Association of Dietary Patterns With Testicular Function in Young Danish Men

This cross-sectional study assesses whether adherence to specific dietary patterns is associated with testicular function and semen quality in young Danish men.


Introduction
Semen quality has decreased substantially in the last few decades. According to a recent metaanalysis that included more than 185 studies, 1 total sperm count has declined by 50% to 60% from 1973 to 2011 in Western countries, in line with a continued decline since the 1940s. 2 In addition, some investigators have also reported a concomitant secular decline in serum testosterone levels. 3 Although debate is ongoing about the underlying causes for these declines, there is a growing concern and evidence that environmental exposures such as endocrine-disrupting chemicals and air pollution or behavioral factors such as smoking and alcohol consumption could explain this decline. 4 Nutritional factors and eating habits are potential behavioral factors contributing to the secular decline in semen quality that have received comparatively little attention. Diet quality has changed dramatically in the last 50 years in Western countries, with a tendency toward higher intakes of total calories, meat, cheese, added fats and sugars, and refined grains. 5 Many studies have examined the association of isolated nutrients-such as zinc, folate, and antioxidants (positive) and saturated and trans-fats (negative)-with semen quality and other markers of testicular function. 6,7 More recently, however, interest has shifted to focus on the role of overall diet patterns. 7,8 Although this literature is still expanding, these studies suggest that adherence to generally healthy diet patterns is associated with better semen quality parameters in North America, Europe, the Middle East, and East Asia. [7][8][9][10][11][12][13][14][15][16] However, the extent to which these findings may generalize beyond the studied countries is unclear when taking into account local variations in dietary behavior, which are key when designing clinical and public health recommendations. Therefore, we examined the association between diet patterns reflecting contemporary eating behavior among young men in Denmark and markers of testicular function, including semen quality, testicular volume, and serum reproductive hormone levels. We hypothesized that dietary patterns aligned with dietary recommendations for the prevention of chronic diseases would be associated with better semen quality and other markers of testicular health.

Participants
In Denmark, all men are required to undergo a physical examination at 18 years of age to determine fitness for military service. Starting in 1996, research staff at the University Department of Growth and Reproduction at Rigshospitalet, Copenhagen, Denmark, have approached men presenting for the physical examination in Copenhagen and invited them to participate in an ongoing study aimed at understanding the determinants of male fertility and reproductive potential. 17 After providing written informed consent, men answered questionnaires about their demographic characteristics, lifestyle, and medical history; provided semen and blood samples; and underwent a physical examination. During the physical examination, weight and height were measured, body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared, and testis size was assessed by ultrasonography. 18 The local ethical committee approved the study, which follows the

Diet Assessment
The food frequency questionnaire used was a modified version of a previously validated food frequency questionnaire used in the Danish National Birth Cohort 19 and the Danish Diet, Cancer and Health Studies. 20 Men reported how often, on average, they consumed specified amounts of 136 food items in the 3 months before enrollment. Portion sizes for individual food items were estimated with the help of photographs, and nutrient intakes were quantified on the basis of the Danish food composition tables. 21 We grouped individual food items into 40 food groups similar to those used in other studies of Western men 22 and based on the similarity of nutrient profiles or Danish culinary use (eTable 1 in the Supplement).

Semen Analysis and Testicular Volume
All men provided a semen sample by masturbation in a room close to the andrology laboratory. The men had been asked to abstain from ejaculation for at least 48 hours before sampling but were still included if abstinence time was shorter. Abstinence time was calculated based on self-reported time of previous ejaculation and time of study sample collection. Semen samples were analyzed in accordance with the current World Health Organization (WHO) guidelines 23 as previously described. 24 Briefly, we assessed semen volume, sperm concentration, sperm motility, and sperm morphology. 23-27 Since 1996, our laboratory has conducted a quality assurance/quality control in comparison with 2 other laboratories. 24,26 Testicular volume was measured using ultrasonography during the physical examination, and we calculated the mean volume for the right and left testes.

