1 140 Earl Warren Hall, School of Public Health, University of California, Berkeley, CA 94720-7360, 2 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Avenue, L-448, Livermore, CA 94550 and 3 Genetics and Developmental Biology Program, West Virginia University, Morgantown, WV 26506, USA
4 To whom correspondence should be addressed. Email: eskenazi{at}berkeley.edu
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Abstract |
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Key words: age/antioxidants/diet/semen quality/sperm
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Introduction |
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In clinical trials, vitamin E supplementation has been found to increase fertilization rates (Geva et al., 1996) possibly by reducing oxidative damage (Comhaire et al., 2000
) and lipid peroxidation potential (Geva et al., 1996
). Ascorbic acid concentration in the seminal plasma has also been found to be negatively associated with reactive oxygen species activity in sperm of infertile men, and the depletion of ascorbic acid intake has been associated with an increase in oxidative damage in the sperm of healthy men (Fraga et al., 1991
). There is only limited evidence, however, that supplementation will improve clinical measures of semen quality. In one small trial of nine infertile men receiving 400 mg/day of
-tocopherol in combination with selenium (Vezina et al., 1996
), there were significant increases in sperm motility, percentage of live sperm and percentage of normal sperm, whereas in other studies, there was no effect of even higher levels of vitamin E supplementation (3001200 mg/day) (Moilanen et al., 1993
; Kessopoulou et al., 1995
; Moilanen and Hovatta, 1995
) alone or in combination with vitamin C (Rolf et al., 1999
).
The micronutrients folate and zinc have also been associated with semen quality. Folate levels in blood plasma (Wallock et al., 1997) and in seminal plasma (Wallock et al., 2001
) have been positively associated with sperm concentration and count. Zinc levels in seminal plasma have been positively associated with sperm concentration and motility in some studies (Fuse et al., 1999
; Chia et al., 2000
), but not others (Lewis-Jones et al., 1996
; Lin et al., 2000
). In clinical trials of men with round cell idiopathic syndrome, folinic acid supplementation (15 mg/day) improved both sperm count and motility (Bentivoglio et al., 1993
). Sperm count also increased after combined zinc sulfate (66 mg) and folic acid (5 mg) treatment in a randomized clinical trial of subfertile men, but not with either alone (Wong et al., 2002
). However, in a trial where infertile men were given a much higher dose of 500 mg/day of zinc sulfate alone, there was a significant improvement of count and progressive motility (Omu et al., 1998
).
Although high dose supplementation may impact semen quality, no study has examined whether variation in intake over the normal dietary and supplement intake range is also associated with semen quality. Also, there remains a question as to whether diet can reverse the decrements in semen quality that occur with age (Eskenazi et al., 2003). The purpose of the present analysis is to determine whether normal dietary and supplement intake of specific micronutrients (zinc and folate) and antioxidants (vitamins C, E and
-carotene) is associated with semen quality in a population of healthy non-smoking men over a wide age range and without a previous history of reproductive problems. In addition, we aim to determine whether nutrient intake can modify the age-related decrements in semen quality we have observed previously in this population.
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Materials and methods |
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Dietary assessment
The men were mailed a questionnaire and a semen collection container with instructions. The questionnaire asked about medical and reproductive history, socio-demographic characteristics, occupation, and lifestyle habits and characteristics. Participants also completed a 100-item self-administered Modified Block Food Frequency Questionnaire (Huang et al., 2002). This food frequency questionnaire (FFQ) estimates average daily intake of nutrients based on usual eating habits over the past year with questions about frequency and portion size of each listed food item. Average daily nutrient intake is derived from this questionnaire for: total calories, protein, fat, carbohydrate and micronutrients; average daily intake of vitamins and minerals from supplements; and frequency per day of food groups. The FFQ took
30 min to complete and was received within 1 week of producing the semen sample. Participants received the results of their semen analysis only after the FFQ was received. We excluded one participant whose nutrient analysis indicated intake of too few foods and calories per day (659 kcal/day), and therefore lacked credibility.
Semen analysis
Semen specimens were produced by masturbation into sterile containers and analysed within 2 h of collection (mean=45 min). Donors were instructed to abstain for 25 days but recorded the actual duration at collection. Replicate specimens were requested when there was indication of loss, low motility or presence of red or white blood cells in the semen. Coded specimens were analysed by established protocols (Shrader et al., 1992) with enhanced quality control (Eskenazi et al., 2003). Semen volume was measured to the nearest 0.1 ml. Sperm concentration was determined in triplicate. Total sperm count was calculated by multiplying the semen volume by the sperm concentration. Motility was assessed visually under 400x phase contrast magnification with a 5 x 5 ocular grid for 150 sperm per sample. Progressively motile sperm were the number of forward-moving sperm that exceeded 25 µm/s (approximately five times the length of the sperm head/s). Total progressively motile sperm (TPMS) was defined as the product of total sperm count and percentage progressive motility.
