Background It is published that after 4-6 hours in explant culture, the human syncytiotrophoblast is unable to exclude viability stains like propidium iodide (PI). This is interpreted as the death of the syncytiotrophoblast, which is subsequently shed and replaced by a new syncytium. We confirm that the syncytiotrophoblast does not exclude non-viability stains after 24 hours but provide evidence that this is not due to the death of the syncytiotrophoblast.
Methods Replicate first trimester placental explants, obtained with written informed consent, were incubated for up to 24 hours. The explants were then; 1)stained with PI, or 2)mRNA was extracted and connexins and pannexins quantified by qRT-PCR, or 3)stained by immunohistochemistry with antibodies reactive with pannexin 1 or connexin 43.
Results At 0 or 4 hours, explants excluded PI but at 24 hours the syncytiotrophoblast nuclei stained strongly for PI. Quantitative RT-PCR indicated multiple connexins as well as, pannexins-2 and -3 were expressed at low levels in the explants. The levels of connexin-43 (p=0.004) and pannexin-1 (p=0.016) mRNA increased significantly between 0 and 24 hours of culture. Immunohistochemistry confirmed the increased expression of connexin-43 and pannexin-1 at 24 hours, and showed increased expression of pannexin-1 at four hours. Incubation with a pannexin-1 channel blocker, resulted in the exclusion of PI from the syncytiotrophoblast.
Discussion We show the uptake of PI by the syncytiotrophoblast of placental explants is mediated by pannexin-1 hemi-channels. Since the syncytiotrophoblast is a multinucleated cell, covering the entire placenta, we believe that the uptake of PI is not a marker of cell death but rather, a reflection of leakiness. In normal pregnancy there are extensive areas of damage to the syncytiotrophoblast which also presumably induce leakiness. The extent to which this leakiness disrupts the function of the syncytiotrophoblast is not yet clear.