Antioxidants are currently being marketed to treat male factor infertility. Indeed, biologic evidence supports the hypothesis that antioxidants would improve male fertility. A variety of pathologic conditions may increase oxida tive stress in semen (1–3). Oxidative stress can cause lipid peroxidation, thereby producing structural modifications to the sperm plasma membrane, which have been shown to interfere with sperm motility, acrosome reactions, and sperm– oocyte fusion (4). Oxidative stress may also damage the nuclear and mitochondrial genome by causing single and double DNA breaks, chemical modifications of bases, DNA crosslinks, and DNA protein crosslinks (5). In semen, antioxidants decrease oxidative stress (6), potentially improving sperm motility and reducing DNA fragmentation (7).
Studies of supplements have tended to show an improve ment in semen parameters with the use of antioxidants. Ben efits of vitamin E (8), selenium (9), N-acetylcysteine (10), or carnitine (7) on sperm motility have been seen after 3 months of treatment. Unfortunately, most of these studies have been small and heterogeneous. Although most of the studies included only infertile men, some included those with normal baseline semen parameters and some with abnormal baseline semen parameters. Treatment with vitamin C and vitamin E has been shown to reduce DNA fragmentation compared with placebo (11).
A recent meta-analysis concluded
that antioxidant supple mentation taken by subfertile males may
increase the chance of live birth; however, large randomized,
well-designed, placebo controlled trials have been needed
(7). A number of the included trials used
antioxidants in combination with in vitro fertilization (IVF); it
is certainly possible that the response to antioxidants would
differ with IVF. In addition, the meta-analysis included trials of
‘‘substances with antioxi dant properties’’ (myo-inositol,
polyunsaturated acids, resvera trol, vitamin B, and vitamin D). A
variety of antioxidant formulations are commercially available, but
trials using anti oxidant formulations have been limited by sample
size and by use of
secondary end points. The Males, Antioxidants, and Infertility
(MOXI) trial was designed to test the hypothesis that antioxidants
would improve male fertility without the use of assisted
reproductive technology (ART).
MATERIALS AND METHODS
The MOXI clinical trial was conducted by the Eunice Kennedy Shriver National Institute of Child Health and Human Develop ment (NICHD) Cooperative Reproductive Medicine Network. The Collaborative Center for Statistics in Science at Yale Uni versity served as the data coordinating center. The trial was conducted at nine clinical sites throughout the United States.
A full description of the trial
with inclusion and exclusion criteria is listed on
(NCT02421887). This was a
multicenter, randomized clinical trial involving couples with male
factor infertility. Heterosexual couples with at least 12 months of
infertility were eligible. Male partners were 18 years of age or
older with at least one abnormal semen param eter on a semen
analysis in the preceding 6 months: sperm concentration %15
million/mL (oligospermia), total motility%40% (asthenospermia),
normal morphology %4% (teratospermia), or DNA fragmentation R25%.
Female part ners were between 18 and 40 years of age with regular
men strual cycles (defined as 25 to 35 days in duration), evidence
of ovulation (by biphasic basal body temperature, ovulation
predictor kits, or luteal serum progesterone level R3 ng/ mL), and
a normal uterine cavity with at least one patent fal lopian tube.
Women over the age of 35 had a normal ovarian reserve, defined as
an early follicular phase follicle stimulating hormone (FSH) level
of %10 IU/L, an antimuller- € ian hormone (AMH) level of R1.0
ng/mL, or antral follicle count >10. Male partners were excluded
if they had a sperm concentration <5 million/mL on the screening
semen anal ysis or if they were taking fertility medication or
testosterone. Men were required to refrain from taking any vitamins
for 4 weeks before randomization.
VOL. 113 NO. 3 / MARCH
ORIGINAL ARTICLE: ANDROLOGY
Approval for the study was obtained from the University of Pennsylvania, which served as the single institutional re view board for each site, with additional local site review (12). Written informed consent was obtained from all male and female participants.
Men received a placebo or an antioxidant formulation con taining 500 mg of vitamin C (ascorbic acid), 400 mg of vitamin E (d-a tocopheryl), 0.20 mg of selenium (L-selenome thionine), 1,000 mg of L-carnitine, 20 mg of zinc, 1,000 mg of folic acid, 10 mg of lycopene, and 2,000 IU of vitamin D daily (IND #125753) for at least 3 months and up to 6 months. The antioxidant and placebo were purchased from and packaged by a commercial manufacturer for the study. This formulation was selected because it was commercially available and each component at comparable doses had been previously studied in a randomized, controlled trial and found to positively impact sperm structure or function and/or pregnancy rates af ter ART (13). The study medications were assigned in a double-blind fashion. The randomization scheme was gener ated using a computer-generated random number sequence in randomly varying blocks of four and six stratified by site and female age (<35 years and R35 years of age) with allocation 1:1 by the data-coordinating center through a Web-based, secured randomization service. Pill counts were conducted at each study visit to monitor compliance.
