MSc in Clinical Embryology
Preconception malnutrition among women and girls in south Asia: prevalence, determinants, and association with pregnancy and birth outcomes
This review highlights the growing double burden of malnutrition among women of reproductive age in South Asia. Using nationally-representative survey data, we highlight that the prevalence of overweight now exceeds that of underweight, while anaemia remains persistently high despite intervention efforts. Underweight and anaemia are more common among unmarried women, whereas overweight is more prevalent among parous women, underscoring the need for life-stage-specific preconception nutrition programs. In our systematic review, micronutrient deficiencies vary widely between and within countries, reflecting regional disparities in nutritional status and inconsistencies in diagnostic methods. Associations of preconception underweight, overweight, anaemia and micronutrient deficiencies with health, nutrition, socio-demographic, and WASH indicators are mixed, emphasising the need for tailored, context-specific interventions. The lack of longitudinal studies limits our understanding of associations between preconception nutritional status and subsequent birth outcomes, underscoring the need for comprehensive, longitudinal studies across South Asia to inform and monitor targeted nutrition programs.
Incidence, outcomes and management of spontaneous haemoperitoneum in pregnancy: a UK population-based study
Background Spontaneous haemoperitoneum in pregnancy (SHiP) is the occurrence during pregnancy of sudden intra-abdominal haemorrhage unrelated to extrauterine pregnancy, trauma or uterine rupture. SHiP is uncommon but is associated with preterm birth, high perinatal mortality and, more rarely, maternal mortality. We investigated the incidence of SHiP in the UK and its diagnosis, management and outcomes. Methods This two-year, prospective surveillance study used the UK Obstetric Surveillance System to collect anonymous data on all women who gave birth in a UK consultant-led maternity unit in 2016 and 2017 and who experienced SHiP. Results We confirmed 20 cases of SHiP, giving an estimated incidence of 1.3 cases per 100,000 maternities, or 1 per 75,614 maternities. The median gestational age at diagnosis was 35.7 weeks (IQR 29.9–38.4 weeks). A minority of affected women were receiving anticoagulant agents for prophylaxis (2/20) or treatment (4/20). The most common initial suspected diagnosis was placental abruption (7/20), followed by intra-abdominal bleeding, uterine rupture, or infection. SHiP was diagnosed using ultrasound in four women, using CT in five, and solely at surgery in 14. Aneurysms (4/20) and organ rupture or haematoma (5/20) were the most common bleeding source, and the condition was most commonly diagnosed and treated by laparotomy (11/20). Perinatal morbidity and mortality were high, with 16% of infants stillborn, an over 80% admission rate to the neonatal unit among the 16 live-born infants, major complications in a third of these infants, and one neonatal death. Maternal morbidity was also high, with 60% of women admitted to the intensive care unit, over half of whom experienced major morbidity, and one maternal death. Conclusions SHiP is rare in the UK but when it occurs, it can be associated with major maternal morbidity and mortality, and perinatal outcomes are poor. International comparisons are complicated by differing definitions of SHiP.
Rapid identification of bacterial isolates using microfluidic adaptive channels and multiplexed fluorescence microscopy.
We demonstrate the rapid capture, enrichment, and identification of bacterial pathogens using Adaptive Channel Bacterial Capture (ACBC) devices. Using controlled tuning of device backpressure in polydimethylsiloxane (PDMS) devices, we enable the controlled formation of capture regions capable of trapping bacteria from low cell density samples with near 100% capture efficiency. The technical demands to prepare such devices are much lower compared to conventional methods for bacterial trapping and can be achieved with simple benchtop fabrication methods. We demonstrate the capture and identification of seven species of bacteria with bacterial concentrations lower than 1000 cells per mL, including common Gram-negative and Gram-positive pathogens such as Escherichia coli and Staphylococcus aureus. We further demonstrate that species identification of the trapped bacteria can be undertaken in the order of one-hour using multiplexed 16S rRNA-FISH with identification accuracies of 70-98% with unsupervised classification methods across 7 species of bacteria. Finally, by using the bacterial capture capabilities of the ACBC chip with an ultra-rapid antimicrobial susceptibility testing method employing fluorescence imaging and convolutional neural network (CNN) classification, we demonstrate that we can use the ACBC chip as an imaging flow cytometer that can predict the antibiotic susceptibility of E. coli cells after identification.
