ABSTRACT
Background
Randomized clinical trials show that men's use of antioxidant supplements during infertility treatment may improve clinical outcomes. However, important limitations in the design of most trials make it difficult to draw firm conclusions on their findings.
Objective
We examined whether men's intake of antioxidants and biologically related compounds without direct antioxidant capacity is associated with outcomes of assisted reproductive technologies (ARTs).
Methods
We conducted a prospective cohort study of men in couples who underwent infertility treatment with ART using their own gametes between 2007 and 2017. We followed 171 couples who presented at Massachusetts General Hospital Fertility Center and underwent 294 autologous ART cycles for infertility treatment. Diet was assessed in both partners using an FFQ. The primary study outcome was the probability of achieving a live birth as a result of infertility treatment. Secondary outcomes were fertilization, implantation, and clinical pregnancy rates. Generalized linear mixed models with random intercepts were fitted to account for multiple ART cycles per woman while adjusting for confounding.
Results
Men's vitamin C intake was positively associated with fertilization rate. The adjusted fertilization rate (95% CI) for couples in the lowest and highest quartiles of men's vitamin C intake were 69% (61–76%) and 81% (74–86%) (P-trend = 0.02). Men's β-carotene intake was positively associated with fertilization rate in intracytoplasmic sperm injection cycles but not in conventional in vitro fertilization cycles (P-interaction = 0.01). Men's α-carotene intake was inversely related to the probability of live birth. The adjusted probabilities of live birth for men in the lowest and highest quartiles of α-carotene intake were 43% (28–60%) and 22% (12–36%), respectively.
Conclusions
Men's intake of vitamin C and β-carotene is positively related to fertilization rate but this does not translate into higher pregnancy or live birth rates in couples undergoing infertility treatment.
Keywords: in vitro fertilization, probability of live birth, assisted reproductive technology, vitamins, carotenoids
Introduction
An estimated 48.5 million couples worldwide are infertile (1), making infertility not only a clinical challenge but also a major public health concern. Male factors are identified in a high percentage of couples evaluated for infertility (2–4) and oxidative stress may be an important underlying cause of male infertility (4). Oxidative stress occurs when reactive oxygen species (ROS) overcome the natural antioxidant defenses of semen and cause cellular damage to sperm (4). Oxidative parameters are higher in the semen of men with a diagnosis of idiopathic infertility than in men with proven fertility (5). In addition, ROS concentrations are significantly higher in semen samples from infertile men when compared with those from healthy controls, which has prompted the suggestion that infertile men may benefit from antioxidant supplementation (6).
A Cochrane review of randomized clinical trials (RCTs) in couples undergoing infertility treatment found that antioxidant supplementation may improve sperm motility as well as clinical pregnancy and live birth rates during infertility treatment (7). However, the Cochrane reviewers also indicated important gaps, which complicate the interpretation of these data. Most importantly, few trials included in the meta-analysis evaluated the same intervention (i.e., the same dose and combination of active ingredients) against the same comparator (i.e., placebo or same active comparator). In addition, the expansive definition of antioxidant used in this meta-analysis, which included trials testing the effects of N-acetyl-cysteine, pentoxifylline, L-arginine, myo-inositol, D-chiro-inositol, L-acetyl-carnitine, L-carnitine, selenium, vitamin E, vitamin B complex, vitamin C, vitamin D, CoQ10, pentoxifylline, zinc, folic acid, vitamin B-12, and omega-3 fatty acids, alone or in combination, resulted in the inclusion of trials evaluating the effects of very dissimilar agents, making it difficult to specifically attribute any observed effect to protection against oxidative stress. Furthermore, many of the included studies had a limited sample size, which prevents a thorough analysis of relevant clinical outcomes, in particular, the probability of achieving a live birth as a result of treatment. Complicating the issue of sample size, 3 of the 4 trials evaluating live birth as an outcome were published in the mid 1990s and reported live birth rates of 10% or lower (8–10), calling into question their relevance to the current practice of reproductive medicine. These issues make it difficult to draw strong conclusions regarding the effects of antioxidants on assisted reproductive technology (ART) outcomes. To address these important gaps, we conducted a prospective study to examine the association of men's micronutrient intake with the direct ability to neutralize free radicals (vitamins C and E), carotenoids with varying degrees of direct (11, 12) and indirect antioxidant capacity (13) (α-carotene, β-carotene, β-cryptoxanthin, lycopene, and lutein and zeaxanthin), and biologically related compounds without direct antioxidant capacity (retinol) with outcomes of infertility treatment using ART.
Methods
Study population
Participants in this study are couples who presented to Massachusetts General Hospital Fertility Center (Boston, MA, USA) for evaluation and treatment of infertility and who enrolled as a couple in the Environment and Reproductive Health (EARTH) Study, an ongoing prospective cohort study investigating environmental and nutritional determinants of fertility (14). Men aged 18–55 y with no history of vasectomy and in a couple anticipating use of their own gametes for infertility treatment were invited to participate. An estimated 60% of potential participants referred to the study and approached by the research nurses agreed to join the study. Here, we report on couples who enrolled between January 2007 and December 2017, in which male partners completed a comprehensive dietary assessment prior to starting infertility treatment with ART.
Trained research nurses measured participants’ height and weight at enrollment. Participants also completed a take-home questionnaire regarding medical and reproductive history, as well as lifestyle factors. Clinical data, including ART cycle characteristics and infertility diagnoses, were abstracted by research nurses from electronic medical records. Research nurses explained the study procedures before participants provided written informed consent. The current analysis includes 171 heterosexual couples who completed diet assessments and underwent 294 autologous ART cycles (Supplemental Figure 1). Cycles that started prior to diet assessment (7 cycles) and oocyte donation cycles (25 cycles) were excluded from the analysis. The study was approved by the Institutional Review Boards of the Massachusetts General Hospital and the Harvard TH Chan School of Public Health.
