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Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 American Journal of Epidemiology Vol. 187, No. 9 Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2018. DOI: 10.1093/aje/kwy101 This work is written by (a) US Government employee(s) and is in the public domain in the US. Advance Access publication: May 14, 2018 Original Contribution Shorter Time to Pregnancy With Increasing Preconception Carotene Concentrations Among Women With 1–2 Previous Pregnancy Losses Keewan Kim, Enrique F. Schisterman, Robert M. Silver, Brian D. Wilcox, Anne M. Lynch, Neil J. Perkins, Richard W. Browne, Laurie L. Lesher, Joseph B. Stanford, Aijun Ye, Jean Wactawski- Wende, and Sunni L. Mumford* * Correspondence to Dr. Sunni L. Mumford, Epidemiology Branch, Division of Intramural Population Health Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 6710B Rockledge Drive, MSC 7004, Bethesda, MD 20892 (e-mail: email@example.com). Initially submitted August 18, 2017; accepted for publication April 30, 2018. Although maternal nutrition may affect fecundity, associations between preconception micronutrient levels and time to pregnancy (TTP) have not been examined. We assessed the relationship between preconception fat-soluble micronutrient concentrations and TTP among women with 1–2 prior pregnancy losses. This was a prospective cohort study of 1,228 women set within the Effects of Aspirin in Gestation and Reproduction (EAGeR) Trial (United States, 2007–2011), which as- sessed the association of preconception-initiated daily low-dose aspirin with reproductive outcomes. We measured precon- ception levels of zeaxanthin, cryptoxanthin, lycopene, α- and β-carotene, and α- and γ-tocopherol in serum. We used discrete Cox regression models, accounting for left-truncation and right-censoring, to calculate fecundability odds ratios and 95% conﬁdence intervals. The models adjusted for age, body mass index, race, smoking, alcohol, physical activity, income, vitamin use, cholesterol, treatment arm, and study site. Serum α-carotene levels (per log unit (μg/dL) increase, fecundability odds ratio (FOR) = 1.17, 95% conﬁdence interval (CI): 1.00, 1.36) and serum α-carotene concentrations at or above the US average (2.92 μg/dL) versus below the average (FOR = 1.21, 95% CI: 1.02, 1.44) were associated with shorter TTP. Compared with levels below the US average (187 μg/dL), γ-tocopherol concentrations at or above the average were associ- ated with longer TTP (FOR = 0.83, 95% CI: 0.69, 1.00). The potential for these nutrients to inﬂuence fecundability deserves further exploration. antioxidants; carotenes; fecundability; lipophilic micronutrients; time to pregnancy; tocopherols Abbreviations: BMI, body mass index; CI, conﬁdence interval; FOR, fecundability odds ratio; hCG, human chorionic gonadotropin; TTP, time to pregnancy. Time to pregnancy (TTP) is a widely accepted measure of synthesized in the body system. The most common dietary couple fecundity and is of great interest for couples seeking preg- sources of carotenoids are yellow- and orange-colored fruits and nancy. Factors affecting couple fecundity include the ages of both vegetables and green leafy vegetables, whereas tocopherols are partners, body mass index (BMI), and various environmental ex- abundant in nuts, seeds, and nut and seed oils. These micronutri- posures (1–4). Modiﬁable lifestyle risk factors, such as cigarette ents are transported by lipoproteins in the body system and have smoking and consumption of alcohol and caffeinated beverages, antioxidant properties, as they scavenge reactive oxygen species have also been suggested as potential factors inﬂuencing fecun- (8). They are critical to normal cellular metabolism (9–11)and dity (5, 6). However, though the overall nutritional quality of have been shown to be important for reproductive hormonal func- the diet has been associated with female fertility (7), relation- tion (12). Antioxidant status (assessed by concentrations of carote- ships between speciﬁc dietary components and couple fecun- noids, retinol, and tocopherols in blood (13) and follicular ﬂuid dity are less understood. (13–15)) in women undergoing in vitro fertilization has been asso- Fat-soluble micronutrients, including carotenoids, retinol, and ciated with embryo quality, which supports a potential role for tocopherols, are obtained exclusively via the diet, as they are not antioxidants in the early stages of human reproduction. 