Fetal sex modulates placental microRNA expression, potential microRNA-mRNA interactions, and levels of amino acid transporter expression and substrates: INFAT study subpopulation analysis of n-3 LCPUFA intervention during pregnancy and associations with offspring body composition

Abstract

Background: Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition.

Results: We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks' gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age.

Conclusions: Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.

Keywords: Adipose tissue; Amino acid transport; Fetal programming; MicroRNA; N-3 long-chain polyunsaturated fatty acids; Placenta; Sexual dimorphism.

Fig. 1

Flowchart of analyses. a, b Maternal and umbilical cord blood samples, placentas and offspring anthropometric parameters from mother-offspring pairs of the subpopulation control (n = 20) and n-3 LCPUFA intervention group (n = 21) of the INFAT cohort, as well as respective placental mRNA transcriptome datasets from our previous study [10] were used in this study for molecular analyses (microRNA, mRNA, protein, amino acids), bioinformatics and association analyses as depicted (b - h)

Fig. 2

Venn diagram of placental microRNAs identified by microRNA profiling. Venn diagram showing the numbers of detected and differentially expressed microRNAs identified in pooled probes from female placentas of each group (Con, control group; N3, intervention group) by qPCR TaqMan low density human microRNA assay. Each pool (n = 1) representing 3 female placentas from each group was screened for 667 human microRNAs. Venn diagram represents the number of separately detected microRNAs in each group and the number of different microRNAs in both groups together (numbers outside of the circles). Total number of different microRNAs are separated in (1) microRNAs detected in one group but not in the other group (numbers exclusively inside the group-specific circle but outside of the intersection and (2) microRNAs common in both groups (numbers in the circle intersection). Within the group of microRNAs common in both groups, the numbers for microRNAs showing differential expression levels between the groups are depicted with upwards and downwards arrow for upregulation and downregulation of microRNAs of the intervention versus control group, respectively. Percent values for respective numbers of detected microRNAs related to 667 microRNAs (total number of screened microRNAs) for each group are shown in parentheses

Fig. 3

Placental LAT1 protein expression. Western blot analysis of placental LAT1 and GAPDH protein levels from female (Con-F) and male (Con-M) offspring of the control group and female (N3-F) and male (N3-M) offspring of the n-3 LCPUFA intervention group are shown (for total blots see Additional file 3: Figure S3). GAPDH was used for protein normalization. Relative optical densities were calculated with the mean for the respective groups and relative expression levels and effects for group, sex, and interaction of group and sex are shown in Table 3