The lack of evidence behind over-the-counter antioxidant supplements for male fertility patients: a scoping review

Abstract STUDY QUESTION What is the evidence for over-the-counter antioxidant supplements for male infertility? SUMMARY ANSWER Less than half of over-the-counter antioxidant supplements for male fertility patients have been tested in a clinical trial, and the available clinical trials are generally of poor quality. WHAT IS KNOWN ALREADY The prevalence of male infertility is rising and, with this, the market for supplements claiming to improve male fertility is expanding. Up to now, there is limited data on the evidence for these over-the-counter supplements. STUDY DESIGN, SIZE, DURATION Amazon, Google Shopping and other relevant shopping websites were searched on 24 June 2022 with the following terms: ‘supplements’, ‘antioxidants’, ‘vitamins’, AND ‘male fertility’, ‘male infertility’, ‘male subfertility’, ‘fertility men’, ‘fertility man’. All supplements with a description of ingredients in English, Dutch, French, Spanish, or German were included. Subsequently, Pubmed and Google Scholar were searched for studies that included the supplements. PARTICIPANTS/MATERIALS, SETTING, METHODS Inclusion criteria were supplements with antioxidant properties, of which the main purpose was to improve male fertility. Included supplements must be available without a doctor’s prescription. Supplements containing plant extracts were excluded, as well as supplements of which the content or dosage was not clear. The ingredients, dosage, price and health claims of the supplements were recorded. We assessed whether substances in the supplements exceeded the recommended dietary allowance (RDA) or tolerable upper intake level (UL). All clinical trials and animal studies investigating included supplements were selected for this review. Clinical trials were assessed for risk of bias with a risk of bias tool appropriate for the study design. MAIN RESULTS AND THE ROLE OF CHANCE There were 34 eligible antioxidant supplements found, containing 48 different active substances. The average price per 30 days was 53.10 US dollars. Most of the supplements (27/34, 79%) contained substances in a dosage exceeding the recommended daily allowance (RDA). All manufacturers of the supplements made health claims related to the improvement of sperm quality or male fertility. For 13 of the 34 supplements (38%), published clinical trials were available, and for one supplement, only an animal study was found. The overall quality of the included studies was poor. Only two supplements were tested in a good quality clinical trial. LIMITATIONS, REASONS FOR CAUTION As a consequence of searching shopping websites, a comprehensive search strategy could not be formulated. Most supplements were excluded because they contained plant extracts or because supplement information was not available (in an appropriate language). WIDER IMPLICATIONS OF THE FINDINGS This is the first review that gives an insight into the market of male fertility supplements as available to infertility patients and other men seeking to improve their fertility. Earlier reviews have focused only on supplements with published clinical trials. However, we show that more than half of the supplements have not been tested in a clinical trial. To our knowledge, this review is the first to assess the dosage of supplements in relation to the RDA. In agreement with the literature, we found that the evidence on male fertility supplements is generally of poor quality. This review should urge pharmaceutical companies to evaluate their products in randomized controlled trials in order to provide people with substantiated information. STUDY FUNDING/COMPETING INTEREST(S) The research position of W.R.d.L. is funded by an unrestricted grant from Goodlife Pharma. W.R.d.L., K.F., and J.P.d.B. are in the research team of a clinical trial on Impryl®, one of the supplements included in this review. REGISTRATION NUMBER N/A.