1907 Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 1908 Kim et al. Outcome assessment Despite their potential importance to human fertility, only 1 study has evaluated the associations between intake of vitamins The primary outcome was TTP or the number of menstrual via supplements and diet and TTP among women with unex- cycles needed to achieve pregnancy over 6 consecutive months plained infertility (16). To our knowledge, no studies to date of follow-up. Pregnancy was determined by positivity for urinary have investigated the association of preconception fat-soluble mi- human chorionic gonadotropin (hCG) and ultrasonography (20). cronutrients measured in blood with fecundability. Therefore, we In brief, urine hCG tests (Quidel Quickvue; Quidel Corporation, evaluated the relationship between preconception fat-soluble San Diego, California), which were sensitive to 25 mIU/mL micronutrient concentrations and fecundability among women hCG, were conducted at home or at the clinic when participants with proven fecundity and no history of infertility. reported missing menses at the end of each cycle during follow- up. For a more sensitive detection of early pregnancy, we also measured free β-hCG levels in daily ﬁrst-morning urine samples METHODS collected on the last 10 days of each woman’s ﬁrst and second Study design cycles of study participation and in spot urine samples collected at clinic visits that took place at the end of each cycle. Among We used data from the Effects of Aspirin in Gestation and women with positive urinary hCG, a 6- to 7-week ultrasound Reproduction (EAGeR) Trial, whichenrolled1,228 womenat4 examination was performed to conﬁrm pregnancy. university medical centers in the United States (2007–2011). The study design of the original trial is described elsewhere (17). Statistical analysis Brieﬂy, participants were 18–40 years of age, had 1 or 2 docu- mented prior pregnancy losses, had up to 2 prior live births, had We determined demographic and reproductive history charac- regular menstrual cycles (i.e., 21–42 days), and were attempting teristics by tertiles of selected micronutrients and compared them pregnancy without the use of infertility treatment. Women with a (using Fisher’s exact test or the χ test as appropriate) among known history of infertility treatment or the presence of major 1,207 women with available micronutrient measurements. Con- medical disorders were excluded. centrations of fat-soluble micronutrients were log-transformed for At baseline, participants completed questionnaires on demo- normality. Geometric mean values and standard deviations of mi- graphic factors, lifestyle, medical and reproductive history, and cronutrients were calculated overall, by number of previous losses, family medical history (17). Height and weight were measured, 2 by number of previous live births, and by pregnancy status and and BMI was calculated (weight (kg)/height (m) ). Participants were compared using Student’s t test or analysis of variance, as were followed for 6 menstrual cycles while attempting preg- appropriate. Pearson correlation coefﬁcients were obtained for nancy or throughout pregnancy for those who became pregnant. log-transformed fat-soluble micronutrient concentrations to assess We provided fertility monitors (Clearblue Easy Fertility Moni- correlations. Distributions of measured micronutrient concentra- tor; Inverness Medical Innovations, Waltham, Massachusetts) to tions were compared with the US average levels reported for assist couples with the timing of intercourse and timing of study womenaged20–39 years in the 2005–2006 National Health and visits. Nutrition Examination Survey. Reported geometric mean values The institutional review board at each study site approved for each micronutrient in the National Health and Nutrition Exam- the trial protocol. All participants provided written informed ination Survey were: 13.8 μg/dL (95% conﬁdence interval (CI): consent. The trial was registered on ClinicalTrials.gov (trial 13.2, 14.5) for zeaxanthin; 8.0 μg/dL (95% CI: 7.5, 8.6) for cryp- NCT00467363). toxanthin; 41.3 μg/dL (95% CI: 40.2, 42.4) for lycopene; 2.9 μg/dL (95% CI: 2.5, 3.4) for α-carotene; 12.2 μg/dL (95% CI: 10.7, 13.9) for β-carotene; 1,010 μg/dL (95% CI: 981, 1,040) for α-tocopherol; Analysis of fat-soluble micronutrients and 187 μg/dL (95% CI: 173, 202) for γ-tocopherol (21). Blood specimens were collected at baseline, processed, and We used Cox proportional hazards regression models for stored at −80°C prior to analysis. Preconception levels of serum discrete survival time, accounting for left-truncation and right- fat-soluble micronutrients, including zeaxanthin, cryptoxanthin, censoring, to estimate