Statistical analysis
Sperm concentration, total sperm count, progressive sperm motility and TPMS were transformed to achieve normality by taking the square root. Unadjusted and adjusted means and 95% confidence intervals (CIs) for square-root-transformed semen parameters were back-transformed for presentation in the text and tables. Motility-related analyses were performed excluding four participants with azoospermia (men aged 63, 77, 77 and 78 years). For analyses on both semen volume and TPMS, a single outlier (>3 SDs from the mean) was excluded from the population.
Intakes of micronutrients and antioxidants based on the FFQ were each divided into three categories (based on the intake of the entire sample of 96 men): the lowest quartile of intake (low intake), the middle two quartiles of intake (moderate intake) and the highest quartile of intake (high intake). An antioxidant composite variable was created a priori based on the intake of vitamins C, E and -carotene. Low antioxidant composite intake was defined as intake in the lowest quartile of two or more antioxidants; high antioxidant composite intake was defined as intake in the highest quartile of two or more antioxidants; and moderate intake was defined as all other combinations of intake (there were no participants in this category with a low intake ranking in two antioxidants and a high intake ranking in another antioxidant, and vice versa).
Analysis of covariance (ANCOVA) was used to examine the relationship between micronutrients and semen parameters. Multivariate ANCOVA models were created with continuous semen parameters as dependent variables, and micronutrient and antioxidant categories and potential confounders as independent covariates. Age (years) and duration of abstinence (5 versus
6 days) were included as covariates in all final models, and time (in minutes) from sample collection to sample analysis was also included in all final models for the motility-related parameters (i.e. for sperm motility, progressive sperm motility and TPMS). Based on information from the literature which suggested associations with semen parameters, the following additional covariates were considered: body mass index (BMI; kg/m2); history of cigarette smoking; season of sample collection (autumn, winter, springsummer); alcohol and coffee use in the 3 months prior to sample collection; hot tub use in the 3 months prior to sample collection; history of urinary tract infection and fertility-related problems; and exposure to work hazards and occupational exposure to ionizing radiation (measured from LLNL badge dosimetry records). A covariate was included in the multivariate model if it was related (P
0.20) to both the nutrient categories in univariate analyses and to the measure of semen quality controlling for age and abstinence, and if when placed in a regression model (with age and abstinence, as well as time to processing for motility measures), the coefficient for the nutrient changed by
10%. Least squares means were computed to obtain the predicted value of each intake category with covariates set at their mean value. The final regression models were also re-specified with the three intake groups as ordinal variables (i.e. 1=low intake, 2=moderate, 3=high) to test for trend.
To assess whether higher micronutrient or antioxidant intake reduced the adverse relationships previously observed with age, models were run with interaction terms for age and nutrient levels. In addition, each final adjusted model was stratified by intake level (low, moderate and high), and the coefficients for age in the three strata were compared by calculating differences and 95% CIs from the combined standard errors. If the 95% CI for any two-way comparison did not include zero, we concluded that there was a significant difference in the association of age with the outcome in the two nutrient intake levels (Altman and Bland, 2003).
Final models were re-specified with daily kilocalorie intake in order to examine relative versus absolute intake. Models were also rerun excluding men reporting very low (<1000 kcal; n=2) or very high (>3000 kcal; n=2) daily intake. Because recent vitamin or supplement use may be a response to an illness, the analyses were reanalysed without those men who began taking multivitamins or antioxidant supplements in the past 2 years (n=12). Similarly, although no men currently had fertility or genitourinary problems, the analyses were analysed again excluding men who reported any history of an abnormal semen analysis (n=5). Because the results in these additional analyses were similar to the final models, they are not presented.
All P-values presented are two-sided. The SAS statistical software package version 9.0 (SAS Institute Inc., Cary, NC) and Stata version 8.0 (STATA Corporation, College Station, TX) were used in the analyses.
