Antioxidant therapy for infertile couples: a comprehensive review of the current status and consideration of future prospects

The term oxidative stress (OS) applies when increased amounts of reactive oxygen species (ROS) surpass the antioxidant (AOX) capacity that naturally offers the protective balance ( 1 ). While OS plays a major role in the pathogenesis of male infertility ( 2 – 4 ), it is equally important to recognize the critical physiological roles of ROS in normal reproductive functions. ROS are indispensable for processes such as sperm capacitation, hyperactivation, and the acrosome reaction, which are essential for successful fertilization ( 5 , 6 ). However, an imbalance favoring excessive ROS production can lead to oxidative damage, negatively affecting male fertility potential and reproductive outcomes ( 7 , 8 ). Also, OS has been linked with many female infertility conditions such as polycystic ovary syndrome (PCOS) ( 9 , 10 ), endometriosis ( 11 – 13 ), premature ovarian failure (POF) ( 14 ), and recurrent miscarriages (RM) ( 15 ). Hence, AOX therapy to enhance fertility by protecting against the harmful effects of ROS is theoretically feasible. AOXs are not regulated as pharmaceutical drugs, thus allowing their utilization without prescription. Indeed, AOXs are extensively consumed due to their easy availability through different retail outlets ( 16 ). Also, the relatively low cost and good safety profile of AOX products are appealing to both healthcare providers and infertile couples. However, a significant diversity exists among practitioners in terms of prescribing AOXs for the purpose of infertility treatment ( 17 ). In this review, we comprehensively discussed the types, mechanisms of action and safety profile of AOXs that are commonly prescribed in infertility clinics. We also highlight the attitudes and practice patterns of clinicians towards AOX prescription during the course of infertility treatment, and compare them with current recommendations by professional societies. Additionally, we discuss the impact of AOX therapy on fertility outcomes in men and women under natural and assisted reproduction conditions. Furthermore, we highlight limitations of the current research on utility of AOXs for treatment of infertile couples and the need for well-designed studies that can bridge the gap between research and practice in this regard.

An AOX is a natural substance that provides protection to the living cell against oxidative damage and deleterious effects of ROS ( 18 ). Under normal circumstances, there is a fine balance that enables AOXs to neutralize excess ROS, and maintain a small amount of ROS necessary for normal cell functions. Thus, when ROS production increases to harmful levels, AOXs play a key role in neutralizing them. In the living cell, the AOX defense system is generally classified into enzymatic and non-enzymatic elements. Enzymatic AOXs include glutathione (GSH) peroxidase, glutathione reductase, catalase (CAT) and superoxide dismutase (SOD) ( 19 ). Nonenzymatic AOXs provide protection against ROS by binding to enzymatic AOXs and ions that helps subsequent suppression of enzymes involved in the oxidation process ( 16 ). They function by scavenging free radicals, stabilizing enzymatic AOXs, and chelating metal ions, thereby mitigating OS. Prominent non-enzymatic AOXs include vitamins such as Vitamin C and Vitamin E ( 17 , 20 , 21 ), glutathione ( 22 , 23 ), coenzyme Q10 ( 24 – 27 ), carotenoids (e.g., beta-carotene, lycopene) ( 28 ), polyphenols ( 29 ), and trace elements like selenium and zinc ( 24 , 30 ). AOXs are available in the market as individual or combined products. Generally, AOXs utilized in clinical practice are categorized as substances with direct AOX actions or substances with AOX properties ( 16 ). Table 1 summarizes the types, natural dietary sources and the mechanism(s) of action of AOXs. Types, dietary sources and mechanism of action of antioxidants. AOX, Antioxidant; CoQ10, Coenzyme Q10; NAC, N-acetyl cysteine; ROS, Reactive oxygen species.

