Volume 113, Issue 3, Pages 552–560.e3
Authors:
Anne Z. Steiner, M.D., M.P.H., Karl R. Hansen, M.D., Ph.D., Kurt T. Barnhart, M.D., Marcelle I. Cedars, M.D., Richard S. Legro, M.D., Michael P. Diamond, M.D., Stephen A. Krawetz, Ph.D., Rebecca Usadi, M.D., Valerie L. Baker, M.D., R. Matthew Coward, M.D., Hao Huang, M.D., M.P.H., Robert Wild, M.D., M.P.H., Ph.D., Puneet Masson, M.D., James F. Smith, M.D., M.S., Nanette Santoro, M.D., Esther Eisenberg, M.D., M.P.H., Heping Zhang, Ph.D. for the Reproductive Medicine Network
Abstract:
Objective
To determine whether antioxidants improve male fertility, as measured by semen parameters and DNA fragmentation at 3 months and pregnancy resulting in live birth after up to 6 months of treatment, among couples with male factor infertility.
Design
Multicenter, double-blind, randomized, placebo-controlled trial with an internal pilot study.
Setting
Nine fertility centers in the United States from December 2015 to December 2018.
Patient(s)
Men (N = 174) with sperm concentration ≤15 million/mL, motility ≤40%, normal morphology ≤4%, or DNA fragmentation >25%, and female partners who were ovulatory, ≤40 years old, and had documented tubal patency.
Intervention(s)
Males randomly assigned to receive an antioxidant formulation (n = 85) containing 500 mg of vitamin C, 400 mg of vitamin E, 0.20 mg of selenium, 1,000 mg of l-carnitine, 20 mg of zinc, 1,000 μg of folic acid, 10 mg of lycopene daily, or placebo (n = 86). Treatment lasted for a minimum of 3 months and maximum of 6 months, and couples attempted to conceive naturally during the first 3 months and with clomiphene citrate with intrauterine insemination of the female partner in months 4 through 6.
Main Outcome Measure(s)
Primary outcome was live birth; secondary outcomes included pregnancy within 6 months of treatment. For the internal pilot, the primary outcomes were semen parameters and sperm DNA fragmentation index after 3 months of treatment.
Result(s)
In the Males, Antioxidants, and Infertility (MOXI) study, after 3 months of treatment, the change in sperm concentration differed between the antioxidant group (median −4.0 [interquartile range−12.0, 5.7] million/mL) and placebo group (+2.4 [−9.0, 15.5] million/mL). However, there were no statistically significant differences between the two groups for changes in sperm morphology, motility, or DNA fragmentation. Among the 66 oligospermic men at randomization, sperm concentration did not differ at 3 months between the antioxidant and control groups: 8.5 (4.8, 15.0) million/mL versus 15.0 (6.0, 24.0) million/mL. Of the 75 asthenospermic men, motility did not differ at 3 months: 34% ± 16.3% versus 36.4% ± 15.8%. Among the 44 men with high DNA fragmentation, DNA fragmentation did not differ at 3 months: 29.5% (21.6%, 36.5%) versus 28.0% (20.6%, 36.4%). In the entire cohort, cumulative live birth did not differ at 6 months between the antioxidant and placebo groups: 15% versus 24%.
Conclusion(s)
Antioxidants do not improve semen parameters or DNA integrity among men with male factor infertility. Although limited by sample size, this study suggests that antioxidant treatment of the male partner does not improve in vivo pregnancy or live-birth rates.
Clinical Trial Registration Number
NCT02421887
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Making Sense of the MOXI Study
The MOXI trial attempted to determine whether antioxidant treatment of subfertile men would lead to an increase in pregnancy and birth rate. The study was designed with an internal pilot phase, which measured changes in the 3-month semen parameters of the first 120 men enrolled. The results of the pilot phase were intended to determine whether the fully powered, 790-couple study—with live birth as its endpoint—would proceed. “Success” in the pilot phase required statistically significant improvements in both motility and DNA Fragmentation Index (DFI) among those first 120 subjects.
Study design is arguably the most important aspect of any scientific manuscript, as it determines the level of evidence and conclusions that can be drawn from the data. It also helps determine how the data can be extrapolated and provides a template for reproducibility. In the MOXI study, couples were enrolled in the pilot study on the basis of a single S/E [semen evaluation], which demonstrated any abnormality in one of 4 parameters: 1) count; 2) motility; 3) morphology; 4) or % DFI (as determined by Sperm Chromatin Structural Assay [SCSA]).
The FDA provides guidance in regard to investigational agents used for the treatment of male factor infertility. Drug exposure must be for at least 26 weeks (i.e., two consecutive spermatogenesis cycles or longer), with semen analysis every 13 weeks, and at least two semen collections per time point.1
Given this primary inclusion criteria (“any abnormality at all on a single S/E”), it is unclear how the investigators could prospectively predict the relative frequency or severity of the different baseline semen abnormalities which would occur in the study subjects. This fact, along with other factors in the study’s design and methodology, raise important questions about its conclusions.
