Each of the 102 samples was run on the same plate in triplicate. All mRNA levels are presented relative to the geometric mean of the three control genes. PHLDA2 expression levels were quantified by Real-time PCR (QPCR) against three reference genes: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), ubiquitin C (UBC) and topoisomerase

(TOP1) [28]. Summary data are presented as mean (SD) or median (inter-quartile range) depending on whether or not the data were normally distributed. Variables not normally distributed were transformed logarithmically. To investigate associations between PHLDA2 expression and parental body composition, fetal growth rates and infants body composition, Pearson’s and Spearman’s Erastin cell line AG 14699 correlation coefficients were calculated where appropriate. Differences in PHLDA2 expression levels between different categories of maternal lifestyle were tested by t-test or one-way

analysis of variance. Neonatal anthropometric measurements were adjusted for sex and gestational age and neonatal DXA measurements were adjusted for sex, gestational age and age at DXA. As there was a question regarding sex differences in mRNA levels between male and female placentas all mRNA data were adjusted for the sex of the baby [29]. Within group Z-scores were generated for femur length and abdominal circumference at 19 and 34 weeks. Royston models were fitted to fetal measurements to create z-scores for size and conditional growth rates [30]. To investigate whether there were sex differences in the relationship between PHLDA2 expression and the variables sex was included in regression analyses as appropriate and where an interaction was found data were analyzed separately by sex. Data were analyzed using Stata

version 11.0 (Statacorp, Texas, USA). In this study, PHLDA2 gene expression was examined in the placentas from 102 infants collected as part of the Southampton Women’s Survey. All were singleton, term deliveries (37 weeks gestation or greater). 53 of the infants were male and 49 were female. Descriptive statistics are given in Table 1. Within this cohort of 102 infants, no association was click here found between the placental expression level of PHLDA2 and birth weight, placental weight or other neonatal anthropometric or body composition measurements at birth ( Table 2). Longitudinal fetal ultrasound data was available at both 19 and 34 weeks for 58 fetuses within the cohort of 102 infants. There were no differences in the birth parameters between this subset of 58 pregnancies and the 43 pregnancies without full fetal scan data (data not shown). A lower 19–34 week femur length z-score change (linear growth velocity) was significantly associated with higher term placental PHLDA2 mRNA levels ( Table 3, Fig. 1).