fecundability odds ratios and 95% lycopene, α-carotene, β-carotene, α-tocopherol, and γ-tocopherol, conﬁdence intervals for relationships between fat-soluble micro- were measured at the University of Minnesota (Minneapolis, nutrients and TTP. A fecundability odds ratio greater than 1 is in- Minnesota) using high-performance liquid chromatography terpreted as indicating increased fecundability. We imputed (18, 19) in 1,207 women (21 women did not have sufﬁcient missing values for fat-soluble micronutrient concentrations (n = sample volume). The mobile phase used for tocopherol was 21; 1.7%) and covariates (BMI, 1.6%; alcohol drinking, 1.3%; 100% methanol (19). For carotenoids, acetonitrile/methylene physical activity, 0.1%; income, 0.1%; vitamin use, 1.5%; total chloride/methanol (70:20:10 by volume) was used as the mobile cholesterol concentration, 1.7%; number of cycles of attempting phase (19), with modiﬁcation by adding 0.015% diisopropyl- pregnancy before study entry, 7.9%) using a multiple imputation ethylamine to aid in analyte recovery. The interassay laboratory model with the fully conditional speciﬁcation method and 5 impu- coefﬁcients of variation ranged from 5.9% for γ-tocopherol to tations (22), with PROC MIANALYZE being used to combine 13.3% for α-tocopherol. Total cholesterol concentration was the results. In addition to the aforementioned variables, the impu- measured using a Roche COBAS 6000 chemistry analyzer tation models included age, race, cigarette smoking, treatment (Roche Diagnostics, Indianapolis, Indiana), with coefﬁcients arm, and study site. The models adjusted for age, BMI, race, ciga- of variation of 2.1% and 2.2% at mean concentrations of rette smoking, alcohol drinking, physical activity, income, vita- 178.6 mg/dL and 258.9 mg/dL, respectively. min use, treatment arm, study site, and serum cholesterol level Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 Preconception Micronutrients and Fecundability 1909 (17). Serum cholesterol was included because lipids are one of α-carotene by treatment arm, differences by study site were de- the major factors that could affect the bioavailability of fat- tected (P = 0.001). For γ-tocopherol, we observed an opposite soluble micronutrients (23) and are also associated with TTP trend in α-carotene levels for most demographic characteristics. (24). We investigated treatment arm and BMI as potential Women whose last pregnancy loss had occurred less than 8 effect-measure modiﬁers. months previously were more likely to be in the middle or upper Because 128 women did not have complete outcome informa- tertile of γ-tocopherol concentrations than in the lower tertile. tion due to early withdrawal from the study and because lower Unlike the case for α-carotene, women who achieved pregnancy levels of micronutrients were observed in those women (see Web after randomization were more likely to be in the lower tertile of γ- Figure 1, available at https://academic.oup.com/aje), we performed tocopherol compared with the higher tertiles. Most women re- ported having taken vitamin supplements (92.4%). Subsequent several sensitivity analyses to investigate potential bias. We com- to inception of the study, 22.0%, 16.2%, 10.8%, 7.3%, 5.1%, and pared complete-case results (n = 1,100) with those from an analy- 3.8% of women achieved pregnancy during menstrual cycles sis where the potential pregnancy outcomes of women who withdrew were imputed using 3 strategies: 1) the survival probabil- 1–6, respectively; 34.8% of women did not achieve pregnancy ity of no pregnancy achieved derived from the complete cases within 6 menstrual cycles of study follow-up or withdrew from using Kaplan-Meier multiple imputation (1,000 imputations), the study. Levels of all measured fat-soluble micronutrients were 2) achievement of pregnancy in 1 cycle after withdrawal, and positively correlated (ranging from r = 0.11 for zeaxanthin and 3) no pregnancy achieved after 6 cycles. The Kaplan-Meier–based γ-tocopherol to r = 0.68 for α-carotene and β-carotene), except for imputation is a missing-at-random–like principled approach that is correlations between α-carotene and γ-tocopherol (r = −0.08) plausible and considers covariates (25), while the latter 2 methods and between β-carotene and γ-tocopherol (r = −0.20). are extreme possibilities of the inﬂuence of potential unobserved Serum concentrations of fat-soluble micronutrients did not dif- outcomes. fer signiﬁcantly by number of previous pregnancy losses (Table 2). We also considered several sensitivity analyses to evaluate the Mean cryptoxanthin concentrations were