Reproductive Hormones
Venous blood samples were drawn from the participants in the morning of the day of participation, and serum was isolated and stored at −20°C. Testosterone concentrations were assessed using a time-resolved fluoroimmunoassay (DELFIA; Wallac) until 2013 and from 2014 onward using an enzyme-linked immunosorbent assay (Access 2; Beckman Coultier Ltd). Free testosterone level was calculated based on the measured serum concentrations of the total testosterone and sex hormone-binding globulin and assumed fixed albumin concentration. 28 Inhibin B concentrations throughout were determined by a specific 2-sided enzyme-immunometric assay (Inhibin B Gen II; Beckman Coulter Ltd). Concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin, and estradiol were measured using a time-resolved immunofluorometric assay (DELFIA; Wallac). From 2014 onward, estradiol concentrations were assessed with a radioimmune assay (Pantex). All hormone levels were analyzed in the same laboratory. The hormone levels were analyzed yearly in batches, including reanalysis of a number of controls from the previous year to ensure comparability over time. We also calculated the ratios of inhibin B to FSH, total testosterone to LH, free testosterone to LH, estradiol to total testosterone, and (estradiol to total testosterone) × 100.

Statistical Analysis
We used principal component analysis to derive diet patterns based on the 40 predefined food groups (eTable 1 in the Supplement). We used orthogonal transformations (Varimax) to achieve a simpler structure with uncorrelated factors to enhance interpretability. In determining the number of factors to retain, we considered eigenvalues, the scree plot, and the interpretability of the factors based on knowledge of Danish culinary traditions and eating behaviors. The substantive meanings of the rotated factors were considered in conjunction with the above empirical criteria, and the derived factors were labeled based on our interpretation of the data. For every person, we estimated factor scores for each of the 4 retained factors by summing the frequency of consumption multiplied by factor loadings across all food items. Thus, each participant was assigned a score for each of the 4 dietary patterns. Factor loadings for all dietary patterns are presented in Figure 1 with positive loading representing high consumption and negative loading representing avoidance of the corresponding food group in relation to each factor. We conducted sensitivity analyses by further adjusting for use of muscle-enhancing products (primarily whey protein and creatine supplements) and without adjusting for BMI (owing to possible mediation). Furthermore, we conducted sensitivity analyses after restricting the data to men who did not report use of muscle-enhancing products and men who never smoked and never used marijuana or other recreational drugs. Results were statistically significant when 2-sided α < .05 for the evaluation of trends across adherence to specific diet patterns and when the 95% CI excluded the null value for pairwise comparisons between categories of adherence. We conducted all statistical analyses using SAS, version 9.4 (SAS Institute Inc). We used principal components analysis as an extraction method in which the factor loading of a food group represents the contribution of that food group to the factor (diet pattern) identified. Positive scores indicate higher consumption of the food group; negative scores, avoidance of the food group.

Results
The 2935 men included in the analysis had a median age of 19 (interquartile range, [19][20] years, and 2290 (78.0%) had a BMI within the normal range ( Table 1 and eTable 2 in the Supplement). We identified 4 dietary patterns: the Western pattern (generally unhealthy), characterized by greater intake of pizza, french fries, processed and red meats, snacks, refined grains, sugar-sweetened beverages, and sweets; the prudent pattern (generally healthy), characterized by greater intake of fish, chicken, vegetables, fruit, and water; the open-sandwich pattern, characterized by greater intake of cold processed meats, whole grains (primarily whole-grain breads), mayonnaise, cold fish, condiments, and dairy; and the vegetarianlike pattern, characterized by intake of vegetables, soy milk, and eggs and avoidance of red meats and chicken ( Figure 1 and eTable 3 in the Supplement).
Men with higher adherence to the Western pattern were less physically active and vice versa for the prudent and the open-sandwich patterns ( Table 1 and eTable 2 in the Supplement). Although no major differences in total carbohydrate intake were noted with increasing adherence to each of the 4 patterns, we found important differences in intakes of fiber, total sugars, and added sugars with increasing adherence to the Western (less fiber and more added sugars), prudent (less added sugars), open-sandwich (more fiber), and vegetarianlike (more added sugars) patterns. Similarly, although no major differences in intakes of total fat were observed, greater adherence to the prudent pattern was associated with higher intake of long-chain ω-3 fatty acids, mostly reflecting differences in fish intake as part of this pattern. Also, major differences in intakes of carotenoids and vitamins C and B reflected differences in fruit and vegetable intake as adherence to the different patterns increased ( In the multivariable analysis, the median total sperm count for men in the highest quintile of adherence to the Western pattern was 26 (95% CI, -42 to -9) million lower than that of men in the lowest quintile of adherence to this pattern. Conversely, the median total sperm count of men in the highest quintile of adherence to the prudent pattern was 43 (95% CI, 23-63) million higher than that of men in the lowest quintile (Figure 2 and eTable 6 in the Supplement). Similar patterns were observed with all sperm parameters and testicular volume. In addition, the median percentage of motile spermatozoa for men in the highest quintile of adherence to the open-sandwich pattern was 2.3% (95% CI, 0.8%-3.9%) higher than that of men in the lowest quintile, and the median percentage of morphologically normal sperm for men in the highest quintile of adherence to the vegetarianlike pattern was 0.8% (95% CI, 0.2%-1.4%) higher than that of men in the lowest quintile (eTable 6 in the Supplement). Time of day of sample collection, median (IQR), h 10 (9-11) 10 (10-11) 10 (10-11) 10 (9-11) 10 (9-11) 10 (9-11) 10 (9-11) 10 (10-11) e Adjusted for energy intake using the residual method making nutrient intake independent from energy intake.  Models were adjusted for age, body mass index, height, smoking, use of marijuana and other recreational drugs, moderate-to-vigorous physical activities (hours per week), history of reproductive diseases, reproductive surgical procedures, sexually transmitted diseases, season and calendar year of the sample, mother's educational level, total energy intake, and abstinence time. Sperm motility models were further adjusted for time elapsed between specimen collection and analysis. Error bars indicate 95% CIs.

