We appreciate the comments raised by Dr. Coward and colleagues and in fact share much of the skepticism expressed over the efficacy of commercially available antioxidant therapies and questionable practices of the dietary supplement industry. Indeed, CellOxess biotechnology was founded to bring about the much needed clarity on the impact severity of oxidative stress in the etiology of many preventable diseases with our initial focus on male and female infertility and the roles antioxidants may play in alleviating the rapid decline of human fertility.

It is a fact that optimum gametogenesis is highly dependent on the redox homeostasis of the reproductive tissue. Therefore, the molecular interplay or the balance between oxidants (or toxic metabolites) generated by cells and antioxidants (the cellular guardians) become an important consideration when the gamete is evidenced to be unhealthy and fertility treatment is then sought. However, poor sperm motility, morphology, concentration, and even damage are not always indicators of chronic oxidative stress. Treating such patients with no evidence of oxidative stress with large doses of an untested combination of antioxidants, such as those used in MOXI or FAZST trials, will have virtually no chance of demonstrating the expected beneficial therapeutic effects and will in fact add more confusion to the benefits of the antioxidant therapy.

Fertilix was developed according to a stringent set of medicinal chemistry principles closely mirroring that of a pharmaceutical. Indeed, the detailed review of oxidative stress as a male infertility factor, the significance of oral antioxidant therapy1,2 (supported by Craig Niederberger3), the rationale for formulation design, as well as the preclinical evidence for Fertilix efficacy have all been covered by several publications.4 The preclinical evidence was also presented at ESHRE and ASRM.

Back in 2013, we also reached out to Dr. Eisenberg, a co-author on the MOXI publication, seeking her assistance to organize a human trial for Fertilix. Unfortunately, no progress was possible at that time. Following Dr. Eisenberg’s suggestion, we applied for NIH grants on two separate occasions in 2014 and 2016 but were turned down both times, without a clear explanation.

We ask Dr. Coward and colleagues to kindly note that, for a variety of reasons, such as market fragmentation, without the financial assistance from NIH or the cooperation of the fertility community in the trial, it is almost impossible to conduct a large, high-quality clinical study with pregnancy and live-birth outcomes as end-points. We point to the extant literature in the field of male infertility to support this statement. 

There are also other misconceptions and inaccuracies in Dr. Coward’s et al recent comments, which we would like to address as bullet points below.

• Fertilix is broadly marketed as a vitamin supplement “for every man…”: Indeed, this is true. For reasons discussed above and in our previous comment, there are three Fertilix formulations for men, differing in doses to minimize the risk of over-supplementation, which can be just as damaging to gametes as a deficiency in certain circumstances. As seen on the product website, these formulations are personalized for men with low, moderate and severe oxidative stress. In the absence of a reliable, commercially available test to accurately measure the severity of oxidative damage, we developed a simple algorithm to estimate the risk exposure of individuals to oxidative stress, derived from published epidemiological studies, with recommendations for dosing. This assessment is far from perfect, but the tool does provide qualitative information to reduce the risk of over-supplementation. Moreover, the lowest dose version, preconceptual, is designed to protect from acute or transient forms of oxidative insult (such as cold or flu infections, excess alcohol consumption, etc.), which may or may not occur in the period prior to conception. This situation is more analogous to that of female preconceptuals/prenatals, where nutritional deficiencies are addressed as a prophylactic measure. In this context, this risk of over supplementation is highest and low doses are essential.

• Composition of Fertilix and ConceptionXR: Coward and colleagues believe that the two formulations are similar, but the differences are actually substantial. As an example, ConceptionXR uses alpha-tocopherol succinate as its Vitamin E ingredient. This synthetic form of Vitamin E delivers only a single isoform of Vitamin E. At 400IU used by ConceptionXR, this form has been associated with serious side-effects reported by high quality studies5,6. Fertilix uses all natural, full-spectrum Vitamin E that contains eight chemically distinct, naturally occurring isoforms of Vitamin E, with a price tag of over 20-fold that of the synthetic version. The full scientific rationale for its inclusion has been published.4 There are significant other differences either in the isoformic nature of the ingredients/doses and delivery forms, but a full explanation would be outside the scope of this letter. Moreover, ConceptionXR is not personalized; like everything in the market, it is a one size fits all approach. 

• Human Clinical trials with Fertilix: As Dr. Samuel Santos-Ribeiro commented, antioxidant therapy has become more-or-less routine among couples seeking fertility treatment. As the confusion over the importance of antioxidant therapy persists and in absence of a clear consensus from researchers and clinicians, increasing numbers of infertile men, particularly those with repeated IVF failures, will consider self-medicating with random, untested antioxidant formulations. The status quo will not change unless the research community can come together and perform studies focused on understanding the heterogeneity of the infertile/subfertile male. Once a more complete picture can be generated and biomarkers developed to specify patient subtypes, only then can treatments be accurately assessed.  The MOXI trial is the antithesis of this approach, an intentionally mixed patient population treated with an arbitrary formulation.  This is the key message to take away from the MOXI trial, and we firmly believe that we as researchers and clinicians can and should do better for the benefit of patients. So, take the criticism of the MOXI trial to heart and let’s improve the study of this subject!