Male participants provided a semen sample on the day of randomization and after 90 days of treatment. The semen analysis included standard measurements such as volume, pH, count, and motility. Semen smears were prepared from each sample and shipped to the University of Utah School of Medicine Andrology and IVF Laboratory for centralized assessment of sperm morphology using World Health Organi zation 5.0 criteria. In addition, 1 mL of semen was stored at 80 C and subsequently shipped frozen to the Utah Androl ogy Laboratory for DNA fragmentation assessment using the sperm chromatin structure analysis (SCSA) test (14), when 10 million sperm were present. A blood sample was obtained at randomization and after 3 months of treatment. The samples were shipped to ARUP Laboratories in Salt Lake City, UT, where they were analyzed for selenium, vitamin E-a tocoph erol, vitamin E-g tocopherol, and zinc.
The couples were provided with free ovulation predictor tests and were instructed on timing their intercourse during the first 3 months of treatment (phase 1). Couples who had not conceived after 3 months of timed intercourse received up to three cycles of ovarian stimulation with clomiphene cit rate with intrauterine insemination (phase 2). Women who conceived were observed through pregnancy and delivery.
The primary outcome for the trial was live birth, defined as a delivery of a live infant after 20 weeks’ gestation. The second ary outcomes included pregnancy, defined as a positive home pregnancy test, within 6 months of treatment. A prespecified, internal pilot was created to examine the effect of the antiox
idant formulation on male semen parameters and DNA frag mentation at 3 months of treatment compared with controls. The protocol was designed such that if the pilot failed to reject the null hypothesis that motility and DNA fragmentation did not differ between the two treatment groups (antioxidant and placebo) after 3 months of treatment, the MOXI trial would stop enrollment.
The primary outcome was a live birth resulting from a preg nancy occurring within the 6 months of treatment. For the po wer analysis, a live-birth rate of 35% in the antioxidant group and 25% in the placebo group with a 17% dropout was assumed. A sample size of 395 in each group would yield 80% power using a two-sided chi-square test with a¼0.05. For the internal pilot, we assumed 50% of the males would have low motility (<40%) at baseline. For sample size calcu lations for the pilot study, we assumed that after 3 months of treatment sperm motility would differ by 13% (95% confi dence interval [CI], 3.45%–23.49%) (13) between the antiox idant and placebo groups, and DNA fragmentation would be 9.1% 7.2% in the antioxidant group and 22.1% 7.7% pla cebo group (11). Assuming a 20% dropout rate, a sample size of 60 in each group would yield R80% power at a¼0.05 for both outcomes.
Intention-to-treat analyses were
performed to compare the two groups. Categorical data are reported
as frequencies and percentages, and analysis conducted using
chi-square analysis and Fisher’s exact test where appropriate.
Nonpara metric data are expressed as median with interquartile
range and bivariate analyses completed using the Wilcoxon rank sum
test. Parametric data are expressed as mean standard
deviation (SD); Student’s t-tests were used for analyses. Ana lyses
were performed with SAS, version 9.4 (SAS Institute). A two-sided
P<.05 was considered statistically significant.
We prescreened 822 couples. Of the 264 couples who provided written informed consent and completed the screening, 171 were eligible and were randomly assigned to a treatment group (Supplemental Fig. 1, available online), and 144 of these couples completed the study. The frequency of dropouts was not statistically significantly different between the study groups (21% in the antioxidant group, and 11% in the placebo group; P¼.055). Adherence, defined as intake of 80% or more of study drug during phase 1, was 88% among the antioxidant users and 82% among the placebo users (P¼.26).
Baseline characteristics are
presented in Table 1
for the male participants and
in Supplemental Table
1 (available on line) for
the female participants. The mean ( SD) selenium levels at
randomization were 160.3 19.8 mg/L, mean vitamin E-a
tocopherol levels were 9.6 2.7 mg/L, and mean zinc levels
were 89.3 12.1 mg/dL. Baseline character istics were no
different between the two groups, except men in the placebo group
were more likely to have fathered a preg nancy in the past. The
baseline semen characteristics (Table 2)
were similar in the two groups, except men in
the available online).