Dietary assessment
Diet was assessed before ART treatment with an extensively validated, self-administered FFQ (15, 16). All participants reported how often they consumed specified amounts of 131 food items during the previous year. We estimated nutrient intake by summing the nutrient contribution of each food and supplement included in the questionnaire, taking into consideration the brands of specific supplements and breakfast cereals. The nutrient content for each item in the questionnaire was obtained from the USDA (17) with supplemental data from food manufacturers. Nutrient intake was energy adjusted using the nutrient residual model (18). Total vitamin A intake was expressed as μg/d of Retinol Activity Equivalents (RAEs), which includes the intake of preformed retinol and provitamin A carotenoids weighed by their retinol-forming capacity. In a validation study, the correlation between FFQ-assessed carotenoid intake and plasma concentrations of the corresponding carotenoid ranged between 0.59 and 0.81 (19). Similar results were obtained for vitamins A, C, and E and carotenoids when FFQ assessments were compared with prospectively collected diet records or 24-h recalls (16). To account for potential confounding due to overall dietary choices, we derived 2 diet patterns using principal components analysis: a “Prudent” pattern characterized by a high intake of fish, fruits, cruciferous vegetables, yellow vegetables, tomatoes, leafy green vegetables, and legumes; and a “Western” pattern characterized by a high intake of processed meat, full-fat dairy, fries, refined grains, pizza, and mayonnaise (20).
Clinical management and assessment of outcomes
Trained study staff abstracted clinical information from the patients’ electronic medical records. Details of patient clinical management have been described elsewhere (21) and are summarized in Supplemental Figure 2. Briefly, after completing a cycle of oral contraceptives, participants underwent 1 of 3 stimulation protocols as clinically indicated: 1) luteal-phase gonadotropin-releasing hormone (GnRH) agonist protocol, 2) follicular-phase GnRH-agonist/flare protocol, or 3) follicular-phase GnRH-antagonist protocol. Clinical staff monitored patients during gonadotropin stimulation for serum estradiol (E2), follicle size and counts, and endometrial thickness, and administered human chorionic gonadotropin (hCG) ∼35–36 h before the scheduled oocyte retrieval to induce oocyte maturation. Oocytes were classified by embryologists as germinal vesicle (GV), metaphase I (MI), metaphase II (MII), or degenerated. Embryologists then determined fertilization rate as the number of oocytes with 2 pronuclei divided by the number of MII oocytes at 17 to 20 h after either insemination or intracytoplasmic sperm injection (ICSI). Patients undergoing cryo-thaw cycles underwent endometrial preparation protocols as clinically indicated. We assessed clinical outcomes identically for fresh and cryo-thaw cycles. Specifically, in all cycles in which insemination was attempted – the first step in assisted reproduction where male exposures could play a role – we assessed implantation (defined as a serum β-hCG concentration >6 mIU/mL, measured ∼17 d after oocyte retrieval), clinical pregnancy [defined as the presence of intrauterine gestational sac(s) on ultrasonography at 6 wk], and live birth (defined as the birth of a neonate on or after 24 weeks of gestation).
Statistical analysis
Men were divided into quartiles of intake of each nutrient of interest. The association of baseline demographic, dietary, and reproductive characteristics with quartiles of nutrient intake was evaluated using Kruskal–Wallis tests for continuous variables and chi-square tests for categorical variables. We used generalized linear mixed models with binomial distribution, logit link function, and random intercepts to evaluate the associations of vitamins A (RAE), C, and E, preformed retinol, and carotenoid (α-carotene, β-carotene, β-cryptoxanthin, lycopene, and lutein and zeaxanthin) intake with ART outcomes. We conducted tests for linear trend across quartiles of intake using the median nutrient intake in each quartile as a continuous variable in the regression models. Spearman correlations were used to describe within-couple similarities in baseline characteristics. Population marginal means were used to present population marginal probabilities of fertilization, implantation, clinical pregnancy, and live birth adjusted for covariates (22). Confounding was evaluated using previous knowledge and taking into consideration the associations between nutrient intake with baseline characteristics (21, 23–27). Multivariable adjusted models included terms for the other nutrients (categorical), age (continuous), BMI (continuous), smoking (categorical), folate and vitamin B-12 from supplements (continuous), calorie intake (continuous), dietary patterns (continuous), treatment protocol (categorical), and initial infertility diagnosis (male factor compared with not); female partner's age (continuous), BMI (continuous), and smoking status (categorical) were also included. We tested whether the association between male intake of the nutrients of interest and the probability of live birth was modified by age (y) (≥35 compared with <35), BMI (kg/m2) (≥25 compared with <25), smoking status (ever compared with never), infertility diagnosis (male factor compared with not), and IVF or ICSI procedures by introducing cross product terms to the multivariable adjusted models. Stratified analyses were conducted when there was a suggestive interaction effect (P value < 0.10). Statistical analyses were performed using SAS 9.4 (SAS Institute).
Results
Men's mean ± SD age and BMI were 36.8 ± 5.1 y and 27.2 ± 3.9, respectively (Table 1). The majority were white (91%), had a college degree or higher (96%), and never smoked tobacco (65%). The initial primary infertility diagnosis was unexplained infertility (39%), followed by male factor (36%). Men's intakes of vitamins A (RAE), C, and E, preformed retinol, and carotenoids were positively correlated with each other, as well as with folate and vitamin B-12 intake from supplements and both dietary patterns (Table 1). Intakes of vitamin A (RAE) and β-carotene were inversely related to total caloric and alcohol intake. No other associations with personal, partner, or reproductive characteristics were found (Table 1; Supplemental Table 1). Age, BMI, and dietary characteristics were positively correlated within couples (Supplemental Table 2).
