Disruption of Zfh3 abolishes mulberry-specific monophagy in silkworm larvae.

Feeding behavior is critical for insect survival and fitness. Most researchers have explored the molecular basis of feeding behaviors by identifying and elucidating the function of olfactory receptors (ORs) and gustatory receptors (GRs). Other types of genes, such as transcription factors, have rarely been investigated, and little is known about their potential roles. The silkworm (Bombyx mori) is a well-studied monophagic insect which primarily feeds on mulberry leaves, but the genetic basis of its monophagy is still not understood. In this report, we focused on a transcription factor encoded by the Zfh3 gene, which is highly expressed in the silkworm central and peripheral nervous systems, including brain, antenna, and maxilla. To investigate its function, Zfh3 was abrogated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) mutagenesis. Since Zfh3 knockout homozygotes are not viable, we studied feeding behavior in heterozygotes, and found that disruption of Zfh3 affects both gustation and olfaction. Mutant larvae lose preference for mulberry leaves, acquire the ability to consume an expanded range of diets, and exhibit improved adaptation to the M0 artificial diet, which contains no mulberry leaves. These results provide the first demonstration that a transcription factor modulates feeding behaviors in an insect.

Preconception caffeine metabolites, caffeinated beverage intake, and fecundability.

BACKGROUND: Caffeine is the most frequently used psychoactive substance in the United States and >90% of reproductive-age women report some amount of intake daily. Despite biological plausibility, previous studies on caffeine and fecundability report conflicting results. Importantly, prior studies measured caffeine exposure exclusively by self-report, which is subject to measurement error and does not account for factors that influence caffeine metabolism. OBJECTIVES: Our objective was to examine associations between preconception serum caffeine metabolites, caffeinated beverage intake, and fecundability. METHODS: Participants included 1228 women aged 18-40 y with a history of 1-2 pregnancy losses in the EAGeR (Effects of Aspirin in Gestation and Reproduction) trial. We prospectively evaluated associations of preconception caffeine metabolites (i.e., caffeine, paraxanthine, and theobromine) measured from 1191 serum samples untimed to a specific time of day, self-reported usual caffeinated beverage intakes at baseline, and time-varying cycle-average caffeinated beverage intake, with fecundability. Using Cox proportional hazards models, we estimated fecundability odds ratios (FORs) and 95% CIs according to each metabolite. Follow-up was complete for 89% (n = 1088) of participants. RESULTS: At baseline, 85%, 73%, and 91% of women had detectable serum caffeine, paraxanthine, and theobromine, respectively. A total of 797 women became pregnant during ≤6 cycles of preconception follow-up. After adjusting for potential confounders, neither serum caffeine [tertile (T)3 compared with T1 FOR: 0.87; 95% CI: 0.71, 1.08], paraxanthine (T3 compared with T1 FOR: 0.92; 95% CI: 0.75, 1.14), nor theobromine (T3 compared with T1 FOR: 1.15; 95% CI: 0.95, 1.40) were associated with fecundability. Baseline intake of total caffeinated beverages was not associated with fecundability (>3 compared with 0 servings/d adjusted FOR: 0.99; 95% CI: 0.74, 1.34), nor was caffeinated coffee (>2 compared with 0 servings/d adjusted FOR: 0.93; 95% CI: 0.45, 1.92) or caffeinated soda (>2 servings/d adjusted FOR: 0.92; 95% CI: 0.71, 1.20). CONCLUSIONS: Our findings are reassuring that caffeine exposure from usual low to moderate caffeinated beverage intake likely does not influence fecundability.This trial was registered at clinicaltrials.gov as NCT00467363.

The Safety of Low-Dose Aspirin on the Mode of Delivery: Secondary Analysis of the Effect of Aspirin in Gestation and Reproduction Randomized Controlled Trial.

