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10: Fetuin-mediated aggregation of amniotic fluid proteins into calcifying nanoparticles (CNP) and preterm premature rupture of membranes (PPROM)

      Objective

      Disturbances in mineral-protein composition of biological fluids result in fetuin-mediated nucleation of hydroxyapatite (insoluble CaP), depletion of essential proteins and formation of CNPs. This process is analogous to the formation of prions and amyloid and results in deposition of mature CNPs in surrounding tissues and cytotoxicity. Here, we explored potential mechanisms through which amniotic fluid (AF) CNP formation may lead to PPROM.

      Study Design

      The kinetics of CNP formation in AF was studied in a long-term culture method of cell-free sterile-filtered amniocentesis samples from 10 preterm women (GA: 27±2 weeks). Known CNP constituents [fetuin, ionized Ca, and inorganic phosphate (Pi)] were assessed in immature (soluble) and mature (insoluble) CNP aggregates. IL-6 was explored by ELISA as an endogenous AF protein trapped during the process of CNP formation. Biological activity of immature and mature CNPs was evaluated in an amniochorion explant system by conventional histological criteria of necrosis. CNP protein co-aggregation was imaged by immuno-electron microscopy. Glucose metabolism was studied via a kinetic assay. For proof of concept, we examined in-vivo the levels of soluble fetuin and IL-6 in AF of 50 women with (n=27) and without (n=23) PPROM. All delivered preterm in the absence of infection.

      Results

      1) Compared to uncultured AF, soluble fetuin, Ca, Pi and IL-6 levels were significantly decreased by CNP formation (all P<.05); 2) Double immuno-gold staining of AF CNPs demonstrated co-aggregation of fetuin and IL-6 in a non-random lattice; 3) Fetal membranes exposed to immature CNPs display a significantly faster rate of glucose metabolism (P<.001) and necrosis; 4) AF of women with PPROM showed significantly lower IL-6 (P<.03) and fetuin (P<.01) levels compared to intact membranes group, with a significant direct correlation between fetuin and IL-6 (R=.413, P=.003).

      Conclusion

      This study supports the premise that CNP formation alters the protein composition of AF and disrupts the function of fetal membranes leading to PPROM and preterm birth.