TABLE 1.
Baseline characteristics of study participants1
| Quartile (range) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Vitamin A (RAE; μg/d) | Vitamin C (mg/d) | Vitamin E (mg/d) | ||||||||
| Characteristics | All participants | Q1 (<748) | Q4 (>1800) | P value | Q1 (<83.4) | Q4 (>186) | P value | Q1 (<8.0) | Q4 (>23.6) | P value |
| Quartile, median | 530.7 | 2365.5 | 64.1 | 302.0 | 6.3 | 33.5 | ||||
| n | 171 | 42 | 43 | 42 | 43 | 42 | 43 | |||
| Male partner characteristics | ||||||||||
| Age, y | 36.8 ± 5.1 | 36.7 ± 4.9 | 36.8 ± 6.0 | 0.90 | 36.4 ± 4.6 | 38.1 ± 5.3 | 0.18 | 35.9 ± 4.5 | 37.8 ± 4.9 | 0.09 |
| BMI, kg/m2 | 27.2 ± 3.9 | 27.5 ± 4.1 | 27.6 ± 3.9 | 0.70 | 26.7 ± 4.0 | 27.3 ± 4.5 | 0.65 | 26.8 ± 3.6 | 28.2 ± 4.4 | 0.52 |
| Ever smoker | 59 (34.5) | 15 (35.7) | 11 (25.6) | 0.54 | 14 (33.3) | 12 (27.9) | 0.69 | 14 (33.3) | 15 (34.9) | 0.97 |
| White | 155 (90.6) | 38 (90.5) | 40 (93.0) | 0.28 | 37 (88.1) | 38 (88.4) | 0.76 | 38 (90.5) | 38 (88.4) | 0.91 |
| College degree or higher | 164 (95.9) | 38 (90.5) | 42 (97.7) | 0.15 | 40 (95.2) | 41 (95.4) | 0.93 | 39 (92.9) | 42 (97.7) | 0.63 |
| Frequency of multivitamin supplement use | 97 (56.7) | 5 (11.9) | 42 (97.7) | <0.0001 | 6 (14.3) | 33 (76.7) | <0.0001 | 2 (4.8) | 40 (93.0) | <0.0001 |
| Dietary characteristics | ||||||||||
| Calorie intake, kcal/d | 2030 ± 654 | 1840 ± 529 | 1860 ± 673 | <0.01 | 1940 ± 541 | 1930 ± 731 | 0.24 | 1980 ± 527 | 1960 ± 829 | 0.34 |
| Alcohol, g/d | 15 ± 14 | 17 ± 14 | 10 ± 10 | 0.07 | 16 ± 14 | 11 ± 12 | 0.03 | 17 ± 14 | 15 ± 16 | 0.55 |
| Caffeine, mg/d | 201 ± 135 | 199 ± 128 | 156 ± 120 | 0.03 | 231 ± 138 | 172 ± 127 | 0.07 | 223 ± 152 | 186 ± 125 | 0.72 |
| Folate from supplements, DFE mg/d | 555 ± 476 | 178 ± 237 | 1000 ± 411 | <0.0001 | 204 ± 245 | 910 ± 581 | <0.0001 | 138 ± 162 | 992 ± 448 | <0.0001 |
| Vitamin B-12 from supplements, μg/d | 26 ± 106 | 32 ± 142 | 13 ± 19 | <0.0001 | 15 ± 85 | 41 ± 139 | <0.0001 | 13 ± 85 | 40 ± 112 | <0.0001 |
| Prudent pattern | 0.12 ± 1.06 | 0.16 ± 1.01 | −0.29 ± 0.80 | 0.01 | 0.15 ± 1.05 | −0.17 ± 0.94 | <0.0001 | 0.29 ± 1.04 | 0.13 ± 1.30 | 0.14 |
| Western pattern | −0.01 ± 0.98 | −0.46 ± 0.60 | 0.14 ± 1.12 | 0.02 | −0.22 ± 0.79 | −0.01 ± 0.95 | 0.60 | −0.30 ± 0.81 | −0.03 ± 1.08 | 0.11 |
| Vitamin A (RAE), μg/d | 1380 ± 894 | 512 ± 127 | 2570 ± 861 | <0.0001 | 679 ± 310 | 2040 ± 1190 | <0.0001 | 655 ± 282 | 2170 ± 1000 | <0.0001 |
| Vitamin C, mg/d | 182 ± 179 | 111 ± 106 | 256 ± 176 | <0.0001 | 62 ± 14 | 398 ± 247 | <0.0001 | 119 ± 162 | 284 ± 181 | <0.0001 |
| Vitamin E, mg/d | 24 ± 34 | 10 ± 15 | 42 ± 50 | <0.0001 | 9 ± 4 | 47 ± 56 | <0.0001 | 6 ± 1 | 57 ± 55 | <0.0001 |
| Preformed retinol, μg/d | 1000 ± 811 | 307 ± 121 | 2000 ± 908 | <0.0001 | 434 ± 283 | 1560 ± 1140 | <0.0001 | 413 ± 241 | 1670 ± 974 | <0.0001 |
| α-Carotene, μg/d | 538 ± 430 | 388 ± 236 | 704 ± 649 | 0.03 | 436 ± 254 | 596 ± 428 | 0.02 | 447 ± 269 | 644 ± 592 | 0.42 |
| β-Carotene, μg/d | 4040 ± 2520 | 2250 ± 859 | 6080 ± 3250 | <0.0001 | 2720 ± 1260 | 5100 ± 2960 | <0.0001 | 2640 ± 1350 | 5380 ± 3220 | <0.0001 |
| β-Cryptoxanthin, μg/d | 106 ± 84 | 101 ± 111 | 103 ± 75 | 0.25 | 66 ± 43 | 145 ± 124 | <0.0001 | 116 ± 118 | 97 ± 60 | 0.21 |
| Lycopene, μg/d | 5280 ± 3020 | 6030 ± 3490 | 5110 ± 2640 | 0.26 | 5630 ± 2930 | 5570 ± 3310 | 0.49 | 5770 ± 3810 | 5430 ± 2910 | 0.71 |