Introduction
Infertility is a widespread problem affecting 8-12% of the population worldwide (Vander Borght and Wyns, 2018). In around 50% of these couples, a male factor is involved, and in 20-30%, it is the main cause of infertility (Agarwal et al., 2021). Some studies have reported a decline in semen quality over the past decades (Sengupta et al., 2017;Agarwal et al., 2021;Levine et al., 2023). Researchers have hypothesized chemical substances (e.g. pesticides), radiation and heat, increases in BMI, advanced male age, and consumption of alcohol and tobacco, as possible causes (Sengupta et al., 2017;Darbandi et al., 2018). However, the metaanalyses reporting a decline in semen quality have been criticized for their methodological limitations (Bonde and Te Velde, 2017). Other studies have not found evidence for this decline or have shown large geographical variations (Swan et al., 1997(Swan et al., , 2000Cipriani et al., 2023). Either way, the recognition of the importance of male (in)fertility has increased and in reaction to this, various (pharmaceutical) companies have developed over-the-counter (OTC) nutritional supplements.
The use of supplements, antioxidant supplements in particular, to improve male fertility is based on the process of oxidative stress . Oxidation occurs in all cells in the human body. In oxidation, a substance loses electrons, which can result in the production of free radicals or reactive oxygen species (ROS). ROS, in turn, can damage (parts of) the sperm cell. Reduction is the process of substances gaining electrons, the opposite of oxidation. The reaction between an oxidative agent and a reductive agent is called a redox reaction.
Antioxidants can prevent the production of ROS or inactivate ROS before they can damage the cell, its DNA or other cell components. When there is an imbalance between oxidation (i.e. the production of ROS) and the level of antioxidants, oxidative stress occurs. Sperm cells are particularly vulnerable to oxidative stress. This is firstly because the sperm cell membrane is rich in polyunsaturated fatty acids, which oxidize easily. Secondly, sperm cells contain little antioxidant-rich cytoplasm.
Building on this theory, antioxidant supplements could prevent or treat oxidative stress in sperm cells and thus improve male fertility. The updated Cochrane review on antioxidants for male subfertility showed that live birth rate slightly increased when antioxidants were used. However, this effect could not be shown when studies with a high risk of bias were removed from the analysis (de Ligny et al., 2022).
It is important to note that a crucial role of oxidative stress is still a matter of debate among male fertility experts. Moreover, it has been reported that the excessive use of antioxidants can result in impaired fertility through the process of reductive stress (Dattilo et al., 2016;Henkel et al., 2019). Antioxidants should therefore be handled with caution in the treatment of male infertility.
As mentioned above, many companies have developed OTC antioxidant supplements that claim to improve male fertility. Some companies have even been founded to focus only on supplements for improving female and/or male fertility. These companies meet the need of fertility patients searching for alternative or additional treatment options. However, it is often unknown whether there is scientific evidence for these specific supplements. If evidence is lacking, this could lead to overtreatment or even impairment of male fertility when reductive stress occurs (Dattilo et al., 2016;Henkel et al., 2019). Moreover, it can lead to serious costs for patients.
Ideally, all supplements that are available to patients and that claim to improve fertility should have been tested in large randomized controlled trials (RCTs). It is of great importance that these trials have relevant outcomes such as clinical pregnancy rates and live birth rates, as these are the ultimate goals for fertility patients and their health care providers. This is also reflected in the infertility core outcome set as developed by Duffy et al. (2021).
The aim of this scoping review is to assess the available OTC antioxidant supplements for male subfertility, and to evaluate the scientific basis of health claims.

Search
On 24 June 2022, the websites of Google Shopping ('Google Shopping') and Amazon ('Amazon') were searched with the following terms: 'supplements', 'antioxidants', 'vitamins' AND 'male fertility', 'male infertility', 'male subfertility', 'fertility men', 'fertility man'. While conducting this search, different websites for supplements were identified and subsequently searched for new supplements. The websites of manufacturers of included supplements were searched for other eligible products. Reference lists of reviews and studies of included supplements were explored as well.

Inclusion and exclusion criteria
Supplements were included based on the following criteria. (i) Supplements must consist of substances with antioxidant properties as defined in the Cochrane review updated in 2022 (de Ligny et al., 2022): 'Antioxidants are substances that inhibit or delay the oxidation of biologically relevant molecules, either by directly scavenging free radicals or by chelation of redox metals'. (ii) The main purpose of the supplement, according to the manufacturer, must be to improve male fertility. More specifically, this means that the package or container should mention at least one of the following terms: fertility, fertile, infertility, subfertility, preconception or reproduction, and male, men, man, him. (iii) The supplement is available without a doctor's prescription.
Exclusion criteria were as follows. (i) Supplements containing plant extracts were excluded because the dosage as well as the antioxidant properties of plant extracts are often unclear. (ii)