OBJECTIVE: This study aimed to examine whether prenatal low-dose aspirin (LDA) therapy affects risk of cesarean versus vaginal delivery. STUDY DESIGN: This study is a secondary analysis of the randomized clinical effects of aspirin in gestation and reproduction (EAGeR) trial. Women received 81-mg daily aspirin or placebo from preconception to 36 weeks of gestation. Mode of delivery and obstetric complications were abstracted from records. Log-binomial regression models estimated relative risk (RR) of cesarean versus vaginal delivery. Data were analyzed among the total preconception cohort, as well as restricted to women who had a live birth. RESULTS: Among 1,228 women, 597 had a live birth. In the intent-to-treat analysis, preconception-initiated LDA was not associated with risk of cesarean (RR = 1.02; 95% confidence interval [CI]: 0.98-1.07) compared with placebo. Findings were similar in just women with a live birth and when accounting prior cesarean delivery and parity. CONCLUSION: Preconception-initiated daily LDA was not associated with mode of delivery among women with one to two prior losses. KEY POINTS: · Aspirin was not associated with risk of cesarean section.. · Aspirin was not associated with mode of delivery.. · No increased risk of bleeding with use of aspirin..

Exposure assessment for Cox proportional hazards cure models with interval-censored survival data.

Mixture cure models have been developed as an effective tool to analyze failure time data with a cure fraction. Used in conjunction with the logistic regression model, this model allows covariate-adjusted inference of an exposure effect on the cured probability and the hazard of failure for the uncured subjects. However, the covariate-adjusted inference for the overall exposure effect is not directly provided. In this paper, we describe a Cox proportional hazards cure model to analyze interval-censored survival data in the presence of a cured fraction and then apply a post-estimation approach by using model-predicted estimates difference to assess the overall exposure effect on the restricted mean survival time scale. For baseline hazard/survival function estimation, simple parametric models as fractional polynomials or restricted cubic splines are utilized to approximate the baseline logarithm cumulative hazard function, or, alternatively, the full likelihood is specified through a piecewise linear approximation for the cumulative baseline hazard function. Simulation studies were conducted to demonstrate the unbiasedness of both estimation methods for the overall exposure effect estimates over various baseline hazard distribution shapes. The methods are applied to analyze the interval-censored relapse time data from a smoking cessation study.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome. RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

The role of maternal preconception vitamin D status in human offspring sex ratio.

Evolutionary theory suggests that some animal species may experience shifts in their offspring sex ratio in response to maternal health and environmental conditions, and in some unfavorable conditions, females may be less likely to bear sons. Experimental data in both animals and humans indicate that maternal inflammation may disproportionately impact the viability of male conceptuses; however, it is unknown whether other factors associated with both pregnancy and inflammation, such as vitamin D status, are associated with the offspring sex ratio. Here, we show that among 1,228 women attempting pregnancy, preconception 25-hydroxyvitamin D concentrations are positively associated with the live birth of a male infant, with notably stronger associations among women with elevated high sensitivity C-reactive protein, a marker of systemic low-grade inflammation. Our findings suggest that vitamin D may mitigate maternal inflammation that would otherwise be detrimental to the implantation or survival of male conceptuses in utero.

Adiposity is associated with anovulation independent of serum free testosterone: A prospective cohort study.

BACKGROUND: Obesity, a body mass index (BMI) ≥30 kg/m2 , is linked to infertility, potentially through a greater risk of anovulation due to elevated androgens. Yet, previous studies have not directly assessed the impact of adiposity, or body fat, on anovulation in the absence of clinical infertility. OBJECTIVE: To characterise the associations between adiposity and anovulation among women menstruating on a regular basis. METHODS: Women from the EAGeR trial (n = 1200), a randomised controlled trial of low-dose aspirin and pregnancy loss among women trying to conceive, were used to estimate associations between adiposity and incident anovulation. Participants completed baseline questionnaires and anthropometry, and provided blood specimens. Women used fertility monitors for up to six consecutive menstrual cycles, with collection of daily first morning voids for hormone analysis in the first two menstrual cycles for prospective assessment of anovulation. Anovulation was assessed by urine pregnanediol glucuronide or luteinising hormone concentration or the fertility monitor. Weighted mixed-effects log-binomial regression was used to estimate associations between measures of adiposity and incident anovulation, adjusted for free (bioavailable) testosterone, anti-Mullerian hormone (AMH), serum lipids, and demographic and life style factors. RESULTS: 343 (28.3%) women experienced at least one anovulatory cycle. Anovulation risk was higher per kg/m2 greater BMI (relative risk [RR] 1.03, 95% confidence interval (CI) 1.01, 1.04), cm waist circumference (RR 1.01, 95% CI 1.00, 1.02), mm subscapular skinfold (RR 1.02, 95% CI 1.01, 1.03), and mm middle upper arm circumference (RR 1.04, 95% CI 1.01, 1.06) adjusted for serum free testosterone, AMH, lipids, and other factors. CONCLUSIONS: Adiposity may be associated with anovulation through pathways other than testosterone among regularly menstruating women. This may account in part for reported associations between greater adiposity and infertility among women having menstrual cycles regularly. Understanding the association between adiposity and anovulation might lead to targeted interventions for preventing infertility.

