P0715 - Biomarkers from minimally invasive sampling that reflects the placental immune environment

Placental miRNAs, such as certain placenta-specific members of the human chromosome 19 miRNA cluster (C19MC), play an important role in maintaining a balanced immune environment in utero to promote a healthy pregnancy. Additionally, placental cells selectively secrete miRNAs to the extracellular space within circulating extracellular vesicles (EVs), which act as vehicles to transport miRNA to distant tissues. The objective of this study was to identify if certain C19MC members are contained in maternal saliva EVs and plasma, and whether the levels of these miRNA correlate with their levels in the placenta. We hypothesize that saliva EVs will contain the placenta specific miR-519c, and that its levels may reflect the intrauterine environment.

Maternal plasma, amniotic fluid, saliva, and placenta tissue were collected simultaneously during term normal cesarean section deliveries (n=8). Total miRNA was extracted from placenta tissue and plasma, and EV miRNA was extracted from saliva (following EV isolation). RNA was then reverse-transcribed using the qScript microRNA cDNA Synthesis kit and miRNA expression was determined by Real-Time PCR using the PerfeCta SYBR Green kit. The C19MC members observed were miR-519c (an anti-inflammatory miRNA implicated in immune tolerance), miR-517a (a highly expressed C19MC member that has been implicated in preeclampsia), and miR-1323 (a highly expressed C19MC member). Correlations of miRNA levels between placental tissue and the studied biofluids were determined using the nonparametric Spearman’s correlation coefficient with a p-value <0.05 required for significance. Additionally, preliminary regression models were created to observe the association between miR-519c expression in placenta versus plasma, placenta versus saliva EVs, and plasma versus saliva EVs.

We observed a statistically significant correlation between miRNA levels in placenta and plasma for miR-519c (Spearman R=0.7832, p=0.0038) and miR-1323 (Spearman R=0.7842, p=0.0096). The correlation between miRNA levels in placenta and plasma for miR-517a failed to meet statistical significance (Spearman R=0.1215, p=0.1215). Interestingly, our preliminary regression modeling also showed significant associations between the levels of miR-519c in placenta versus plasma (R-square=0.5888, p=0.0036), placenta versus saliva EVs (R-square=0.4294, p=0.0207), and plasma versus saliva EVs (R-square=0.3683, p=0.0364); however, this association was not seen between the placenta and amniotic fluid samples (R-square=0.1291, p=0.2513).

The results demonstrate that the expressions of the C19MC members miR-519c and miR-1323 in placenta and maternal plasma are significantly correlated. Furthermore, based on preliminary regression modeling, miR-519c expression was associated between the placenta and maternal plasma samples, as well as between the placenta and saliva EVs samples. To our knowledge, these results are the first to correlate placenta-specific miRNA levels between placenta and different maternal biofluids collected simultaneously during delivery. This is an important step in determining whether specific C19MC members circulating in EVs, such as saliva EVs, can be utilized as biomarkers for pathologic pregnancy conditions.