(Note that the antioxidant formulation contained vitamin E-a
tocopherol and did not contain vitamin E-g
In this randomized controlled trial of couples with male factor infertility, the use of an antioxidant combination in the male partner did not result in a statistically significant improve ment in semen parameters after 3 months of therapy compared with placebo. Furthermore, men with asthenosper mia or high DNA fragmentation did not exhibit an improve ment in motility or a decrease in DNA fragmentation, as had been hypothesized. Although the internal pilot was not pow ered to examine differences in pregnancy rates, couples whose male partner received an antioxidant were not more likely to conceive during natural cycles or with intrauterine insemination.
Treatment with an antioxidant
formulation did not in crease motility among the entire cohort nor
in the subgroup with asthenospermia at baseline. Although
benefits of vitamin C (15), selenium (9), N-acetylcysteine (10),
L-carnitine (16), and zinc (17) on sperm motility have been found
after 3 months of treatment, most of those studies were small and
heterogeneous. Most studies have included only infertile men,
although some included men with normal baseline semen parameters
and some with abnormal baseline semen parameters. A recent Cochrane
meta-analysis by Smits et al. (7), which included only with men
with abnormal sperm, found that only N-acetylcysteine, selenium,
and vitamin E alone improved sperm motility. Given the degree of
heteroge neity, pooling of all antioxidant results was not
possible. However, the Cochrane meta-analysis did find a 12%
absolute increase in motility in men treated with combination
antiox idants for 3 months compared with controls (7). In the trial
by Raigani et al. (18), 84 men with oligoasthenoteratospermia using
a combination of folic acid and zinc for 14 weeks showed no
improvement in sperm motility after 14 weeks of therapy, similar to
Treatment with an antioxidant formulation did not decrease DNA fragmentation as measured by the SCSA among the entire cohort or among men with high DNA frag mentation at baseline. Only a few clinical trials have compared sperm DNA fragmentation between those treated with and without antioxidants. Greco et al. (11) enrolled 64 men with DNA fragmentation levels R15%, as measured us ing terminal deoxynucleotidyl transferase dUTP nick end la beling (TUNEL) assay. After 2 months of treatment with vitamin E and vitamin C, the DNA fragmentation index decreased from 22% to 9%, with no change noted in the pla cebo group. Although the DNA fragmentation levels were no different in our cohort at baseline, we did not observe a similar reduction over 3 months, despite using a combination that included both vitamin E and C. Our cohort was over twice the size, and we used SCSA, not TUNEL, to quantify DNA fragmentation. Just as we noted in men with high DNA frag mentation (>25%), Stenqvist et al. (19) found no improve ment in DNA fragmentation as measured by SCSA after 6 months of therapy with an antioxidant combination. The recent Cochrane meta-analysis, which included five trials with a variety of antioxidants with a total of 254 participants, found that men treated with antioxidants
had on average 5% lower DNA fragmentation, but the confidence interval was broad and crossed 0 (7). Taken together with previous find ings, our results indicate that while antioxidants may reduce reactive oxygen species (ROS), this does not appear to trans late into reduced sperm DNA fragmentation.
Couples in which the male partner was treated with anti oxidants were not more likely to have a pregnancy resulting in a live birth in the first 3 months of treatment with timed in tercourse, nor in the second 3 months of treatment with ovarian stimulation with intrauterine insemination. Antioxi dants did not improve pregnancy or live-birth rates.
Given that our trial also found no improvement in semen parameters, the data and safety monitoring board concluded that continuing the trial in light of the lack of response was not justifiable. The trial-stopping rule had the strong underly ing hypothesis that the effect of the intervention on live births is (at least partially) mediated through improvements in sperm motility and a reduction in sperm DNA fragmentation. The in ternal pilot study was designed to provide further evidence that antioxidants could improve semen quality to justify an investment in a trial of sufficient magnitude to study the outcome of live birth. However, conventional semen quality parameters and even sperm DNA fragmentation are, at best, modest predictors of a couple’s fertility when trying with or without medical assistance.
The recent Cochrane Review found that antioxidant use increased the odds of pregnancy by 2.97-fold and the odds of live birth by 1.8-fold (7). The meta-analysis included nine studies of six antioxidant or antioxidant combinations for a total of 750 participants in the live-birth analysis. Two of the trials, which strongly favored antioxidants, were in couples undergoing IVF (20, 21). Follow-up evaluation in the natural conception trials was not systematic (22, 23). In the Omu trial (23), the couples were evaluated for 6 months after cessation of antioxidant therapy. Similar to our MOXI
trial, the high-quality trials included in the Cochrane review did not find a benefit to antioxidants on live birth (24, 25). Our negative findings contradict the overall conclusions from the Cochrane Review and meta-analysis. This could be due to many factors. Henkel et al. (26) suggest that excessive use of antioxidants may upset the balance between oxida tion and reduction, leading to reductive stress. Although this is a theoretical concern, the antioxidant formulation used in our study did not include excessive amounts of any given antioxidant; the doses aligned with those used in prior trials.