Available research on AOX therapy is surrounded by several limitations and biases, including small sample sizes, heterogenous patient populations and inclusion criteria, poor study designs, and inconsistent reporting of clinical outcomes. Additionally, the use of a wide range of AOXs (either single or in combinations) and the lack of standardized dosages in different studies preclude the ability to draw a firm conclusion on the efficacy of AOX supplementation in solving infertility problem. This has led to the skepticism of the current guidelines by professional societies regarding AOX therapy for infertility. This may also explain the considerable variability that currently exists in practitioners’ attitude towards the prescription of AOX therapy for the purpose of overcoming infertility issues. Such variability surrounds many aspects of AOX therapy including pre-testing the patient for OS, the choice of a specific AOX or AOX combination, proper dose, optimum duration of therapy and the desired outcome. Therefore, there is a great demand for high-quality research to clarify the exact role of AOXs in the context of infertility treatment. Future studies should focus on careful diagnostic work-up to assess OS indices as an essential pre-request before starting AOX therapy ( 206 ). Such test would be helpful for selecting patients with high levels of OS who are candidates for AOX therapy, and for monitoring the treatment response. This in turn requires the availability of reliable tools for confirming the diagnosis of OS and evaluation its severity. Indeed, this is technically difficult in women. However, assessment of seminal OS in men is feasible using some tools such as the MiOXSYS System that can help assess oxidation-reduction potential (ORP) levels in semen and seminal plasma ( 207 ). The test would help personalize treatment for cases with high seminal ORP such as idiopathic infertility, varicocele or those with genital tract infection. The 6th Edition of the World Health Organization (WHO) manual for human semen analysis classifies seminal OS testing as a research tool, emphasizing the lack of robust evidence to confirm its clinical utility ( 208 ). Despite this designation, OS testing is extensively utilized in andrology research, clinical laboratories, and ART centers, underscoring its recognized importance in advancing the understanding of male fertility. Future studies are also warranted to investigate the impact of AOX on LBR, MR, and SDF since only few well-designed studies have analyzed these outcomes in infertile patients following AOX therapy. Additionally, future studies are needed to determine the proper treatment duration, optimum dosage as well as the ideal supplement (single vs. combined AOX) ( 206 ). Furthermore, it is essential to ensure that there are no negative consequences of utilizing AOXs as a therapeutic option for infertile men.

AOXs are generally well tolerated and have a good safety profile. However, the fact that AOXs are largely available over-the-counter with a reasonable price increases the risk that some patients can overmedicate with AOXs leading to detrimental effects. It has been suggested that administration of large amounts or improper combinations of AOXs may lead to shift of the redox balance towards a ‘reductive stress’ status ( 31 ). The term ‘antioxidant paradox’ highlights the potential negative impact of over-utilization of AOX supplementation ( 32 , 33 ). Recent reports indicate that the intake of excess AOXs may even cause impairment of male fertility potential due to reductive stress ( 34 , 35 ). However, there is no clear understanding of the exact mechanism of action of reductive stress on male reproduction, and further research is required. Additionally, high doses of AOXs may be associated with some adverse effects. Furthermore, it is imperative to consider the role of excipients or fillers in commercially available supplements. For instance, titanium dioxide, previously regarded as a safe additive, has recently been implicated in genotoxicity, raising concerns about its safety. These findings underscore the need for a judicious and evidence-based approach in the formulation and administration of AOX supplements to ensure both their safety and therapeutic efficacy ( 36 , 37 ). A summary of potential adverse effects of AOXs frequently used in the treatment of infertile couples is provided in Table 2 ( 38 – 54 ). Potential adverse effects of antioxidants. Doses given are for adult male. AOX, antioxidant; CoQ10, Coenzyme Q10; NAC, N-acetyl cysteine; PUFAs, Polyunsaturated fatty acids.

OS plays a significant role in couples’ infertility. The theoretical basis for AOX therapy to counteract the harmful effects of ROS on fertility is feasible. In infertile men, it is hypothesized that cases with elevated seminal OS may benefit from AOX therapy. Hence, several AOXs, including vitamins E and C, carotenoids, carnitine, cysteine, CoQ10, selenium, zinc, and folate, have been employed as potential therapeutic interventions for male infertility. However, results of clinical trials remain controversial, and current guidelines from major professional societies offer no definitive recommendations regarding AOX use in male infertility. AOXs have shown promise in the management of PCOS, endometriosis, poor ovarian reserve and unexplained infertility. Also, AOX supplements such as inositol, L-carnitine, ALC and CoQ10 have shown potential for women undergoing ICSI, particularly those with previous ICSI failure. However, current evidence supporting AOX in improving important reproductive outcomes such as LBR remains low, necessitating further studies. Additionally, further research is needed to evaluate the exact role of AOX therapy and to identify the most effective single AOX or AOX combinations in these conditions.

