Background Multifetal pregnancies are associated with increased risk for preeclampsia (PreE), but the underlying pathogenesis may differ from singleton gestations. It remains unclear whether both placentas in twin pregnancies complicated by PreE exhibit molecular signatures of the disease simultaneously or in isolation.

Methods We performed RNA sequencing on 32 individual placental samples from twin gestations grouped by clinical PreE diagnosis: 24 dichorionic (DT) and 8 monochorionic (MT). Additionally, 10 newly sequenced singleton PreE placentas were analyzed. These were integrated with a benchmark dataset comprising 15 early-onset PreE and 10 control singleton placentas from our prior study (GSE203507), along with 102 samples from two independent studies (GSE114691 and GSE1482410). Differential expression analysis and a machine learning pipeline were used to identify a 98-transcript classification signature for PreE, achieving 97% accuracy in the benchmark dataset.

Results Twin placentas partially aligned with the molecular spectrum of PreE observed in singleton gestations. Signature scoring revealed transcriptional similarities between twin and singleton PreE placentas across varying clinical severities. Intertwin transcriptional divergence was more pronounced in DT than MT samples, regardless of clinical diagnosis. Functional pathway analysis showed consistent dysregulation patterns in PreE, but with notable heterogeneity among twin samples. Some twin placentas without clinical PreE displayed PreE-like transcriptional signatures, suggesting molecular changes may precede clinical manifestation.

Conclusions These results highlight complexity in PreE pathology in twin gestations. In DT pregnancies complicated by PreE, the placentas may be affected unequally, suggesting that PreE in twins may be associated with a single diseased placenta.

The authors have declared no competing interest.

Research reported in this publication was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (NIH) under Award Number R01 HD084628 (to IAB). The funding source had no role in study design, data analysis, interpretation of data, writing of the report, or decision to submit for publication. The content is the exclusive responsibility of the authors and should not be construed as representing an official view of the NIH.

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This study was approved by the IRBs of Yale University, The Ohio State University, the Abigail Wexner Research Institute at Nationwide Children's Hospital, and the University of Illinois at Chicago. All participants provided written informed consent.

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Supporting data are available in the article and its Supplement. The sequencing data generated for this manuscript were deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through accession number GSE272342.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272342