Early pregnancy prediction of spontaneous preterm birth before 32 completed weeks of pregnancy using plasma RNA: transcriptome discovery and initial validation of an RNA panel of markers

Abstract

Objective To compare the second‐trimester plasma cell‐free (PCF) transcriptome of women who delivered at term with that of women with spontaneous preterm birth (sPTB) at or before 32 weeks of gestation and identify/validate PCF RNA markers present by 16 weeks of gestation. Design Prospective case–control study. Setting Academic tertiary care centre. Population Pregnant women with known outcomes prospectively sampled. Methods PCF RNAs extracted from women at 22–24 weeks of gestation (five sPTB up to 32 weeks and five at term) were hybridised to gene expression arrays. Differentially regulated RNAs for sPTB up to 32 weeks were initially selected based on P value compared with control (P < 0.01) and fold change (≥1.5×). Potential markers were then reordered by narrowness of distribution. Final marker selection was made by searching the Metacore™ database to determine whether the PCF RNAs interacted with a reported set of myometrial Preterm Initiator genes. RNAs were confirmed by quantitative reverse transcription polymerase chain reaction and tested in a second group of 40 women: 20 with sPTB up to 32 weeks (mean gestation 26.5 weeks, standard deviation ±2.6 weeks), 20 with spontaneous term delivery (40.1 ± 0.9 weeks) sampled at 16–19+5 weeks of gestation. Main outcome measure Identification of PCF RNAs predictive of sPTB up to 32 weeks. Results Two hundred and ninety‐seven PCR RNAs were differentially expressed in sPTB up to 32 weeks of gestation. Further selection retained 99 RNAs (86 mRNAs and 13 microRNAs) and five of these interacted in silica with seven Preterm Initiator genes. Four of five RNAs were confirmed and tested on the validation group. The expression of each confirmed PCF RNA was significantly higher in sPTB up to 32 weeks of gestation. In vitro study of the four mRNAs revealed higher expression in placentas of women with sPTB up to 32 weeks and the potential to interfere with myometrial quiescence. Conclusions The PCF RNA markers are highly associated with sPTB up to 32 weeks by 16 weeks of gestation. Tweetable abstract Women destined for spontaneous preterm birth can be identified by 16 weeks of gestation with a panel of maternal plasma RNAs.

Detection of Embryonic Trisomy 21 in the First Trimester Using Maternal Plasma Cell-Free RNA

“…We believe the proprietary PCR RNA isolation protocol used in the current study is an important component for reproducibility. Follow-on studies using RNA isolated from placenta-derived extracellular vesicles should provide additional insight into the source of these RNA T21 biomarkers [ 26 , 65 ] and support our thesis [ 42 ] and that of others [ 26 , 40 , 66 ] that dysregulation of placental physiology is an antecedent to many pregnancy complications.…”

“…While the maternal plasma transcriptome has not changed over the last decade, the analytic tools have improved. We posited that the PCR RNA isolation and normalization protocol we developed for the prediction of preterm birth [ 42 ] would be useful for predicting other pregnancy complications such as T21. Other researchers have suggested the identification of a standardized RNA extraction and normalization protocol that provides both high yield and high-quality RNA is essential for downstream analysis to become reproducible and validated across RNA analysis methods and laboratories (e.g., see discussion in [ 41 ]).…”

“…First, we do not know the origin of the RNA markers (embryo/placenta, mother, or some combination). While our experience with preterm birth [ 42 ] suggests they are likely from the placenta, it is possible unrecognized maternal mosaicism might lead to a false-positive test. Second, we narrowed our outcomes to normal “controls” and T21 “cases”.…”

“…We described a panel of five PCF RNAs that effectively identified by 16 weeks, women destined for extreme preterm birth (e.g., birth ≤ 32 weeks) whether due to labor or preterm premature rupture of membranes [ 42 ]. Our in vitro work suggested the prognostic RNAs originate from the placenta [ 42 ]. Here, we hypothesize that transcriptional abnormalities in maternal plasma will reflect T21 and can be predicted reproducibly by qRT-PCR.…”

The LINC00261/MiR105-5p/SELL axis is involved in dysfunction of B cell and is associated with overall survival in hepatocellular carcinoma

“…Differentially expressed genes (DEGs) (or lncRNA (DELs) and miRNAs (DEMs)) were identified between different liver tissues using R package ‘limma.’ For DEGs, the threshold of absolute fold-change was more than 2. For DELs and DEMs, the threshold of absolute fold-change was more than 1.5 ( Li et al, 2021 ; Weiner et al, 2021 ). For DEGs/DELs/DEMs, the adjusted p-value was less than 0.05.…”