higher in nulliparous assumptions of our underlying causal framework. In this setting women (8.1 μg/dL) than in parous women (7.4 μg/dL in women we were interested in investigating the association between whohad1previouslivebirth and7.5 μg/dL in women who had 2 fat-soluble micronutrient concentrations and TTP among women previous live births; P < 0.01), whereas no signiﬁcant differences with 1–2 prior losses, without generalizing our ﬁndings to all across parity were detected for the other micronutrients. reproductive-age women. If, however, one were interested in Mean concentrations of fat-soluble micronutrients differed by answering this question for the broader population, there may be pregnancy status, except for lycopene (Web Figure 1). Except for potential concerns regarding selection bias, as our participants γ-tocopherol, higher concentrations were detected for all mea- were restricted to women with a speciﬁc reproductive history, and sured micronutrients in pregnant women relative to nonpregnant because past micronutrient levels may be associated with repro- women and women who withdrew early from the study. Com- ductive history (Web Figure 2). Under this framework, we con- pared with the US average level reported for women aged 20–39 sidered 3 possible sources of confounding. First, we evaluated the years (21), levels of zeaxanthin, α-carotene, and β-carotene were potential impact of an unmeasured dietary factor (U ) that is asso- higher among the women in our study, whereas mean concentra- ciated with past and current micronutrient concentrations across a tions of cryptoxanthin, lycopene, α-tocopherol, and γ-tocopherol range of correlations from −0.8 to 0.8. Second, we evaluated the were lower than the US average. impact of adjustment for previous fecundability (TTP )toblock A log unit increase in α-carotene (μg/dL) was associated with the pathway using a proxy for previous infertility (i.e., attempting a shorter TTP (fecundability odds ratio (FOR) = 1.17, 95% CI: pregnancy for longer than 1 year). Third, we assessed an unmea- 1.00, 1.36) after adjustment for potential confounders (Table 3). sured confounder of the relationship between reproductive history For α-carotene, concentrations at or above the US average (≥2.9 (S)and currentTTP, U , as this may introduce selection bias if it μg/dL) were associated with a shorter TTP (FOR = 1.21, 95% is strongly correlated with S and TTP (26). We simulated a vari- CI: 1.02, 1.44) than concentrations below the US average. Simi- able which could represent genetic factors (27, 28) across a range larly, higher lycopene and β-carotene concentrations were associ- of correlations between U and S (odds ratios of 1.7 and 2.7) and ated with a shorter TTP, although the association was imprecise U and TTP (fecundability odds ratios of 0.5 and 0.7). SAS, ver- or attenuated after adjusting for the covariates. A log unit increase sion 9.4 (SAS Institute, Inc., Cary, North Carolina), was used for in γ-tocopherol concentration (μg/dL) was associated with a lon- all statistical analysis. ger TTP (FOR = 0.66, 95% CI: 0.52, 0.84), and a consistent yet attenuated association was detected for concentrations at or above the US average (≥187 μg/dL) (FOR = 0.73, 95% CI: 0.62, 0.87), compared with those below the US average (<187 μg/dL). After RESULTS adjustment for covariates, however, a log unit increase in γ- tocopherol was not associated with TTP, whereas γ-tocopherol Women of normal weight (BMI <25), nonsmoking women, concentrations at or above the US average remained associated women with more than a high school education, and women in higher income categories were more likely to be in the middle or with a longer TTP (FOR = 0.83, 95% CI: 0.69, 1.00) relative upper tertile of serum α-carotene concentrations than in the to those below the US average. No other signiﬁcant associations ﬁrst tertile (Table 1). Women whose last pregnancy loss had were identiﬁed for other measured fat-soluble micronutrients occurred less than 4 months previously or who achieved preg- and TTP. We did not detect any effect-measure modiﬁcation nancy after randomization also tended to be in the middle and by treatment arm or BMI in our data. upper tertiles of α-carotene concentration compared with the For our sensitivity analysis, we used 3 different methods to ﬁrst. Though we did not detect differences in tertiles of impute pregnancy during our 6 cycles of follow-up. By design, all Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 1910 Kim et al. Table 1. Demographic Characteristics of 1,207 Study Participants With Fat-Soluble