Infection Inspection: using the power of citizen science for image-based prediction of antibiotic resistance in Escherichia coli treated with ciprofloxacin.
Antibiotic resistance is an urgent global health challenge, necessitating rapid diagnostic tools to combat its threat. This study uses citizen science and image feature analysis to profile the cellular features associated with antibiotic resistance in Escherichia coli. Between February and April 2023, we conducted the Infection Inspection project, in which 5273 volunteers made 1,045,199 classifications of single-cell images from five E. coli strains, labelling them as antibiotic-sensitive or antibiotic-resistant based on their response to the antibiotic ciprofloxacin. User accuracy in image classification reached 66.8 ± 0.1%, lower than our deep learning model's performance at 75.3 ± 0.4%, but both users and the model were more accurate when classifying cells treated at a concentration greater than the strain's own minimum inhibitory concentration. We used the users' classifications to elucidate which visual features influence classification decisions, most importantly the degree of DNA compaction and heterogeneity. We paired our classification data with an image feature analysis which showed that most of the incorrect classifications happened when cellular features varied from the expected response. This understanding informs ongoing efforts to enhance the robustness of our diagnostic methodology. Infection Inspection is another demonstration of the potential for public participation in research, specifically increasing public awareness of antibiotic resistance.
Prediction Models for Maternal and Offspring Short- and Long-Term Outcomes Following Gestational Diabetes: A Systematic Review.
OBJECTIVES: Gestational diabetes mellitus (GDM), affecting one in seven pregnant women worldwide, can have short- and long-term adverse outcomes for both the mother and her baby. Despite a raft of prognostic models aiming to predict adverse GDM outcomes, very few have impacted clinical practice. This systematic review summarizes and critically evaluates prediction models for GDM outcomes, to identify promising models for further evaluation. METHODS: We searched EMBASE, MEDLINE, Web of Science, CINAHL, and CENTRAL for studies that reported the development or validation of predictive models for GDM outcomes in mother or offspring (PROSPERO: CRD42023396697). RESULTS: Sixty-four articles detailing 103 developed and 12 validated models were included in this review. Of these, 45% predicted long term, 31% birth, and 23% pregnancy outcomes. Most models (87%) had a high risk of bias, lacking sufficient outcome events, internal validation, or proper calibration. Only eight models were found at low risk of bias. CONCLUSIONS: Our findings highlight a gap in rigorously developed prediction models for adverse GDM outcomes. There is a need to further validate existing models and evaluate their clinical utility to generate risk prediction tools capable of improving clinical decision-making for women with GDM and their children.
Adverse drug reactions that arise from the use of medicinal products outside the terms of the marketing authorisation.
BACKGROUND: New European (EU) pharmacovigilance (PV) legislation, introduced in 2012, widened the scope of an Adverse Drug Reactions (ADR) definition so that it also includes noxious and unintended response to a medicinal product arising from the use outside the terms of the marketing authorisation (MA), whereby the use outside the MA also includes off-label use, overdose, misuse, abuse and medication errors (MEs). OBJECTIVES: To explore the ADRs arising from the use outside the terms of the MA reports in the Croatian pharmacovigilance database. METHODS: A retrospective, observational study of the HALMED PV database was undertaken before and after the implementation of the new legislation in Croatia. The outcome measure included ADRs arising from the use of the products outside the terms of the MA. An assessment was performed based on the information provided in a reference document, an SmPC, using predefined criteria. RESULTS: Among 679 ADRs included in the analysis, 162 (23,9%) ADR reports were related to the use outside of the MA, 370 (54,5%) were related to the use within the MA and 147 (21,6%) were adjudged as not-assessable. Our study demonstrated a significant increase in the number of ADRs arising from the use outside the terms of the MA after the implementation of the new legislation (P = 0,039), primarily due to a notable increase in the number of overdose reports received by the poisoning centre, while the number of ADRs caused by MEs did not change significantly (p = 0,672). CONCLUSION: This study elucidated partial implementation of the new EU PV legislation and the need for instilling proper education for patients and HCPs, improving reporting systems and strengthening collaboration between relevant stakeholders.
