Universal First-Trimester Screening Biomarkers for Diagnosis of Preeclampsia and Placenta Accreta Spectrum

Abstract

Background: Disruptions in epigenetic mechanisms regulating placentation, particularly imbalances in the levels of small non-coding RNAs, contribute to various pregnancy complications, including preeclampsia (PE) and placenta accreta spectrum (PAS). Given that abnormal trophoblast differentiation, invasiveness, and angiogenesis-reduced in PE and excessive in PAS-are central to the pathogenesis of these conditions, this study aimed to identify universal circulating piRNAs and their targets.

Methods: Small RNA deep sequencing, quantitative reverse transcription combined with real-time polymerase chain reaction, magnetic bead-based multiplex immunoassay, ELISA, and Western blotting were employed to quantify circulating piRNAs and proteins in the blood serum of pregnant women during the 11th-14th weeks of gestation.

Results: Statistically significant negative correlations were identified between PE- and PAS-associated piRNAs (hsa_piR_019122, hsa_piR_020497, hsa_piR_019949, and piR_019675) and several molecules, including Endoglin, IL-18, VEGF-A, VEGF-C, Angiopoietin-2, sFASL, HB-EGF, TGFα, and Clusterin. These molecules are involved in processes such as angiogenesis, inflammation, the epithelial-mesenchymal transition, cell proliferation, adhesion, and apoptosis. A first-trimester pregnancy screening algorithm was developed using logistic regression models based on Clusterin concentration and the levels of hsa_piR_020497, hsa_piR_019949, piR_019675, and hsa_piR_019122.

Conclusions: The proposed screening tool for early pregnancy monitoring may enable the prediction of PE or PAS in the first trimester, allowing timely interventions to reduce maternal and perinatal morbidity and mortality.

Keywords: Clusterin; ELISA; blood serum; extracellular vesicles; first-trimester screening; piRNA; placenta accreta spectrum; preeclampsia; real-time PCR.

Figure 1

PLS-DA of RT-PCR data (-∆Ct values) for piRNAs in the blood serum of pregnant women at 11–14 weeks of gestation: (A) Score plot comparing Norm and PE groups. (B) Score plot comparing Norm and PAS groups. The VIP scores for specific piRNAs are listed to the right of the score plots.

Figure 2

Logistic regression models for diagnosing pregnancy complications in the first trimester based on piRNA levels in maternal blood serum. (A) Comparison of normal pregnancy and PE groups. (B) Comparison of normal pregnancy and PAS groups. (C) Comparison of normal pregnancy and all complications (PE and PAS). (D) Comparison of PE and PAS groups. Sensitivity (Se) and specificity (Sp) values are indicated.

Figure 3

Protein analysis of maternal blood serum at 11–14 weeks of gestation using the Bio-Plex Pro human cancer biomarker assay.

Figure 4

Logistic regression models for first-trimester pregnancy complication screening. (A) Model distinguishing Norm and PE groups. (B) Model distinguishing PE and PAS groups.

Figure 5

Clusterin levels in the vesicular fraction of maternal blood serum at 11–14 weeks of pregnancy. (A) Relative clusterin levels from Western blotting. A representative blot (top-right inset) shows chemiluminescent signal intensity at ~40 kDa, corresponding to the α-subunit of the secretory form of clusterin. (B) Clusterin concentration (ng/mL) measured by ELISA. Original Western blot images can be found in Supplementary File S1.

Figure 6

Logistic regression models for diagnosing pregnancy complications (PE or PAS) in the first trimester based on clusterin levels in the vesicular fraction of maternal blood serum at 11–14 weeks of pregnancy.

Figure 7

Correlation matrix based on the non-parametric Spearman rank correlation method. Significant (p < 0.05) correlations are indicated by a dot, non-significant correlations are indicated by a cross, positive correlations are marked in blue, and negative correlations in red—the more significant the correlation, the larger the dot size.