Oxidative stress and reproductive function

We have been aware that oxidative stress is a key factor in the aetiology of infertility since the importance of alpha-tocopherol for male reproductive function was first established in the 1930s and 1940s (Mason 1942). With the passage of time, it has become apparent that this form of stress impacts every aspect of the reproductive process from gametogenesis to the birth of healthy offspring. In this special issue of Reproduction, we have highlighted the impact of oxidative stress at the earliest stages of development from the primary generation of spermatozoa and oocytes, through their union at fertilization and the subsequent development of a preimplantation embryo (Video-1). Of course, this is not the whole story. Redox mechanisms are centrally involved throughout the reproductive process and are known to be instrumental in the orchestration of such processes as luteolysis, the preparation of an endometrial nidus for the embryo, blastocyst implantation, placental development and the progress of pregnancy to parturition (Maiti et al. 2017, Hussain et al. 2021). Oxidative stress can disrupt any and all of these processes, leading to a range of pathological conditions that compromise reproductive function, including endometriosis, polycystic ovarian disease, infertility, gestational diabetes mellitus, intrauterine growth restriction, preeclampsia, preterm labour and fetal death (Aitken et al. 2022).

Across all of these different elements of the reproductive process, a central theme is that a functional reproductive system depends on the need to maintain an appropriate cellular redox balance. The term ‘oxidative stress’ refers to the emergence of pathology when excessive levels of reactive oxygen species generation overwhelm the cells’ intrinsic antioxidant defence capacity, disturbing the redox balance in favour of oxidation, i.e. electron loss. The antioxidant protection designed to protect cells from such attacks comprises a wide range of small molecular mass free radical scavengers like vitamin E, vitamin C, taurine, uric acid and thiols such as glutathione. It also encompasses a range of antioxidant enzymes, many of which are widespread in biological systems such as superoxide dismutase, catalase and glutathione peroxidase. However, every cell type is different in terms of the range and balance of antioxidant factors needed to maintain normal cellular function. In this special issue, O’Flaherty and Scarlata (2022) expertly summarize the antioxidant systems employed by spermatozoa and highlight the versatility and importance of peroxiredoxins in this context, particularly peroxiredoxin 6. Similarly, Deluao et al. (2022) provide a detailed description of the antioxidant systems employed by embryos to prevent them from entering into a state of oxidative stress, while Martin et al. (2022) emphasize the importance of antioxidants in defending the functional integrity of oocytes, particularly in the context of ageing.

While the focus of such reviews is inevitably on oxidative stress, we should not lose sight of the fact that imbalance in the redox systems underpinning the reproductive process may also pivot in the opposite direction, creating a state of ‘reductive stress’. In this case, the presence of reductants (electron donors) is so overwhelming that the oxidative processes that drive cell function are suppressed. Excessive levels of reduced NAD⁺ (NADH), reduced NADP⁺ (NADPH) and reduces glutathione (GSH) can be as harmful as oxidative stress in eliciting a pathological change and, biomedically, have been implicated in such clinical conditions as cardiovascular disease and diabetes mellitus (Xiao and Loscalzo 2020). It is uncertain how often reductive stress disrupts the reproductive process; however, it may be a significant factor in the clinical confusion generated by the indiscriminate administration of antioxidants to patients who are not suffering from oxidative stress.

All of the reviews highlighted in this special issue advocate the use of antioxidant therapy to counteract pathologies dependent on oxidative stress. Despite the indisputable logic of such therapeutic intervention, the results of clinical trials where antioxidants have been used to treat either male or female infertility have generally been disappointing. Successive Cochrane reviews hint at a possible therapeutic benefit from antioxidant therapy but fall short of a confident recommendation. Thus, a recent review addressing the use of antioxidants to treat male infertility concluded that the data reveal ‘very low-certainty evidence from … randomised controlled trials suggesting that antioxidant supplementation in subfertile males may improve live birth rates for couples attending fertility clinics’ and ‘low-certainty evidence suggests that clinical pregnancy rates may increase’ (de Ligny et al. 2022). An analysis of the use of antioxidants to treat female infertility similarly concluded that ‘there was low- to very low- quality evidence to show that taking an antioxidant may benefit subfertile women’(Showell et al. 2020). These detailed reviews are certainly suggestive of a positive effect, but they hardly amount to ringing endorsements. Clearly, the clinical trials conducted to date have failed to deliver the kind of high-quality data that can either refute or confirm the benefits of antioxidant therapy in the promotion of reproductive success. Apart from being appropriately organized in terms of patient number, randomization, controls and, ideally, a cross-over design, there is one basic flaw in most of the studies conducted to date – they have failed to select patients on the basis of any evidence of oxidative stress. Giving antioxidants to patients who are not suffering from an oxidative attack runs the risk of driving them into a state of reductive stress that may have a detrimental impact on their fertility. To give one example of many, the administration of antioxidant vitamins, zinc and selenium to males suffering from high levels of DNA damage in their spermatozoa was found to induce reductive stress characterized by a dramatic decondensation of the sperm chromatin, presumably due to the reduction of disulphide bridges in the protamine networks responsible for stabilizing the sperm nucleus (Ménézo et al. 2007).