| Lutein and zeaxanthin, μg/d | 3060 ± 2400 | 1680 ± 651 | 4350 ± 3520 | <0.0001 | 1980 ± 1070 | 3930 ± 3290 | <0.001 | 1880 ± 1080 | 4050 ± 3350 | <0.0001 |
| Female partner characteristics | ||||||||||
| Age, y | 34.9 ± 3.7 | 34.9 ± 3.1 | 34.9 ± 3.9 | 0.51 | 35.0 ± 3.3 | 35.7 ± 4.1 | 0.28 | 35.3 ± 3.6 | 35.4 ± 3.9 | 0.19 |
| BMI, kg/m2 | 23.9 ± 4.4 | 23.4 ± 3.5 | 24.5 ± 4.3 | 0.03 | 23.2 ± 3.4 | 23.9 ± 4.0 | 0.51 | 22.7 ± 2.7 | 25.0 ± 4.7 | 0.18 |
| Ever smoker | 46 (26.9) | 15 (35.7) | 6 (14.0) | 0.07 | 14 (33.3) | 8 (18.6) | 0.21 | 17 (40.5) | 6 (14.0) | 0.05 |
| Initial infertility diagnosis | ||||||||||
| Male factor | 61 (35.7) | 16 (38.1) | 19 (44.2) | 0.13 | 13 (31.0) | 14 (32.6) | 0.32 | 17 (40.5) | 20 (46.5) | 0.29 |
| Female factor | 43 (25.2) | 7 (16.7) | 10 (23.3) | 7 (16.7) | 13 (30.2) | 11 (26.2) | 11 (25.6) | |||
| Unexplained | 67 (39.1) | 19 (45.2) | 14 (32.5) | 22 (52.3) | 16 (37.2) | 14 (33.3) | 12 (27.9) | |||
Values are represented as means ± SDs for continuous variables, or n (%) for categorical variables. DFE, dietary folate equivalent; Q, quartile; RAE, Retinol Activity Equivalent.
Men's vitamin C intake was positively associated with fertilization rate. The adjusted fertilization rate (95% CI) for couples in increasing quartiles of men's vitamin C intake was 69% (61–76%), 74% (68–80%), 74% (64–77%), and 81% (74–86%), respectively (P-trend = 0.02). Men's intakes of vitamins A (RAE) and E, preformed retinol, and carotenoids were not associated with fertilization rates (Table 2). β-carotene intake was positively associated with fertilization rate in ICSI cycles but not in IVF cycles (P-interaction = 0.01; Figure 1). We found no differences in the relation between intake of these nutrients and fertilization rate when the intake from foods and supplements was considered separately (Supplemental Table 3).
TABLE 2.
Men's baseline intake of vitamins A, C, and E, carotenoids, and preformed retinol in relation to fertilization rate (n = 171 couples, 243 cycles)1
| Q1 | Q2 | Q3 | Q4 | P-trend | |
|---|---|---|---|---|---|
| Vitamin A (RAE) (range, μg/d) | (<748) | (748–1140) | (1150–1800) | (>1800) | |
| 2PN/MII | 261/374 | 268/367 | 281/383 | 320/432 | 0.17 |
| Adjusted fertilization rate (95% CI)2 | 0.76 (0.69–0.83) | 0.75 (0.68–0.81) | 0.75 (0.68–0.81) | 0.69 (0.60–0.77) | |
| Vitamin C (range, mg/d) | (<84) | (84–126) | (127–186) | (>186) | |
| 2PN/MII | 269/389 | 289/405 | 255/362 | 317/400 | 0.02 |
| Adjusted fertilization rate (95% CI)2 | 0.69 (0.61–0.76) | 0.74 (0.68–0.80) | 0.74 (0.64–0.77) | 0.81 (0.74–0.86) | |
| Vitamin E (range, mg/d) | (<8) | (8–13) | (14–24) | (>24) | |
| 2PN/MII | 277/379 | 247/355 | 284/404 | 322/418 | 0.98 |
| Adjusted fertilization rate (95% CI)2 | 0.76 (0.69–0.83) | 0.71 (0.64–0.78) | 0.73 (0.66–0.80) | 0.75 (0.66–0.82) | |
| Preformed retinol (range, μg/d) | (<397) | (397–779) | (786–1340) | (>1340) | |
| 2PN/MII | 248/368 | 271/367 | 279/383 | 332/438 | 0.77 |
| Adjusted fertilization rate (95% CI)2 | 0.72 (0.63–0.79) | 0.76 (0.69–0.82) | 0.77 (0.70–0.82) | 0.71 (0.62–0.79) | |
| α-Carotene (range, μg/d) | (<251) | (251–433) | (437–704) | (>704) | |
| 2PN/MII | 283/385 | 301/410 | 263/367 | 283/394 | 0.29 |
| Adjusted fertilization rate (95% CI)2 | 0.76 (0.69–0.82) | 0.76 (0.70–0.82) | 0.74 (0.67–0.80) | 0.71 (0.64–0.78) | |
