When does a difference in human intrauterine growth of singletons conceived after IVF and embryo culture in two different culture media appear? Differences in fetal development after culture of embryos in one of two IVF media were apparent as early as the second trimester of pregnancy. Abnormal fetal growth patterns are a major risk factor for the development of chronic diseases in adult life. Previously, we have shown that the medium used for culturing embryos during the first few days after fertilization significantly affects the birthweight of the resulting human singletons. The exact onset of this growth difference was unknown. In this retrospective cohort study, all 294 singleton live births after fresh embryo transfer in the period July 2003 to December 2006 were included. These embryos originated from IVF treatments that were part of a previously described clinical trial. Embryos were allocated to culture in either Vitrolife or Cook commercially available sequential culture media. We analysed ultrasound examinations at 8 (n 290), 12 (n 83) and 20 weeks (n 206) gestation and used first-trimester serum markers [pregnancy-associated plasma protein-A (PAPP-A) and free -hCG]. Differences between study groups were tested by the Students t-test, (2) test or Fishers exact test, and linear multivariable regression analysis to adjust for possible confounders (for example, parity, gestational age at the time of ultrasound and fetal gender). A total of 294 singleton pregnancies (Vitrolife group n(VL) 168, Cook group: n(C) 126) from 294 couples were included. At 8 weeks gestation, there was no difference between crown-rump length-based and ovum retrieval-based gestational age (GA) (n(VL) 163, n(C) 122, adjusted mean difference, 0.04 days, P 0.84). A total of 83 women underwent first-trimester screening at 12 weeks gestation (n(VL) 45, n(C) 38). GA, nuchal translucency (multiples of median, MoM) and PAPP-A (MoM) did not differ between the study groups. Free -hCG (MoM) SEM differed significantly (1.55 0.19 in Vitrolife versus 1.06 0.10 in Cook; P 0.031, Students t-test). At 20 weeks gestation, a more advanced GA, reflecting an increased fetal growth, was seen at ultrasound examination in the Vitrolife group (n 115) when compared with the Cook group (n 91). After adjustment for confounding factors, both the difference between GA based on three biparietal diameter dating formulas minus the actual (ovum retrieval based) GA (adjusted mean difference 1.14 days (P 0.04), 1.14 days (P 0.04) and 1.36 days (P 0.048)), as well as head circumference (HC) and trans-cerebellar diameter (TCD) were significantly higher in the Vitrolife group (HCvl 177.3 mm, HCc 175.9 mm, adjusted mean difference 1.8, P 0.03; TCDvl 20.5 mm, TCDc 20.2 mm, adjusted mean difference 0.4, P 0.008). A first trimester (12 weeks) fetal screening was not yet offered routinely during the study period, therefore only 28 of women in our study participated in this elective screening programme. Although all sonographers were experienced and specially trained to perform these ultrasound examinations and were unaware of the randomization procedure, we cannot totally rule out possible intra- and inter-observer variability. Despite being indispensable in daily practice, sonographic weight formulas have a limited accuracy. According to the fetal origins hypothesis, many adult diseases originate in utero owing to adaptations made by the fetus to the environment it encounters. This study indicates that the embryonic environment is already important for fetal development. Therefore, our study emphasizes the need to investigate fetal growth patterns after assisted reproduction technologies and long-term health outcomes of IVF children, especially in relation to the culture medium used during the first few days of preimplantation development. Not applicable.