Recalled maternal lifestyle behaviors associated with anti-müllerian hormone of adult female offspring.

Anti-müllerian hormone (AMH) is an established marker of ovarian reserve that decreases with age. Though the pool of ovarian follicles is established during fetal development, impacts of in utero exposures on AMH are uncertain. Thus, we sought to evaluate associations of in utero exposures with AMH of adult daughters with a prospective cohort study of adult daughters at university medical centers. Women noted their mother's reported use of diethylstilbestrol (DES), vitamins, tobacco, alcohol, and caffeine during pregnancy, and their mother's occupation during pregnancy. All participants were reproductive age women (18-40 years) enrolled in the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial. Serum AMH concentrations were measured at baseline prior to conception and categorized using clinical guidelines. Multinomial regression models estimated associations between each exposure and high (>3.5 ng/mL) and low (<1.0 ng/mL) versus normal AMH (1.0-3.5 ng/mL), adjusting for participant's age, mother's age, mother's history of fertility treatment, and mother's use of vitamins. In 1202 women with available data, maternal caffeine use was associated with an increased risk of low AMH, compared to normal (relative risk [RR] 1.90, 95 % confidence interval [CI] 1.09, 3.30). Vitamins were associated with an increased risk of high AMH compared to normal (RR 1.93, 95 % CI 1.24, 3.00). Other exposures were not associated with AMH concentrations in offspring. Maternal caffeine and vitamin use during pregnancy may be associated with ovarian reserve in adult offspring, highlighting the potential importance of pregnancy lifestyle on the reproductive health of daughters.

The influences of sleep duration, chronotype, and nightwork on the ovarian cycle.

Despite research indicating that sleep disorders influence reproductive health, the effects of sleep on reproductive hormone concentrations are poorly characterized. We prospectively followed 259 regularly menstruating women across one to two menstrual cycles (the BioCycle Study, 2005-2007), measuring fasting serum hormone concentrations up to eight times per cycle. Women provided information about daily sleep in diaries and chronotype and night/shift work on a baseline questionnaire. We evaluated percent differences in mean hormone concentrations, the magnitude of shifts in the timing and amplitude of hormone peaks, and the risk for sporadic anovulation associated with self-reported sleep patterns and night/shift work. We estimated chronotype scores - categorizing women below and above the interquartile range (IQR) as "morning" and "evening" chronotypes, respectively. For every hour increase in daily sleep duration, mean estradiol concentrations increased by 3.9% (95% confidence interval [CI] 2.0, 5.9%) and luteal phase progesterone by 9.4% (CI 4.0, 15.2%). Receiving less than 7 hours of sleep per day was associated with slightly earlier rises in peak levels for several hormones. Women reporting night/shift work (n = 77) had lower testosterone relative to women employed without night/shift work (percent difference: -9.9%, CI -18.4, -0.4%). Women with morning chronotypes (n = 47) had earlier rises in estradiol during their cycles and potentially an earlier rise in luteinizing hormone. Compared to those who had intermediate chronotypes, women with evening chronotypes (n = 42) had a later luteinizing hormone peak of borderline statistical significance. A reduced risk for sporadic anovulation was suggested, but imprecise, for increasing hours of daily sleep leading up to ovulation (risk ratio 0.79, CI 0.59, 1.06), while an imprecise increased risk was observed for women with morning chronotypes (risk ratio 2.50, CI 0.93, 6.77). Sleep-related hormonal changes may not greatly alter ovarian function in healthy women, but have the potential to influence gynecologic health.