Micronutrient Measurements Taken at Baseline, by Tertiles (μg/dL) of α-Carotene and γ-Tocopherol, Effects of Aspirin in Gestation and Reproduction Trial, 2007–2011 a b α-Carotene γ-Tocopherol Tertile 1 Tertile 2 Tertile 3 Tertile 1 Tertile 2 Tertile 3 Demographic Characteristic (n = 403) (n = 402) (n = 402) (n = 404) (n = 404) (n = 399) P Value P Value No. % No. % No. % No. % No. % No. % Age, years 0.71 0.69 <35 359 89.1 356 88.6 351 87.3 361 89.4 353 87.4 352 88.2 ≥35 44 10.9 46 11.4 51 12.7 43 10.6 51 12.6 47 11.8 Body mass index <0.0001 <0.0001 <25 145 36.6 210 53.2 268 67.7 266 67.0 212 53.3 145 37.0 ≥25 251 63.4 185 46.8 128 32.3 131 33.0 186 46.7 247 63.0 Race <0.01 0.42 White 368 91.3 387 96.3 388 96.5 385 95.3 385 95.3 373 93.5 Nonwhite 35 8.7 15 3.7 14 3.5 19 4.7 19 4.7 26 6.5 Frequency of cigarette smoking <0.0001 <0.01 Never smoker 319 79.4 358 90.4 372 93.2 361 89.6 364 90.6 324 82.7 Fewer (<6 times/week) 40 10.0 24 6.1 21 5.3 25 6.2 25 6.2 35 8.9 Daily 43 10.7 14 3.5 6 1.5 17 4.2 13 3.2 33 8.4 Frequency of alcohol drinking 0.36 <0.0001 Never drinker 261 65.1 264 66.7 267 67.6 297 74.1 266 66.3 229 58.7 Sometimes (up to 2–3 times/week) 135 33.7 123 31.1 116 29.4 100 24.9 123 30.7 151 38.7 Often (4–6 times/week or more) 5 1.3 9 2.3 12 3.0 4 1.0 12 3.0 10 2.6 Physical activity level 0.02 0.17 High 145 36.0 120 29.9 133 33.1 147 36.4 122 30.2 129 32.3 Moderate 141 35.0 177 44.0 179 44.5 165 40.8 177 43.8 155 38.9 Low 117 29.0 105 26.1 90 22.4 92 22.8 105 26.0 115 28.8 Educational level <0.0001 0.02 More than high school 305 75.9 363 90.3 374 93.0 365 90.4 346 85.6 331 83.2 High school 84 20.9 32 8.0 24 6.0 34 8.4 52 12.9 54 13.6 Less than high school 13 3.2 7 1.7 4 1.0 5 1.2 6 1.5 13 3.3 Annual income, dollars <0.0001 0.61 <20,000 41 10.2 22 5.5 29 7.2 30 7.4 28 6.9 34 8.5 20,000–39,999 143 35.5 89 22.2 77 19.2 94 23.3 105 26.0 110 27.6 40,000–74,999 53 13.2 60 15.0 63 15.7 69 17.1 51 12.6 56 14.0 75,000–99,999 24 6.0 60 15.0 64 15.9 49 12.2 55 13.6 44 11.0 ≥100,000 142 35.2 170 42.4 169 42.0 161 40.0 165 40.8 155 38.9 Employment 0.44 0.03 Yes 285 74.6 301 78.0 296 74.6 285 73.1 290 73.8 307 80.4 No 97 25.4 85 22.0 101 25.4 105 26.9 103 26.2 75 19.6 Time since last pregnancy loss, months 0.02 0.04 ≤4 187 46.8 226 57.4 226 57.4 210 52.4 218 55.3 211 53.7 5–8 79 19.8 72 18.3 66 16.8 71 17.7 72 18.3 74 18.8 9–12 42 10.5 30 7.6 26 6.6 39 9.7 40 10.2 19 4.8 >12 92 23.0 66 16.8 76 19.3 81 20.2 64 16.2 89 22.7 No. of pregnancies, not including losses 0.14 0.55 0 167 41.4 168 41.8 180 44.8 157 38.9 183 45.3 175 43.9 1 162 40.2 139 34.6 126 31.3 152 37.6 138 34.2 137 34.3 2 70 17.4 86 21.4 88 21.9 89 22.0 77 19.1 78 19.6 3 4 1.0 9 2.2 8 2.0 6 1.5 6 1.5 9 2.3 Table continues Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 Preconception Micronutrients and Fecundability 1911 Table 1. Continued a b α-Carotene γ-Tocopherol Tertile 1 Tertile 2 Tertile 3 Tertile 1 Tertile 2 Tertile 3 Demographic Characteristic (n = 403) (n = 402) (n = 402) (n = 404) (n = 404) (n = 399) P Value P Value No. % No. % No. % No. % No. % No. % No. of previous live births 0.36 0.28 0 186 46.2 183 45.5 190 47.3 170 42.1 193 47.8 196 49.1 1 156 38.7 148 36.8 133 33.1 160 39.6 144 35.6 133 33.3 2 61 15.1 71 17.7 79 19.7 74 18.3 67 16.6 70 17.5 No. of previous pregnancy losses 0.46 0.12 1 261 64.8 277 68.9 269 66.9 258 63.9 285 70.5 264 66.2 2 142 35.2 125 31.1 133 33.1 146 36.1 119 29.5 135 33.8 Treatment arm 0.24 0.14 Low-dose aspirin 201 49.9 190 47.3 214 53.2 218 54.0 199 49.3 188 47.1 Placebo 202 50.1 212 52.7 188 46.8 186 46.0 205 50.7 211 52.9 Study site 0.001 0.57 Utah 311 77.2 325 80.9 348 86.6 340 84.2 325 80.5 319 80.0 New York 39 9.7 24 6.0 13 3.2 20 5.0 26 6.4 30 7.5 Colorado 20 5.0 27 6.7 25 6.2 21 5.2 29 7.2 22 5.5 Pennsylvania 33 8.2 26 6.5 16 4.0 23 5.7 24 5.9 28 7.0 Pregnancy after randomization <0.0001 0.001 Yes 215 53.4 283 70.4 289 71.9 283 70.1 272 67.3 232 58.2 No 188 46.7 119 29.6 113 28.1 121 30.0 132 32.7 167 41.9 Abbreviation: IPAC, International Physical Activity Questionnaire. Range (minimum–maximum) of α-carotene levels: tertile 1, 0.21–2.22 μg/dL; tertile 2, 2.23–4.31 μg/dL; tertile 3, 4.33–46.95 μg/dL. Range (minimum–maximum) of γ-tocopherol levels: tertile 1, 65.0–143.0 μg/dL; tertile 2, 144.0–192.0 μg/dL; tertile 3, 193.0–476.0 μg/dL. c 2 Weight (kg)/height (m) . Physical activity, assessed by means of the IPAQ–Short Form, was categorized on the basis of standard IPAQ cutoffs (42). 