| β-Carotene (range, μg/d) | (<2370) | (2370–3530) | (3548–5130) | (>5130) | |
| 2PN/MII | 273/377 | 240/337 | 299/433 | 318/409 | 0.29 |
| Adjusted fertilization rate (95% CI)2 | 0.75 (0.65–0.82) | 0.71 (0.63–0.78) | 0.70 (0.63–0.77) | 0.79 (0.70–0.86) | |
| β-Cryptoxanthin (range, μg/d) | (<52) | (52–74) | (75–145) | (>145) | |
| 2PN/MII | 328/442 | 262/383 | 272/361 | 268/370 | 0.84 |
| Adjusted fertilization rate (95% CI)2 | 0.77 (0.70–0.82) | 0.70 (0.62–0.76) | 0.76 (0.69–0.81) | 0.74 (0.66–0.80) | |
| Lycopene (range, μg/d) | (<3230) | (3230–4630) | (4640–6290) | (>6290) | |
| 2PN/MII | 320/453 | 250/347 | 326/429 | 234/327 | 0.64 |
| Adjusted fertilization rate (95% CI)2 | 0.73 (0.66–0.79) | 0.76 (0.69–0.81) | 0.77 (0.70–0.82) | 0.72 (0.64–0.78) | |
| Lutein and zeaxanthin (range, μg/d) | (<1500) | (1500–2540) | (2550–3660) | (>3660) | |
| 2PN/MII | 295/401 | 256/364 | 292/410 | 287/381 | 0.25 |
| Adjusted fertilization rate (95% CI)2 | 0.79 (0.71–0.85) | 0.77 (0.69–0.83) | 0.71 (0.63–0.77) | 0.71 (0.61–0.79) |
Models for vitamins A, C, and E were mutually adjusted for each other. Models for carotenoids and preformed retinol were mutually adjusted for each other and additionally adjusted for the intake of vitamins C and E. 2PN, 2-pronuclear zygote; MII, metaphase II; Q, quartile; RAE, Retinol Activity Equivalent.
All models were adjusted for age, BMI, smoking, calorie intake, folate and vitamin B-12 from supplements, calorie intake, the Prudent and Western dietary pattern, in vitro fertilization treatment protocol, initial infertility diagnosis (male factors or not), partner's age, BMI, and smoking status.
FIGURE 1.
Men's baseline total β-carotene intake in relation to fertilization rate in (A) in vitro fertilization (IVF) cycles (n = 93 cycles) and (B) intracytoplasmic sperm injection (ICSI) cycles (n = 150 cycles). All models were adjusted for vitamins C and E, other carotenoids, age, BMI, smoking, calorie intake, folate and vitamin B-12 from supplements, calorie intake, the Prudent and Western dietary pattern, in vitro fertilization treatment protocol, initial infertility diagnosis (male factors or not), partner's age, BMI, and smoking status.
We found no relation between men's intake of vitamins A (RAE), C, and E, and preformed retinol with the probabilities of implantation, clinical pregnancy, or live birth (Table 3). However, men's α-carotene intake was inversely related to the probabilities of implantation and live birth (Table 3). The adjusted probability of live birth for couples with men in the lowest and highest quartiles of α-carotene intake was 43% (28–60%) and 22% (12–36%), respectively (P value = 0.04). Similarly, we found an inverse relation between intake of β-carotene from supplements and probability of implantation, although intake of β-carotene from foods was unrelated to implantation or the other clinical outcomes (Supplemental Table 4). Most (74%) α-carotene intake in this population was accounted for by carrot intake, which had a nonstatistically significant inverse relation with probability of implantation. The adjusted probabilities of implantation (95% CI) for couples in the lowest and highest quartiles of men's carrot intake were 61% (47–74%) and 47% (34–62%) (P-trend = 0.12). Carrot intake was unrelated to the probability of clinical pregnancy or live birth.
TABLE 3.