Rhythmic Fluctuations in Levels of Liver Enzymes During Menstrual Cycles of Healthy Women and Effects of Body Weight.

BACKGROUND & AIMS: Female sex hormones affect several non-reproductive organs, but little is known about their effects on the liver during a normal menstrual cycle. We aimed to investigate the association between sex hormones and liver enzymes in healthy menstruating women. METHODS: We performed a post-hoc analysis of data from the BioCycle study, a longitudinal cohort study designed to determine the association of sex hormones with markers of oxidative stress during the menstrual cycle. We analyzed data collected from 259 menstruating women, over 1-2 menstrual cycles, who had as many as 16 separate office visits, timed by fertility monitors. Levels of liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase, and alkaline phosphatase (ALKP), bilirubin, and lipids were measured by laboratory assays. RESULTS: We found a natural cyclic pattern for liver enzymes, with transaminases and ALKP peaking in the mid-follicular phase and reaching a trough in the late luteal phase; the peak to trough differences were 4.0 ± 4.9 U/L for ALT and 8.8 ± 4.0 U/L for ALKP. Levels of ALT were significantly and negatively associated with levels of progesterone on the preceding visit (P = 5x10-4), whereas level of ALKP was negatively associated with level of estrogen (P = .007) and progesterone (P = 1x10-11). Food and alcohol intake did not modify the association. The amplitude of ALT fluctuation was greater in African Americans and decreased with age. Fluctuations in levels of ALT were smaller in women with body mass indices >30 kg/m2 (P = .03). During menstrual fluctuation, 49% of participants had ALT values both above and below the normal cut-off value (19 U/L). CONCLUSIONS: Levels of liver enzymes fluctuate during the normal menstrual cycle, possibly mediated by progesterone, and the fluctuation varies with age and body mass index. These findings indicate the importance of accounting for phase of menstrual cycle when interpreting liver enzyme measurements in menstruating women.

Shorter Time to Pregnancy With Increasing Preconception Carotene Concentrations Among Women With 1-2 Previous Pregnancy Losses.

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 assessed the association of preconception-initiated daily low-dose aspirin with reproductive outcomes. We measured preconception levels of zeaxanthin, cryptoxanthin, lycopene, α- and ß-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 associated with longer TTP (FOR = 0.83, 95% CI: 0.69, 1.00). The potential for these nutrients to influence fecundability deserves further exploration.

Maternal Weight Gain During Pregnancy: Comparing Methods to Address Bias Due to Length of Gestation in Epidemiological Studies.

BACKGROUND: Studies examining total gestational weight gain (GWG) and outcomes associated with gestational age (GA) are potentially biased. The z-score has been proposed to mitigate this bias. We evaluated a regression-based adjustment for GA to remove the correlation between GWG and GA, and compared it to published weight-gain-for-gestational-age z-scores when applied to a study sample with different underlying population characteristics. METHODS: Using 65 643 singleton deliveries to normal weight women at 12 US clinical sites, we simulated a null association between GWG and neonatal mortality. Logistic regression was used to estimate approximate relative risks (RR) of neonatal mortality associated with GWG, unadjusted and adjusted for GA, and the z-score, overall and within study sites. Average RRs across 5000 replicates were calculated with 95% coverage probability to indicate model bias and precision, where 95% is nominal. RESULTS: Under a simulated null association, total GWG resulted in a biased mortality estimate (RR = 0.87; coverage = 0%); estimates adjusted for GA were unbiased (RR = 1.00; coverage = 94%). Quintile-specific RRs ranged from 0.97-1.03. Similar results were observed for site-specific analyses. The overall z-score RR was 0.97 (84% coverage) with quintile-specific RRs ranging from 0.64-0.90. Estimates were close to 1.0 at most sites, with coverage from 70-94%. Sites 1 and 6 were biased with RRs of 0.66 and 1.43, respectively, and coverage of 70% and 80%. CONCLUSIONS: Adjusting for GA achieves unbiased estimates of the association between total GWG and neonatal mortality, providing an accessible alternative to the weight-gain-for-gestational-age z-scores without requiring assumptions concerning underlying population characteristics.