As mentioned, the full study’s primary endpoint was live birth. The power calculation for that endpoint required enrollment of 790 couples in order to demonstrate a statistically significant difference between the antioxidant and placebo arms. However, the study only enrolled 171 couples, of which only 144 of those completed the study. Not surprisingly, there was no statistical difference between the placebo and antioxidant arms for the live birth endpoint. Given that less than 20% of the required enrollment was achieved, it is unclear why the authors state any conclusion about this endpoint.
The pilot design required the antioxidant group to show a statistically significant improvement in both motility and DFI percentages after three months of treatment. As neither parameter showed significant improvement, the study was ended. However, several issues confound the pilot phase data:
Over the years, many studies have demonstrated the ability of antioxidant supplementation to reduce seminal oxidative stress and improve semen parameters in sub-fertile men.3-4 Studies seeking to determine whether those improvements translate into higher birth rates and, in which clinical settings (i.e. IUI, IVF, natural conception, etc.), that might be the case, have presented unique challenges and yielded mixed results. We do not believe that the MOXI study was able to answer that question. Unfortunately, that puzzle remains unsolved, since a careful review of the MOXI data appears to raise more questions than it resolves.
The antioxidant supplement and placebo used in the MOXI study were provided by Theralogix, LLC at the investigator’s request. The authors provided the company with a prepublication copy of the manuscript, which, as members of the Medical Advisory Board at Theralogix, we had the opportunity to review. Our review of that manuscript forms the basis for the concerns expressed in this communication.
Respectfully,
Marc Goldstein, MD, FACS
Wayne Hellstrom, MD, FACS
Glenn Schattman, MD, FACOG
Robert Stillman, MD, FACOG
References:
We would like to thank the medical advisory board of Theralogix for their interest in the MOXI trial. We understand that the findings might be disconcerting to Theralogix as the company currently markets and sells a product containing this antioxidant formulation “to promote sperm structure and function” (https://theralogix.com/products/conceptionxr-motility-support-supplement).
We appreciate that the medical advisory board read the pre-published version of paper that was provided by the RMN as a courtesy to Theralogix. It appears that some of the commentary (internal pilot sample size) is also based on the protocol, which is publically available on clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT02421887?term=MOXI&draw=2&rank=2). While we appreciate that the medical advisory board of Theralogix has an opinion about the quality of the MOXI trial, we do not share that opinion. We would like to point out that this protocol was vetted through peer review by study section, an advisory board at the NIH, and a DSMB. In addition, an IND was obtained from the FDA. The trial was designed to mimic how antioxidants are currently being marketed and prescribed by physicians, and used by patients.
Regarding the issues put forth by the medical advisory board for Theralogix:
As noted in your letter, the protocol called for 120 subjects to be included in the internal pilot. Based on the assumption that 50% of men would have low motility, we needed 30 men in each group (total 60) to achieve >80% power at an alpha of 0.05. MOXI ultimately enrolled 171 (51 men were enrolled while the internal pilot samples and data were being analyzed). There were 74 men enrolled in the trial with low motility. Thus this published study was adequately powered to look at changes in semen parameters among men with low motility.
The power analysis was not based on the assumption that the change in DNA fragmentation would be observed only among men with abnormal DNA fragmentation at baseline, but on all subjects.
We assessed DNA fragmentation by TUNEL in addition to SCSA. The data was not presented due to space limitations. There was no significant difference between the two groups in change in DNA fragmentation (assessed by TUNEL). Change in DNA fragmentation by TUNEL (%) is 2.3 (-3.4 to 7.8) and -0.7 (-5.7 to 6.0) for anti-oxidant group and placebo group, respectively (p=0.283).
Among men with abnormal semen parameters, all semen parameters improved between baseline and visit 3. This improvement was observed among both the placebo and antioxidant groups (although more commonly “statistically significant” in the placebo group). This is likely due to regression to the mean. This is one reason why placebos are needed in clinical trials (aka “placebo effect”). It should be pointed out, though, that these subgroups have very small numbers and “improvements” should be interpreted with caution. Additionally, there were no significant differences in change in semen parameters between groups.
However, in the full cohort, change in concentration did significantly differ between the placebo and antioxidant group, favoring the placebo. Of note, better semen parameters and lower DNA fragmentation was also observed among the placebo group in the FAZST trial (1) which randomized 2370 men to the antioxidants, folic acid and zinc, or placebo.
While we note the cited systematic review published in the Arab Journal of Urology (2), we would note that our trial findings are consistent with the Cochrane systematic review (3), which found no benefit on live birth when studies at high risk of bias were removed from the analysis. In addition, it is consistent with the FASZT trial, which showed that antioxidants did not improve live birth among the 2370 men enrolled in that RCT (1).
