Circular RNA Sequencing of Maternal Platelets: A Novel Tool for the Identification of Pregnancy-Specific Biomarkers Cees Oudejans, a, * Vera Manders, a,b Allerdien Visser, a Remco Keijser, b Naomi Min, a,b Ankie Poutsma, a Joyce Mulders, a Tarah van den Berkmortel, a Di-Jan Wigman, a Britt Blanken, a Aldo Jongejan, c Eva Pajkrt, d Marjon de Boer, e Erik A. Sistermans, f Daoud Sie, f Myron G. Best, g,h,i Tom Wu ¨ rdinger, g,i and Gijs Afink b BACKGROUND: In the first trimester of pregnancy, the maternal platelet is directly involved in a positive feed- back mechanism that facilitates invasion of the extravil- lous trophoblast into the maternal spiral arteries. Dysfunctional trophoblast invasion with defective deep placentation is primordial in the etiology of the “great obstetrical syndromes.” METHODS: In this proof-of-concept study, using tran- scriptome analysis of circular RNA (circRNA) following RNA sequencing of maternal platelets, we tested whether pregnancy-specific circRNA markers could be identified in the first trimester of normal pregnancies. Differential transcript expression analysis of circRNAs, as predicted by Accurate CircRNA Finder Suite, CircRNA Identifier (version 2), and Known and Novel Isoform Explorer, was done using thromboSeq.R with variation of multiple settings. Test performance was checked for (a) de novo circRNA identification using the novel platelet-specific Plt-circR4 as a positive con- trol, (b) complete segregation of groups (pregnant vs nonpregnant) after heat map–dendrogram clustering, (c) identification of pregnancy-specific circRNA markers at a false discovery rate (FDR) <0.05, and (d) confirma- tion of differentially expressed circRNA markers with an FDR <0.05 by an independent method, reverse transcription–quantitative PCR. RESULTS: Of the differentially expressed circRNAs with P values <0.05, 41 circRNAs were upregulated (logFC >2), and 52 circRNAs were downregulated (logFC less than 2) in first-trimester platelet RNA. Of these, nuclear receptor-interacting protein 1 circRNA covering exons 2 and 3 of the 5 0 -untranslated region was preg- nancy specific with upregulation in first-trimester mater- nal platelets compared to nonpregnant controls. CONCLUSION: CircRNA sequencing of first-trimester maternal platelets permits the identification of novel pregnancy-specific RNA biomarkers. Future use could include the assessment of maternal and fetal well-being. Introduction Maternal plasma RNA sequencing allows noninvasive in vivo monitoring of placental function (1–4). Besides cell-free RNA from maternal plasma, additional RNA sources are available for this purpose, such as fetal tro- phoblast cells recovered from Pap smears (trophoblast retrieval and isolation from cervix) and maternal plate- lets (5). These RNA sources differ in origin (fetal vs maternal), cell-type (villous vs extravillous trophoblast, platelet), and content (cell-free vs cellular RNA) and thus in the biological and clinical information retrieved. The majority of cell-free RNA of fetal origin in maternal plasma is derived from the placenta and all of its compo- nents, both the inner layers (stroma, vessels, Hofbauer cells) and outer layers (trophoblast and all its subtypes: syncytiotrophoblast, cytotrophoblast, and extravillous trophoblast) (3). Cellular RNA retrieved from HLA-G– positive cells in the endocervix (trophoblast retrieval and isolation from cervix) originates from the extravil- lous trophoblast (6). Cellular RNA obtained from the a Department of Clinical Chemistry, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; b Reproductive Biology Laboratory, Amsterdam UMC, Academic Medical Center, Amsterdam, the Netherlands; c Department of Clinical Epidemiology, Biostatistics, and Bioinformatics, Amsterdam UMC, Academic Medical Center, the Netherlands; d Department of Obstetrics/Gynecology, Amsterdam UMC, Academic Medical Center, Amsterdam, the Netherlands; e Department of Obstetrics/ Gynecology, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; f Department of Clinical Genetics, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; g Department of Neurosurgery, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; h Department of Pathology, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; i Brain Tumor Center Amsterdam, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands. *Address correspondence to this author at: Amsterdam University Medical Centers, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Fax þ31-20-444-3895; e- mail cbm.oudejans@amsterdamumc.nl. Received May 26, 2020; accepted September 29, 2020. DOI: 10.1093/clinchem/hvaa249 V C American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com. 508 Clinical Chemistry 67:3 Molecular Diagnostics and Genetics 508–517 (2021) Downloaded from https://academic.oup.com/clinchem/article/67/3/508/6012968 by guest on 18 November 2023