The Positive Roles for Reactive Oxygen Species in Human Reproduction; Implications for the Therapeutic Application of Antioxidants

1. Introduction

Oxidative stress (OS) is a well-recognized pathological phenomenon that plays a central role in the aetiology of human infertility. OS can occur as a result of a variety of factors including pathological conditions such as varicocele, polycystic ovary syndrome, endometriosis, fibroids and inflammation [1,2,3], exposure to environmental stressors such as air pollutants, pesticides and radiofrequency electromagnetic radiation [4,5,6,7] and the impact of a wide variety of lifestyle factors including diet, obesity, and smoking [8,9,10,11,12]. Fundamentally, OS occurs because of an imbalance between reactive oxygen species (ROS) production and the body’s ability to neutralise those ROS through the action of enzymatic and non-enzymatic antioxidants [13]. This imbalance can lead to cellular damage because their unstable and reactive nature allows ROS to abduct electrons from nearby molecules including proteins, nucleic acids and carbohydrates leading to lipid peroxidation, protein degradation and the formation of advanced glycation products respectively. In addition, ROS are instrumental in the activation of cellular defence mechanisms such as apoptosis/ferroptosis, which can lead to the large scale depletion of reproductive cells, including the male and female germ lines [12]. Within the microcosm of reproductive health, oxidative stress is recognized as having a key role in disrupting such fundamental biological processes such as fertilization, oocyte maturation, ovulation, decidualisation, embryo development, blastocyst implantation, pregnancy and parturition [13,14,15]. Given all these negative impacts of ROS on the reproductive process, it is reasonable to question why evolution would have encouraged the generation of these potentially toxic molecules in cells that are so critically important for procreation and transgenerational carriage of the genome. This review addresses the physiological roles of ROS in the reproductive biology of mammals, with particular reference to human fertility and the ability of men and women to participate in the procreative process.

2. The Positive Role for ROS in Reproductive Cells

ROS are biologically important signalling molecules that are constantly being generated in cells. Their biochemical significance is built upon on three fundamental biochemical principles:

(1)

ROS can oxidize cysteine thiols to generate the corresponding sulfenic acid (SOH) or create disulfide bridges, thereby fundamentally altering protein conformation and function. In the following review of the reproductive process, this property of ROS is exemplified in numerous ways from the cross linking of chromatin in the sperm head to the activation of zymogens (e.g. metalloproteases) and the suppression of protein phosphatase activity [16,17]. The latter is an extremely powerful consequence of ROS exposure that results in phosphorylation-dependent signal transduction cascades being maintained in an activated state. A reproductively important example is the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway, which is essential for cell proliferation, protein synthesis, cell cycle progression, the suppression of apoptosis, the resumption of meiosis and implantation [18,19]. Similarly, mitogen-activated protein kinase (MAPK) activity is promoted by ROS and drives several processes central to reproductive fitness including spermatogenesis and oocyte maturation [20,21]. Thiol oxidation may also affect cellular activity via changes in transcription factors such as Nrf2/Keap1 or NF-κB that regulate the responsiveness of reproductive cells and tissues to oxidative stress and are critical for the survival of the germ line and critical processes such as ovulation, implantation and labour [22,23,24].

(2)

At high levels of intensity, ROS can be very destructive, attacking a variety of critical biomolecules and inducing physiological cell death [12,25]. This property is an essential element in cellular remodelling processes encountered in reproduction including luteolysis, menstruation, implantation as well as the deletion of defective gametes and embryos. Under these circumstances, ROS are being used as positive mediators of cellular turnover, mediating such key processes as apoptosis, ferroptosis and autophagy [12,19,26].

A third fundamental property of ROS is that they can interact with metal centres at the core of many key proteins and control redox-switching activities. Typically, such metal centres are occupied by transition metals such as iron and copper, the redox status of which controls the overall function of the protein. For example, the redox regulation of heme-iron centres in key proteins such as cytochrome P450s, cytochrome C, nitric oxide synthase, catalase and a variety of peroxidases is important for such diverse reproductive processes as sperm hyperactivation, progesterone synthesis by the corpus luteum and the generation of prostaglandins at parturition. Lipoxygenase activity is also dependent on the oxidation of an iron atom at its active site, triggering such vital biological processes as eicosanoid generation and ferroptosis [27]. Similarly, the redox cycling of bound copper supports the functionality of proteins such as superoxide dismutase (SOD) and ceruloplasmin that play critical protective roles at all stages of the reproductive process from gametogenesis to foetal development [28,29].