Men's baseline intakes of vitamins A, C, and E, carotenoids and preformed retinol in relation to clinical assisted reproductive technology outcomes (n = 171 couples, 294 cycles)1
| Q1 | Q2 | Q3 | Q4 | P-trend | |
|---|---|---|---|---|---|
| Vitamin A (RAE) (range, μg/d) | (<748) | (748–1140) | (1150–1800) | (>1800) | |
| Implantation (events/cycles) | 33/67 | 46/75 | 41/84 | 41/68 | 0.23 |
| Adjusted implantation rate (95% CI)2 | 0.42 (0.27–0.60) | 0.66* (0.50–0.79) | 0.47 (0.33–0.61) | 0.64 (0.45–0.79) | |
| Pregnancy (events/cycles) | 30/67 | 43/75 | 35/84 | 37/68 | 0.61 |
| Adjusted pregnancy rate (95% CI)2 | 0.38 (0.23–0.56) | 0.61* (0.45–0.75) | 0.36 (0.24–0.50) | 0.50 (0.32–0.68) | |
| Live birth (events/cycles) | 28/67 | 30/75 | 27/84 | 31/68 | 0.45 |
| Adjusted live birth rate (95% CI)2 | 0.30 (0.17–0.48) | 0.38 (0.23–0.54) | 0.24 (0.15–0.37) | 0.40 (0.23–0.60) | |
| Vitamin C (range, mg/d) | (<84) | (84–126) | (127–186) | (>186) | |
| Implantation (events/cycles) | 43/75 | 44/72 | 35/71 | 39/76 | 0.32 |
| Adjusted implantation rate (95% CI)2 | 0.62 (0.46–0.76) | 0.61 (0.47–0.74) | 0.42 (0.29–0.57) | 0.50 (0.35–0.65) | |
| Pregnancy (events/cycles) | 37/75 | 39/72 | 34/71 | 35/76 | 0.51 |
| Adjusted pregnancy rate (95% CI)2 | 0.50 (0.34–0.66) | 0.51 (0.37–0.65) | 0.37 (0.24–0.52) | 0.42 (0.28–0.58) | |
| Live birth (events/cycles) | 32/75 | 32/72 | 25/71 | 27/76 | 0.35 |
| Adjusted live birth rate (95% CI)2 | 0.43 (0.27–0.60) | 0.37 (0.24–0.52) | 0.21* (0.13–0.34) | 0.29 (0.18–0.45) | |
| Vitamin E (range, mg/d) | (<8) | (8–13) | (14–24) | (>24) | |
| Implantation (events/cycles) | 42/79 | 39/71 | 44/78 | 36/66 | 0.78 |
| Adjusted implantation rate (95% CI)2 | 0.51 (0.35–0.68) | 0.56 (0.40–0.71) | 0.57 (0.41–0.71) | 0.50 (0.31–0.69) | |
| Pregnancy (events/cycles) | 37/79 | 33/71 | 41/78 | 24/66 | 0.66 |
| Adjusted pregnancy rate (95% CI)2 | 0.39 (0.24–0.55) | 0.44 (0.29–0.60) | 0.50 (0.34–0.66) | 0.48 (0.29–0.67) | |
| Live birth (events/cycles) | 31/79 | 24/71 | 33/78 | 28/66 | 0.86 |
| Adjusted live birth rate (95% CI)2 | 0.30 (0.18–0.47) | 0.29 (0.17–0.44) | 0.35 (0.21–0.51) | 0.33 (0.18–0.54) | |
| Preformed retinol (range, μg/d) | (<397) | (397–779) | (786–1340) | (>1340) | |
| Implantation (events/cycles) | 39/66 | 43/86 | 38/72 | 41/70 | 0.60 |
| Adjusted implantation rate (95% CI)2 | 0.55 (0.37–0.72) | 0.51 (0.35–0.67) | 0.49 (0.34–0.64) | 0.62 (0.42–0.79) | |
| Pregnancy (events/cycles) | 37/66 | 36/86 | 36/72 | 36/70 | 0.82 |
| Adjusted pregnancy rate (95% CI)2 | 0.50 (0.32–0.68) | 0.41 (0.27–0.58) | 0.40 (0.26–0.56) | 0.52 (0.32–0.71) | |
| Live birth (events/cycles) | 31/66 | 25/86 | 29/72 | 31/70 | 0.33 |
| Adjusted live birth rate (95% CI)2 | 0.33 (0.19–0.52) | 0.23 (0.13–0.38) | 0.30 (0.18–0.45) | 0.44 (0.25–0.65) | |
| α-Carotene (range, μg/d) | (<251) | (251–433) | (437–704) | (>704) | |
| Implantation (events/cycles) | 43/65 | 38/73 | 42/71 | 38/85 | 0.03 |
| Adjusted implantation rate (95% CI)2 | 0.67 (0.51–0.80) | 0.51 (0.36–0.66) | 0.58 (0.43–0.72) | 0.40* (0.26–0.56) | |
| Pregnancy (events/cycles) | 36/65 | 33/73 | 40/71 | 36/85 | 0.39 |
| Adjusted pregnancy rate (95% CI)2 | 0.52 (0.36–0.68) | 0.39 s (0.26–0.55) | 0.51 (0.36–0.66) | 0.39 (0.26–0.55) | |
| Live birth (events/cycles) | 33/65 | 26/73 | 34/71 | 23/85 | 0.07 |
| Adjusted live birth rate (95% CI)2 | 0.43 (0.28–0.60) | 0.28 (0.16–0.42) | 0.37 (0.24–0.53) | 0.22* (0.12–0.36) | |
| β-Carotene (range, μg/d) | (<2370) | (2370–3530) | (3550–5130) | (>5130) | |
| Implantation (events/cycles) | 41/67 | 32/67 | 45/79 | 43/81 | 0.52 |
| Adjusted implantation rate (95% CI)2 | 0.50 (0.30–0.69) | 0.44 (0.29–0.60) | 0.58 (0.42–0.73) | 0.61 (0.40–0.79) | |
| Pregnancy (events/cycles) | 37/67 | 28/67 | 41/79 | 39/81 | 0.84 |
| Adjusted pregnancy rate (95% CI)2 | 0.47 (0.28–0.67) | 0.35 (0.21–0.52) | 0.48 (0.33–0.64) | 0.50 (0.30–0.70) | |
| Live birth (events/cycles) | 34/67 | 23/67 | 31/79 | 28/81 | 0.94 |
| Adjusted live birth rate (95% CI)2 | 0.35 (0.19–0.56) | 0.22 (0.12–0.37) | 0.32 (0.20–0.48) | 0.35 (0.18–0.57) | |
| β-Cryptoxanthin (range, μg/d) | (<52) | (52–74) | (75–145) | (>145) | |
| Implantation (events/cycles) | 37/70 | 41/68 | 47/90 | 36/66 | 0.47 |
| Adjusted implantation rate (95% CI)2 | 0.49 (0.33–0.64) | 0.55 (0.40–0.69) | 0.52 (0.39–0.65) | 0.59 (0.42–0.74) | |
| Pregnancy (events/cycles) | 34/70 | 37/68 | 39/90 | 35/66 | 0.30 |
| Adjusted pregnancy rate (95% CI)2 | 0.41 (0.27–0.57) | 0.47 (0.33–0.62) | 0.39 (0.27–0.53) | 0.55 (0.38–0.71) | |
| Live birth (events/cycles) | 28/70 | 33/68 | 30/90 | 25/66 | 0.54 |
