Arthurs, A. L. | Search bioRxiv

bioRxiv2026

Engineered trophoblast organoids recapitulate molecular and functional features of preeclampsia

Preeclampsia is a major pregnancy complication driven by placental dysfunction, yet research is limited by reliance on patient-derived tissues and models that do not fully capture human disease. Here, we develop a genetically engineered human trophoblast organoid model of preeclampsia that can be generated without access to placental tissue. Using a CRISPR-based Prime Integrase strategy, we engineered induced trophoblast stem cells to express the preeclampsia-associated soluble fms-like tyrosine kinase-1 (sFlt-1) exon 15a isoform. Engineered organoids showed a transcriptional shift towards primary preeclamptic placentae and developed several features of disease. These included reduced PlGF, increased IL-6 and soluble endoglin, oxidative stress, impaired growth and an elevated sFlt-1/PlGF ratio comparable to primary preeclamptic trophoblast organoids. These broader changes were not reproduced by adding recombinant human sFlt-1 to control organoids. Conditioned media from engineered organoids impaired endothelial network formation, demonstrating a functional effect of the altered trophoblast secretome. Treatment with sulfasalazine and metformin also restored angiogenic balance and organoid growth. Together, these findings establish a tractable human model that reproduces molecular and functional features of preeclampsia and provides a platform to study disease mechanisms and test potential therapies.

cell biology↗

bioRxiv2025

Late-Onset Preeclampsia is characterised by Accelerated Placental Aging

Late-onset preeclampsia (LOPE) is a major pregnancy complication characterised by hypertension and placental dysfunction, resolving only upon delivery. Here, we show that LOPE placentae undergo accelerated molecular aging, marked by telomere attrition, DNA damage and trophoblast senescence. Using primary placental tissue and trophoblast organoids, we demonstrate oxidative stress as a driver of telomere shortening and angiogenic imbalance. Inflammation did not alter placental aging trajectories. Antioxidant treatment (superoxide dismutase) preserved telomere length, reduced DNA damage and restored angiogenic balance, highlighting oxidative stress as a modifiable determinant of placental aging. We identify reduced expression of telomeric repeat-containing RNAs (TERRAs) as a molecular hallmark of LOPE, and show that antisense oligonucleotide-mediated TERRA depletion exacerbates telomere erosion and senescence. Together, these findings delineate oxidative stress and TERRA loss as mechanisms driving placental decline, establish trophoblast organoids as a tractable model of placental aging, and reveal potential therapeutic avenues for mitigating preeclampsia-associated placental dysfunction.

developmental biology↗

bioRxiv2024

Circular RNAs correlate with DNA damage in ageing human placental tissue and in stillbirth