Relationship between physical occupational exposures and health on semen quality: data from the Longitudinal Investigation of Fertility and the Environment (LIFE) Study.

OBJECTIVE: To study the relationship among occupation, health, and semen quality in a cohort of men attempting to conceive. DESIGN: Observational prospective cohort. SETTING: Not applicable. PATIENT(S): A total of 501 couples discontinuing contraception were followed for 1 year while trying to conceive; 473 men (94%) provided one semen sample, and 80% provided a second sample. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen data obtained through at-home semen collection with next-day analysis/quantification. RESULT(S): In all, complete data were available for 456 men, with a mean age of 31.8 years. Work-related heavy exertion was consistently associated with lower semen concentration and total sperm count. Thirteen percent of men who reported heavy exertion displayed oligospermia, compared with 6% who did not report workplace exertion. Shift work, night work, vibration, noise, heat, and prolonged sitting were not associated with semen quality. Men with high blood pressure had significantly lower strict morphology scores compared with normotensive men (17% vs. 21%). In contrast, hyperlipidemia, diabetes, and composite of total comorbidities were not associated with semen quality. The number of medications a man was taking as a proxy of health status was associated with semen quality. There was a negative association between number of medications and sperm count. CONCLUSION(S): A negative relationship among occupational exertion, hypertension, and the number of medications with semen quality was identified. As these are potentially modifiable factors, further research should determine whether treatment or cessation may improve male fecundity.

The effect of a very short interpregnancy interval and pregnancy outcomes following a previous pregnancy loss.

OBJECTIVE: We sought to assess the relationship between a short interpregnancy interval (IPI) following a pregnancy loss and subsequent live birth and pregnancy outcomes. STUDY DESIGN: A secondary analysis of women enrolled in the Effects of Aspirin in Gestation and Reproduction trial with a human chorionic gonadotropin-positive pregnancy test and whose last reproductive outcome was a loss were included in this analysis (n = 677). IPI was defined as the time between last pregnancy loss and last menstrual period of the current pregnancy and categorized by 3-month intervals. Pregnancy outcomes include live birth, pregnancy loss, and any pregnancy complications. These were compared between IPI groups using multivariate relative risk estimation by Poisson regression. RESULTS: Demographic characteristics were similar between IPI groups. The mean gestational age of prior pregnancy loss was 8.6 ± 2.8 weeks. The overall live birth rate was 76.5%, with similar live birth rates between those with IPI ≤3 months as compared to IPI >3 months (adjusted relative risk [aRR], 1.07; 95% confidence interval [CI], 0.98-1.16). Rates were also similar for periimplantation loss (aRR, 0.95; 95% CI, 0.51-1.80), clinically confirmed loss (aRR, 0.75; 95% CI, 0.51-1.10), and any pregnancy complication (aRR, 0.88; 95% CI, 0.71-1.09) for those with IPI ≤3 months as compared to IPI >3 months. CONCLUSION: Live birth rates and adverse pregnancy outcomes, including pregnancy loss, were not associated with a very short IPI after a prior pregnancy loss. The traditional recommendation to wait at least 3 months after a pregnancy loss before attempting a new pregnancy may not be warranted.

Accuracy loss due to selection bias in cohort studies with left truncation.