| Adjusted live birth rate (95% CI)2 | 0.25 (0.14–0.40) | 0.38 (0.25–0.53) | 0.27 (0.17–0.40) | 0.35 (0.21–0.53) | |
| Lycopene (range, μg/d) | (<3230) | (3230–4630) | (4640–6290) | (>6290) | |
| Implantation (events/cycles) | 36/70 | 43/72 | 38/69 | 44/83 | 0.77 |
| Adjusted implantation rate (95% CI)2 | 0.47 (0.33–0.62) | 0.62 (0.47–0.75) | 0.47 (0.33–0.62) | 0.55 (0.41–0.68) | |
| Pregnancy (events/cycles) | 33/70 | 38/72 | 35/69 | 39/83 | 0.94 |
| Adjusted pregnancy rate (95% CI)2 | 0.42 (0.28–0.56) | 0.54 (0.39–0.68) | 0.39 (0.26–0.55) | 0.45 (0.31–0.59) | |
| Live birth (events/cycles) | 25/70 | 31/72 | 30/69 | 30/83 | 0.76 |
| Adjusted live birth rate (95% CI)2 | 0.27 (0.17–0.41) | 0.41 (0.27–0.56) | 0.28 (0.17–0.43) | 0.29 (0.18–0.43) | |
| Lutein and zeaxanthin (range, μg/d) | (<1500) | (1500–2540) | (2550–3660) | (>3660) | |
| Implantation (events/cycles) | 35/63 | 41/75 | 39/76 | 46/80 | 0.51 |
| Adjusted implantation rate (95% CI)2 | 0.49 (0.30–0.68) | 0.57 (0.40–0.72) | 0.48 (0.34–0.63) | 0.59 (0.40–0.75) | |
| Pregnancy (events/cycles) | 32/63 | 35/75 | 36/76 | 42/80 | 0.50 |
| Adjusted pregnancy rate (95% CI)2 | 0.42 (0.25–0.62) | 0.44 (0.28–0.61) | 0.42 (0.28–0.57) | 0.51 (0.33–0.69) | |
| Live birth (events/cycles) | 29/63 | 30/75 | 25/76 | 32/80 | 0.78 |
| Adjusted live birth rate (95% CI)2 | 0.33 (0.18–0.53) | 0.34 (0.20–0.51) | 0.23 (0.14–0.37) | 0.34 (0.20–0.53) |
Models of vitamins were all adjusted for the other vitamins. Models of carotenoids and preformed retinol were all adjusted for vitamins C, E, and the other carotenoids. Q, quartile; RAE, Retinol Activity Equivalent.
All models were adjusted for age, BMI, smoking, calorie intake, folate and vitamin B-12 from supplements, calorie intake, the Prudent and Western dietary pattern, in vitro fertilization treatment protocol, initial infertility diagnosis (male factors or not), partner's age, BMI, and smoking status. *P value <0.05 compared with the first quartile.
We found no evidence that smoking, age, BMI, or infertility diagnosis modified the relation between intake of the nutrients of interest and outcomes of infertility treatment (data not shown).
Discussion
In this prospective cohort of couples undergoing infertility treatment with ART, we identified a positive relation of men's vitamin C intake with fertilization rate and β-carotene intake with fertilization rate in ICSI cycles but not in conventional IVF cycles. Nevertheless, these relations did not result in significant differences in clinical outcomes. We also found unexpected associations between higher α-carotene intake and lower probability of implantation and live birth, and between higher β-carotene from supplements and lower probability of implantation, but the clinical significance of these findings is uncertain.
We found positive associations between men's vitamin C and β-carotene intake with fertilization rate. Spermatozoa DNA integrity plays a crucial role in achieving fertilization in natural and assisted conception (28–30). A study showed that oxidative parameters in the semen of infertile men were significantly higher than in fertile men, and a high correlation was found between oxidative parameters, sperm ROS formation, and DNA fragmentation concentrations (5). Vitamin C and carotenoids are naturally found in semen (4) and can act as free radical scavengers that help overcome ROS (31). Vitamin C and β-carotene have also been related to semen quality among young healthy men (32). This literature and our data suggest that vitamin C and β-carotene intake might reduce the ROS concentration, which in turn decreases DNA fragmentation concentrations and results in a better fertilization rate. The association between β-carotene and fertilization rate in ICSI but not IVF cycles may indicate that the benefits of protection against ROS are particularly important among men with documented abnormalities in semen quality. The association with vitamin C and β-carotene but not with vitamin E is still consistent with the overall hypothesis of the beneficial effects of antioxidants on male fertility. This pattern may reflect the different distribution of antioxidants in seminal plasma (vitamin C and β-carotene) compared with spermatozoa (vitamin E) and highlight the heightened susceptibility to oxidative damage of activated ejaculated spermatozoa for which unspecific protection present in seminal plasma may be more important than other mechanisms to protect against oxidative damage. It is important to note that this apparent benefit in fertilization rates did not result in higher probability of live birth. Hence, the overall clinical relevance of these findings is in question.