In the following sections, the positive contributions of ROS to all stages of reproductive process from gamete maturation to childbirth, are examined. The review is focused on human reproduction although the concepts expressed are often supplemented with data obtained in animal models, particularly laboratory rodents. Furthermore, this review focuses on ROS-mediated events and only gives limited consideration to other forms of redox-induced change, including S-nitrosylation, sulfhydration, glutathionylation, CoAlation, and protein carbonylation, which have been considered in detail elsewhere [30]. The results of this analysis clearly illustrate the important biological role of ROS in reproductive biology and highlight why caution should be exercised in determining the type and dose of antioxidants deployed in the therapeutic management of human infertility.

3. ROS and Sperm Function

3.1. ROS and Chromatin Cross-Linking

Human spermatozoa feature 3 major sources of ROS including electron leakage from their mitochondria, the activation of specialized calcium-dependent NADPH oxidases such as NOX5 and the activity of L-amino acid oxidases such as IL4I1 [12] (Figure 1; Step 1). These ROS are then used to drive various aspects of sperm biology starting with their fundamental architecture. As spermatozoa mature, cysteine groups in an important protective enzyme, glutathione peroxidase 4 (GPx4), become oxidized leading to the formation of multiple intermolecular disulphide bridges and the formation of a polymer that stabilizes the spiral arrangement of the mitochondria around the axoneme. In this way, an enzyme that started as a powerful antioxidant becomes transformed, via oxidation, to a key structural element in the sperm midpiece, the mitochondrial sheath [31].

Another redox-mediated change in maturing spermatozoa involves chromatin cross linking in the sperm head. This change compacts the DNA into a near-crystalline state and is thought to be important for safeguarding the integrity of the male genome during its long journey from the male to the female reproductive tract [32,33]. The cross linking of sperm chromatin is achieved by replacement of nuclear histones with cysteine-rich protamines during spermatogenesis, followed by ROS-mediated formation of inter- and intra- molecular disulfide bridges, mediated by GPx4 during epididymal transit (Figure 1; Step 2) [34,35,36,37]. Although all Eutherian mammals use 6–9 cysteines for disulfide cross-linking, the specific placement and subsequent intra- vs inter- molecular bonding patterns vary. Interestingly, variation in packaging efficiency and DNA integrity appears to correlate with sperm competition levels within a species. Higher competition often selects for more robustly packaged and damage-resistant chromatin to ensure successful fertilisation in a competitive environment. In this context, it is interesting to note that human spermatozoa, which have not evolved to cope with high levels of sperm competition, have notoriously poor chromatin cross linking and, as a result, are far more susceptible to DNA damage than other Eutherian spermatozoa [36]. Such deficiencies in chromatin packaging correlate with incomplete thiol oxidation during epididymal transit. As a result, spermatozoa from infertile males are often found to possess a higher thiol content (fewer disulfide bonds) compared with normozoospermic, control samples [38,39,40].

3.2. ROS and Capacitation

Another crucial pathway regulated by redox-responsive mechanisms is capacitation, the process by which mammalian spermatozoa undergo a final maturation during their ascent of the female reproductive tract and gain the capacity to fertilize the oocyte. Capacitation is characterised by increases in pH, intracellular calcium, HCO₃, cyclic AMP (cAMP) and protein phosphorylation [41]. ROS are positively involved in many of these processes as indicated in Figure 1; Step 3. Thus, the increase in tyrosine phosphorylation that characterises the attainment of a capacitated state, is a major redox-driven event in human spermatozoa. This enhancement is achieved via the ability of H₂O₂ to oxidize catalytic cysteine residues (often protected by glutathionylation or CoAlation) at the active site of tyrosine phosphatases to generate sulfenic acids which can then react with adjacent cysteines to form disulfide bonds or with nearby amides to form sulfenyl-amide linkages [30,42].

Additionally, ROS promote tyrosine phosphorylation by stimulating generation of the second messenger that drives this activity, cAMP, via the ability of O₂^•− to stimulate soluble adenylyl cyclase activity [43,44,45]. While the biochemical mechanisms responsible for redox-regulated cAMP generation in spermatozoa are not fully understood, the adenylyl cyclase within these cells contain cysteine residues that are susceptible to oxidation forming disulfide bonds or sulfenic acids. These modifications may induce a structural shift in the catalytic domain, increasing the enzyme's affinity for its substrate, ATP, or its essential cofactors, Mg²⁺ or Mn²⁺. In addition, ROS facilitate capacitation by enhancing the rate of cholesterol efflux from the plasma membrane, thereby contributing to an increase in membrane fluidity [46]. This phenomenon reflects the ability of ROS to directly induce the formation of oxysterols, which are more hydrophilic that the parent sterols and, as a result, move closer to the sperm surface where they are removed by binding proteins such as albumin (Figure 1; Step 4). The disruption of this redox process using hydrophobic antioxidants such as vitamin E or A, suppresses the tyrosine phosphorylation events associated with sperm capacitation and also interferes with sperm-zona interaction [47].

