how this is related with the fact that nowadays infertility is becoming a worldwide health problem, where one out of six couples are under fertility treatment and thus the use of assisted reproductive technologies (ART) to overcome this problem is growing exponentially. Nevertheless, ART is not harmless and is also associated with an increase of ROS production [7]. Although there is literature focused on the e ffects of consumption of oral substances with antioxidant properties on sperm parameters, the purpose of this review is to discuss the e fficiency of antioxidant intake as a dietary supplement as well as an additive through ART procedures to counteract excessive ROS production that leads to infertility. We will also focus on the molecular mechanisms of action of those compounds with antioxidant activity in the male reproductive system, mainly reviewing literature that relates antioxidant treatment with ART, clinical pregnancy, and live birth as final outcomes.

### **2. Sources of ROS in Spermatozoa**

Several situations result in nonphysiological levels of ROS overwhelming the natural scavenger systems (Figure 1). For example, lifestyle habits, such as alcohol consumption, smoking, exposure to toxicants, or pathologies such as obesity, varicocele, stress, and ageing have been associated with increased production of ROS in seminal plasma [8]. Presence of leucocytes in semen, as well as high percentage of spermatozoa with morphological anomalies [9] or immature spermatozoa with cytoplasmatic droplets containing high amount of enzymes are some examples associated to high ROS levels [9–12].

Currently, human infertility is a global health problem that has led to an exponential grown in the use of ART in the last years to overcome fertility problems. However, ART protocols imply sample centrifugation, light exposure, change of oxygen concentration, pH, or temperature, and the use of culture media with metals content that can produce hydroxyl radicals by Haber–Weiss and Fenton reactions (see explanation below). Hence, optimization of ART protocols has been proposed to minimize artificial ROS production, for instance, by decreasing *g*-force during sperm selection [13], decreasing spermatozoa incubation time during in vitro fertilization (IVF), which in turn decreases the time where aberrant spermatozoa that produce more ROS are in contact with the oocyte, and by decreasing sperm concentration or atmospheric oxygen concentration during embryo culture under in vitro conditions [7]. In order to reduce human leucocyte contamination on raw semen, paramagnetic bead technology (Dynabead ®) can be used. Thus, magnetic beads coated with leukocyte antigen CD45 decrease leukocyte contamination [14,15], doubling the percentage of spermatozoa–oocyte penetration, as shown in a heterologous assay using hamster oocytes [16].

**Figure 1.** Potential stimuli that cause reactive oxygen species (ROS) production in spermatozoa.

### **3. Bivalent Role of ROS on Sperm Function**

Mammalian spermatozoa are extraordinary cells able to survive in a different body from where they were created. They are very specialized cells having the sole purpose to deliver the paternal genome into the oocyte. However, after ejaculation, spermatozoa must undergo a complex process within the female reproductive tract named capacitation, which allows spermatozoa to fertilize the oocyte [17,18]. Capacitation is a cascade of different cellular events that imply high production and consumption of energy. Although there is controversy on the preponderant metabolic pathways, glycolysis or OXPHOS, used by spermatozoa to generate energy in the form of ATP, it seems that there are sperm species preferences [1]. OXPHOS is the most efficient pathway, obtaining about 30 molecules of ATP by oxidizing one molecule of glucose, while during glycolysis, only two molecules of ATP are obtained per molecule of glucose. It has been described that OXPHOS is the major source of ROS in spermatozoa [19]. Furthermore, ROS might play a bivalent role in sperm function: mild ROS levels boost different intracellular events that culminate on oocyte fertilization, while higher ROS levels induce sperm DNA damage and embryo miscarriage [20,21]. In a comprehensive review, Ford summarized ROS physiological functions on sperm capacitation [22]. It is known that soluble adenylyl cyclase (sAC) is activated by bicarbonate and Ca2+, converting ATP into cAMP, subsequently activating the PKA pathway that mediates the phosphorylation of protein in tyrosine residues, which is used as a hallmark of sperm capacitation [23,24]. It has been proposed that ROS participate in the activation of the cAMP/PKA pathway by increasing cAMP levels, although the mechanism of cAMP production is still not clear in spermatozoa [22]. In adipocytes, it has been proposed that the mechanism of action is through inhibition of phosphodiesterase activity [25]. In human spermatozoa, it was proven that ROS action is mediated by PKA [26]. Thus, the induction of tyrosine phosphorylation was suppressed by a PKA inhibitor (H89) and the responsiveness to progesterone (sperm-oocyte fusion) when spermatozoa were coincubated with NADPH proved it to be a ROS generator [26]. In a different study, capacitated human spermatozoa showed increased levels of cAMP that was mimicked in vitro by exposure of spermatozoa to superoxide anions (O2−). Superoxide dismutase (SOD) addition inhibited cAMP levels and the sperm acrosome reaction in a concentration-dependent manner [27]. These results were confirmed by others where superoxide anions increased cAMP concentration and capacitated spermatozoa produced H2O2, leading to an increase in protein tyrosine phosphorylation [28]. Nevertheless, when ROS production overcomes antioxidant defenses, detrimental effects on spermatozoa can be summarized as increased LPO

