Despite decades of research in the field of human reproduction, the mechanisms responsible for human parturition still remain elusive. The objective of this study was to describe the changes in the exosomal miRNA concentrations circulating in the maternal plasma between mothers delivering term and preterm neonates, across gestation using a longitudinal study design. This descriptive study identifies the miRNA content in exosomes present in maternal plasma of term and preterm birth (PTB) (n = 20 and n = 10 per each gestational period, respectively) across gestation (i.e., first, second, and third trimesters and at the time of delivery). Changes in exosomal miRNA signature in maternal plasma during term and preterm gestation were determined using the NextSeq 500 high-output 75 cycles sequencing platform. A total of 167 and 153 miRNAs were found to significantly change (P < 0.05) as a function of the gestational age across term and PTB pregnancies, respectively. Interestingly, a comparison analysis between the exosomal miRNA profile between term and PTB reveals a total of 173 miRNAs that significantly change (P < 0.05) across gestation. Specific trends of changes (i.e., increase, decrease, and both) as a function of the gestational age were also identified. The bioinformatics analyses establish that the differences in the miRNA profile are targeting signaling pathways associated with TGF-β signaling, p53, and glucocorticoid receptor signaling, respectively. These data suggest that the miRNA content of circulating exosomes in maternal blood might represent a biomolecular “fingerprint” of the progression of pregnancy.

Our objective was to determine the amniotic fluid–derived exosomal proteomic profile in patients who had spontaneous preterm birth (PTB) or preterm premature rupture of membranes (pPROM) compared with those who delivered at term. A cross-sectional study of a retrospective cohort was used to quantify and determine the protein content of exosomes present in amniotic fluid, in PTB or pPROM, and normal term labor (TL) or term not in labor (TNIL) pregnancies. Exosomes were isolated by differential centrifugation and quantified using nanocrystals (Qdot) coupled to CD63 and placental alkaline phosphatase (PLAP) by fluorescence nanoparticle tracking analysis. The exosomal proteomic profile was identified by liquid chromatography–tandem mass spectrometry, and a small ion library was constructed to quantify the proteomic data by Sequential Window Acquisition of All Theoretical analysis. Ingenuity Pathway Analysis determined canonical pathways and biofunctions associated with dysregulated proteins. Amniotic fluid exosomes have similar shape and quantity regardless of the conditions; however, the PLAP/CD63 ratios for TL, PTB, and pPROM were significantly higher (∼3.8-, ∼4.4-, and ∼3.5-fold, respectively) compared with TNIL. The PLAP/CD63 ratio was also significantly higher (∼1.3-fold) in PTB compared with pPROM. Biological functions primarily indicated nonspecific inflammatory response regardless of condition, but unique profiles were also identified in cases (PTB and pPROM) compared with term. Amniotic fluid exosomes provide information specific to normal and abnormal parturition. Inflammatory marker enrichment and its uniqueness in term and preterm pregnancies support the value of exosomes in determining underlying physiology associated with term and preterm parturition.

Endocrine factors and signals of fetal organ maturation are reported determinants of birth timing. To test the hypothesis that paracrine signaling by exosomes are key regulators of parturition, maternal plasma exosomes from CD-1 mice were isolated and characterized throughout gestation and the biological pathways associated with differentially-expressed cargo proteins were determined. Results indicate that the shape and size of exosomes remained constant throughout gestation; however, a progressive increase in the quantity of exosomes carrying inflammatory mediators was observed from gestation day (E)5 to E19. In addition, the effects of late-gestation (E18) plasma exosomes derived from feto-maternal uterine tissues on parturition was determined. Intraperitoneal injection of E18 exosomes into E15 mice localized in maternal reproductive tract tissues and in intrauterine fetal compartments. Compared to controls that delivered at term, preterm birth occurred in exosome-treated mice on E18 and was preceded by increased inflammatory mediators on E17 in the cervix, uterus, and fetal membranes but not in the placenta. This effect was not observed in mice injected with early-gestation (E9) exosomes. This study provides evidence that exosomes function as paracrine mediators of labor and delivery.

At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i) exosomes act as carriers of signals in utero-placental compartments and ii) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05). Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined AEC exosome characteristics and their cargo reflected the physiologic status of the cell of origin and suggests that AEC-derived exosomal p38 MAPK plays a major role in determining the fate of pregnancy. Understanding the propagation of fetal signals and their mechanisms in normal term pregnancies can provide insights into pathologic activation of such signals associated with spontaneous preterm parturitions.