Hyperactivated motility is another feature of capacitation that is redox regulated. This type of movement is characterised by a change in the flagellar waveform to deliver a high amplitude, high intensity, asymmetrical beat pattern. This vitally important change is dependent on associated changes in tyrosine phosphorylation and is stimulated by ROS via the MAPK/ERK (Extracellular Signal-Regulated Kinase) pathway [48,49,50]. Another ROS-regulated factor involved in the control of hyperactivation is the Epidermal Growth Factor Receptor (EGFR); a transmembrane protein found in many cell types, mediating differentiation, cell proliferation and migration. EGFR activity has been shown to be directly enhanced by ROS through the inhibition of the receptor’s internalization and, indirectly, via the suppression of protein phosphatase activity [51,52,53]. In spermatozoa, ROS-dependent EGFR activation leads to actin polymerization which, in turn, leads to hyperactivated motility by mechanically facilitating flagellar bending and acting as a regulatory scaffold for key signalling molecules such as Catsper [54,55,56]. In keeping with this model, studies have shown that light-induced ROS production successfully activates EGFR leading to hyperactivated motility, contrasting with the suppression of this process by the presence of SOD [54].

3.3. ROS and Fertilization

Following successful hyperactivation and capacitation, further redox-regulated changes lead to egg recognition, the acrosome reaction and finally sperm-oocyte fusion [41,45,55,56]. The ROS mediated increase in membrane fluidity during capacitation enables egg-recognition complexes to move anteriorly within the plasma membrane to a location where sperm-zona recognition can be achieved [57]. This cell-cell interaction then activates signal transduction processes in the spermatozoa leading to the induction of the acrosomal exocytosis and, ultimately, fusion with the vitelline membrane.

The mechanistic underpinnings of these membrane fusion events appear to involve the induction of lipid peroxidation via the lipoxygenase pathway, followed by activation of phospholipase A2, where the removal of the oxidized fatty acid from position 2 (sn2) of the phospholipid, generates a lysophospholipid [58,59]. The latter event results in membrane instability, thereby supporting fusion of the plasma and outer acrosomal membranes during acrosomal exocytosis. ROS might also be involved in the IP3-mediated activation of actin severing proteins that promote the acrosome reaction by further destabilizing the plasma membrane [54,55]. The increase in membrane instability might also explain the observed positive role of ROS in the generation of a fusogenic equatorial segment in the spermatozoa, primed for interaction with the oocyte’s plasma membrane [60].

3.4. ROS and Sperm Vitality

Finally, ROS are also involved in the mediation of both sperm survival and senescence. In the context of sperm vitality, ROS have been suggested to act directly on AMP kinase (AMPK), activating the enzyme via the oxidation of key cysteines (299 and 304) on the α-subunit and upregulating both the cells’ antioxidant defences, as well as their capacity for ATP generation [61,62]. On the other hand, ROS are also central to the induction of apoptosis in human spermatozoa, facilitating the apoptotic removal of aged, damaged spermatozoa and ensuring that they do not participate in the fertilization process [63]. In light of these observations, it has been suggested that sperm capacitation and senescence represent a physiological continuum mediated by ROS [64]. During the early stages of sperm capacitation ROS are positive mediators of biological change promoting signal transduction pathways, elevating intracellular calcium and increasing membrane fluidity, under the protective control of antioxidants, notably peroxiredoxin-6 [65]. However, if capacitated spermatozoa do not find an oocyte, these same changes will ultimately generate senescence and seal their apoptotic fate [64].

4. ROS and Oocyte Function

4.1. Oocyte Recruitment and Maturation

Oocyte recruitment, development and maturation involve a series of complex molecular signalling processes within the follicular microenvironment, many of which are mediated by ROS [66,67,68,69]. Oocytes spend much of their lifespan reposing within primordial follicles and arrested at prophase I. Following follicle selection and growth, a pre-ovulatory surge of gonadotrophins (LH and FSH) triggers ROS production through NOX enzymes, precipitating germinal vesicle breakdown (GVB) and meiotic resumption (Figure 2; Step 1). Within cumulus-oocyte complexes (COCs), cumulus cells mediate transmission of the gonadotrophin-driven signals that coordinate meiotic resumption, partly by modulating cyclic AMP (cAMP) generation. Concurrently, ATP production in the mitochondria rise to meet the energetic demands of oocyte maturation, with ROS produced as a by-product (Figure 2; Step 2). At physiological levels, ROS, especially H₂O₂, can activate signalling pathways such as AMPK and EGFR, which are involved in energy sensing, cumulus expansion, and ovulation [70]. AMPK and calcium-activated pathways are both implicated in meiotic resumption from diplotene arrest [71,72]. LH itself stimulates ROS production in the ovary, as suggested by ascorbic acid depletion studies [73], and this transient oxidative stress is functionally critical. Thus, normal cumulus expansion and mucification are effectively inhibited in vitro in both mice and rats by membrane permeant antioxidants such as BHA (butylated hydroxyanisole) [74,75]. In this situation, ROS serve as a primary signal for the activation of AMPK (Figure 2; Step 3).