John Aitken (RJA), Parviz Gharagozloo (PG), Joel Drevet (JD), Jorge Hallak (JH), Alfonso Gutierrez-Adan (AGA). The following co-authors are not linked to the company but support the scientific statements and rationale pertaining to a large human clinical trial: Juan Alvarez, Gihan Bareh, Sheryl Homa, Sergey I. Moskovtsev, Mohammad Hossein Nasr-Esfahani, Cristian O'Flaherty, Suresh Sikka, Steven Somkuti, Armand Zini.

DECLARATION OF INTEREST

PG is CEO of a company, CellOxess biotechnology, involved in the design and manufacture of antioxidant formulations for male and female infertility.  RJA, JD, JH and AGA are honorary advisors to this company.

* Correspondence: john.aitken@newcastle.edu.au and parviz.gharagozloo@celloxess.com

REFERENCES

1. Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod 2011;26:1628–1640.
2. Aitken RJ, Gibb Z, Baker MA, Drevet JR and Gharagozloo P. Causes and consequences of oxidative stress in spermatozoa. Reproduction, Fertility and Development, 2016, 28, 1–10.
3. Niederberger C. Re: The Role of Sperm Oxidative Stress in Male Infertility and the Significance of Oral Antioxidant Therapy. J Urol. 2012 Apr;187(4):1377.
4. Gharagozloo P, Gutiérrez-Adán A, Champroux A, Noblanc A, Kocer A, Calle A, et al. A novel antioxidant formulation designed to treat male infertility associated with oxidative stress: promising preclinical evidence from animal models. Hum Reprod 2016;31:252-62.
5. Klein EA, Thompson IM Jr, Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes HL, Gaziano JM et al. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 2011;306:1549–1556.
6. Miller ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage Vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005;142:37–46.

Yet another flawed antioxidant trial; a plea for rigorous evaluation of antioxidant therapy for male infertility involving oxidative stress

The role of oxidative stress in the aetiology of male infertility has been appreciated since the 1920’s when it was demonstrated that depriving rats of natural vitamin E, the major antioxidant vitamin, generated testicular degeneration and infertility (1,2). Subsequently, a great deal of research has been conducted on the role of oxidative stress in male infertility and there is now a general consensus that reactive oxygen species play a significant role in the creation of defective sperm function and the induction of sperm DNA damage (3-6). Recent papers clearly reveal roles for oxidative stress in a variety of clinical situations including the aetiology of non-obstructive azoospermia (7), defective sperm quality in teratozoospermia (8), the origins of sperm DNA damage (9), the toxic impact of parabens (10) and the pathophysiology of varicocele (11). If oxidative stress is such an important cause of male infertility, then surely antioxidant therapy should be part of the cure (12).

In animal models, this is certainly the case. For example, the GPx5 (glutathione peroxidase 5) knockout mouse suffers from localized oxidative stress in the epididymis (13). As a consequence of this stress, the spermatozoa exhibit clear signs of oxidative damage including impaired DNA compaction and high levels of oxidative DNA damage. When Gpx5-deficient males were mated to normal females, the presence of oxidatively damaged spermatozoa was associated with higher incidences of miscarriage and developmental defects in the offspring. However, when such GPx5-/- males were treated with a carefully engineered antioxidant formulation, oxidative DNA damage in the spermatozoa was found to return to control levels (14). Similarly, treatment with the same antioxidant formulation almost completely restored the fertility of mice rendered infertile by a transient testicular hyperthermia (14). Indeed there is a wealth of animal literature demonstrating that antioxidants can restore fertility and testicular function in animals treated with a wide range of factors capable of causing oxidative stress in the male reproductive tract including cadmium (15), phthalate esters (16), lead, organophosphate pesticides (17), induced diabetes (18), acrylamide exposure (19), mental stress (20) and so on.

Against such a background of supportive data in animal models, it is perhaps surprising that extension of these studies into the clinical domain has not provided evidence of a clear therapeutic benefit of antioxidant administration in man. Several meta-analyses addressing this question have been performed.  Recent systematic Cochrane analyses, for example, (21,22) concluded that there was low quality evidence for a positive effect on clinical pregnancy and live birth rates but emphasized the flaws in existing studies based on ‘on serious risk of bias due to poor reporting of methods of randomisation, failure to report on the clinical outcomes, live birth rate and clinical pregnancy, often unclear or even high attrition, and also imprecision due to often low event rates and small overall sample sizes’. Both Cochrane analyses called for large, well-designed, randomised placebo-controlled trials to clarify these results. It was, therefore, with interest that we encountered the paper in Fertility and Sterility by Steiner et al. (23) reporting the results of a large randomized placebo-controlled trial of antioxidant therapy in male infertility. While this study has many excellent features, disappointingly, it turned out to be as fundamentally flawed as its predecessors.  

