ARTICLE ABSTRACTBackground: Prenatal estrogen exposure is thought to contribute to later life diseases such as breast cancer. However, few studies have directly measured prenatal estrogens and most have relied on proposed “markers” of estrogen exposure. We used a large population-based birth cohort to directly measure the relationship between prenatal estrogens and perinatal characteristics, including putative markers of estrogen exposure.Methods: Total estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4) were assayed by liquid chromatography/tandem mass spectrometry from archived mixed arterial and venous serum from 860 umbilical cord blood samples.Results: Values for all estrogens were strongly intercorrelated. Cord estrogen concentrations did not differ between males and females. Levels of all estrogens were reduced in twins and concentrations increased with gestational age. Neither E1 nor E2 was correlated with birth weight, but E3 and E4 levels correlated weakly, whereas onset of labor was associated with higher estrogen concentrations. E1 and E2 concentrations were not associated with preeclampsia in the current pregnancy, but E3 and E4 concentrations were lower in pregnancies complicated by preeclampsia and antepartum hemorrhage.Conclusions: Umbilical cord estrogen concentrations vary with gestational age, mode of delivery, pregnancy complications, and twinning, but not with infant sex. Putative markers of prenatal estrogen exposure, preeclampsia, and birth weight did not correlate with direct fetal measures of the most potent estrogen (E2) but were associated with weaker estrogens (E3 and E4). Twins had lower concentrations of all estrogens.Impact: This is the largest and best characterized dataset of prenatal estrogen concentrations, measured using highly accurate mass spectrometry/spectroscopy. These observations represent the new “gold standard” for umbilical cord estrogens, and will inform the interpretation of other datasets and the early life origins of health and disease. Cancer Epidemiol Biomarkers Prev; 23(6); 946–52. ©2014 AACR.