Activated AMPK can then increase phosphodiesterase 3A (PDE3A) activity, thereby facilitating reduced generation and accelerated breakdown of cAMP and cGMP. The decline in cAMP, in particular, leads to dephosphorylation of maturation promoting factor (MPF) and resumption of meiosis. However, if the metabolic activity of oocytes fails to meet mitochondrial thresholds, as reflected in poor AMPK activation, then ROS-mediated apoptotic pathways are initiated by cytochrome C release, inducing large-scale apoptosis and a state of follicular atresia (Figure 2. Step 3) [75]. In addition to ROS mediated activation of AMPK, EGFR activation by H₂O₂ stimulates the calpain-2 pathway, leading to loosening of granulosa cell adhesions thereby allowing these cells to expand and reorganise as the follicle grows [76] (Figure 2; Step 4).

Ovarian follicles are therefore much like spermatozoa in that they rely significantly on redox regulation. ROS signalling pathways are crucial for the stimulation of follicle growth and the resumption of meiosis. While ROS are potentially toxic, the short half-life and limited diffusion capacity of these molecules, helps confer spatial precision to these signalling events within the ovarian follicle. However, if ROS generation overwhelms the follicle’s defensive capacity, then these same metabolites can trigger pathways leading to apoptosis and cell death. Thus, the ultimate fate of each follicle, and the oocyte it encases, critically depends upon its capacity to manage ROS, divining whether it will successfully achieve ovulation or succumb to atresia [77,78,79,80].

4.2. ROS and Ovulation

Ovulation is similarly modulated by redox activity. As indicated above, the LH surge that precedes ovulation, is associated with a sharp rise in inflammatory precursors in the ovary and an increase in ROS generation (Figure 3; Step 1) [81]. A causal relationship between acute inflammation, ROS and ovulation has been suggested by the ability of anti-inflammatory strategies [82] as well as antioxidants to successfully suppress ovulation in vivo [83,84]. Critical in this regard is the ability of ROS to promote the activation of proteases (matrix metalloproteinases [MMPs-14 and 16s] and plasminogen activators) that actively digest the collagen and connective tissues of the follicular wall (the theca externa), creating a weakened area called the stigma, which ultimately becomes the point of follicular rupture (Figure 3; Step 2). MMP activation involves a cysteine switch whereby ROS oxidise a thiol group in the pro-domain of the inactive zymogen, displacing a zinc ion, thus activating the enzyme. ROS also promote MMP activation by inhibiting key phosphatases allowing activation of the MAPK pathway and facilitating activation of transcription factors such as activator protein-1 (AP-1), which bind to the promoter region of MMP genes [16,17,84,85]. Such redox regulated protease activation in conjunction with ROS-induced apoptosis of granulosa cells, promotes detachment of the cumulus cells from the follicular wall, further contributing to follicular wall breakdown and ovulation of the mature oocyte [84]. LH also triggers the activation of EGFs (Epidermal Growth Factor like factors), in granulosa cells via ROS-mediated activation of MMPs [86,87]. EGFs trigger mucification of the cumulus cells [88] and cause an increase in the generation of PGE2 leading to a localized inflammatory response necessary for ovulation [89].

Evidence of NOX4 and 5 in human granulosa cells also supports a role for ROS in follicular rupture and ovulation [90], while animal studies using Drosophila, support this claim by demonstrating that O₂^∙- generating NOX enzymes work in conjunction with SOD3 to generate H₂O₂ which then acts as a secondary messenger triggering the apoptotic pathway leading to follicular rupture [91]. Indeed, in vivo studies in mice as well as ex vivo studies in rabbit also corroborate the essential role for ROS, such as H₂O₂, in follicular rupture and ovulation [75,87,91].

The generation of estrogen is another crucial function of the ovulating follicle. According to the two-cell/two gonadotrophin theory, LH stimulates follicular thecal cells to generate androgens which are then converted into estrogen by the granulosa cells under the influence of FSH. This is a redox regulated process catalysed by cytochrome P450 side chain cleavage activity within the mitochondria, to actively convert cholesterol to pregnenolone and other products. These events depend on the movement of electrons from the mitochondrial electron transport chain (ETC) to the P450 enzyme mediated by adrenodoxin and NADPH-dependent FAD flavoprotein, adrenodoxin reductase (Figure 3; Step 4).The reliance on electrons transfer elevates the risk of ROS production [92] which is in turn quickly controlled by peroxidase and catalase and other non-enzymatic antioxidants in granulosa cells to ensure redox balance and limit oxidative damage [68,69].