The antioxidant formulation was selected based on input from the steering committee, advisory board, and data and safety monitoring board. Although one or two individual an tioxidants could have been selected for the study, a combina tion formulation was selected because  there are multiple antioxidants,  antioxidant formulations are being mar keted and prescribed, and  there was no single ‘‘superior’’ antioxidant. A commercially available antioxidant formula tion was selected to reduce the potential for opposing effects of antioxidants, reductive stress due to excessive antioxi dants, or poor or impure product selection. Unfortunately, the design of the MOXI trial does not allow the differentiation of effects of individual nutrients and inherently assumes there are no interacting effects between the different antioxidants in the formulation. Because this assumption may not be true, future randomized controlled trials could study individ ual components through a factorial design.
Another theoretical concern is that we selected patients who would be unlikely to benefit from antioxidants. For example, only men with elevated levels of ROS should have been included. However, this is not how antioxidants are currently marketed or prescribed. Our inclusion criteria were similar if not more selective compared with prior trials. We also evaluated subgroups who were more likely to have ROS damage—those with asthenospermia and high DNA fragmen tation—and did not see any evidence of benefit.
This multisite, randomized, double-blind, placebo controlled trial was designed with adequate power to
determine the extent to which antioxidants improve semen parameters and DNA fragmentation. Prior trials have been small and of low or very low quality (13). All men enrolled in the MOXI trial had male factor infertility, with at least one abnormal semen parameter and a partner with normal fertility testing. Plasma vitamin levels confirm that men in the antioxidant group complied with the regimen, and the men randomized to placebo did not cross over. The trial was powered to examine changes in semen parameters in the entire cohort and in subgroups with specific sperm abnormalities.
Although MOXI was not powered to determine group differences in live birth, it is the largest, appropriately de signed trial to date to examine the impact of antioxidant treatment in the male partner on subsequent non-ART out comes; we found no increase in live birth either with timed intercourse or with intrauterine insemination. Future studies may seek to determine whether there are subpopulations (e.g., men with low vitamin levels, men with high levels of ROS in their semen) for which antioxidants may improve semen parameters. Larger trials are needed to examine live birth as an outcome.
Antioxidant treatment does not improve semen parameters or DNA integrity in infertile males. Although limited by sample size, this study suggests that combination antioxidant treat ment of the male partner does not improve in vivo pregnancy or live-birth rates in couples with male factor infertility, but larger trials are needed to confirm this finding.
Acknowledgments: Esther Eisenberg, M.D., M.P.H.
served as Project Scientist for the network, was a member of the RMN steering committee along with the principal investiga tors, and as such had a role in the design and conduct of the study; interpretation of the data; and review and approval of the manuscript. Dr. Eisenberg did not have any role in the funding decisions nor oversight of the grants that funded the RMN or RMN investigators. The authors thank the other mem bers of the NICHD Reproductive Medicine Network: Univer sity of North Carolina, Chapel Hill: S.L. Young; C. Nagle; University of Oklahoma, Oklahoma City: M.C. Lindgren, L.B. Craig, L.Y. Turner, M.R. Starkey-Scruggs; Yale Univer sity: F. Sun, T. Thomas, H. Carlson, D. DelBasso, L. Sakai; Uni versity of Pennsylvania: C. Coutifaris; University of California–San Francisco: R. Wong, T Leung, L Jalalian; Penn State College of Medicine, Hershey, PA: W.C. Dodson, S.J. Estes, J. Ober, P. Rawa; Augusta University, Augusta, GA: L.C. Layman, L. Gavrilova-Jordan, L.A. Ogden, C. La serna; Wayne State University, Detroit, MI: A. Awonuga, K.L. Collins, M. Yoscovits, E. Puscheck; Eunice Kennedy Shriver National Institute of Child Health and Human Devel opment: L. DePaolo; Advisory Board: D. Guzick (Chair), A. Branum, M. Thomas, J. Redmon, M. Goldman; Data and Safety Monitoring Board: F. Witter (Chair), P. Coney, S. Mis smer, P. Cato, L. Inoue, R. Brannigan. The authors also thank E. Schisterman and S. Mumford of the NIH/NICHD for their input.
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