How is it possible to design a study determining the efficacy of antioxidant therapy when no attempt is made to determine levels of oxidative stress in the patients before or after the administration of treatment? This makes no sense. It is like giving insulin to everyone who comes into hospital in a coma. Some will improve, some will continue to deteriorate and, overall, any therapeutic benefit will be lost in the noise. The males selected in this study exhibited at least one abnormal semen parameter in the previous 6 months [sperm concentration <15 million/mL (oligospermia), total motility <40% (asthenospermia), normal morphology <4% (teratospermia), or DNA fragmentation >25%]. Naturally, there are many different reasons for males to exhibit such defects and only in a proportion will this be due to oxidative stress. The authors clearly recognise this fundamental defect in their study but justify the omission of oxidative stress measurements on the basis that the random allocation of antioxidant treatment to infertile patients, irrespective of need, represents the current standard of clinical practice. While this statement is clearly correct, surely we should be encouraging higher standards of clinical implementation, rather than just reinforcing the status quo.

Similar arguments could be directed towards the DNA damage component of the study which focused on the vulnerability of the chromatin to low pH (the sperm chromatin structure assay) rather than the formation of oxidative base adducts (8-Oxo-2'-deoxyguanosine) which has been recently shown to be rather a common trait of infertile male patients (24) and to concern specific paternal chromosomal regions that could be linked to developmental defects in the offspring (25).

The other major factor that needs to be considered is the choice of formulation used in the trial. There are numerous male fertility supplements available in the market worldwide, greatly varying by both the nature of antioxidants incorporated into the formulation and the doses used. So why this particular composition? The authors justify the ingredients and doses used because similar ones had been previously evaluated individually in clinical trials and demonstrated some benefit. However, other studies have demonstrated serious concerns with either the isoformic nature of the ingredients or high doses of the individual ingredients used (14). As an example, high doses of antioxidants such as vitamin C or Zinc, especially in the situation where oxidative stress is absent or minimal, can lead to reductive stress (12) causing sperm DNA decondensation (26-28). Reductive stress arising from over-supplementation can therefore be just as damaging to spermatozoa as oxidative stress and should be avoided. This is another reason why understanding a patient’s oxidative stress status is important to the success of a clinical trial, so patients with low or no oxidative stress can be excluded from the trial.

The field desperately needs to support large scale placebo-controlled studies of the type conducted by Steiner et al (23). However, we shall learn nothing if we do not select the patients on the basis of criteria reflecting levels of oxidative stress in the male reproductive tract, treat accordingly with an evidence-based formulation and measure the performance of the treatment against those oxidative stress markers. Whether such treatment will also increase pregnancy rates will depend on a variety of other factors that will not necessarily be impacted by antioxidant therapy.  However, if antioxidant therapy can, as the animal models demonstrate, reverse levels of oxidative damage in both the male and female germ lines (29) then at least one component of the overall pregnancy equation will have been successfully addressed. Furthermore, reducing levels of oxidative DNA damage in the male germ line prior to conception is not just about establishing pregnancies per se, but making sure that the mutational load carried by the resultant offspring is kept as low as possible.

In conclusion, the paper by Steiner et al (23) clearly does indicate that the prevailing clinical practice of randomly prescribing antioxidant therapy to the subpopulation of males attending infertility clinics is unlikely to generate any overall benefit. However, this does not mean that carefully formulated antioxidant preparations are without significant therapeutic merit. They just have to be given to patients where there is evidence of oxidative stress as a causative factor in the patients’ infertility profile. This simple requirement has been met in previous small-scale studies and positive outcomes observed (30).  The time has now arrived not for us to abandon this approach to therapy but to replicate such studies at scale, using a randomized, placebo-controlled, double-blinded study design, ensuring that the appropriate patients are selected for treatment and the appropriate assessment criteria are in place to measure the impact of antioxidant intervention.

John Aitken (RJA), Parviz Gharagozloo (PG), Joel Drevet (JD), Jorge Hallak (JH), Alfonso Gutierrez-Adan (AGA), Juan Alvarez, Rafael Ambar, Gihan Bareh, Sheryl Homa, Sergey I. Moskovtsev, Mohammad Hossein Nasr-Esfahani, Cristian O'Flaherty, Suresh Sikka, Steven Somkuti, Paul Turek, Armand Zini.