A method to isolate syncytiotrophoblast-derived medium-large extracellular vesicle small RNA from maternal plasma.
Syncytiotrophoblast-derived extracellular vesicles (STB-EVs) have an important role in placental research: both as mediators of feto-maternal signalling and as liquid biopsies reflecting placental health. Recent evidence highlights the importance of STB-EV RNA. Isolation of STB-EV RNA from maternal blood is therefore an important challenge. We describe a novel technique where we first separate medium-large particles from plasma using centrifugation then use a highly specific bead-bound antibody to placental alkaline phosphatase to separate STB-EVs from other similar-sized particles. We demonstrate the yield and size profile of small RNA obtained from plasma STB-EVs. We present data confirming isolation of placenta-derived micro RNA from maternal plasma using this method. The technique has been successfully applied to validate novel RNA discoveries from placental perfusion models. We propose it could offer new insights through transcriptomic analyses, providing a syncytiotrophoblast-specific signal from maternal blood.
Neuropilin-1 is uniquely expressed on small syncytiotrophoblast extracellular vesicles but not on medium/large vesicles from preeclampsia and normal placentae.
UNLABELLED: Preeclampsia (PE) is a multisystem progressive hypertensive disorder unique to human pregnancy. The placenta is fundamental to its pathogenesis and releases placental factors as well as extracellular vesicles (small and medium/large syncytiotrophoblast extracellular vesicles (STB-EVs)) as a response to syncytiotrophoblast stress such as tissue factor and plasminogen activator inhibitors 1. Neuropilin 1 (NRP-1) is an anti-angiogenic factor involved in development, angiogenesis, arteriogenesis, and vascular permeability. NRP-1 acts as a co-receptor for growth factors such as vascular endothelial growth factor (VEGF), placenta growth factor (PLGF), and epidermal growth factor (EGF). Given the documented pro and anti-angiogenic roles of STB-EVs, we hypothesized that 1) STB-EVs might express NRP-1; and 2) the expression of NRP-1 might differ between normal and preeclampsia STB-EVs. METHODS: We isolated STB-EVs (both small and medium/large) from PE and NP placentae using the physiologic ex vivo dual lobe perfusion model. The enriched STB-EVs were characterized by Western blot, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA) according to the international society of extracellular vesicles (ISEV) guidelines. We assessed for NRP-1 expression with Western blot (placenta and STB-EVs) and immunohistochemistry (placenta). We performed co-expression analysis for placenta alkaline phosphatase (PLAP - a known STB-EV marker) and NRP-1 with immunoprecipitation followed by Western blot. RESULTS: We confirmed NRP-1 expression in NP and PE placenta. We showed that NRP-1 Expression was limited to small syncytiotrophoblast membrane extracellular vesicles (S STB-EVs) but not medium/large STB-EVs and that NRP-1 is co-expressed with PLAP. CONCLUSION: Neuropilin-1 is uniquely expressed on small syncytiotrophoblast extracellular vesicles but not on medium/large vesicles from preeclampsia and normal placentae.
Syncytiotrophoblast Extracellular Vesicles From Late-Onset Preeclampsia Placentae Suppress Pro-Inflammatory Immune Response in THP-1 Macrophages
Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe ex-vivo placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy, and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As expected, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-β compared to PE medium/large-STBEV. However, there was no significant difference in the small STBEV population between the two groups, although in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE.
