O-163 The influence of aquaporin 3 on dynamics of cavitation, implantation and re-expansion after vitrification/warming

Abstract

Abstract Study question We wished to investigate whether aquaporin 3 (AQP3) is involved in trophectoderm-related events: cavity formation and maintenance, implantation and re-expansion after the vitrification/warming process. Summary answer AQP3 regulates blastocoel formation and expansion. It is also required for implantation (as assessed by in vitro implantation assay) and maintaining developmental potential after vitrification. What is known already During blastocoel expansion blastocyst pulsates, i.e. contracts and re-expands in an oscillatory way. Recent studies utilizing a mouse model have shown, that the dynamics of cavitation and biomechanical properties of the trophectoderm are intertwined, and our results suggest, that it may, to a certain extent, reflect the developmental capabilities of blastocysts. Moreover, the dynamics of blastocyst re-expansion after vitrification/warming is also suggested to be a promising biomarker of embryo quality. As AQP3 is involved in water influx and transportation of small molecules (such as cryoprotectants) through the epithelial layer, it is a good candidate for a regulator of blastocoel expansion. Study design, size, duration We analyzed 142 mouse blastocysts to correlate the dynamics of their cavitation with their ability to implant in vitro (an outgrowth assay). We investigated the expression of Aqp3 throughout the preimplantation development. We tested how inhibition of AQP3 affects the dynamics of cavitation (n = 37; control n = 118) and formation of outgrowths (n = 77; control n = 81). We vitrified blastocysts with a limited activity of AQP3 (n = 41; control n = 38) and investigated their ability to re-expand and implant in vitro. Participants/materials, setting, methods The dynamics of cavitation was assessed by time-lapse imaging. After filming, mouse blastocysts were cultured for additional 4 days to test their ability to form outgrowths. Inhibition of AQP3 was performed by adding 100µM DFP00173 (a selective AQP3 inhibitor) to the culture medium. AQP3-inhibited blastocysts were analyzed for the dynamics of cavitation and outgrowth formation. Additionally, embryos with a limited activity of AQP3 were vitrified, warmed and then the outgrowth assay was performed. Main results and the role of chance Blastocysts unable to implant in vitro properly (i.e. forming too small outgrowths or not forming outgrowths at all) contract longer and more frequently than embryos that form correct outgrowths. Moreover, in such blastocysts, we observed shorter and weaker re-expansion phases. As AQP3 may be one of the regulators of cavitation, we followed its expression in embryos and showed that Aqp3 mRNA level increases from the 2-cell to the morula stage. AQP3 protein can be found in the cytoplasm during cleavage and translocates to basolateral membranes in trophectodermal cells in blastocysts. We revealed that inhibition of AQP3 resulted in delayed cavity formation. Moreover, inhibition of AQP3 significantly weakened the blastocyst’s ability to (re-)expand and also extended the duration and decreased frequency of the blastocoel contractions. Importantly, changes in the duration of contractions and amplitude of re-expansions were similar to those observed in blastocysts not able to implant in vitro properly. This accords with our observation that blastocysts with a limited activity of AQP3 are mostly unable to create outgrowths. Finally, we proved that inhibition of AQP3 strongly weakened re-expansion of the cavity after vitrification/warming. Furthermore, such blastocysts had a significantly lower ability to implant in vitro after vitrification. Limitations, reasons for caution We examined the role of AQP3 in blastocoel (re)-expansion and the relationship between cavitation dynamics and embryo’s ability to implant only in mice, so additional studies on other mammalian species, including humans, are needed. Moreover, our in vitro implantation assay does not fully reflect the complexity of implantation in vivo. Wider implications of the findings Our data indicate, that AQP3 regulates the dynamics of blastocoel expansion in mouse embryos. Impaired dynamics of cavitation, unsuccessful implantation or vitrification failures might be a result of decreased AQP3 activity, possibly not only in a mouse but also in other mammalian species, including humans. Trial registration number not applicable