BACKGROUND: Selection is a common problem in paediatric and perinatal epidemiology, and truncation can be thought of as missing person time that can result in selection bias. Left truncation, also known as late or staggered entry, may induce selection bias and/or adversely affect precision. There are two kinds of left truncation: fixed left truncation where the start of follow-up is initiated at a set time, and variable left truncation where follow-up begins at a stochastically varying time-point. METHODS: Using data from a time-to-pregnancy study, augmented by a simulation study, we demonstrate the effects of fixed and variable truncation on estimates of the hazard ratio. RESULTS: First, fixed or variable non-differential left truncation results in a loss of precision. Fixed or variable differential left truncation results in a bias either towards or away from the null as well as a loss of precision. The extent and direction of this bias is a function of the size and direction of the association between exposure and outcome, and occurs in common scenarios and under a wide range of conditions. CONCLUSIONS: As demonstrated in simulation studies, selection bias due to left truncation could have a serious impact on inferences, especially in the case of fixed or variable differential left truncation. When present in epidemiologic studies, proper accounting for left truncation is just as important as proper accounting for right censoring.

Validation of different instruments for caffeine measurement among premenopausal women in the BioCycle study.

Effects of caffeine on women's health are inconclusive, in part because of inadequate exposure assessment. In this study we determined 1) validity of a food frequency questionnaire compared with multiple 24-hour dietary recalls (24HDRs) for measuring monthly caffeine and caffeinated beverage intakes; and 2) validity of the 24HDR compared with the prior day's diary record for measuring daily caffeinated coffee intake. BioCycle Study (2005-2007) participants, women (n = 259) aged 18-44 years from western New York State, were followed for 2 menstrual cycles. Participants completed a food frequency questionnaire at the end of each cycle, four 24HDRs per cycle, and daily diaries. Caffeine intakes reported for the food frequency questionnaires were greater than those reported for the 24HDRs (mean = 114.1 vs. 92.6mg/day, P = 0.01) but showed high correlation (r = 0.73, P < 0.001) and moderate agreement (К = 0.51, 95% confidence interval: 0.43, 0.57). Women reported less caffeinated coffee intake in their 24HDRs compared with their corresponding diary days (mean = 0.51 vs. 0.80 cups/day, P < 0.001) (1 cup = 237 mL). Although caffeine and coffee exposures were highly correlated, absolute intakes differed significantly between measurement tools. These results highlight the importance of considering potential misclassification of caffeine exposure.

Energy-containing beverages: reproductive hormones and ovarian function in the BioCycle Study.

BACKGROUND: Energy-containing beverages are widely consumed among premenopausal women, but their association with reproductive hormones is not well understood. OBJECTIVE: The objective was to assess the association of energy-containing beverages, added sugars, and total fructose intake with reproductive hormones among ovulatory cycles and sporadic anovulation in healthy premenopausal women. DESIGN: Women (n = 259) in the BioCycle Study were followed for up to 2 menstrual cycles; they provided fasting blood specimens during up to 8 visits/cycle and four 24-h dietary recalls/cycle. RESULTS: Women who consumed ≥1 cup (1 cup = 237 mL) sweetened soda/d had 16.3% higher estradiol concentrations compared with women who consumed less sweetened soda (86.5 pg/mL compared with 74.4 pg/mL, P = 0.01) after adjustment for age, BMI, race, dietary factors, and physical activity. Similarly elevated estradiol concentrations were found for ≥1 cup cola/d and noncola soda intake. Neither artificially sweetened soda nor fruit juice intake ≥1 cup/d was significantly associated with reproductive hormones. Added sugar above the average US woman's intake (≥73.2 g/d) or above the 66th percentile in total fructose intake (≥41.5 g/d) was associated with significantly elevated estradiol but not consistently across all models. No associations were found between beverages, added sugars, or total fructose intake and anovulation after multivariate adjustment. CONCLUSIONS: Even at moderate consumption amounts, sweetened soda is associated with elevated follicular estradiol concentrations among premenopausal women but does not appear to affect ovulatory function. Further research into the mechanism driving the association between energy-containing beverages and reproductive hormones, and its potential implications for women's health, is warranted.

Assessment of skewed exposure in case-control studies with pooling.