The administration of antioxidants is therefore not a benign therapeutic strategy; it is capable of inducing as much harm as good. Clearly the field is now desperate for a simple point-of-care assay in order to determine the levels of oxidative stress suffered by any given patient and to gauge the level of antioxidant supplementation needed to bring the redox system back into a state of balance. This is a point that has been made many times, and yet we still do not have a simple validated diagnostic technique capable of delivering a rapid assessment of a given patient’s redox status (Aitken 2020, 2021). The MiOXSYS system has recently been introduced to fill this clinical need, at least in the context of male infertility. This solid state system gives a read out of redox potential in unprocessed human semen samples. The method is simple, rapid and based upon well-established electrochemical principles. While this diagnostic system is thought to measure the balance of ROS generation by the spermatozoa and antioxidant protection provided by seminal plasma (Agarwal et al. 2016), recent data suggest that removal of the spermatozoa from the semen has no impact on the redox potential measurements obtained (Joao et al. 2022). It therefore seems as if such measurements are dominated by the antioxidant properties of seminal plasma. A second problem with this system appears to be the way in which the results are expressed. The manufacturer’s recommendation is to take the redox potential measurement secured with the MiOXSYS device and then divide this value by sperm count to generate an index expressed in terms of mV/million spermatozoa per millilitre. Unfortunately, this strategy creates some confusion because it is uncertain whether any correlations observed with clinical outcomes are a function of sperm count and/or oxidoreductive potential. The measurement of oxidoreductive potential in semen or blood may well have clinical relevance, as has been found to be the case in certain areas of cardiovascular medicine for instance. However, the way in which the results of such determinations are validated and expressed may need further attention.

In addition to the use of antioxidants to treat systemic antioxidant stress in the patient as a whole, several of the papers presented in this special issue mention the potential use of antioxidants in vitro as a component of the media used to culture spermatozoa, oocytes and embryos in the context of assisted reproduction (Aitken et al. 2022, Deluao et al. 2022, Martin et al. 2022). Such strategies appear to show great promise as a means of preserving the structural and functional integrity of gametes and embryos during the in vitro phase of their existence. Again, we are probably at the beginning of this field rather than the end. We do not yet have any consensus as to which antioxidants to add and in what concentrations they should be presented. There does however appear to be agreement that the major source of ROS in these different cell types involves electron leakage from the mitochondria (Aitken et al. 2022, Deluao et al. 2022, Martin et al. 2022). Refining the development of antioxidants that target the mitochondrial compartment might be beneficial in this regard, either via efficient scavenging of ROS within these organelles (Koppers et al. 2008, Cobley 2020), removing lipid aldehydes such as 4-hydroxynonenal, which are known to promote ROS formation by the mitochondrial electron transport chain (Aitken et al. 2012, Lord et al. 2015), or by controlling the mitochondrial permeability transition pore (Vercesi et al. 1997, Daiber 2010). As ever, the major task with the design of antioxidants is to suppress the overproduction of toxic reactive oxygen metabolites while allowing the redox processes that drive normal cellular function to flow freely.

Another theme to emerge from the collection of papers in this special issue is that the consequences of oxidative stress in gametes and embryos are not just confined to the question of fertility. It is also powerfully associated with the normality of the developing embryo and its capacity to develop to term and have a normal health trajectory thereafter.

As far as the oocyte is concerned, oxidative stress appears to be a significant factor in the non-disjunction of chromosomes in meiosis 1 that play a major role in precipitating a loss of developmental potential in the eggs of ageing women (Martin et al. 2022). The genetic integrity of spermatozoa is also well known to be vulnerable to oxidative attack, leading to the creation of highly mutagenic oxidative lesions in the form of 8-hydroxy-2′-deoxyguanosine (Vorilhon et al. 2018). The presence of these lesions in the fertilizing spermatozoon may affect the developmental normality of any resultant embryo in a number of ways. For example, inadequate or erroneous repair of such lesions by the oocyte following fertilization may make a significant contribution to the genetic and epigenetic mutational load subsequently carried by the offspring, which is known to be predominantly paternally determined (Aitken 2022, Aitken et al. 2022). Another consequence of oxidative stress in the male germline may be an impact on telomere length as elegantly summarized in this issue by Moazamian et al. (2022). Telomere length is known to be a paternally inherited trait that is highly dependent on paternal age. Thus, the older your father when you were born, the longer your telomeres. Moreover, the effects are cumulative across generations; so, if your grandfather was old when your father was conceived, and your father was old when you were conceived then you will have very long telomeres. Long telomeres are traditionally thought of as a buffer against the attrition that accompanies repeated cell division and to be associated with longevity. An innovative hypothesis elaborated by Moazamian et al. (2022) is that the mild oxidative stress associated with paternal ageing results in an adaptive lengthening of telomeres in the male germline in order to provide the developing spermatozoa and the progeny they sire, with a measure of protection against a stressful environment. This is possible in the developing male germline because telomerase is still active in these cells (Ozturk 2015). By contrast, in mature spermatozoa, an adaptive, telomere-lengthening response to oxidative stress is not possible because, by this stage of development, telomerase activity has been lost. However, in mature spermatozoa, telomeres can become shortened as a result of an oxidative attack, because the guanine residues in the telomeres’ TTAGGG repeats are vulnerable to such free radical-induced damage. It may be for this reason that male infertility appears to be frequently associated with a shortening of telomere length (Moazamian et al. 2022). By implication, infertile men possessing short telomeres because of an oxidative insult on the father’s spermatozoa may not only experience subfertility but a shortened life span as well. Such associations may conceivably underpin the emerging associations between male infertility and somatic health and add credence to the fascinating concept that semen quality may serve as ‘the canary in the coal mine’, providing an early warning sign of pending defects in somatic health that might otherwise remain undetected. (Hanson et al. 2017, Burke et al. 2022).

In summary, this special issue brings together a series of papers written by internationally acclaimed experts in the field that effectively captures the fundamental principles that underpin oxido-reductive stress in the reproductive system and the implications such stress has for the early stages of mammalian development. These papers also highlight that oxidative damage experienced by gametes and embryos at the beginning of development can have significant implications for the long-term health trajectory of the progeny. Developing robust diagnostic procedures to diagnose and treat such stress is a priority task for the future that will impact the reproductive efficiency of our species as well as domestic animals on whose fertility we depend.