P2D387: Consumption of Native Amniotic Fluid Mesenchymal Stem Cells in a Model of Intrauterine Growth Restriction (IUGR): Further Biological Basis for Transamniotic Stem Cell Therapy (TRASCET) as a Potential Novel Treatment for This Disease

Background: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) derived from amniotic fluid or placenta has been shown experimentally to minimize placental inflammation in the setting of intrauterine growth restriction (IUGR) resulting in various benefits to the fetus. Given that MSCs are naturally occurring in the amniotic fluid, we sought to determine the impact of IUGR on the cellularity of that fluid, with a particular focus on the MSC population, in the hypoxic model of that disease.

Methods: After IACUC approval, four time-dated pregnant Sprague Dawley dams were exposed to alternating 12-hour hypoxia (10.5% O2) cycles in late gestation from gestational day 15 (E15) until term (E21; IUGR group). Additional three time-dated pregnant Sprague Dawley dams not exposed to hypoxia served as normal controls. At term, all dams underwent procurement of all recoverable amniotic fluid from each amniotic cavity (total n=88, equally divided between the two groups) under direct visualization via a laparotomy. This was followed by gross morphometry of each fetus and placenta. Fresh amniotic fluid samples underwent quantitative multicolor flow cytometry for the detection of cells expressing 4′,6-diamidino-2-phenylindole (DAPI, a marker of live viable cells), as well as of cells concomitantly expressing DAPI, CD29, and CD44 (the latter two markers of MSCs), while being also negative for CD45, utilizing standard gating strategies (figure). Statistical analysis was performed using Wilcoxon rank-sum tests and median regression. Results were reported as median (interquartile range) with significance set at p< 0.05.

Results: Placental efficiency (defined as fetal/placental weight ratio) was significant lower in the IUGR group compared to controls (p < 0.001), confirming reproduction of the disease model. Three amniotic fluid samples had to be excluded due to contamination (final IUGR n=42 and control n=43). There was no significant difference in the median individual amniotic fluid volume between the groups (p < 0.792). Compared to controls, IUGR fetuses had statistically significant lower densities of both total live cells as well as of live MSCs in the amniotic fluid (both p< 0.001). There was no significant difference in the ratio of MSCs/live cells between IUGR and control fetuses (p < 0.512). None of these analyses were affected by accounting or not for nesting of fetuses within the same dam. There was a significant decrease in the total number of MSCs in the IUGR group vs. controls in a comparison without maternal nesting (p < 0.001) and a non-significant trend lower in a comparison with nesting (p=0.113).

Conclusion: Native amniotic fluid mesenchymal stem cells appear to be consumed in the setting of IUGR. This provides further biological basis for transamniotic stem cell therapy as a potential novel treatment for this disease while also suggesting that targeted amniotic cell profiling could become a useful diagnostic adjunct in patient stratification and management.