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Results |
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For the most part, the demographic characteristics of the men were not associated with overall diet and supplement intake (data not shown). However, supplement users compared with non-supplement users tended to be older (P=0.03), to consume no alcohol in the previous 3 months (P=0.05) and to consume fewer calories (mean intake 1650 versus 1844 kcal; P=0.06), although their dietary intake of nutrients was similar. Increasing levels of folate intake were associated with lower BMI (r=0.26; P=0.01), but increasing kilocalorie intake (r=0.23; P=0.02). Intake of folate (r=0.26; P=0.01), zinc (r=0.33; P=0.001) and vitamin E (r=0.25; P=0.01) from diet alone was negatively correlated with age, but nutrient intake from diet and supplement use together did not differ by age for any of the nutrients.
As shown in Table II, >65% of men based on food intake alone and >40% based on supplement use and food intake combined reported intakes for vitamin E, zinc and folate below the dietary reference intake (DRI) (Institute of Medicine, 2000a, b
, 2002
). About 40% of the men had intakes which fell below the DRI for vitamin C intake based on dietary intake alone and 20% based on supplements and diet combined. For the micronutrients in Table II, 92100% of those in the high intake groups took supplements (multiple or single-entity) versus 4864% in the moderate intake groups versus 538% in the low intake groups. In the respective high intake group, 100% took single-entity vitamin C supplements, 83% took vitamin E and 29% took
-carotene supplements, and in the moderate intake group, 17% took vitamin C, 10% took vitamin E and none took
-carotene supplements; in the low intake group, no one took single-entity supplements.
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Discussion |
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We assessed dietary and supplement intake based on self-report on an FFQ. By categorizing intake into low, moderate and high, we were able to guard against effects being driven by unusually high intake by some men who took vitamin supplements. Although the FFQ ascertains eating habits over the last year, participants generally telescope their report so that their dietary report may reflect recent patterns of intake (Willett, 1998). Such a reporting bias would be to the benefit of this study, since the critical period for spermatogenesis was the preceding 3 month period.
Although we did control for a number of important potential confounders, men with high antioxidant intake may differ from men with lower intake in ways which remained unmeasured and/or unknown. For example, although the men in the higher intake group had similar overall caloric intake compared with men with lower antioxidant intake (data not shown), they were of lower body mass, suggesting that men with higher intake may be more physically active. Also, men with higher intake (and those with moderate intake) were less likely to have smoked in the past, although this difference was not significant. Thus, it remains possible that uncontrolled residual confounding reflecting other health behaviours may explain the better semen quality of men with higher antioxidant intake.
The results of our analyses are biologically plausible. Antioxidants may play a critical role in protecting male germ cells against oxidative damage (Fraga et al., 1991). The production of reactive oxygen species has been associated with loss of motility and a decreased capacity for spermoocyte fusion (Aitken et al., 1989
; Agarwal et al., 2003
). Kessopoulou et al. (1995)
showed that vitamin E levels after treatment improved the in vitro function of the human sperm as evidenced by the zona-binding test. Fraga et al. (1991)
found that seminal plasma levels of vitamin C were directly associated with the level of oxidative damage in human sperm DNA.
Although several clinical intervention trials of antioxidants have found improvement in semen characteristics, the health and habits of the men in our study may also contribute to differences in findings from some of the clinical trials. Rolf et al. (1999) hypothesized that the length of vitamin C and E administration in their study (i.e. 8 weeks) may have been too short to improve semen quality in infertile men if the effect is on the testis. In contrast, in our study, the men's intake of antioxidants reflected their normal diets and supplement use behaviour, although the association was seen in only the high intake group where almost all were taking supplements. The effects of small differences in intake may have greater impact in our study, because all the men in this study were non-smokers. Smokers normally need two to three times the intake of vitamin C to maintain blood plasma levels comparable with those of non-smokers (Fraga et al., 1996
), and some studies include both smokers and non-smokers (Comhaire et al., 2000
).
In summary, we found that higher antioxidant intake over the normal dietary and supplement use range was associated with higher sperm numbers and higher motility in a sample of healthy non-smoking volunteers, and that antioxidant intake, to some extent, may attenuate the impact of age on sperm motility. At present, a large proportion of the US population ingests insufficient amounts of antioxidants, e.g. >75% of adult American men do not meet the 15 mg/day RDA for vitamin E (Institute of Medicine, 2000a), and only 17.7% of men ate at least the recommended five fruit and vegetable servings per day in 2002 [Centers for Disease Control and Prevention (CDC), 2002
]. Our findings support the suggestion that a healthy diet with supplement use may be an inexpensive and safe way to improve semen quality and fertility.
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Acknowledgements |
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Submitted on June 14, 2004; resubmitted on November 11, 2004; accepted on December 7, 2004.