OS in an important factor in the success of medically assisted reproduction (IUI, IVF and ICSI) ( 112 , 128 , 154 ). In these techniques, OS can result from endogenous as well as exogenous sources. These factors and the effect of AOXs on ART are discussed in this section ( Figure 1 ). One of the main indications of IUI is the presence of oligozoospermia, asthenozoospermia or teratozoospermia or a combination of the three parameters in the male partner of the infertile couple. As mentioned previously, male partners with these defects exhibit OS in their semen ( 206 – 208 ). In addition, culture media used in the preparation of the semen as well as those used in cell cultures have varying amounts of ROS which adds to the OS burden to which spermatozoa are exposed ( 209 , 210 ). Moreover, the process of sperm preparation deprives the spermatozoa from the natural AOXs present in the seminal plasma leading to accumulation of ROS. This is even more pronounced when centrifugation is used in the preparation (e.g. Percoll gradient separation). Cryopreservation and thawing are extra sources of OS when frozen semen is used for IUI ( 211 ). Accordingly, AOXs are used to treat the male partners of the infertile couples as mentioned previously ( 16 , 66 ). In addition, AOX supplementation of the sperm preparation medium for IUI was used in an attempt to counteract the effects of OS. Pentoxifylline exerts several AOX activities, such as the maintenance of GSH levels and mitochondrial viability ( 212 ). The addition of pentoxifylline to the sperm preparation medium resulted in improvement of semen characteristics in asthenozoospermic patients ( 213 ). Additionally, CPR was significantly increased by adding pentoxifylline to the semen preparation medium for IUI (27.5% versus 11.5% in the control group) ( 214 ). Similarly, the addition of caffeine to the semen preparation medium for IUI was described ( 215 ). Finally, adjusting the sperm and embryo cryopreservation media by adding AOXs is used to mitigate the effect of OS ( 216 ). The techniques of IVF and ICSI exert a considerable amount of OS on the gametes and embryos. In addition to the endogenous and exogenous sources of OS mentioned previously to which spermatozoa are subjected, the oocytes and embryos are also subjected to similar OS ( 217 ). For example, oocytes lose their AOX protection contained in the cumulus cells during the denudation process. The changes of temperature and the osmotic and pH changes during the oocyte and embryo handling (denudation, pipetting, etc.) are all sources of OS ( 100 ). In addition, exogenous sources of OS to which the gametes and embryos are subjected include exposure to the atmospheric O 2 , the high oxygen concentration in the incubator (compared to the 2% concentration in the fallopian tube), volatile organic compounds present in the laboratory air or released from the consumables (plastic ware), visible light, humidity in the incubator and the type of mineral oil used. The processes of cryopreservation and thawing are also sources of OS ( 100 ). Consequently, many steps are taken in the IVF laboratory in order to counteract or diminish the effect of this OS ( 128 ). Accordingly, men and women undergoing IVF or ICSI can be treated with AOXs in an attempt to improve the clinical results. Treatment of infertile men with the AOX “Menevit” for 3 months prior to IVF resulted in higher viable pregnancy rate (38.5%) compared to the control group (16%). The study enrolled 60 couples undergoing IVF-ICSI treatment, who were randomly assigned into two groups in a 2:1 ratio: one receiving the AOX Menevit and the other a placebo. Male participants were included based on evidence of OS, indicated by impaired sperm parameters such as poor morphology, reduced motility, or compromised membrane integrity, in conjunction with significant SDF (>25% TUNEL positivity). Female partners were excluded if they exhibited diminished ovarian reserve, defined as fewer than five oocytes retrieved in a prior IVF cycle or elevated early follicular phase FSH levels, or were aged over 39 years ( 218 ).