Syncytiotrophoblast‐derived extracellular vesicles carry apolipoprotein‐E and affect lipid synthesis of liver cells in vitro
AbstractIn normal pregnancy, hepatic metabolism adaptation occurs with an increase in lipid biosynthesis. Placental shedding of syncytiotrophoblast‐derived extracellular vesicles (STBEVs) into the maternal circulation constitutes a major signalling mechanism between foetus and mother. We investigated whether STBEVs from normal pregnant women might target liver cells in vitro and induce changes in lipid synthesis. This study was performed at the Nuffield Department of Women's & Reproductive Health, Oxford, UK. STBEVs were obtained by dual‐lobe placental perfusion from 11 normal pregnancies at term. Medium/large and small STBEVs were collected by ultracentrifugation at 10,000g and 150,000g, respectively. STBEVs were analysed by Western blot analysis and flow cytometry for co‐expression of apolipoprotein‐E (apoE) and placental alkaline phosphatase (PLAP). The uptake of STBEVs by liver cells and the effect on lipid metabolism was evaluated using a hepatocarcinoma cell line (HepG2 cells). Data were analysed by one‐way ANOVA and Student's t test. We demonstrated that: (a) STBEVs carry apoE; (b) HepG2 cells take up STBEVs through an apoE‐LDL receptor interaction; (c) STBEV incorporation into HepG2 cells resulted in (i) increased cholesterol release (ELISA); (ii) increased expression of the genes SQLE and FDPS (microarray) involved in cholesterol biosynthesis; (iii) downregulation of the CLOCK gene (microarray and PCR), involved in the circadian negative control of lipid synthesis in liver cells. In conclusion, the placenta may orchestrate the metabolic adaptation of the maternal liver through release of apoE‐positive STBEVs, by increasing lipid synthesis in a circadian‐independent fashion, meeting the nutritional needs of the growing foetus.
Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia
Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age–matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N G -nitro- l -arginine methyl ester (eNOS inhibitor; P <0.05) but not by N -(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P <0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P <0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P <0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease.
Placental extracellular vesicles express active dipeptidyl peptidase IV; levels are increased in gestational diabetes mellitus
ABSTRACTGestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy and is characterized by insulin resistance and decreased circulating glucagon‐like peptide‐1 (GLP‐1). GDM resolves rapidly after delivery implicating the placenta in the disease. This study examines the biological functions that cause this pathology. The placenta releases syncytiotrophoblast‐derived extracellular vesicles (STB‐EVs) into the maternal circulation, which is enhanced in GDM. Dipeptidyl peptidase IV (DPPIV) is known to play a role in type 2 diabetes by breaking down GLP‐1, which in turn regulates glucose‐dependent insulin secretion. STB‐EVs from control and GDM women were analysed. We show that normal human placenta releases DPPIV‐positive STB‐EVs and that they are higher in uterine than paired peripheral blood, confirming placental origin. DPPIV‐bound STB‐EVs from normal perfused placentae are dose dependently inhibited with vildagliptin. DPPIV‐bound STB‐EVs from perfused placentae are able to breakdown GLP‐1 in vitro. STB‐EVs from GDM perfused placentae show greater DPPIV activity. Importantly, DPPIV‐bound STB‐EVs increase eightfold in the circulation of women with GDM. This is the first report of STB‐EVs carrying a biologically active molecule that has the potential to regulate maternal insulin secretion.
Protein Profiling of Placental Extracellular Vesicles in Gestational Diabetes Mellitus
Throughout pregnancy, some degree of insulin resistance is necessary to divert glucose towards the developing foetus. In gestational diabetes mellitus (GDM), insulin resistance is exacerbated in combination with insulin deficiency, causing new-onset maternal hyperglycaemia. The rapid reversal of insulin resistance following delivery strongly implicates the placenta in GDM pathogenesis. In this case–control study, we investigated the proteomic cargo of human syncytiotrophoblast-derived extracellular vesicles (STBEVs), which facilitate maternal–fetal signalling during pregnancy, in a UK-based cohort comprising patients with a gestational age of 38–40 weeks. Medium/large (m/l) and small (s) STBEVs were isolated from GDM (n = 4) and normal (n = 5) placentae using ex vivo dual-lobe perfusion and subjected to mass spectrometry. Bioinformatics were used to identify differentially carried proteins and mechanistic pathways. In m/lSTBEVs, 56 proteins were differently expressed while in sSTBEVs, no proteins reached statistical difference. Differences were also observed in the proteomic cargo between m/lSTBEVs and sSTBEVs, indicating that the two subtypes of STBEVs may have divergent modes of action and downstream effects. In silico functional enrichment analysis of differentially expressed proteins in m/lSTBEVs from GDM and normal pregnancy found positive regulation of cytoskeleton organisation as the most significantly enriched biological process. This work presents the first comparison of two populations of STBEVs’ protein cargos (m/l and sSTBEVs) from GDM and normal pregnancy isolated using placenta perfusion. Further investigation of differentially expressed proteins may contribute to an understanding of GDM pathogenesis and the development of novel diagnostic and therapeutic tools.