Structured AbstractO_ST_ABSBackgroundC_ST_ABSUnexplained stillbirth may occur due to premature placental ageing, with unexpected deterioration of placental function for gestational age. Circular RNAs (circRNAs) are enzyme-resistant RNA molecules that accumulate in ageing tissues. Furthermore, circRNAs bind gDNA directly, forming circRNA:DNA complexes which can induce DNA breaks and genomic instability. ObjectivesThis study investigated tissue ageing and circRNA accumulation with gestational age in healthy and stillbirth placentae, and determined whether circRNAs directly interact with placental DNA causing DNA damage and cellular senescence. Study designPlacenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, 23, 26, 31, 34 weeks gestation) were assessed. Abundance of 7 candidate circRNAs (circ_0009000, circ_0024157, circ_0061017, circ_0036877, circ_0054624, circ_0111277 and circ_0000284), and their linear transcripts, was quantified using qPCR. Total RNA, in the presence and absence of RNase R and RNase H1, was determined using the Qubit fluorimeter. Physical interaction of candidate circRNAs with DNA was confirmed by DNA:RNA ImmunoPrecipitation (DRIP)-qPCR. Telomere length was assessed using real-time PCR. Relative abundance of senescence-associated genes was quantified using qPCR. DNA damage was assessed using an alkaline Comet Assay. Patient-derived trophoblast stem cells (TSCs) differentiation into syncytiotrophoblasts or extravillous trophoblasts was confirmed using immunofluorescence microscopy. The effect of circ_0000284 knockdown was assessed following transfection with either a siRNA (designed to knockdown circ_0000284) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. Abundance of circRNAs in maternal blood sampled between 15-16 weeks gestation (n=12 control, n=6 women who went on to have a stillbirth) was determined using qPCR. Appropriate statistical analyses were undertaken (SPSS). ResultsPlacental DNA damage, senescence and expression of 7 candidate circRNAs, but not their linear transcripts, were increased in 40 and 41+ weeks gestation samples, and in stillbirth, compared with earlier gestations (37-39 weeks). DRIP-qPCR signal size was significantly larger in term placentae than in enzyme-treated controls, confirming that all candidate circRNA loci bind to placental DNA. Abundance of circRNA was significantly decreased with the addition of RNase H1, compared with all healthy gestation samples, indicating that stillbirth placentae may lack RNase H1. Telomere length is shorter in placentae from stillbirths compared with healthy 37 weeks placentae. Depletion of circ_0000284 by specific siRNA in primary cells significantly reduced DNA damage and increased expression of senescence-associated genes compared to control. Abundance of candidate circRNAs are increased in maternal blood at 16 weeks gestation for women who went on to have a stillbirth compared with women who had live births. ConclusionsStillbirth placentae show accelerated ageing with shortened telomeres, premature DNA breaks, increased cellular senescence and accumulation of candidate circRNAs, at levels consistent with older gestation tissue. These circRNAs bind to DNA in the placenta, and circ_0000284 knockdown reduces DNA breaks and senescence in primary placental cells. Therefore, circRNAs play a role in placental ageing and associate with stillbirth, likely via decreased RNase H1 abundance, preventing circRNA degradation and facilitating circRNA accumulation, and subsequent circR-loop formation. circRNAs present a viable method of stillbirth risk screening. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/610398v4_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@4a5a15org.highwire.dtl.DTLVardef@160231borg.highwire.dtl.DTLVardef@a0d0aeorg.highwire.dtl.DTLVardef@1e4590f_HPS_FORMAT_FIGEXP M_FIG C_FIG AJOG at a GlanceO_LIThis study was conducted to confirm premature placental ageing in cases of unexplained stillbirth, and to investigate the role of circular RNAs in the process of placental ageing. C_LIO_LICircular RNAs accumulate in ageing healthy placenta and accumulate prematurely in stillbirth placentae. Circular RNAs bind directly to placental DNA, inducing DNA breaks and cellular senescence, thereby contributing to overall functional decline of the placenta, reducing its ability to support the fetus. Circular RNAs in maternal blood represents a novel screening tool for detection of stillbirth risk. C_LIO_LIPremature placental ageing has previously been associated with stillbirth, however circular RNAs have never before been implicated in this process. Circular RNA accumulation in ageing tissues has been shown in model species (i.e. C. elegans, Drosophila Melanogaster, mice) but this is the first evidence in human tissues and in the placenta. C_LI

molecular biology↗

bioRxiv2024

Genetically edited placental organoids cast new light on the role of ACE2 in placental development

ACE2 expression is altered in pregnancy disorders and ACE2 gene variants are associated with several major pregnancy complications including small-for-gestational-age, fetal growth restriction and preeclampsia. This study utilised gene-editing to generate both ACE2 knockout and ACE2 rs2074192 placental organoids, facilitating mechanistic studies into the role of ACE2 in placental development, and the effect of fetal carriage of ACE2 rs2074192 CC, CT and TT genotypes. Parameters of cell and organoid growth were measured, together with qPCR, Western Blotting, and ELISA assessments, in all groups from both organoid models. Here, we report that ACE2 knockout results in delayed placental cell growth and increased cell death. ACE2 knockout organoids had lower ACE protein expression, reduced organoid diameters and asymmetrical growth. Placental organoids with the ACE2 rs2074192 TT genotype had significantly higher expression of ACE2 mRNA and ACE2 protein with elevated ACE2:ACE expression ratio and no change in ACE protein expression. Despite increased expression of ACE2 protein, ACE2 enzyme activity was significantly decreased in ACE2 rs2074192 TT placental organoids. TT organoids also had reduced diameters and asymmetrical growth. Our research provides new molecular understanding of the role of ACE2 in placental development, with potential implications for pregnancy in carriage of the ACE2 rs2074192 gene variant.

molecular biology↗