Dylan McCullough

Background: Preeclampsia is a hypertensive disorder of pregnancy with major maternal and fetal consequences. While the molecular basis of early-onset preeclampsia is well studied, the mechanisms underlying late-onset disease—and how they differ by fetal sex—remain poorly understood. Placental transcriptomic profiling at term can reveal persistent molecular alterations reflecting cumulative disease processes. Methods: We conducted a cross-sectional observational analysis of placental gene expression using RNA sequencing in a subset of 58 term placentas (21 male-bearing and 37 female-bearing pregnancies) drawn from two large prospective birth cohorts. Pregnancies were classified based on a clinical diagnosis of late-onset preeclampsia (diagnosed ≥ 20 weeks’ gestation according to ISSHP criteria) or as uncomplicated pregnancies. We then assessed for differential gene expression. Cell type proportions were estimated using CIBERSORTx from a placenta-specific reference single-cell dataset. Weighted gene co-expression network analysis identified modules of co-expressed genes associated with late-onset preeclampsia and fetal sex. Results: Differential gene expression analysis identified 150 genes with altered expression in male-bearing placentas from pregnancies with late-onset preeclampsia compared to those from uncomplicated pregnancies. No differentially expressed genes were identified in female-bearing placentas. Cell type deconvolution revealed increased abundance of CD14 + monocytes and CD8 + activated T cells (log odds of 1.42 and 1.44 respectively) and reduced fetal GZMK natural killer cells (log odds of 0.60) in male-bearing placentas from affected pregnancies. In female-bearing placentas, late-onset preeclampsia was associated with increased fetal nucleated red blood cells and maternal plasma cells (log odds of 1.33 and 1.40 respectively). Male-specific co-expression analysis identified gene modules enriched for biological processes including RNA processing, immune regulation, and metabolism. Conclusions: Placental transcription and cellular responses to late-onset preeclampsia differ by fetal sex. Evidence of altered immune cell composition and gene co-expression in male-bearing placentas suggests a sex-specific vulnerability. These findings highlight the importance of considering fetal sex in molecular investigation and clinical management of preeclampsia. Plain english summary: Preeclampsia is a common pregnancy complication marked by high blood pressure, but how it affects the placenta, especially in later pregnancy and depending on the baby’s sex, is not well understood. In this study, we analysed placental tissue from pregnancies with and without late-onset preeclampsia using RNA sequencing. By separating the data based on whether the neonate was male or female, we found striking differences in gene expression. Only placentas from male-bearing pregnancies showed significant changes in gene expression linked to preeclampsia. These changes involved genes related to immune response, metabolism and vascular function. We also used computational tools to estimate what types of cells were present in each placental sample. In male-bearing pregnancies affected by late-onset preeclampsia, there was a notable increase in certain immune cells, suggesting an altered immune response and increased inflammation. In contrast, female-bearing pregnancies affected by late-onset preeclampsia showed an increase in cell composition for two blood related cell types, but no significant gene expression differences. By grouping genes that worked together into networks, we identified several groups, especially in placentas from male-bearing pregnancies, that were strongly associated with biological processes known to be disrupted in preeclampsia, such as blood vessel formation, extracellular matrix remodelling, and hormone regulation. These findings emphasise the importance of considering fetal sex in pregnancy research and could help guide future sex-specific diagnostic or treatment strategies.

Background/Objectives: Gestational diabetes mellitus (GDM) prevalence in Australia has increased from 5.6% (2010) to 19.3% (2022), coinciding with the introduction of mandatory folic acid (FA) food fortification and increased supplementation. Animal studies show that high FA intake in pregnancy impairs maternal glucose regulation, but the underlying mechanisms are unknown. We investigated whether fortification has altered maternal folate status to increase GDM risk, and whether key hormones that regulate maternal glucose homeostasis are affected following FA fortification. Methods: Serum folate, red cell folate (RCF), prolactin (PRL), human placental lactogen (hPL) and placental growth hormone (GH2) were measured in early pregnancy samples from women enrolled in prospective cohorts: SCOPE (N = 1164; pre-fortification) and STOP (N = 1300; post-fortification). Associations with GDM were assessed. Results: Compared to pre-fortification, women post-fortification had a higher GDM incidence (5.0% vs. 15.2%), serum folate (↑ 18%), RCF (↑ 259%), hPL (↑ 29%), and GH2 (↑ 13%) concentrations. RCF concentrations above the clinical reference range were found in 57.6% of women post-fortification. Causal mediation analysis suggests that higher RCF contributed to increased GDM risk. Women with RCF excess had 48% more GDM cases, and higher PRL (↑ 24.2%) and hPL (↑ 12.7%) levels compared to those within the reference range. Conclusions: Maternal folate excess is likely contributing to the rising prevalence of GDM in Australia. These findings highlight the need to evaluate excess FA/folate safety in pregnancy, particularly in countries with mandatory fortification. Placental hormones may represent a mechanistic link between excess folate and GDM, warranting further investigation.

Structured Background: Unexplained 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. Objectives: This 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 design: Placenta 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). Results: Placental 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. Conclusions: Stillbirth 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 AJOG at a Glance: This 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. Circular 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. Premature 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.

