In this study, we conduct a large-scale profiling of maternal circulating miRNAs at four stages during and after healthy pregnancies. By comparing with the corresponding expression patterns in healthy non-pregnant women, our findings suggest the existence of subtle variations in miRNA profiles involving distinct but small subpopulations of transcripts potentially associated to specific pregnancy-related tissues and fluids at all stages during gestation (from the first through to third trimester) and after birth.
We identify 1,449 miRNAs in maternal circulation, which represents one of the largest sets of mature miRNAs reported in circulation , particularly during pregnancy . While no gross differences in the global profiles were evident neither between pregnant and non-pregnant women, nor between pregnancy stages, direct differential expression analysis revealed significant differences in a relatively small number of individual miRNAs, many of which have also been described in other studies of plasma miRNAs during pregnancy [36, 37].
Limited changes in miRNA expression have indeed been reported in a variety of body fluids in numerous conditions [5, 6]. Based on these observations, and our own findings in this study, we hypothesized that changes in c-miRNAs during pregnancy, if any, are more likely to be found at the level of subtle collective changes in defined miRNA subpopulations.
Along this line, we started by looking at subsets of miRNAs known to be associated with specific events of pregnancy. In this regard, miRNAs members belonging to the C14MC and C19MC families have attracted considerable attention due to their known involvement in placental function and their potential as biomarkers of placental health and embryo development [13, 22, 38–41]. Our finding of highly significant changes in the collective expression of these two signatures during all three stages of pregnancy, but not after birth, further supports the notion of a key involvement of these c-miRNA populations in healthy pregnancy.
Significantly increased expression of C14MC and C19MC miRNAs during pregnancy, in placental tissue, have been documented [20, 42], and it has been suggested that these transcripts are either actively or passively transported from the placental compartment to the maternal plasma during the natural progression of pregnancy [22, 43, 44]. This notion, however, is in direct conflict with our finding that C14MC transcripts were collectively down-regulated in maternal circulation during pregnancy; a pattern of expression that opposed what has been described in placenta. This observation suggests instead that, far from being passively transported from the placental compartment, C14MC miRNAs are selectively excluded of maternal circulation and probably maintained inside of the intrauterine compartment, suggesting the existence of selective uptake mechanisms exhibited by the placenta and/or their active down regulation in maternal tissues contributing to c-miRNAs. Consistent with the notion of an active removal or down regulation of placental miRNAs, transcripts most highly expressed in placenta were also found collectively reduced throughout pregnancy but not after birth. C19MC miRNAs, on the other hand, displayed a collective increased expression during pregnancy, suggesting different mechanisms regulating the presence of C19MC and C14MC miRNAs in maternal plasma during the progression of normal pregnancy [14, 45].
Following the same approach, we examined the expression of c-miRNAs associated with other specific pregnancy related compartments, such as those present in amniotic fluid, umbilical cord serum and early breast milk which are related to the normal progression of pregnancy . These reproductive compartments are involved in a wide range of adaptations to events such as implantation, maintenance, labor, lactation [16, 46], or even events associated with the control of inflammation and tolerance at the maternal-fetal interface [47, 48]. Our findings demonstrated a down regulation of the collective expression of these miRNAs during all stages of pregnancy, again suggesting the possibility that the placenta or other reproductive tissues may regulate and restrict the passage of these transcripts from the intrauterine environment to the maternal circulation, or alternatively suggesting the active down regulation of these transcripts by other maternal tissues.
By looking at fluctuations in the proportions of up and down regulated miRNAs, between women giving birth to female and male babies, we document, to the best of our knowledge for the first time, highly significant biases in the expected proportions of differentially expressed c-miRNAs as a function of fetal sex at all stages during pregnancy, as well as after birth. These findings suggest possible existence of specific molecular signatures associated to fetal sex, as early as the first trimester, and opens the possibility of detecting fetal sex much earlier than it is currently possible using other methods [49, 50] and solely based on patterns of c-miRNAs. In this regard, no differential expression of miRNAs has been detected between male and female derived umbilical cord after a normal pregnancy. However, alterations in miRNAs between male and female fetuses have been linked to perinatal complications [45, 51].
Finally, by combining our longitudinal transcriptome profiling with ultrasonographic measurements of fetal growth derived from the exact same women and conducted at the exact same stages, we identify a circulating miRNA signature of fetal growth, occurring in maternal plasma, during normal pregnancy. Our randomization analyses demonstrate that the observed association between this signature and fetal growth was significantly higher than expected by chance, and that it was not related to changes in maternal body weight. To the best of our knowledge, this is the first report of a miRNA signature of fetal growth present in maternal plasma during the normal course of pregnancy.
The functional significance of the observed collective changes in c-miRNA subpopulations remains unclear. It is also not clear whether these changes reflect systemic maternal demands or whether they are secondary to physiological adaptations taking place in other organs or whether this reflects a new, as yet, undescribed placental function (i.e., selective take of maternal circulating transcripts). Future additional work will be needed to identify the functional roles of these changes as, in general, the functional significance of extracellular circulating miRNAs in other biofluids remains unclear.