5. ROS, Corpus Luteum Function and Menstruation

Following ovulation, the corpus luteum (CL) is formed from the remaining follicular structure and is tasked with producing progesterone to delay the shedding of the endometrium, thereby allowing implantation to occur. Nitric oxide (NO) promotes blood flow and angiogenesis within the newly formed CL. Low levels of ROS generation also stabilize Hypoxia-Inducible Factor 1-alpha which triggers the release of Vascular Endothelial Growth Factor (VEGF), which is the primary engine driving the massive influx of blood vessels into the CL (Figure 3; Step 5) [93]. Whilst ROS and RNS are crucial in the establishment of the corpus luteum, there is a simultaneous shift towards the upregulation of antioxidant protection. Thus, the CL, literally the ‘yellow body’, acquires its colour from the presence of a powerful antioxidant, β-carotene, designed to limit the amount of oxidative damage incurred by the CL during the luteal phase. Together with a parallel increase in SOD activity, ROS are actively scavenged during the lifespan of the CL in order to prolong the generation of progesterone.

However, in the absence of a conceptus, SOD expression decreases, and ROS accumulate leading to the initiation of luteolysis [94,95]. During this process, ROS uncouple the LH receptor from adenylyl cyclase and inhibit steroidogenesis by interrupting transmitochondrial cholesterol transport. ROS (particularly lipid peroxides) also activate the cyclooxygenase (COX-2) responsible for regulating the production of prostaglandin F2α (PGF2α) and trigger the induction and expression of a transcription factor, nuclear factor-kappa B (NF-κB), that binds to the promoter region of the PTGS2 gene encoding COX-2, dramatically enhancing PGF2α synthesis [95,96]. The latter then acts on both luteal cells and inflammatory cells such as macrophages and neutrophils in the immediate vicinity, prompting them to upregulate ROS production in a self-perpetuating cycle to complete the destruction of the CL in preparation for the initiation of another menstrual cycle.

In concert with luteolysis, ROS are also mediators of the endometrial regression that characterises menstruation. In this context, the fall in progesterone levels resulting from the lack of human chorionic gonadotrophin (hCG) signalling from the foetus, creates a pro-inflammatory state that activates NF-κB and triggers the expression of several genes with a key role in menstruation including COX-2, MMPs and cytokines [97,98]. The latter then trigger the infiltration of leukocytes and the generation of more ROS. Oxidative stress, exacerbated by local hypoxia caused by PGF2α-induced vasoconstriction, triggers widespread apoptosis in the upper layer of the endometrium (stratum functionalis) allowing endometrial shedding, while minimizing damage to the underlying tissues and preventing development of a chronic inflammatory condition that might damage future fertility [97,98].

6. ROS Involvement in the Establishment of Pregnancy

6.1. ROS and Early Embryonic Development

Following ovulation, ROS are centrally involved in the rapid senescence of the unfertilized oocyte thereby ensuring that an ageing egg is rapidly eliminated and cannot contribute a potentially damaged genome the next generation [99]. If fertilization does occur, physiological concentrations of ROS are involved in pronuclear formation, initiation of cleavage and subsequent cell proliferation [100,101]. However, it is important to note that the needs of the embryo change as embryo development progresses and therefore reasonable to infer that ROS production would also vary. During the cleavage stages of embryonic development, energy production relies on the oxidation of pyruvate via carboxylic acid metabolism with lactate being produced as a by-product [101,102]. At this stage of embryogenesis, ROS production is relatively low, and the embryo is extremely vulnerable to oxidative stress. This offers a mechanistic explanation for the limitations experienced in early embryo culture systems including the 2-cell block in mice and the 4-cell block in human embryos [102,103]. Such blocks occur at the moment of zygotic genome activation due to ROS accumulation in the high oxygen tension environments employed in traditional embryo culture systems, impacting effective activation of the embryonic genome and thus hindering cellular division [104,105].

When the embryo develops into a blastocyst, the mitochondria increase their contribution to overall metabolism and ROS generation is accelerated due to electron leakage from the ETC (Figure 4; Step 1). The ROS generated at this time influence patterns of gene expression, generating proteins that support embryo growth and differentiation [106,107]. Central to this developmental response to oxidative stress is the ability of ROS to activate signal transduction pathways such as MAPK, which is centrally involved in the process of lineage determination [108,109,110]. Through their ability to cross link cysteines on the redox-sensing protein KEAP1, ROS are also able to stabilise the transcription factor NRF2, which regulates genes involved in maintaining redox homeostasis [111]. Other transcription factors important for development including hypoxia-inducible factors and proteins in the FoxO subfamily are also responsive to ROS and, again, play a key role in ensuring that the developing embryo does not suffer from oxidative stress [112]. The generation of ROS in the early embryo largely involves NOX, complexes I-III of the mitochondrial ETC and xanthine oxidase, while superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins are the major defences against oxidative damage [112,113,114,115].

