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Study Question: To what extent does fecundability vary across seasons?Summary Answer: After accounting for seasonal patterns in pregnancy planning, we observed higher fecundability in the fall and lower fecundability in the spring, particularly at lower latitudes.What Is Known Already: In human populations, there are strong seasonal patterns of births that vary across geographic regions and time periods. However, previous studies of seasonality and fecundity are limited because they examine season of birth rather than season of conception and therefore neglect to account for seasonal variation in initiating attempts to conceive or pregnancy loss or differences in gestational length.Study Design, Size, Duration: We conducted a preconception cohort study of 14 331 women residing in North America (June 2013-May 2018: n = 5827) and Denmark (June 2007-May 2018: n = 8504). Participants were attempting to conceive without fertility treatment and had been attempting pregnancy for ≤6 menstrual cycles at enrolment.Participants/material, Setting, Methods: We collected information on season of each pregnancy attempt using last menstrual period dates over the study period. Pregnancy was reported on female bi-monthly follow-up questionnaires. We fit log-binomial models with trigonometric regression to examine periodic variation in fecundability. We accounted for seasonal variation in initiation of pregnancy attempts by including indicator variables for menstrual cycle of attempt in the regression models.Main Results and the Role Of Chance: Initiation of pregnancy attempts peaked in September, with stronger seasonality in North America than in Denmark (48 vs. 16% higher probability initiating attempts in September compared with March). After accounting for seasonal variation in initiation of pregnancy attempts, we observed modest seasonal variation in fecundability, with a peak in the late fall and early winter in both cohorts, but stronger peak/low ratios in North America (1.16; 95% confidence interval [CI]: 1.05, 1.28) than in Denmark (1.08; 95% CI: 1.00, 1.16). When we stratified the North American data by latitude, we observed the strongest seasonal variation in the southern USA (peak/low ratio of 1.45 [95% CI: 1.14, 1.84]), with peak fecundability in late November.Limitations, Reasons For Caution: We estimated menstrual cycle dates between follow-up questionnaires, which may have introduced exposure misclassification, particularly when women skipped follow-up questionnaires. We were unable to measure seasonally varying factors that may have influenced fecundability, including ambient temperature, vitamin D levels or infectious disease.Wider Implications Of the Findings: An understanding of how fecundability varies across seasons could help identify factors that can impair reproductive function. Neglecting to account for seasonal variation in initiation of pregnancy attempts could bias estimates of seasonal patterns in fecundability. This is the first preconception cohort study to examine seasonal variation in fecundability after accounting for seasonality in initiation of pregnancy attempts. Fecundability was highest in the fall and lowest in the spring, with stronger effects in southern latitudes of North America, suggesting that seasonal exposures may affect fecundity.Study Funding/competing Interest(s): This research was funded by the Eunice K. Shriver National Institute of Child Health and Human Development (R21-050264, R01-HD060680, R21-HD072326 and R01-HD086742) and the Danish Medical Research Council (271-07-0338). The authors declare no conflicts of interest. [ABSTRACT FROM AUTHOR]