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Tests for trend were conducted across quintiles using the median value in each quintile of the diet patterns as a continuous variable in the regression models, and the P value was based on the Wald test. a P < .01. b P < .05.  Table 2).

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We then evaluated the association between diet patterns and having any semen parameter below the WHO lower reference limits 23 and the higher semen quality limits of Damsgaard et al. 29 In this analysis, high adherence to the Western diet pattern was associated with a higher estimated Similar associations were observed in sensitivity analyses in which we further adjusted for use of muscle-enhancing supplements, fit models without adjustment for BMI, and restricted analyses to men who did not report use of muscle-enhancing products and who never smoked or used marijuana or other recreational drugs (eTables 9-16 in the Supplement).

Discussion
We studied a group of young men who were unaware of their fertility status and found that men with generally healthier diets had better testicular function. Specifically, greater adherence to a Western pattern was associated with lower semen quality as well as lower serum inhibin B concentrations and ratios of inhibin B to FSH, suggesting reduced spermatogenesis. 30,31 In contrast, adherence to a prudent pattern was associated with higher semen quality. Higher adherence to an open-sandwich pattern (a traditional Danish pattern with whole grain bread) was associated with a higher count of motile spermatozoa, whereas adherence to a vegetarianlike pattern was associated with a higher percentage of morphologically normal spermatozoa. To our knowledge, this is the largest study to date examining the association between diet patterns and markers of testicular function.
Our results are consistent with our hypothesis that adherence to healthy diet patterns is associated with better testicular health, as reflected in semen quality parameters and reproductive hormone concentrations. Healthier dietary patterns, including local variations such as the Mediterranean diet in studies conducted in Spain 16 and Greece, 32 have been consistently associated with better semen quality. In recent reviews, 7,9,10 dietary patterns favoring intakes of seafood, poultry, whole grains, legumes, skim milk, fruits, and vegetables have been consistently associated with better semen parameters in studies in North America, Europe, the Middle East, and Asia. 7,8,[11][12][13][14][15][16]32,33 A study in the Netherlands found that men who adhered to a healthy diet pattern had higher sperm concentration, total sperm count, and motile spermatozoa. 34 In another study in Poland, the prudent dietary pattern was associated with higher sperm concentrations. 15 Associations between unhealthy dietary patterns and lower semen quality have been less consistent, however.
Our findings suggest that local variations to generally healthy diets beyond the Mediterranean diet pattern may also offer benefits that could be missed in studies focused on more traditional diet scores. For example, the only diet pattern associated with sperm motility in our study was the opensandwich pattern, which reflects a Nordic culinary tradition characterized by consumption of whole-grain bread, cured or smoked fish, and high-fat dairy products. However, we cannot exclude           surgical procedures, sexually transmitted diseases, season and calendar year of the sample, mother's educational level, total energy intake, and time of the day of the sample collection (except for testicular volume).

