Absolute and relative quantification of placental specific microRNAs in maternal circulation in placental insufficiency related complications

Abstract
The primary goal of our study has been to identify placental specific microRNAs present in maternal plasma differentiating between normal pregnancies and non-pregnant individuals. The selection of appropriate pregnancy associated microRNAs with the diagnostical potential was based on following criteria: (1) detection rate of 100 % in full-term placentas, (2) detection rate of above 67 % in maternal plasma throughout gestation (at least 4 positive wells out of 6 tested wells) and (3) detection rate of 0 % in whole peripheral blood and plasma samples of non-pregnant individuals. Initially, we tested microRNAs (miR-34c, miR-372, miR-135b and miR-518b) which had been previously identified as pregnancy-associated miRNAs. Additionally we selected 16 other highly specific placental miRNAs (miR-512-5p, miR-515-5p, miR-224, miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d, miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526a and miR-526b) from the miRNAMap database. Seven microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) were newly identified as pregnancy associated with diagnostic potential. Further, we examined if extracellular placental specific microRNAs (miR-520a*, miR-520h, miR-525 and miR-526a) can differentiate pregnancies with placental insufficiency related complications from normal ones. Absolute and relative quantification of placental specific microRNAs (miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 50 normal pregnancies, 32 complicated pregnancies (21 preeclampsia with or without intrauterine growth retardation and 11 IUGR) and 3 pregnancies at various gestational stages who later developed preeclampsia w/o IUGR and/or IUGR using real-time PCR and the comparative Ct method relative to ubiquitous miR-16. Both quantification approaches revealed significant increase of extracellular placental specific microRNAs levels over time in normally progressing pregnancies, however did not differentiate between normal and complicated pregnancies at the time of preeclampsia and/or IUGR onset. Nevertheless, significant elevation of extracellular microRNAs was observed during early gestation (within 12th to 16th weeks) in pregnancies with later onset of preeclampsia and/or IUGR. To avoid instability of extracellular microRNAs in maternal plasma, prenatal monitoring should be performed using only the samples with the storage life below two months. Early gestation extracellular microRNAs screening may differentiate between normal pregnancies and those who will later develop placental insufficiency related complications.
Acknowledgements: This work was supported by grant projects MSM 0021620806 and GAUK 260707/SVV/2010.