Sincerely,
Anne Z. Steiner, MD, MPH
Karl Hansen, MD, PhD
Kurt Barnhart, MD
Marcelle Cedars, MD
Richard Legro, MD
Michael Diamond, MD
Matthew Coward, MD
Nanette Santoro, MD
Esther Eisenberg, MD, MPH
Heping Zhang, PhD
For the Reproductive Medicine Network
References:
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
REFERENCES
Reply to “Yet another flawed antioxidant trial; a plea for rigorous evaluation of antioxidant therapy for male infertility involving oxidative stress” by John Aitken et al.
No doubt there would be immense praise for the MOXI Trial by the fertility supplement industry if the trial had shown a benefit to antioxidants. However, it is not surprising to receive criticism when the results don’t align with the interests of owners and associates of companies with financial interest in the effectiveness of antioxidant formulations for the treatment of male infertility. The CEO, chief scientific officer, advisory board members, and associates of Celloxcess LLC, along with the co-founder and owner of Alphasperm and a part-owner of Yad-Tech, are among the recent critics of MOXI. Celloxcess produces and sells Fertilix, which shares 8 of its 10 vitamin ingredients with all 8 vitamins in the supplement utilized in the MOXI trial. Tremendous planning went into the selection of the commercial antioxidant formulation for MOXI. We performed a complete market analysis of widely available supplements with attention toward identifying a formulation that was both representative of marketed supplements, as well as which contained pure, non-proprietary antioxidants, which had previously been shown to improve semen parameters.
The NICHD Reproductive Medicine Network tested the hypothesis that antioxidants would improve male fertility in a randomized, double blinded fashion. The trial was designed to study a prevailing clinical practice based on dogma, observations from animal studies (dating from 1926!), and underpowered clinical trials. We agree with Aitken, Gharagozloo, and associates that the MOXI trial found no benefit to support the practice of prescribing antioxidant therapy to infertile males, exactly in the manner in which their supplements (Fertilix, Alphasperm, and Fertil Pro) and many others are marketed.
Fertilix is broadly marketed as a vitamin supplement “for every man; those preparing to conceive naturally, or those requiring added support” in order “to support a normal reproductive redox balance, essential for sperm development and DNA integrity” (1). We agree that a trial evaluating the use of antioxidants in a highly select group of patients with high levels of oxidative stress in the male reproductive tract would be interesting and informative. Unfortunately, this approach is neither feasible nor representative of current practice patterns or direct-to-consumer marketing by the supplement industry. Although direct measurement of oxidative stress in semen is possible, it is not widely available nor routinely performed on fresh semen samples. Therefore, the pragmatic MOXI trial protocol implemented the more reliable and widely accepted surrogate measure of oxidative stress in sperm, DNA fragmentation — which is one of the specifically marketed benefits promulgated by Fertilix’s advertising. Antioxidant therapy did not improve the DNA fragmentation index for the entire treatment group or the subgroup of men with high DNA fragmentation at baseline, in a multicentered, double-blind, randomized, placebo-controlled clinical trial.
Given the revenue generated by the antioxidant supplement industry, coupled with the strong opinions and expertise of its owners, scientific officers, and advisory board members, perhaps they can design and fund an appropriately designed, adequately powered, randomized placebo-controlled trial with primary outcomes of pregnancy and live birth in the subgroup they espouse as needed. Until then, the negative MOXI Trial, along with the negative FAZST Trial (2), will be the definitive clinical trials on this subject. In the meantime, will Celloxcess and others only market their supplements to those with documented high levels of reactive oxygen species in the semen and not make claims about their effects on sperm parameters, DNA integrity, and fertility outcomes? Continuing to prescribe these products to all subfertile males, including preconception patients, no longer appears to be appropriate clinical practice.
Sincerely,
R. Matthew Coward, MD FACS
Anne Z. Steiner, MD, MPH
Karl Hansen, MD, PhD
Kurt Barnhart, MD
Marcelle Cedars, MD
Richard Legro, MD
Michael Diamond, MD
Nanette Santoro, MD
Esther Eisenberg, MD, MPH
Heping Zhang, PhD
For the Reproductive Medicine Network
References:
For such a widespread treatment strategy (i.e. the now practically routine use of antioxidants in subfertile men), one needs to perform a pragmatic RCT! I cannot agree more Dr Coward et al, this is exactly the kind of trial that we needed! Many physicians are unconscious "pill mills" for male fertility supplements, without strong evidence to prescribe them. When a subspeciality has such a large drop-out rate following treatment as ours, one needs to seriously ask whether one of the reasons may not be that we tell patients "take this, you need it" and then nothing changes! The frustration caused by untested strategies may be many times more harmful than the temporary (false) hope these supplements give to the patients.
This is not (nor should it be) the end of testing the potential benefit of antioxidants in men, namely in specific subpopulations. However, this study should be a reminder to us all that we should not promote "one size fits all" and untested treatments.
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.
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