Sexually dimorphic patterns in maternal circulating microRNAs in pregnancies complicated by fetal growth restriction

Abstract

Background: Current methods fail to accurately predict women at greatest risk of developing fetal growth restriction (FGR) or related adverse outcomes, including stillbirth. Sexual dimorphism in these adverse pregnancy outcomes is well documented as are sex-specific differences in gene and protein expression in the placenta. Circulating maternal serum microRNAs (miRNAs) offer potential as biomarkers that may also be informative of underlying pathology. We hypothesised that FGR would be associated with an altered miRNA profile and would differ depending on fetal sex.

Methods: miRNA expression profiles were assessed in maternal serum (> 36 weeks' gestation) from women delivering a severely FGR infant (defined as an individualised birthweight centile (IBC) < 3rd) and matched control participants (AGA; IBC = 20-80th), using miRNA arrays. qPCR was performed using specific miRNA primers in an expanded cohort of patients with IBC < 5th (n = 15 males, n = 16 females/group). Maternal serum human placental lactogen (hPL) was used as a proxy to determine if serum miRNAs were related to placental dysfunction. In silico analyses were performed to predict the potential functions of altered miRNAs.

Results: Initial analyses revealed 11 miRNAs were altered in maternal serum from FGR pregnancies. In silico analyses revealed all 11 altered miRNAs were located in a network of genes that regulate placental function. Subsequent analysis demonstrated four miRNAs showed sexually dimorphic patterns. miR-28-5p was reduced in FGR pregnancies (p < 0.01) only when there was a female offspring and miR-301a-3p was only reduced in FGR pregnancies with a male fetus (p < 0.05). miR-454-3p was decreased in FGR pregnancies (p < 0.05) regardless of fetal sex but was only positively correlated to hPL when the fetus was female. Conversely, miR-29c-3p was correlated to maternal hPL only when the fetus was male. Target genes for sexually dimorphic miRNAs reveal potential functional roles in the placenta including angiogenesis, placental growth, nutrient transport and apoptosis.

Conclusions: These studies have identified sexually dimorphic patterns for miRNAs in maternal serum in FGR. These miRNAs may have potential as non-invasive biomarkers for FGR and associated placental dysfunction. Further studies to determine if these miRNAs have potential functional roles in the placenta may provide greater understanding of the pathogenesis of placental dysfunction and the differing susceptibility of male and female fetuses to adverse in utero conditions.

Keywords: Biomarker; FGR; Placenta; Placental dysfunction; Pregnancy; Serum; Sexual dimorphism; Stillbirth; miRNA.

Fig. 1

Volcano plot of differentially expressed microRNAs in maternal serum of FGR versus normal pregnancies. Log2 (fold-change) values for detected miRNAs were plotted against − log10 of the unadjusted p-value. Significantly up-regulated genes shown in red, significantly down-regulated genes shown in blue (p < 0.05). Dotted line represents p = 0.05

Fig. 2

Interacting mRNA networks of all altered miRNAs. A, B A list of all 11 miRNAs altered in FGR was uploaded to miRNET and A networks of interacting genes (pink circles) associated with altered miRNAs (blue square) were determined. B Functional enrichment analysis of network genes was performed using Reactome, GO and KEGG. Key functional effects associated with the network were found in pathways associated with cellular response to stress (red; 77 node genes; Adj p = 4.9 × 10^–6), cell proliferation (yellow; 111 node genes; Adj p value = 0.00012) and vascular development (turquoise; 97 node genes; Adj p value = 0.00037)

Fig. 3

Functional enrichment analysis of miRNAs and predicted target genes. A list of the 11 miRNAs altered in FGR was uploaded to Ingenuity Pathway Analysis and A predicted diseases and disorders and B predicted molecular and cellular functions and functions associated with physiological system development were determined from miRNAs and their experimentally validated and predicted targets. Orange line represents significance threshold value of − log p value = 1.5

Fig. 4

Q-PCR validation of candidate microRNAs in maternal serum in uncomplicated and FGR pregnancies. qPCR was performed on individual microRNAs isolated from maternal serum of women with appropriately grown (AGA; IBC 20–80th) or growth-restricted (FGR; IBC < 5th) infants using specific primers for A miR-28-5p, B miR-29c-3p, C miR-301a-3p, D miR-378a-3p, E miR-409-3p, F miR-454-3p and G miR-526b-5p. Data were normalised to 2 reference miRNAs and expressed as 2^−ΔCt. Individual data points shown (n = 18–31/group); line represents the median. *p < 0.05 Mann–Whitney U test

Fig. 5

Effect of fetal sex on microRNA levels in maternal serum in uncomplicated and FGR pregnancies. qPCR was performed on individual microRNAs isolated from maternal serum of women with appropriately grown (AGA; IBC 20–80th) or growth-restricted (FGR; IBC < 5th) infants using specific primers for A miR-28-5p, B miR-29c-3p, C miR-301a-3p, D miR-378a-3p, E miR-409-3p, F miR-454-3p and G miR-526b-5p. Data were normalised to 2 reference miRNAs and expressed as 2^−ΔCt. Data were stratified into male (n = 15/group) and female (n = 16/group). Individual data points shown, line represents the median. 2-way ANOVA with linear step-up multiple comparison test. Interaction (F) between the groups was considered significant when p < 0.05. Kruskal–Wallis followed by Dunn’s post hoc analysis was used to determine difference between individual groups; *p < 0.05, **p < 0.01

Fig. 6

Relationship between circulating microRNAs and human placental lactogen (hPL) in maternal serum. Correlation between serum miRNA expression measured by qPCR and hPL a hormone marker of placental dysfunction detected by ELISA for A miR-28-5p, B miR-29c-3p, C miR-301a-3p, D miR-378a-3p, E miR-409-3p, F miR-454-3p and G miR-526b-5p. Positive correlations with hPL were detected for B miR-29c-3p (r² 0.3519, p < 0.05) and F miR-454-3p (r² 0.449, p < 0.001). Individual data points shown (n = 34–56), line represents the median. Spearman rank correlations

Fig. 7

Effect of fetal sex on correlation between microRNAs and hPL in maternal serum. Correlation between maternal serum miRNA expression from male or female pregnancies measured by qPCR and hPL a hormone marker of placental dysfunction detected by ELISA for A miR-28-5p, B miR-29c-3p, C miR-301a-3p, D miR-378a-3p, E miR-409-3p, F miR-454-3p and G miR-526b-5p. Positive correlations with hPL were detected for B miR-29c-3p (r² (r = 0.467, p =  < 0.05) in males only and F miR-454-3p (r² r = 0.511, p < 0.010) in females only. Significant difference detected in intercept/elevation for F miR-454-3p (p < 0.01, linear regression). Individual data points shown (n = 17–27 males, n = 18–32 females/group), line represents the median. Spearman rank correlations

Fig. 8

Venn diagram showing the overlapping microRNAs identified from studies comparing maternal serum from uncomplicated and FGR pregnancies. Current study compared with Mouillet et al. [22], Whitehead 2013 [23] and Hromadnikova 2019 [24]