DECLARATION OF INTEREST

PG is CEO of a company, CellOxess biotechnology, involved in the design and manufacture of antioxidant formulations for male and female infertility.  RJA, JD, JH and AGA are honorary advisors to this company.

* Correspondence: john.aitken@newcastle.edu.au and parviz.gharagozloo@celloxess.com

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3. Aitken RJ, De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod 2010;16:3-13.
4. Aitken RJ, Drevet JR. The importance of oxidative stress in determining the functionality of mammalian spermatozoa: a two-edged sword. Antioxidants (Basel) 2020;9: pii: E111.
5. Bisht S, Faiq M, Tolahunase M, Dada R. Oxidative stress and male infertility. Nat Rev Urol. 2017;14: 470-485.
6. Drevet JR, Aitken RJ. Oxidation of sperm nucleus in mammals a physiological necessity to some extends with adverse impacts on oocyte and offspring. Antioxidants (Basel) 2020;9: pii: E95.
7. Cito G, Becatti M, Natali A, Fucci R, Picone R, Cocci A, et al. Redox status assessment in infertile patients with non-obstructive azoospermia undergoing testicular sperm extraction: A prospective study. Andrology 2020;8:364-371.
8. Ammar O, Mehdi M, Muratori M. Teratozoospermia: its association with sperm DNA defects, apoptotic alterations and oxidative stress. Andrology 2020 ( in press).
9. Elbardisi H, Finelli R, Agarwal A, Majzoub A, Henkel R, Arafa M. Predictive value of oxidative stress testing in semen for sperm DNA fragmentation assessed by sperm chromatin dispersion test. Andrology 2019 (in press).
10. Samarasinghe SVAC, Krishnan K, Naidu R, Megharaj M, Miller K, Fraser B et al. Parabens generate reactive oxygen species in human spermatozoa. Andrology 2018;6:532-541.
11. Altintas R, Ediz C, Celik H, Camtosun A, Tasdemir C, Tanbek K, Tekin S, Colak C, Alan C. The effect of varicocoelectomy on the relationship of oxidative stress in peripheral and internal spermatic vein with semen parameters. Andrology 2016;4:442-446.
12. Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod 2011;26:1628–1640.
13. Chabory E, Damon C, Lenoir A, Kauselmann G, Kern H, Zevnik B, et al. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice. J Clin Invest 2009;119:2074-2085.
14. Gharagozloo P, Gutiérrez-Adán A, Champroux A, Noblanc A, Kocer A, Calle A, et al. A novel antioxidant formulation designed to treat male infertility associated with oxidative stress: promising preclinical evidence from animal models. Hum Reprod 2016;31:252-62.
15. Alharthi WA, Hamza RZ, Elmahdi MM, Abuelzahab HSH, Saleh H. Selenium and L-carnitine ameliorate reproductive toxicity induced by cadmium in male mice. Biol Trace Elem Res 2019 (in press)
16. Zhao Y, Li MZ, Shen Y, Lin J, Wang HR, Talukder M1, Li JL. Lycopene prevents dehp-induced leydig cell damage with the Nrf2 antioxidant signaling pathway in mice. J Agric Food Chem 2020;68:2031-2040.
17. Naderi N, Souri M, Nasr Esfahani MH Hajian M, Tanhaei Vash N. Ferulago angulata extract ameliorates epididymal sperm toxicity in mice induced by lead and diazinon. Andrology 2019 (in press).
18. Bahmanzadeh M, Goodarzi MT, Rezaei Farimani A, Fathi N, Alizadeh Z. Resveratrol supplementation improves DNA integrity and sperm parameters in streptozotocin-nicotinamide-induced type 2 diabetic rats. Andrologia 2019;51:e13313.
19. Erdemli Z, Erdemli ME, Turkoz Y, Gul M, Yigitcan B, Gozukara Bag H. The effects of acrylamide and Vitamin E administration during pregnancy on adult rats testis. Andrologia 2019;51:e13292.
20. Fahim AT, Abd El-Fattah AA, Sadik NAH, Ali BM. Resveratrol and dimethyl fumarate ameliorate testicular dysfunction caused by chronic unpredictable mild stress-induced depression in rats. Arch Biochem Biophys 2019;665:152-165.
21. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database Syst Rev 2014:12:CD007411.
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23. Steiner AZ, Hansen KR, Barnhart KT, Cedars MI, Legro RS, Diamond MP, et al. The effect of antioxidants on male factor infertility: the Males, Antioxidants, and Infertility (MOXI) randomized clinical trial. Fertil Steril 2020;pii: S0015-0282:32547-6.
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25. Xavier MJ, Nixon B, Roman SD, Scott RJ, Drevet JR, Aitken RJ. Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa. Hum Reprod 2019;34:1876-90.
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