Pooling-based strategies that combine samples from multiple participants for laboratory assays have been proposed for epidemiologic investigations of biomarkers to address issues including cost, efficiency, detection, and when minimal sample volume is available. A modification of the standard logistic regression model has been previously described to allow use with pooled data; however, this model makes assumptions regarding exposure distribution and logit-linearity of risk (i.e., constant odds ratio) that can be violated in practice. We were motivated by a nested case-control study of miscarriage and inflammatory factors with highly skewed distributions to develop a more flexible model for analysis of pooled data. Using characteristics of the gamma distribution and the relation between models of binary outcome conditional on exposure and of exposure conditional on outcome, we use a modified logistic regression to accommodate nonlinearity because of unequal shape parameters in gamma distributed exposure for cases and controls. Using simulations, we compare our approach with existing methods for logistic regression for pooled data considering: (1) constant and dose-dependent effects; (2) gamma and log-normal distributed exposure; (3) effect size; and (4) the proportions of biospecimens pooled. We show that our approach allows estimation of odds ratios that vary with exposure level, yet has minimal loss of efficiency compared with existing approaches when exposure effects are dose-invariant. Our model performed similarly to a maximum likelihood estimation approach in terms of bias and efficiency, and provides an easily implemented approach for estimation with pooled biomarker data when effects may not be constant across exposure.

Caffeinated beverage intake and reproductive hormones among premenopausal women in the BioCycle Study.

BACKGROUND: Caffeinated beverages are widely consumed among women of reproductive age, but their association with reproductive hormones, and whether race modifies any such associations, is not well understood. OBJECTIVE: We assessed the relation between caffeine and caffeinated beverage intake and reproductive hormones in healthy premenopausal women and evaluated the potential effect modification by race. DESIGN: Participants (n = 259) were followed for up to 2 menstrual cycles and provided fasting blood specimens for hormonal assessment at up to 8 visits per cycle and four 24-h dietary recalls per cycle. Weighted linear mixed models and nonlinear mixed models with harmonic terms were used to estimate associations between caffeine and hormone concentrations, adjusted for age, adiposity, physical activity, energy and alcohol intakes, and perceived stress. On the basis of a priori assumptions, an interaction between race and caffeine was tested, and stratified results are presented. RESULTS: Caffeine intake ≥200 mg/d was inversely associated with free estradiol concentrations among white women (ß = -0.15; 95% CI: -0.26, -0.05) and positively associated among Asian women (ß = 0.61; 95% CI: 0.31, 0.92). Caffeinated soda intake and green tea intake ≥1 cup/d (1 cup = 240 mL) were positively associated with free estradiol concentrations among all races: ß = 0.14 (95% CI: 0.06, 0.22) and ß = 0.26 (95% CI: 0.07, 0.45), respectively. CONCLUSIONS: Moderate consumption of caffeine was associated with reduced estradiol concentrations among white women, whereas caffeinated soda and green tea intakes were associated with increased estradiol concentrations among all races. Further research is warranted on the association between caffeine and caffeinated beverages and reproductive hormones and whether these relations differ by race.

Realignment and multiple imputation of longitudinal data: an application to menstrual cycle data.

Reproductive hormone levels are highly variable among premenopausal women during the menstrual cycle. Accurate timing of hormone measurement is essential, especially when investigating day- or phase-specific effects. The BioCycle Study used daily urine home fertility monitors to help detect the luteinising hormone (LH) surge in order to schedule visits with biologically relevant windows of hormonal variability. However, as the LH surge is brief and cycles vary in length, relevant hormonal changes may not align with scheduled visits even when fertility monitors are used. Using monitor data, measurements were reclassified according to biological phase of the menstrual cycle to more accurate cycle phase categories. Longitudinal multiple imputation methods were applied after reclassification if no visit occurred during a given menstrual cycle phase. Reclassified cycles had more clearly defined hormonal profiles, with higher mean peak hormones (up to 141%) and reduced variability (up to 71%). We demonstrate the importance of realigning visits to biologically relevant windows when assessing phase- or day-specific effects and the feasibility of applying longitudinal multiple imputation methods. Our method has applications in settings where missing data may occur over time, where daily blood sampling for hormonal measurements is not feasible, and in other areas where timing is essential.