Folic acid (FA) food fortification in Australia has resulted in a higher-than-expected intake of FA during pregnancy. High FA intake is associated with increased insulin resistance and gestational diabetes. We aimed to establish whether maternal one-carbon metabolism and hormones that regulate glucose homeostasis change in healthy pregnancies post-FA food fortification. Circulating folate, B12, homocysteine, prolactin (PRL), human placental lactogen (hPL) and placental growth hormone (GH2) were measured in early pregnancy maternal blood in women with uncomplicated pregnancies prior to (SCOPE: N = 604) and post (STOP: N = 711)-FA food fortification. FA food fortification resulted in 63% higher maternal folate. STOP women had lower hPL (33%) and GH2 (43%) after 10 weeks of gestation, but they had higher PRL (29%) and hPL (28%) after 16 weeks. FA supplementation during pregnancy increased maternal folate and reduced homocysteine but only in the SCOPE group, and it was associated with 54% higher PRL in SCOPE but 28% lower PRL in STOP. FA food fortification increased maternal folate status, but supplements no longer had an effect, thereby calling into question their utility. An altered secretion of hormones that regulate glucose homeostasis in pregnancy could place women post-fortification at an increased risk of insulin resistance and gestational diabetes, particularly for older women and those with obesity.

The search for novel microRNA (miRNA) biomarkers in plasma is hampered by haemolysis, the lysis and subsequent release of red blood cell contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multicompartment origin and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to sample. The inclusion of red-blood-cell-derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify posthoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload miRNA expression data from a short-read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. The code, DraculR web tool and its tutorial are freely available as detailed herein.

The abundance of cell-free microRNA (miRNA) has been measured in blood plasma and proposed as a source of novel, minimally invasive biomarkers for several diseases. Despite improvements in quantification methods, there is no consensus regarding how haemolysis affects plasma miRNA content. We propose a method for haemolysis detection in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature we tested differential miRNA abundance between plasma samples with known haemolysis status. Using these miRNAs with statistically significant higher abundance in our haemolysed group, we further refined the set to reveal high-confidence haemolysis association. Given our specific context, i.e., women of reproductive age, we also tested for significant differences between pregnant and non-pregnant groups. We report a novel 20-miRNA signature used to identify the presence of haemolysis in silico in HTS miRNA-sequencing data. Further, we validated the signature set using firstly an all-male cohort (prostate cancer) and secondly a mixed male and female cohort (radiographic knee osteoarthritis). Conclusion: Given the potential for haemolysis contamination, we recommend that assays for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.

The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10–12 weeks’ gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue samples collected across 6–23 weeks’ gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6–10 weeks’ and 11–23 weeks’ gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of individual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126, and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.

The abundance of cell-free microRNA (miRNA) has been measured in many body fluids, including blood plasma, which has been proposed as a source with novel, minimally invasive biomarker potential for several diseases. Despite improvements in quantification methods for plasma miRNAs, there is no consensus on optimal reference miRNAs or to what extent haemolysis may affect plasma miRNA content. Here we propose a new method for the detection of haemolysis in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature in plasma we first identified differentially expressed miRNAs between samples with known haemolysis status and selected miRNA with statistically significant higher abundance in our haemolysed group. Given there may be both technical and biological reasons for differential abundance of signature miRNA, and to ensure the method developed here was relevant outside of our specific context, that is women of reproductive age, we tested for significant differences between pregnant and non-pregnant groups. Here we report a novel 20 miRNA signature (miR-106b-3p, miR-140-3p, miR-142-5p, miR-532-5p, miR-17-5p, miR-19b-3p, miR-30c-5p, miR-324-5p, miR-192-5p, miR-660-5p, miR-186-5p, miR-425-5p, miR-25-3p, miR-363-3p, miR-183-5p, miR-451a, miR-182-5p, miR-191-5p, miR-194-5p, miR-20b-5p) that can be used to identify the presence of haemolysis, in silico , in high throughput miRNA sequencing data. Given the potential for haemolysis contamination, we recommend that assay for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.

Motivation: The search for novel microRNA (miRNA) biomarkers in plasma is hampered by haemolysis, the lysis and subsequent release of red blood cell (RBC) contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multi-compartment origin, and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to sample. The inclusion of RBC derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify post hoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. Results: We present DraculR, an interactive Shiny/R application that enables a user to upload microRNA expression data from short read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. Availability and implementation: DraculR and its tutorial are freely available from ( https://mxhp75.shinyapps.io/shinyVamp/ ). Code is available from ( https://github.com/mxhp75/shinyVamp.git ).

Imbalanced maternal micronutrient status, poor placentation, and oxidative stress are associated with greater risk of pregnancy complications, which impact mother and offspring health. As selenium, iodine, and copper are essential micronutrients with key roles in antioxidant systems, this study investigated their potential protective effects on placenta against oxidative stress. First trimester human placenta explants were treated with different concentrations of selenium (sodium selenite), iodine (potassium iodide), their combination or copper (copper (II) sulfate). The concentrations represented deficient, physiological, or super physiological levels. Oxidative stress was induced by menadione or antimycin. Placenta explants were collected, fixed, processed, and embedded for laser ablation inductively coupled plasma-mass spectrometry (LA ICP-MS) element imaging or immunohistochemical labelling. LA ICP-MS showed that placenta could uptake selenium and copper from the media. Sodium selenite and potassium iodide reduced DNA damage and apoptosis (p < 0.05). Following oxidative stress induction, a higher concentration of sodium selenite (1.6 µM) was needed to reduce DNA damage and apoptosis while both concentrations of potassium iodide (0.5 and 1 µM) were protective (p < 0.05). A high concentration of copper (40 µM) increased apoptosis and DNA damage but this effect was no longer significant after induction of oxidative stress. Micronutrients supplementation can increase their content within the placenta and an optimal maternal micronutrient level is essential for placenta health.