3 methods resulted in dissimilar levels of censoring of women at 6 Moreover, the 2 extreme cases for potential outcomes of with- months of follow-up. The results of the Kaplan-Meier–based drawals also resulted in largely similar results, further supporting imputation were similar to the complete-case results for all fat- the robustness of our ﬁndings (Web Table 1). soluble micronutrients, implying that plausible outcomes of with- To investigate the potential for unmeasured confounders to drawals were unlikely to have substantively changed our results. introduce selection bias, we performed additional sensitivity Table 2. Distributions of Preconception Serum Concentrations (μg/dL) of Fat-Soluble Micronutrients (n = 1,207), Effects of Aspirin in Gestation and Reproduction Trial, 2007–2011 No. of Previous Pregnancy Losses No. of Previous Live Births Micronutrient Total 1(n = 807) 2 (n = 400) P Value 0 (n = 559) 1 (n = 437) 2 (n = 211) P Value Zeaxanthin 15.7 (1.5) 15.8 (1.5) 15.5 (1.5) 0.50 15.9 (1.5) 15.3 (1.5) 16.0 (1.4) 0.31 c c Cryptoxanthin 7.7 (1.6) 7.7 (1.6) 7.6 (1.6) 0.59 8.1 (1.7) 7.4 (1.6) 7.5 (1.6) 0.01 Lycopene 26.7 (1.6) 27.0 (1.7) 26.2 (1.6) 0.31 26.8 (1.6) 26.0 (1.7) 27.9 (1.6) 0.21 c c α-Carotene 4.3 (1.8) 4.3 (1.8) 4.3 (1.8) 0.84 4.3 (1.7) 4.1 (1.8) 4.6 (1.8) 0.11 β-Carotene 15.4 (2.1) 15.5 (2.1) 15.4 (2.1) 0.95 15.9 (2.0) 15.1 (2.1) 15.0 (2.1) 0.48 c c α-Tocopherol 912.6 (1.3) 911. (1.3) 914.2 (1.3) 0.88 925.3 (1.3) 892.9 (1.3) 920.8 (1.3) 0.09 c c γ-Tocopherol 167.6 (1.4) 167.2 (1.4) 168.3 (1.4) 0.74 170.8 (1.4) 163.9 (1.4) 166.7 (1.4) 0.14 Values are presented as geometric mean (geometric standard deviation). Overall group difference calculated using analysis of variance. P < 0.05 for difference between groups. Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 1912 Kim et al. Table 3. Associations Between Log-Transformed Levels of Fat-Soluble Micronutrients and Time to Pregnancy (n = 1,228), Effects of Aspirin in Gestation and Reproduction Trial, 2007–2011 Unadjusted Adjusted Micronutrient FOR 95% CI FOR 95% CI Continuous variable, μg/dL Zeaxanthin 1.19 0.98, 1.45 1.12 0.89, 1.39 Cryptoxanthin 1.12 0.95, 1.32 1.06 0.88, 1.28 Lycopene 1.15 0.98, 1.35 1.19 0.99, 1.43 α-Carotene 1.23 1.08, 1.41 1.17 1.00, 1.36 β-Carotene 1.24 1.11, 1.39 1.12 0.98, 1.28 α-Tocopherol 1.15 0.85, 1.55 1.23 0.85, 1.78 γ-Tocopherol 0.66 0.52, 0.84 0.83 0.62, 1.11 Dichotomous variable (≥US average) Zeaxanthin 1.10 0.94, 1.29 1.04 0.87, 1.24 Cryptoxanthin 1.09 0.93, 1.29 1.04 0.87, 1.24 Lycopene 1.03 0.81, 1.30 1.02 0.79, 1.32 α-Carotene 1.29 1.10, 1.52 1.21 1.02, 1.44 β-Carotene 1.26 1.07, 1.48 1.11 0.92, 1.33 α-Tocopherol 1.04 0.88, 1.23 1.04 0.87, 1.25 γ-Tocopherol 0.73 0.62, 0.87 0.83 0.69, 1.00 Abbreviations: CI, conﬁdence interval; FOR, fecundability odds ratio. Imputed data for missing micronutrient concentrations and covariates were used in the analyses. b 2 Models adjusted for age (years), body mass index (weight (kg)/height (m) ), race (white or nonwhite), frequency of cigarette smoking (never smoker, fewer (<6 times/week), or daily), frequency of alcohol drinking (never drinker, sometimes (up to 2–3 times/week), or often (4–6 times/week or more)), physical activity level (high, moderate, or low; see Table 1), annual income (<$20,000, $20,000–$39,999, $40,000–$74,999, $75,000–$99,999, or ≥$100,000), vitamin use (yes or no), total cholesterol concentration (mg/dL), treatment arm (low dose aspirin or placebo), and study site. A micronutrient level less than the US average (<138 μg/dL for zeaxanthin, <8 μg/dL for cryptoxanthin, <41.3 μg/ dL for lycopene, 2.9 μg/dL for α-carotene, <12.2 μg/dL for β-carotene, <1,010 μg/dL for α-tocopherol, and <187 μg/dL for γ-tocopherol) was used as the referent. analyses. Adjustment for an unmeasured dietary factor (U ), past was only marginally associated with a shorter TTP in our study, TTP (TTP ), or an unmeasured genetic factor (U ), either indi- we observed that α-carotene was signiﬁcantly associated with a 0 2 vidually (Web Figures 3A and 3B) or together (Web Figure 3C), 17% shorter TTP. Although we did not observe any effect- would block the backdoor pathways and did not alter our results. measure modiﬁcation by BMI or age, taken together these data support a beneﬁcial role of carotenoids in TTP and necessitate further exploration of a potential role of carotenoids in fertility. Carotenoids, which are mostly obtained from fruits and vegeta- DISCUSSION bles, have been shown to inhibit free radical reactions in body sys- Overall, we found that increasing preconception serum carot- tems and to protect cellular membranes from lipid peroxidation enoid concentrations were associated with improved fecundabil- (11). Speciﬁc biological mechanisms to account for the associa- ity