In addition to their role in embryonic cell division and differentiation, ROS are also powerful mediators of cell death, ensuring that damaged cells do not contribute to the developing embryo and that defective embryos do not contribute to future generations. The use of ROS in the apoptotic deletion of defective cells is therefore a quality control measure ensuring that the maternal investment in pregnancy will ultimately be productive.

6.2. ROS and Implantation

Implantation involves synchronised interactions between the trophoblast, derived from the trophectoderm of the blastocyst, and the epithelial lining of the uterus. In humans, the process of implantation involves three basic phases: apposition of the activated and hatched blastocyst to the endometrial lining, adhesion, and finally invasion of the decidualised endometrial lining. The decidual cells regulate the invasion of the blastocyst through immune responses, paracrine signalling as well as other molecular signalling pathways. Mounting evidence suggests that physiological concentrations of ROS contribute to the cellular and molecular events underlying the implantation process [116,117].

Once formed, the blastocyst must hatch out of the zona pellucida before implantation can occur. The vigorous pulsing activity of the blastocyst at the time of hatching necessitates a sudden increase in mitochondrial ATP production which, in turn, leads to a spike in local O₂^∙-production as a consequence of electron leakage from the mitochondria, with possible additional input from NADPH and xanthine oxidase activities [118,119] (Figure 4; Step 1). Thomas et al., demonstrated the pivotal role of ROS, specifically O₂^∙-, plays in blastocyst hatching in the mouse [118]. Their studies on murine embryos, involved comparison of blastocysts in pre, peri- and post- hatching stages. While blastocysts in both pre- and post- hatching stages recorded low levels of O₂^∙- and high SOD activity, blastocysts in the peri-hatching stage showed the opposite. The addition of SOD to peri-hatching blastocysts in vitro decreased hatching, while direct exposure of such embryos to O₂^∙- enhanced this process [118]. ROS generated from this oxidative burst activate a family of enzymes called ADAMs (A Disintegrin and Metalloproteinases), particularly ADAM17, which cleaves pro-HB-EGF (Heparin-binding epidermal growth factor-like growth factor) to release the biologically active molecule, HB-EGF (Figure 4; Step 2). The latter then promotes the rapid proliferation of trophectoderm cells and the accumulation of fluid within the blastocoel. This increases the internal hydrostatic pressure, physically stretching the ZP from the inside and forcing this structure to fracture. HB-EGF signalling also triggers the blastocyst to produce and secrete lysins or proteases that chemically digest and weaken the ZP, making it easier for the embryo to escape. ROS also silence protein tyrosine phosphatases which would otherwise suppress HB-EGF signalling by dephosphorylating the cognate receptor (EGFR) [120,121,122,123,124].

A hatched blastocyst must then engage in a series of interactive events with the endometrium to allow for successful implantation, such as apposition, adhesion/attachment and invasion. Hormone synthesis, inflammatory like events, uterine secretions and gene expression all have a part to play in orchestrating these critical changes allowing for interactions between the endometrial lining and the blastocyst [124]. During apposition, the uterine wall secretes lysophosphatidic acid (LPA) the production of which involves the oxidation of low-density lipoprotein (LDL) by ROS [125]. LPA, in turn, triggers a downstream signalling pathway involving G protein-coupled receptors and phospholipase C leading to the release Ca²⁺ and activation of protein kinase C (PKC). This process culminates in the shedding of embryonic HB-EGF into the uterine environment signalling the blastocyst’s presence [126]. LPA also drives COX-2-derived prostaglandin E₂ (PGE₂) production in the luminal epithelium and the stroma at the site of adhesion. In addition, LPA increases nitric oxide synthase activity leading to the production of NO at the implantation site [126,127]. The combination of PGE₂ and NO plays a key role in angiogenesis and vascularisation leading to the evolution of a highly vascularised nidus capable of supporting implantation.