​ The Cochrane review published in 2019 showed that although the use of AOXs for men prior to IVF did not improve the CPR (OR = 2.64, 95% CI 0.94 to 7.41), the LBR increased significantly (OR = 3.61, 95% CI 1.27). However, the authors of the review noted that these data were based on 2 RCTs only and that further studies are needed ( 11 ). Women undergoing IVF and ICSI were also treated with AOXs prior to the procedure with variable claims of success. These treatments include NAC, melatonin, coQ10, thiamine, riboflavin, niacin B3, vitamins B6 and B12, folate, vitamins A, C, D and K, calcium, phosphorus, magnesium, sodium, potassium, chloride, iron, zinc, copper, selenium, iodine, vitamin E, vitamin K, L-arginine, inositol, biotin, pantothenic acid, eicosatetraenoic acid (EPA), docosahexaenoic acid (DHA) and various combinations of the above ( 188 ). The treatment is usually given for 3 to 6 months prior to the procedure. Pre-treatment of IVF patients with AOXs improved embryo quality, particularly in poor responders ( 219 ). Indeed, a significantly lower glutathione-S-transferase (GST) enzyme activity and significantly high MDA levels have been shown in the cumulus cells of poor responders compared to high responders ( 220 ). However, the Cochrane review conducted by Showell et al. in 2020 showed no improvement in the CPR (OR = 1.15, 95% CI = 0.95 – 140) or LBR (0.94, 95% CI = 0.41- 2.15) when AOXs were given to women treated with IVF or ICSI ( 188 ). In this Cochrane review, the LBR was calculated from 2 small RCTs and it is therefore clear that more studies are needed, particularly in view of the multifactorial nature of IVF and ICSI. Finally, supplementation of embryo culture media with AOXs was shown to improve the quality of the embryos, the implantation rate as well as the CPR in IVF/ICSI cycles ( 221 , 222 ). Furthermore, the addition of a combination of AOXs (ALC, NAC and α-lipoic acid) to the cryoprotectant medium of mice embryos resulted in increased blastocyst cryo-survival and viability post-vitrification ( 216 ). The outcome of ICSI is known to be influenced by a myriad of factors. In a recent Cochrane review, it was stated that the chance of live birth with ICSI is between 30% and 41% ( 223 ). Consequently, efforts are directed to maximize ICSI outcomes. Increased OS has been identified as an important factor governing success of ICSI ( 112 ). Thus, it is plausible to use AOX in an endeavor to optimize the outcome of ICSI. Although the list of publications on the use of AOX in couples undergoing ICSI is steadily growing, knowledge on the relationship between AOX and ICSI outcomes is lacking ( 224 ). An RCT including 85 women with different indications for ICSI who have been supplemented with AOX daily from the cycle preceding ICSI cycle, versus 85 women who were not given any AOX acting as controls, concluded that AOX therapy did not change clinical or laboratory outcomes ( 225 ). However, A recent systematic review indicated a favorable impact of AOX on pregnancy outcomes following ICSI ( 224 ). In a recent systematic review and meta-analysis of RCTs, it was concluded that oral supplementation of CoQ10 may increase clinical pregnancy rates, without an effect on LBR, and MR compared with placebo or no-treatment in women with infertility undergoing ICSI ( 226 ). Moreover, a recent meta-analysis concluded that melatonin treatment significantly increases the CPR but not LBR in ART cycles. Melatonin treatment also increased the number of oocytes collected, maturate oocytes, and good quality embryos. There was no evidence suggesting that melatonin treatment increases the adverse events in ART cycles. However, the findings of this meta-analysis should be explained cautiously due to remarkable heterogeneity of the included IVF patients ( 227 ). AOXs such as inositol, L-carnitine, CoQ10, and ALC may be helpful for women undergoing ICSI or in cases of previous ICSI failure ( 226 ). However, the role of AOXs in increasing LBR is limited by low-quality evidence ( 188 ). Indeed, conducting large controlled clinical trials using AOXs supplementation is mandatory to evaluate their influence on ICSI outcome.