Methods: After IACUC approval, 10 time-dated pregnant Sprague-Dawley dams underwent prenatal exposure to 60mg/kg of all-trans retinoic acid on embryonic day 10 for the induction of fetal NTDs. Animals were killed before term for analyses, at which time amniotic fluid samples from all fetuses were procured prior to scrutiny for the presence, type, location, and size of NTDs. Samples from 61 fetuses could be analyzed. Each individual fresh amniotic sample (volume = 0.2-0.7mL) underwent quantitative multicolor flow cytometry for the detection of cells concomitantly expressing Nestin and Sox-2, primary markers of neural stem cells (aNSCs), as well as of cells concomitantly expressing CD 29 and CD44, markers of mesenchymal stem cells (aMSCs). Data were expressed as the ratio of positive cells to the overall number of cells. Statistical analysis included ANOVA and post-hoc Bonferroni adjusted comparisons, with significance set at p<0.05.
Results: A NTD was present in 57% (35/61) of the fetuses, namely either an isolated spina bifida (n=19), an isolated exencephaly (n=6), or a combination of the two (n=10). There was a statistically significant increase in the proportion of aNSCs in fetuses with spina bifida (6.78±1.87%), when compared with fetuses with exencephaly (0.64±0.23%), and with fetuses with both spina bifida and exencephaly (0.22±0.09%). Conversely, there was a statistically significant decrease in the proportion of aMSCs in fetuses with exencephaly, either isolated (1.09±0.42%), or in combination defects (2.37±0.63%), when compared with normal fetuses (8.83±1.38%). No such differences were noted in fetuses with isolated spina bifida. In fetuses with isolated exencephaly, there was a statistically significant inverse correlation between the proportion of aNSCs and defect size. This did not apply to fetuses with spina bifida, or combination defects.
Conclusion: The proportion of neural and mesenchymal stem cells in the amniotic fluid correlate with the type and size of select neural tube defects. Quantitative amniotic cell profiling may become a useful diagnostic tool in the prenatal management of these anomalies. Further analyzes of this concept in neural tube defects, as well as possibly other congenital anomalies, is warranted.