WHAT DOES THIS MEAN FOR PATIENTS?
This review looks at which antioxidant (nutritional) supplements are freely available to men who want to improve their fertility. We assessed whether they are safe and whether research has proved their effect.
In this study, we searched shopping websites for supplements that claim to improve the fertility of men. We found a total of 34 different supplements. Most (79%) of the supplements contained excessive dosages of vitamins or minerals, which may cause lower semen quality. We also found that a minority (38%) of the supplements had been tested in a published study. If a supplement was tested in a study, the study was generally of poor quality.
Supplements of which the content or dosage were not clear or described in a language other than English, Dutch, French, Spanish, or German, were also excluded. Although the English websites of Google Shopping and Amazon were searched, some supplements contained information in another language.

Data collection
After identifying all eligible supplements, predefined data on the product were retrieved. Information on the product was obtained from the website of the manufacturer or, if not available, from the website where the product can be purchased. Predefined outcomes were ingredients and the dosage of the supplement, claims regarding male fertility as stated by the manufacturer (written on the website or on the packaging of the product), and price per 30 days in US dollars.
If available, the dosage of individual ingredients was related to the recommended dietary allowance (RDA) and tolerable upper intake level (UL). All ingredients of which an RDA or UL was available, were assessed. Both values were retrieved from the database of the NIH Office of Dietary Supplements (Medicine 2000(Medicine , 2001(Medicine , 2011(Medicine , 2005(Medicine , 1998. From this database, the highest value for men aged between 18 and 50 years was used.

Search and risk of bias assessment of studies
Scientific evidence on the supplements was searched on the supplement websites, and on Pubmed and Google Scholar, searching for the (brand) name of the supplement. If this retrieved no articles, the search was repeated with the name of the manufacturer. Only studies investigating a supplement with the exact same composition as the included supplement were evaluated in this review. Studies that did not investigate the supplement in relation to male fertility were not included in this review. Studies in a language other than English, Dutch, French, Spanish, or German, were excluded from this review.
Included studies were then assessed for risk of bias with the appropriate risk of bias tool by two of four independent researchers: W.R.d.L. and J.P.d.B., H.G., or L.M.P. Huygen (who provided assistance with the Spanish studies; see Acknowledgements). For each study design, a distinctive tool was used because different study methods require different methodological quality assessment. For RCTs, non-randomized studies of interventions, and pre-post studies with a control group, the Cochrane Risk of Bias 2 (RoB2), the ROBINS-I and the EPOC RoB tool were used, respectively. For pre-post studies without a control group, the NIH tool was used. For cohort studies, the Newcastle-Ottawa Scale (NOS) was used. These tools were assigned to each study design according to Ma et al. (2020). Disagreements were discussed among researchers and if consensus could not be achieved, a third researcher was involved: W.R.d.L., J.P.d.B., H.G., or L.M.P. Huygen (see Acknowledgements).

Search and selection of supplements
The search yielded a total of 89 eligible supplements based on the product name ( Fig. 1). Most of these products (51 supplements) were found on Amazon. Seven supplements were retrieved from the Google Shopping website. Two websites mentioning several supplements for male fertility were also found: Newpharma.nl and Homer News, providing six and seven previously unidentified supplements, respectively. Nine other supplements were found through the websites of included supplements. Reference checking of reviews yielded nine additional supplements.
All 89 supplements were then screened more thoroughly by assessing the package (online) and, if available, the manufacturer's website. This led to the exclusion of 42 supplements for containing plant extracts (e.g. maca root, pine bark, ginseng, green tea, etc.). Eight supplements were excluded due to unknown content or formulation of ingredients. This was either not mentioned on the packaging or website, or the website was only available in Russian. Two products had exactly the same content and manufacturer as another included supplement and were therefore removed as duplicates. Finally, three supplements were

Characteristics of included supplements
The 34 included supplements contained 48 different active substances. Most of the included supplements (27/34, 79%) contained substances in a dosage exceeding the RDA. Two supplements, Belimen V R and FH Pro for men V R , contained dosages exceeding the tolerable UL (Table 1). The price per 30 days ranged from 21.76 to 159.64 US dollars, with an average of 53.10 US dollars.
The general health claim of all manufacturers was that their product 'optimizes', 'normalizes', 'improves', or 'supports' male fertility or reproduction. Both groups of supplements, with and without published studies, made these claims. Three manufacturers with a supplement tested in a clinical trial mentioned the study in their claims with terms as 'shown in a clinical study', 'tested effective', and 'significantly improves'. Three manufacturers selling a supplement without published evidence referred to science in their claims with terms such as 'a science-backed multivitamin', 'scientifically formulated', and 'science-backed formula'.