Decidualisation of the endometrial cells is another important facet of implantation characterized by the morphological remodelling and differentiation of maternal stromal cells during the secretory phase of the menstrual cycle. Decidualisation is mediated by oestrogen, progesterone, transcription factors, cytokines and other complex signal transduction pathways. This process sees the elongated, fibroblast-like cells of the uterine stroma transforming into rounded, epithelioid-like cells. Interestingly, decidualisation not only involves a morphological transformation of existing stromal cells but is also associated with the influx of inflammatory cells into the uterus prior to blastocyst adhesion [127,128]. The initiation of decidualization is a biphasic process involving ROS [129]. While progesterone and cAMP are the primary drivers, they use ROS to activate certain genes. Thus, decidualization triggers the activation of a free radical-generating NADPH oxidase (NOX-4) producing a burst of O₂^∙- and H₂O₂ inside the cell which is needed to activate the transcription factor C/EBPβ (CCAAT/enhancer-binding protein beta) (Figure 4; Step 3). This factor then binds to the promoters of key decidual markers like prolactin and insulin-like growth factor-binding protein-1 (Figure 4; Step 4) [129,130]. Significantly, antioxidants or NOX inhibitors can block the generation of ROS during this initiating phase of decidualization, reducing the expression of decidual markers.

In addition to this positive role for ROS in initiating the decidualisation process, the formation of these cells is associated with the upregulation of antioxidant enzymes like SOD2, catalase, glutathione peroxidase and glucocorticoid-inducible kinase-1 in these differentiated cells [131,132]. This sudden elaboration of defensive enzymes designed to protect against oxidative damage does not impair the ability of ROS to drive positive physiological changes during implantation but rather helps prepare the uterus for the approaching oxidative stress associated with pregnancy.

The final step of implantation is the invasion of the endometrial lining by the blastocyst. To accomplish this step, the trophoblast layer differentiates into different subtypes: villous cytotrophoblasts (vCTBs) and syncytiotrophoblasts (STs). The invasive behaviour and functional characteristics of cytotrophoblasts have often been compared to those observed in malignant cells, especially when considering the expression of Tubulointerstitial nephritis antigen-like 1 (TINAGL1 or lipocalin7),a structural matrix protein that interacts with integrins on the trophoblast surface and endometrial epithelium, enabling adhesion [133]. This adhesion event then triggers intracellular signalling cascades including the FAK (Focal Adhesion Kinase) and MAPK/ERK pathways, which are essential for trophoblast outgrowth and promoted by the local generation of ROS, through their ability to inhibit tyrosine phosphatases [134]. Cytotrophoblasts at the time of invasion also exhibit a down regulation of E-cadherin, a negative regulator of trophoblast invasion. By analogy with cancer cell invasion, the generation of ROS is thought to trigger this change in E-cadherin expression through the upregulation of transcription factors such as Snail (SNAI1) and Slug (SNAI2), which are transcriptional repressors of the CDH1 gene (which encodes E-cadherin) as well as stimulation of the MAPK/ERK kinase pathway [135,136,137]. ROS can also stabilize Hypoxia-Inducible Factor 1-alpha (HIF-1α), which then promotes the expression of E-cadherin repressors to facilitate trophoblast invasion [138,139]. During the invasive stage of embryo implantation, the syncytiotrophoblast expresses endothelial nitric oxide synthase to increase the production of NO, a vasodilator, which is of critical importance in establishing the placentation process [139]. Trophoblast invasion is also facilitated by a range of MMPs, (MMP-1, MMP-2, MMP-3 MMP-9, MMP-11 and MMP-14) that have been identified in human placentae [140,141]. ROS are critical for the activation and production of MMPs via pathways that involve the upregulation of MAPK and PI3K/Akt [142,143,144]. These enzymes are central to the implantation process, promoting degradation of the ECM and allowing the trophoblast to break through the uterine lining.

7. ROS and Parturition

The fertility journey culminates in parturition, a complex process featuring hormonal fluctuation, inflammatory reactions and rapid tissue remodelling. ROS are physiologically involved in several aspects of parturition, including the myometrial contractions that initiate labour and the ripening of the cervix, largely through activation of the MAPK pathway. P38 MAPK proteins, modulated by ROS, initiate the release of pro-inflammatory cytokines and prostaglandins from the uterine lining [145]. Within the same tissues, oxytocin in turn activates NF-κB mediated inflammatory signalling pathways leading to stimulation of ROS generation via NADPH oxidases (primarily NOX 1 and NOX4), the sensitisation of oxytocin receptors and the onset of myometrial contractions leading to labour [146,147]. Further evidence of the crucial role of ROS during parturition can be seen in the ability of non-enzymatic antioxidants to reduce the risk of preterm birth [147]. In this context, N-acetylcysteine was found to decrease COX-2, and prevent the activation of NF-κB, by scavenging local ROS generation and inhibiting some of the key downstream signalling pathways involved in the onset of labour [148].