and a shorter TTP among women with proven fecundity and tion between antioxidant micronutrients and TTP are uncertain. no history of infertility. On the other hand, our data also suggested However, antioxidants have been shown to be associated with that serum γ-tocopherol levels at or above the US average were reproductive hormone concentrations in healthy women (12), associated with a longer TTP. TTP is a widely used measure of suggesting potentially complex hormonal interactions, which couple fecundity, and our data support a possible role of fat- may subsequently lead to improved reproductive function. Our soluble micronutrients, which are easily obtained through ﬁnding highlights a potential role of preconception carotenoids in diet, in fecundability. fecundability, although the clinical implications of these results A positive association between carotenoids and shortened TTP for women attempting pregnancy in the general population war- was also reported in a recent study of women diagnosed with rant further investigation. unexplained infertility (16). In that study, intake of β-carotene In our data, serum γ-tocopherol concentrations at or above from dietary supplements was associated with a shorter TTP in the US average and increased γ-tocopherol concentrations were womenwithBMI ≥25 or age <35 years. Although β-carotene associated with a longer TTP. Our results differed from the Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 Preconception Micronutrients and Fecundability 1913 results of other studies, which observed positive associations bioavailability of other micronutrients (31). Most study parti- between tocopherols and reproductive outcomes. In particular, cipants had taken vitamin supplements prior to enrollment and Browne et al. (13) identiﬁed a positive association between during the trial, which may have affected our study results. γ-tocopherol measured in follicular ﬂuid and better embryo Although we included use of vitamin supplements during the quality in women undergoing in vitro fertilization, suggesting a multivariable regression analysis, we did not have data regard- potential beneﬁcial role of γ-tocopherol in embryo-level out- ing the dose and speciﬁc type of vitamin supplements taken. Data comes. Differences between serum and follicular ﬂuid measures on dietary intake, which inﬂuences levels of micronutrients (34), might explain the discrepant ﬁndings. In a study of women with were not available. However, our fat-soluble micronutrient con- unexplained infertility, vitamin E supplementation was associ- centrations measured in serum are useful markers of intakes of ated with a shorter TTP in women aged ≥35 years (16); however, foods rich in those micronutrients, particularly fruits and vege- serum γ-tocopherol levels were not measured. tables, though correlations vary by speciﬁc micronutrient (35, 36). Though the differences might also be related to differences in Given the short half-lives of fat-soluble micronutrients (37–39), study design, primary outcomes, and assessment of micronutrient our micronutrient concentrations measured at baseline may not status, most importantly, the women in our study have demon- have adequately reﬂected micronutrient status at the time of strated fecundity rather than infertility. Interestingly, serum conception. Our follow-up duration of 6 months was compara- γ-tocopherol concentrations measured in our women at baseline ble to that of other studies that investigated TTP (40, 41)and did not vary by reproductive history characteristics, such as num- allowed us to investigate factors associated with subfertility. How- ber of previous losses or live births; however, all women had a ever, our study was limited in that we may have been able to history of prior losses and no history of infertility treatment. identify infertility had we been able to follow the women for Although mean concentrations differed by pregnancy status, with 12 months, rather than 6 months. lower levels being observed in pregnant women (163.4 μg/dL) Information on pregnancy status and timing of pregnancy than in nonpregnant women (171.3 μg/dL) and women who was not available for approximately 10% of our participants withdrew from the study (186.1 μg/dL), our sensitivity analyses who withdrew early, and their preconception levels of fat-soluble indicated a minimal inﬂuence of the difference in γ-tocopherol micronutrients were different from those who were retained in concentrations by pregnancy and withdrawal status on our re- the study. We performed sensitivity analyses based on dif- sults. Given potentially