Unexpectedly, we found inverse relations of α-carotene intake with implantation and live birth rates. Intake of β-carotene from supplements, but not from foods, was also related to a lower implantation rate. Intakes of preformed retinol and all other carotenoids, regardless of source, were unrelated to these outcomes, as were the intake of vitamins C and E. Intakes of carotenoids in this population are similar to those in the general US population (33). Most (74%) α-carotene intake in this population was accounted for by a single food: carrots. However, carrot intake was unrelated to the probability of implantation or any other outcome of clinical relevance. α-Carotene and β-carotene are provitamin A carotenoids. Although studies have shown that excess intake of vitamin A and retinoids by pregnant women might result in teratogenesis (34–36), no study has reported teratogenicity due to high vitamin A intake by the male partner. The lack of association with vitamins C and E, or with all other carotenoids suggests that these relations may not be due to the effects on oxidative stress. Similarly, the lack of association with preformed retinol and other provitamin A carotenoids suggests that these observed relations may not reflect an underlying biologic association explained by provitamin A activity. Instead, we believe these findings are more consistent with a chance finding although additional, larger studies on this topic are warranted.
There is extensive literature on the relation between antioxidants and outcomes of infertility treatments, including several RCTs addressing this question. Of note, a Cochrane systematic review and meta-analysis of these RCTs concluded that supplementing men in couples undergoing infertility treatment with antioxidants might improve pregnancy and live birth rates (7). Subgroup analyses suggested that supplementation with vitamin E could specifically improve pregnancy and live birth rates. Our findings are not consistent with the Cochrane review. However, as acknowledged by the Cochrane reviewers, their conclusion was based on only 4 small RCTs (8–10, 37) with a combined sample size of 157 couples (compared with 171 couples in our study), 3 of which were published during the 1990s and may not be relevant to current practice. The most recent RCT conducted in 2007 concluded that antioxidant mixtures improve the pregnancy and live birth rates during IVF-ICSI treatment (37). However, some of the antioxidant mixtures evaluated in previous studies, such as garlic and folate, are not technically antioxidants, making it difficult to draw strong conclusions. The vitamin E subgroup analysis included only 2 studies (8, 9) with a combined sample size of 67 couples, but with intervention doses of vitamin E that were much higher than intakes observed in our study (100 mg/d and 600 mg/d, respectively compared with an average of 58 mg/d among men in the highest quartile of intake in our study). Nonrandomized studies also suggest a benefit of vitamin E alone (9, 38) or in combination with vitamin C (39). As was the case for RCTs, however, studies showing benefit used much higher doses than the observed intakes in our population. Clearly, additional prospective studies, and in particular, RCTs sufficiently powered to evaluate the role of supplementation with antioxidants and related biologic compounds among men in couples seeking fertility treatment are needed to clarify their clinical utility.
Our study has multiple strengths, including its prospective design, complete participant follow-up, and larger sample size compared with the previous clinical trials, which aid interpretation of the findings. Comprehensive demographic, lifestyle, and dietary data allowed us to account for a broad range of potential confounders, including highly correlated nutrients. However, there are some limitations to consider in interpreting our results. First, FFQs are good tools for identifying relative rankings of intake, but not for measuring precise intake levels. Second, the dietary assessment tool used in this study assesses a typical diet. Therefore, it may not accurately reflect short-term changes in diet made specifically during treatment cycles. If short-term changes in nutrient intake are related to ART outcomes, our study would not be able to capture these effects. Third, whereas the intake of some of the nutrients examined are higher among men in this population than among men in the general population, we cannot rule out a beneficial effect of these nutrients at intakes such as those previously used in some RCTs. Fourth, as is the case with all observational studies, we cannot rule out unmeasured confounding as an explanation for the observed associations. Lastly, since all couples were undergoing infertility treatment with ART, the findings may not be generalizable to men in couples trying to become pregnant without medical assistance. In addition, couples who seek infertility treatment tend to have more education and higher socioeconomic status than infertile couples who do not, even in states with extensive insurance coverage for infertility treatment as is the case of Massachusetts, where the study was conducted.
In conclusion, in this prospective cohort we found positive relations between men's baseline intake of vitamin C and fertilization rate, and β-carotene intake and fertilization rate in ICSI cycles. These associations are consistent with the hypothesis that protection against oxidative damage in the male partner may result in reproductive benefits for couples trying to become pregnant. We also found unexpected inverse relations between α-carotene intake and β-carotene intake from supplements with implantation and live birth rates. Although intriguing, these latter associations are more consistent with a chance finding but should be re-evaluated in larger studies.
Supplementary Material
Acknowledgments
The authors’ responsibilities were as follows—RH and JEC: were involved in the design of the study, concept of the study, and revision of intellectual content of the manuscript; MCL and YHC: analyzed the data; MCL and JEC: interpreted the data and drafted the manuscript; JP, RH, and JEC: were involved in the acquisition of the data; JEC: had primary responsibility for the final content; and all authors: were involved in the critical revision of the manuscript and read and approved the final manuscript.
Notes
Supported by grants P30ES000002, R01ES009718, R01ES022955, K99ES026648, and P30DK046200 from the NIH. M-CL was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2917-I-564-066) and China Medical University, Taiwan (CMU107-Z-04).
Author disclosures: M-CL, Y-HC, AJG, LM-A, FLN, PLW, JP, RH, and JEC, no conflicts of interest.
Supplemental Figures 1 and 2 and Supplemental Tables 1–4 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/.
Abbreviations used: ART, assisted reproductive technology; GnRH, gonadotropin-releasing hormone; hCG, human chorionic gonadotropin; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; RAE, Retinol Activity Equivalent; RCT, randomized clinical trial; ROS, reactive oxygen species; MII, metaphase II.
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