8. The Positive Role for ROS and Reductive Stress

Given ROS play such an important role in the biology of reproduction, it would be reasonable to ask whether the indiscriminate administration of antioxidants, albeit with the best of therapeutic intentions, might impede rather than promote this process. The over supplementation of healthy individuals who are not suffering from oxidative stress with antioxidants, runs the risk of impairing the myriad cellular signalling pathways that rely on physiological levels of ROS, as detailed in this review, generating a state of reductive stress. The latter is just as associated with infertility issues as oxidative stress [149]. In vitro scavenging of ROS suppresses tyrosine phosphorylation events associated with sperm capacitation, inhibiting hyperactivated motility, disrupting acrosomal exocytosis and, in animal models, reducing fertilization rates [49,150,151,152,153]. Similarly with oocyte maturation, while low doses of antioxidant can facilitate this process, high doses delay or completely suppress the ability of mammalian oocytes to undergo meiotic maturation in vitro and ovulate in vivo [154,155,156,157,158]. High doses of antioxidant have also been found to have a negative impact on both cleavage and blastocyst development rates in vitro, while compounds with antioxidant properties such as sanguinarine and EGCG have also been found to suppress blastocyst implantation and post-implantation embryonic development [159,160]. Later in pregnancy, coenzyme Q10 administration has been found to increase oxidative stress in rats [161]. Furthermore, because ROS play a crucial role in the fetal brain-sparing response (prioritization of oxygen and nutrient delivery to the brain, heart and adrenal glands at the expense of other organs during pregnancy), there are also concerns that excessive antioxidant use by pregnant women could weaken foetal defences against acute hypoxia, increasing the risk of hypoxic–ischaemic encephalopathy [162]. Towards the end of pregnancy, antioxidants have been used in an attempt to address complications such as pre-eclampsia, pre-term labour, foetal death, foetal growth restriction and stillbirth but no positive outcomes have been recorded. Indeed, occasionally detrimental impacts of such treatment have even surfaced including decreases in human chorionic gonadotrophin generation, foetal growth restriction, low birthweight, gestational hypertension and others [163,164,165,166].

In terms of mechanisms, over-supplementation with antioxidants can drive mitochondrial ROS generation by enhancing the reduced status of key electron donors to the ETC. This leads to increases in the NADH:NAD⁺ and FADH₂:FAD ratios to the point that the ETC cannot cope; electrons leak directly, or following reverse electron transport to Complex I, and are swept up oxygen, to generate O₂^∙-, which then rapidly dismutates to H₂O₂ under the influence of SOD. Similarly, increases in the NADPH: NADP⁺ ratio can favour the activation of NADPH oxidases such as NOX 4 and NOX5, generating ROS and impairing cell function [167].

High doses of powerful reductants like vitamin C can also enhance oxidative stress by promoting Fenton chemistry, whereby the reduced form of transition metals such as iron and copper can promote the generation of free radicals. High levels of certain antioxidants can also interfere with cellular homeostasis by disrupting the intricate redox signalling processes highlighted above or by interfering with critical homeostatic mechanisms such as apoptosis or protein folding. Some antioxidants such as polyphenols may also have chemical structures (large planar hydrophobic molecules) that at high concentrations can insinuate themselves into membranes disrupting cellular activity by promoting electron leakage and ROS generation from the mitochondria or disrupting receptor activation and signal transduction at the plasma membrane. They can also intercalate into the DNA, distorting the DNA backbone, disrupting chromatin compaction and inducing DNA fragmentation [168,169,170]. At high doses, instead of protecting the genome from oxidative stress, such molecules can induce DNA strand breaks and/or inhibit repair enzymes like topoisomerase [171].

9. Conclusions

So, ROS are physiologically important at all stages of the reproductive process. While oxidative stress may well contribute towards many of the pathologies affecting human fertility, antioxidants should be utilised with care, with the aim of ensuring that an appropriate redox balance is maintained and that critical biological processes can continue unimpaired. This is especially important as there is now growing evidence for reductive stress negatively impacting the reproductive process from gametogenesis to parturition [172]. To rationalize the use of antioxidants to treat reproductive deficiencies in vivo and in vitro, we need to consider much more carefully the dose and specific structure of the antioxidants used in clinical practice. Too often, antioxidant administration is not calibrated with the level of oxidative stress being experienced by the patient or the cells being targeted. Indeed, antioxidants are frequently administered in vivo and in vitro without any diagnostic assessment of oxidative stress [173]. In addition, too little consideration has been given to the source and type of oxidative stress when selecting antioxidants in terms of their physicochemical properties (charge, size, hydrophobicity) or mode of antioxidant action (one electron-, two electron-, or hydrogen atom- donating) in optimizing their biological action. With the introduction of new ART culture media formulations enriched with antioxidants and an abundance of articles highlighting the negative impacts of ROS on ART outcomes, there is a risk that antioxidants will be used in doses that compromise the physiological role of ROS in driving the reproductive process and inadvertently create a state of reductive stress, which can be just as damaging as its oxidative counterpart.