differential impacts of γ-tocopherol on ferent statistical approaches to address potential bias due to such fertility outcomes, further investigation is necessary to elucidate missingness. Overall, our sensitivity analyses demonstrated con- its potential role in fecundability. sistency of results across several different imputation methods, The differences we observed between carotene and tocopherol conﬁrming the robustness of our results to the impact of partici- levels and TTP in our study may be explained by several factors. pant withdrawal. Because of the original clinical trial enrollment Serum carotene concentrations measured in our study were criteria, the generalizability of our ﬁndings is limited to women inversely correlated with γ-tocopherol concentrations, a result with 1–2 prior losses. Although we are not attempting to gen- consistent with that of a study carried out among postmenopausal eralize our ﬁndings to all reproductive-age women, we investi- womeninthe Women’s Health Initiative (29). Competition for gated the potential for selection bias due to such restriction. micellar solubilization before absorption was noted in an animal Reassuringly, the results were not different from our observed study in which simultaneous administration of large doses of ﬁndings, even after accounting for strong potential unmeasured α-tocopherol reduced the utilization of β-carotene (30). In a confounders. clinical trial conducted among 59 adults, Willett et al. (31) To our knowledge, this was the ﬁrst study assessing associa- reported a reduction in plasma carotenoid levels after 16 weeks tions between preconception fat-soluble micronutrients measured of daily α-tocopherol administration (800 IU) as well, suggest- in blood—reﬂecting an individual’s antioxidant status—and ing competitive absorption and subsequent interactive bioactivity couple fecundity. Our data support a beneﬁcial role for carotenes of carotenes and tocopherols. Almost all demographic factors, in TTP among women who have demonstrated fecundity and are including BMI, cigarette smoking, alcohol drinking, physical attempting pregnancy. However, because the effect sizes tended to be small overall, additional research is needed to better describe activity, education, and income, when characterized by tertiles the clinical signiﬁcance and relevance of these ﬁndings. Fat- of γ-tocopherol and α-carotene, showed opposing trends in our soluble micronutrients are abundant in fruits, vegetables, and study. Thus, it appears that numerous lifestyle variables as well seed oils and are easily obtainable through a healthy diet in the as dietary habits could affect bioavailability of carotenes and to- copherols ( general population. Given the positive associations between 32). Our study had multiple strengths. Measurement of TTP was micronutrients and TTP and the fact that diet is a modiﬁable prospective, which minimized the misclassiﬁcation that can occur lifestyle factor, further exploration of the roles of fat-soluble in many retrospective observational studies (33). The prospective micronutrients and fecundability in the general population of study design particularly strengthens our ﬁndings, as selected mi- reproductive-age women is warranted. cronutrients were measured in women while they were trying to conceive. However, there were several limitations in our study. Although TTP is a measure of couple fecundity, data on the micronutrient status of male partners were not available in our ACKNOWLEDGMENTS study. Therefore, future investigation of a potential contribution of male partners’ macro- and micronutrient status to couples’ Author afﬁliations: Division of Intramural Population TTP is of interest. Further, intake of vitamin supplements tends Health Research, Eunice Kennedy Shriver National Institute to increase plasma levels of micronutrients and may affect the of Child Health and Human Development, Bethesda, Am J Epidemiol. 2018;187(9):1907–1915 Downloaded from https://academic.oup.com/aje/article/187/9/1907/4995887 by DeepDyve user on 18 July 2022 1914 Kim et al. 12. Mumford SL, Browne RW, Schliep KC, et al. Serum Maryland (Keewan Kim, Enrique F. Schisterman, Neil J. antioxidants are associated with serum reproductive hormones Perkins, Sunni L. 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American Journal of Epidemiology – Oxford University Press
Published: Sep 1, 2018
Keywords: pregnancy; fertility; micronutrients; tocopherols; pregnancy history; vitamin e; reproductive physiological process; body mass index procedure
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