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STUDY QUESTION Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? SUMMARY ANSWER Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. WHAT IS KNOWN ALREADY In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. STUDY DESIGN, SIZE, DURATION Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006–2016). PARTICIPANTS/MATERIALS, SETTING, METHODS Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. MAIN RESULTS AND ROLE OF CHANCE Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (−819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: −1265.7, −373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: −21.8%; 95% CI: −37.1%, −2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: −23.8%; 95% CI: −39.9%, −3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. LIMITATIONS, REASONS FOR CAUTION Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Background: Frequent dairy consumption in childhood has been related to higher growth-hormone concentrations that may affect mammary gland and pubertal development.Objective: We evaluated the relation of dairy intake to breast composition at Tanner stage 4 and age at menarche.Design: A total of 515 Chilean girls are included in the Growth and Obesity Cohort Study. The subjects have been followed longitudinally since they were 3-4 y old (from 2006 to the present). Starting in 2013, diet was assessed every 6 mo via a 24-h recall. The breast fibroglandular volume (FGV) was measured with the use of dual-energy X-ray absorptiometry at Tanner stage 4. The date of menarche was reported every 6 mo. Our analysis included 290 girls with data on prospective diet and breast composition and 324 girls with data on prospective diet and age at menarche.Results: The mean ± SD breast FGV and percentage of fibroglandular volume (%FGV) (i.e., FGV divided by total breast volume times 100) at Tanner stage 4 was 81.7 ± 32.2 cm3 and 42.0% ± 16.7%, respectively. Only sweetened, artificially flavored milk-based drinks were associated with the %FGV with girls who consumed >125 g/d having a %FGV that was 4.5% (95% CI: 0.9%, 8.1%) higher than that of girls who consumed none (P-trend = 0.007). Yogurt intake was associated with a lower FGV. Specifically, girls who consumed >125 g yogurt/d had -10.2 cm3 (95% CI: -20.2, -0.3 cm3) less FGV than did girls who consumed no yogurt (P-trend = 0.03). The majority (90.7%) of girls in our cohort attained menarche before the data analyses with a mean ± SD age at menarche of 11.9 ± 0.7 y. In multivariable models, low-fat dairy, low-fat milk, and yogurt intakes were associated with a later age at menarche. In particular, girls who consumed >125 g yogurt/d had menarche, on average, 4.6 mo (95% CI: 1.9, 7.4 mo) later than girls who consumed no yogurt (P-trend = 0.01).Conclusion: More-frequent consumption of sweetened, artificially-flavored milk-based drinks is associated with a higher %FGV, whereas higher yogurt intake is associated with a lower FGV and delayed age at menarche in Chilean girls.

STUDY QUESTION What metabolic pathways and metabolites in the serum and follicular fluid are associated with peak estradiol levels and the number of mature oocytes? SUMMARY ANSWER In the serum metabolome, mostly fatty acid and amino acid pathways were associated with estradiol levels and mature oocytes while in the follicular fluid metabolome, mostly lipid, vitamin, and hormone pathways were associated with peak estradiol levels and mature oocytes. WHAT IS KNOWN ALREADY Metabolomics has identified several metabolic pathways and metabolites associated with infertility but limited data are available for ovarian stimulation outcomes. STUDY DESIGN, SIZE, DURATION A prospective cohort study of women undergoing IVF from 2009 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 125 women undergoing a fresh IVF cycle at a fertility clinic in the Northeast United States who provided a serum and follicular fluid sample. Untargeted metabolomics profiling was conducted using liquid chromatography with high-resolution mass spectrometry in two chromatography columns (C18 and hydrophilic interaction chromatography (HILIC)). The main ovarian stimulation outcomes were peak serum estradiol levels and number of mature oocytes. We utilized adjusted generalized linear regression models to identify significant metabolic features. Models were adjusted for age,BMI, initial infertility diagnosis, and ovarian stimulation protocol. We then conducted pathway analysis using mummichog and metabolite annotation using level-1 evidence. MAIN RESULTS AND ROLE OF CHANCE In the serum metabolome, 480 and 850 features were associated with peak estradiol levels in the C18 and HILIC columns, respectively. Additionally, 437 and 538 features were associated with mature oocytes in the C18 and HILIC columns, respectively. In the follicular fluid metabolome, 752 and 929 features were associated with peak estradiol levels in the C18 and HILIC columns, respectively, Additionally, 993 and 986 features were associated with mature oocytes in the C18 and HILIC columns, respectively. The most common pathways associated with peak estradiol included fatty acids (serum and follicular fluid), hormone (follicular fluid), and lipid pathways (follicular fluid). The most common pathways associated with the number of mature oocytes retrieved included amino acids (serum), fatty acids (serum and follicular fluid), hormone (follicular fluid), and vitamin pathways(follicular fluid). The vitamin D3 pathway had the strongest association with both ovarian stimulation outcomes in the follicularfluid. Four and nine metabolites were identified using level-1 evidence (validated identification) in the serum and follicular fluid metabolomes, respectively. LIMITATIONS, REASONS FOR CAUTION Our sample was majority White and highly educated and may not be generalizable to thewider population. Additionally, residual confounding is possible and the flushing medium used in the follicular fluid could have diluted our results. WIDER IMPLICATIONS OF THE FINDINGS The pathways and metabolites identified by our study provide novel insights into the biologicalmechanisms in the serum and follicular fluid that may underlie follicular and oocyte development, which could potentially be used to improve ovarian stimulation outcomes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the following grants from the National Institute of Environmental Health Sciences (P30-ES019776, R01-ES009718, R01-ES022955, P30-ES000002, R00-ES026648, and T32-ES012870), and National Institute of Diabetes and Digestive and Kidney Diseases (P30DK046200). The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]