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Tests for trend were conducted across quintiles using the median value in each quintile as a continuous variable in the regression models; P value was based on the Wald test.
b Measured using ultrasonography and reported as mean (95% CI) of both testicles. the role of chance of this isolated association with sperm motility. The differences in semen quality observed may reflect the differences in nutritional profiles observed across levels of adherence to the 4 patterns identified. Previous work focused on intake of specific nutrients suggests that the associations observed herein could be explained by differences in intake of long-chain ω-3 fatty acids, [35][36][37] carotenoids, vitamins C and B, 38,39 and possibly differences in carbohydrate quality, including intake of added sugars. [40][41][42] The interpretation of the hormone findings is less straightforward. Men with the highest adherence to the Western pattern had higher testosterone levels, assessed as total and free testosterone, compared with men with less adherence. At the same time, they also had the highest estradiol concentration but unchanged LH levels. Thus, it appears that adherence to the Western pattern may lead to an increased aromatization of testosterone to estradiol. We speculate that this change has resulted in increased negative feedback at the hypothalamic level. If this is correct, it might also explain why FSH is not sufficiently increased as a compensation for the lower inhibin B levels. Thus, we can speculate that adherence to the Western pattern at least partly leads to some degree of reduced hypothalamic activity that in turn leads to a reduction in spermatogenesis. In addition to this speculation, the lower ratio of inhibin B to FSH itself points toward a direct (primary) adverse effect on the testicles. If the associations reflect causation, adherence to a Western diet may lead to combined primary and secondary endocrine reduction of spermatogenesis. However, we cannot clarify that association any further. Similarly, we cannot clarify the biological significance of the slightly lower ratio of testosterone to LH for men with adherence to the open-sandwich pattern.

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Fewer studies have examined the association of dietary patterns with reproductive hormones and testicular size. Unlike our results, Jurewicz and colleagues 15 reported that adherence to the prudent pattern was associated with higher testosterone concentrations, whereas a Western diet was not associated with testosterone levels among 336 men in Poland. However, their study included only men who attended a fertility clinic with semen parameters higher than the 1999 reference limits according to the WHO or with oligozoospermia, which could have introduced selection bias. 15 In addition, there was no observed association of the Western and Mediterranean patterns with testicular volume or reproductive hormones in 215 men in Spain. 16 The lower inhibin B level and ratio Diet Patterns, Quintile The WHO lower reference limits for semen parameters are less than 1.5 mL for semen volume, less than 15 million/mL for sperm concentration, less than 39 million for total sperm count, less than 40% for motile spermatozoa, less than 32% for progressively motile spermatozoa, and less than 4% for normal sperm morphology. Models were adjusted for age, body mass index, height, smoking, use of marijuana and other recreational drugs, moderate-to-vigorous physical activities (hours per week), history of reproductive diseases, reproductive surgical procedures, sexually transmitted diseases, season and calendar year of the sample, mother's educational level, total energy intake, and abstinence time. Sperm motility models were further adjusted for time elapsed between specimen collection and analysis. For the Western pattern, P = .006 for trend; for the prudent pattern, P = .004 for trend; for the open-sandwich pattern, P = .54 for trend; and for the vegetarianlike pattern, P = .39 for trend.

Strengths and Limitations
Strengths of the study include the large sample size, affording high statistical power and precision.
Most importantly, because these men were not aware of their fertility status, the generalizability of the results to the general population of young men regarding testicular function is increased and selection bias is unlikely. In addition, participants not included in this analysis had comparable demographics to men in this analysis 17,24 (eTable 17 in the Supplement) and comparable concentrations of the reproductive hormones. 44 Finally, the use of dietary pattern allows for easier translation of these results into clinical or public health recommendations. 22 Our study has some limitations, including the potential misclassification of self-reported diet and reproductive parameters based on a single measurement. However, previous studies 45, 46 have shown that a single semen sample may suffice for studies aimed at identifying mean differences in semen quality between men. Because this was a cross-sectional study, it limits our ability to determine causality; however, dietary habits tend to be quite stable over time. 47,48 We acknowledge that multiple comparison may be an issue, because we compared 4 main exposures in association with multiple related outcomes. However, even using a Bonferroni-adjusted P = .003, our main conclusions remain unchanged. In addition, we cannot exclude residual confounding, although we did adjust for many potential confounders.

Conclusions
Our findings support the evidence that adhering to generally healthy diet patterns is associated with better semen quality and more favorable markers of testicular function. Because diet is modifiable, these results suggest the possibility of using dietary intervention as a potential approach to improving testicular function in men of reproductive age.