Included studies
We found published studies for 13 of the 34 supplements, while for one supplement (Speramax V R ), we found only an animal study testing the supplement in female mice (Hamzah, 2018). Most study designs were pre-post studies with or without a (retrospective) control group treated with placebo or receiving no treatment. We also identified five RCTs, of which two investigated the supplement Proxeed Plus V R (Tremellen et al., 2007;Clifton and Ellington 2009;Busetto et al., 2018;Tsounapi et al., 2018;Micic et al., 2019). In addition, we found one case report, one retrospective cohort study and one non-randomized clinical study (Junca et al., 2012;Lipovac et al., 2016;Tremellen et al., 2021). Most studies included male fertility patients, based on abnormal semen parameters or high DNA fragmentation. The number of randomized or included patients ranged from one couple to 657 participants. The treatment period ranged from 30 days to 6 months ( Table 2).

Risk of bias in studies
The overall quality of the included studies was poor (Table 2). This was due to small groups or no sample size calculation, no assessment of treatment adherence or the use of other supplements before or during the study, no (description of) blinding of outcome assessors, high or no description of loss to follow-up, and/or failure to report all predefined outcome measures. All five pre-post studies with a control group were graded as having a high risk of bias based on baseline imbalances between the study groups, the use of a retrospective control group, and/or no (report of) blinding of outcome assessors (Imhof et al., 2012;Amar et al., 2015;Lipovac et al., 2016Lipovac et al., , 2021Elmahaishi et al., 2017). One nonrandomized clinical study was graded with the ROBINS II tool as having a critical risk of bias based on no report of baseline parameters, treatment adherence or loss to follow up, and no statistical comparison of end point outcomes between the two study groups (Lipovac et al., 2016). Moderate or high risk of bias was assigned to three of the five RCTs based on failure to report the method of randomization, no blinding of outcome assessors, and loss to follow-up ( . Two RCTs were graded as having a low risk of bias with the Cochrane Risk of Bias tool (Tremellen et al., 2007;Micic et al., 2019).