and DNA damage and reduction of sperm motility, which are associated with lower sperm fertility (Reviewed by [29]). Thus, ROS homeostasis is pivotal for male reproductive potential as they mediate important functions of sperm, such as capacitation, but when ROS levels surpass these biological levels, they readily oxidize lipids and proteins at membranes and compromise sperm quality and fertilization capacity (Figure 2).

**Figure 2.** Proposed model of the bivalent role of reactive oxygen species (ROS) in sperm. (i) High levels of ROS concentration induced by different factors such as assisted reproductive technologies (ART), diseases, medical treatment, life style, etc., overwhelming the sperm antioxidant system induce plasma membrane lipid peroxidation and DNA damage. (ii) Physiological ROS level produced mainly by mitochondria induce production of high levels of cAMP by an undetermined mechanism, activating the PKA pathway, and leading to tyrosine phosphorylation, a hallmark of sperm capacitation.

### **4. Mechanism of ROS Defense in Spermatozoa**

Spermatozoa differentiation is achieved during spermiogenesis as they gradually lose their cytoplasm. By the end of the process, the cytoplasm content is very small compared to other cells, where most of the space is occupied by DNA (sperm head). This special feature results in spermatozoa possessing low intracellular antioxidant activity consisting of superoxide dismutase (SOD), nuclear glutathione peroxidase (GPx), peroxiredoxin (PRDX), thioredoxin (TRX), and thioredoxin reductase (TRD) [30]. Therefore, sperm ROS scavenger activity basically depends on the antioxidant content of the seminal plasma, which is formed mainly by a trio of enzymes where SOD converts superoxide anion (O2−.) to hydrogen peroxide (H2O2), preventing the formation of hydroxyl radical that is an inductor of LPO. However, the H2O2 generated is a strong membrane oxidant that is rapidly eliminated either by catalase (CAT) or GPx activities, giving H2O as a product. Finally, seminal plasma also contains nonenzymatic antioxidant components such as α-tocopherol (vitamin E), ascorbic acid (vitamin C), pyruvate, urate, taurine, and hypotaurine [31].

It should be noted that most ART involves washing steps, meaning that all the natural antioxidant defenses contained in seminal plasma are removed. Likewise, this also happens after natural insemination. During ejaculation, spermatozoa are surrounded by antioxidant molecules coming from seminal plasma but once the ejaculate reaches the vagina, seminal plasma is diluted, leading in both cases to spermatozoa facing ROS. Although spermatozoa possess antioxidant scavenger systems, it seems that they are not strong enough when ROS levels exceed physiological levels, subsequently making spermatozoa highly susceptible to OS.
