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:

• The power calculation for the pilot phase assumed that at least 50% of the enrolled subjects would have low motility at baseline; however, only 43% of the subjects had low motility, meaning that the ‘motility endpoint’ of the internal pilot was statistically underpowered.
• While the paper describes the sample size assumption and power calculation for the motility endpoint of the pilot phase, it does not do the same for the DNA fragmentation endpoint. Only 44 of the 171 subjects (25.7%) had elevated DFI at baseline. Whether this ‘n’ provided adequate statistical power is not disclosed.
• DFI was assessed using the SCSA on cryopreserved specimens. SCSA actually measures chromatin compaction, and only indirectly measures DNA fragmentation. Two other assays, TUNEL and COMET, directly measure DNA fragmentation. A recently published study2 demonstrated that antioxidant treatment will reduce DNA fragmentation when it is measured directly by TUNEL, but that treatment with antioxidants will not demonstrate improvement in DFI assays, such as SCSA,  that measure chromatin structure.2 The authors conclude that SCSA is not a valid assay methodology to assess the impact of antioxidant treatment.
• In the published paper, Table 4 provides the pilot-phase subgroup data, stratified by the individual baseline semen abnormalities, for both the placebo and antioxidant arms. In the placebo arm, all four subgroups (low count, low motility, low morphology and high DFI) demonstrated statistically significant improvement on the 3-month semen analysis. This finding – statistically significant improvement in the placebo arm for every semen parameter - raises serious questions about the design and methodology of the study, yet  the authors do not comment on it.

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:

1. FDA Center for Drug Evaluation and Research. Testicular Toxicity: Evaluation During Drug Development. Guidance for Industry. https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm, Accessed Dec 12, 2018.
2. Le Saint C, Kadoch IJ, Bissonnette F, et al. Beneficial effect of antioxidant therapy on sperm DNA integrity is not associated with a similar effect on sperm chromatin integrity. AME Med J 2019;4:31.
3. Majzoub A & Agarwal A. Systematic review of antioxidant types and doses and male infertility: Benefits on semen parameters, advanced sperm function, assisted reproduction and live birth rate. Arab J Urol 2018;16(1):113-124.
4. Busetto GM, Agarwal A, Virmani A, et al. Effect of metabolic and antioxidant supplementation on sperm parameters in oligo‐astheno‐teratozoospermia, with and without varicocele: A double‐blind placebo‐controlled study. Andrologia. 2018;50:e12927