Discussion
This is the first scoping review on nutritional supplements that are freely available to male fertility patients. It draws an image of the broad selection subfertile men are offered when they are searching for supplements in order to improve their fertility.
There is a great variety of active substances in supplements and nearly 80% of the supplements contain substances in a dosage exceeding the recommended daily allowance. In two supplements, a substance was present in a dosage exceeding the tolerable UL. It is worth noting that both of these values for dietary reference intake are set based on heterogeneous studies in groups of healthy individuals. They should be regarded as guiding instead of binding (Institute of Medicine Subcommittee, on Interpretation, Intakes Uses of Dietary Reference, and Intakes Institute of Medicine Standing Committee on the Scientific Evaluation of Dietary Reference, 2000). Despite this fact, we chose to report these outcomes measures because of the oftenoverlooked risk of reductive stress.
Previous research has shown that high dosages of antioxidants can cause reductive stress (Dattilo et al., 2016;Henkel et al., 2019). One can imagine that if oxidative stress causes the balance to tip over, reductive stress will be able to do this the other way. When this results in very low levels of ROS, physiological processes that ensure sperm maturation and capacitation might be disturbed, leading to impaired fertility. We found no information on this risk on any of the supplement websites.
Our review furthermore shows the costs of supplements for male fertility patients. The supplements need to be taken for at least 3 months to cover the period necessary for sperm production, 72 days. This means that men pay an average of at least 159.30 US dollars, with a maximum price of 478.92 US dollars for a 3-month period.
The numbers are even more remarkable when one considers that only 38% of the supplements were tested in clinical trials which, overall, were poor in quality. Only two RCTs were found to have a low risk of bias (Tremellen et al., 2007;Micic et al., 2019). The recently updated Cochrane review on antioxidants for subfertile men also reports that the evidence for male fertility supplements is of low quality (de Ligny et al., 2022).
Studies on the effect of a single antioxidant substance on clinical outcomes are limited, with often no more than two available studies on one substance. A low-quality study compared zinc with a placebo and found significantly more pregnancies in the intervention group (Omu et al., 1998). Two >25-year-old studies compared vitamin E with placebo and one showed significantly more live births in the intervention group (low quality evidence) (Kessopoulou et al., 1995;Suleiman et al., 1996). L-Carnitine and co-enzyme Q10 each did not have a significant effect on clinical pregnancies or live births (Balercia et al., 2005(Balercia et al., , 2009. All these findings suggest that the subject of male fertility supplements is non-transparent, which could lead to misinformation of patients. It is important to note that (male) fertility patients are in a vulnerable situation, as the cause of their subfertility is often unclear (Fainberg and Kashanian 2019). This may lead to insecurity and the motivation to seek a solution outside conventional health care. It should be a priority to give clear information based on good quality evidence to these patients.
The discrepancy between the health claims and supporting evidence should lead to interference of regulatory institutions.
Antioxidants for subfertile men | 5        Fair study quality NIH EPOC, effective practice and organization of care risk of bias tool; IMSI, intracytoplasmic morphologically selected sperm injection; LBR, live birth rate; na: not applicable; NBT assay, nitroblue tetrazolium assay; NIH, national institutes of health risk of bias tool; NOS, Newcastle-Ottawa scale; ORP, oxidation reduction potential; RCT, randomized controlled trial; RoB, risk of bias; ROBINS, risk of bias in non-randomized studies of interventions; ROS, reactive oxygen species; SCSA, sperm chromatin structure assay; SCD, sperm chromatin dispersion test; sDF, sperm DNA fragmentation; SDI, sperm decondensation index; SHBA, sperm hyaluronic binding assay; TMSC, total motile sperm count; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling assay.
In reality, the regulatory framework of dietary supplement is very complex and, in many ways, different from that of medical drugs or devices (Dwyer et al., 2018). There is no clarity on the definition of dietary supplements and regulation of the market differs even between economically comparable regions such as the USA and the European Union. All these challenges aside, the aim of health care providers, but also of governments and regulatory institutions, is to ensure the safety of consumers and their ability to make an informed decision. Based on the results of this review, we can state that these goals have not yet been achieved. Large RCTs, that check and correct for confounding factors such as lifestyle, diet, and use of other supplements are therefore urgently needed. Primary outcomes should be clinical, with the ultimate outcome being live birth rate. The previously mentioned Cochrane review on this subject concluded that antioxidants may lead to an increased live birth rate (OR 1.43, 95% confidence interval 1.07-1.91) based on 12 studies (de Ligny et al., 2022). Unfortunately, the evidence for this outcome was of very low certainty. In response to this, a few well-designed studies are now being conducted, but the scientific attention still does not reflect the scale of the problem (Smits et al., 2020;NCT04193358).
A limitation of this review is that supplements with ingredients listed in a language other than English, Dutch, French, Spanish, or German were excluded. This led to exclusion of several supplements (with ingredients listed in Russian and Chinese). Many supplements were excluded for containing plant extracts; this may have resulted in a less comprehensive image of available supplements. In this review, we have searched shopping websites to show a patient's view of the market for male fertility supplements. As a consequence, a comprehensive search strategy was not possible.

Conclusion
This review shows that the extensive range of OTC male fertility supplements is not supported by good quality evidence. To make a well-informed decision, patients should be provided with information on harms and benefits of a treatment based on welldesigned RCTs, ideally with the ultimate end point that matters: ongoing pregnancy or live birth. Surveillance of the content of the supplements and the information available for patients and professionals should be improved. Factual information on supplements as provided in this review should be available for patients through professional channels such as websites of fertility clinics and patient organizations.

Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.