Placental small extracellular vesicles from normal pregnancy and gestational diabetes increase insulin gene transcription and content in β cells
Abstract Insulin secretion increases progressively during pregnancy to maintain normal maternal blood glucose levels. The placenta plays a crucial role in this process by releasing hormones and extracellular vesicles into the maternal circulation, which drive significant changes in pregnancy physiology. Placental extracellular vesicles, which are detectable in the plasma of pregnant women, have been shown to signal peripheral tissues and contribute to pregnancy-related conditions. While studies using murine models have demonstrated that extracellular vesicles can modulate insulin secretion in pancreatic islets, it remains unclear whether these effects translate to human biology. Understanding how placental signals enhance insulin synthesis and secretion from β cells could be pivotal in developing new therapies for diabetes. In our study, we isolated placental small extracellular vesicles from human placentae and utilised the human β cell line, EndoC-βH3, to investigate their effects on β-cell function in vitro. Our results indicate that human β cells internalise placental small extracellular vesicles, leading to enhanced insulin gene expression and increased insulin content within the β cells. Moreover, these vesicles up-regulated the expression of Annexin A1, a protein known to increase insulin content. This up-regulation of Annexin A1 holds promise as a potential mechanism by which placental small extracellular vesicles enhance insulin biosynthesis.
In vivo evidence of significant placental growth factor release by normal pregnancy placentas
AbstractPlacental growth factor (PlGF) is an angiogenic factor identified in the maternal circulation, and a key biomarker for the diagnosis and management of placental disorders. Furthermore, enhancing the PlGF pathway is regarded as a promising therapy for preeclampsia. The source of PlGF is still controversial with some believing it to be placental in origin while others refute this. To explore the source of PlGF, we undertook a prospective study enrolling normal pregnant women undergoing elective caesarean section. The level of PlGF was estimated in 17 paired serum samples from the uterine vein (ipsilateral or contralateral to the placental insertion) during caesarean section and from a peripheral vein on the same day and second day post-partum. PlGF levels were higher in the uterine than in the peripheral vein with a median difference of 52.2 (IQR 20.1–85.8) pg/mL p = 0.0006. The difference when the sampled uterine vein was ipsilateral to the placenta was 54.8 (IQR 37.1–88.4) pg/mL (n = 11) and 23.7 (IQR −11; 70.5) pg/mL (n = 6) when the sample was contralateral. Moreover, PlGF levels fell by 83% on day 1–2 post-partum. Our findings strongly support the primary source of PlGF to be placental. These findings will be of value in designing target therapies such as PlGF overexpression, to cure placental disorders during pregnancy.
Genome-wide association study meta-analysis provides insights into the etiology of heart failure and its subtypes.
Heart failure (HF) is a major contributor to global morbidity and mortality. While distinct clinical subtypes, defined by etiology and left ventricular ejection fraction, are well recognized, their genetic determinants remain inadequately understood. In this study, we report a genome-wide association study of HF and its subtypes in a sample of 1.9 million individuals. A total of 153,174 individuals had HF, of whom 44,012 had a nonischemic etiology (ni-HF). A subset of patients with ni-HF were stratified based on left ventricular systolic function, where data were available, identifying 5,406 individuals with reduced ejection fraction and 3,841 with preserved ejection fraction. We identify 66 genetic loci associated with HF and its subtypes, 37 of which have not previously been reported. Using functionally informed gene prioritization methods, we predict effector genes for each identified locus, and map these to etiologic disease clusters through phenome-wide association analysis, network analysis and colocalization. Through heritability enrichment analysis, we highlight the role of extracardiac tissues in disease etiology. We then examine the differential associations of upstream risk factors with HF subtypes using Mendelian randomization. These findings extend our understanding of the mechanisms underlying HF etiology and may inform future approaches to prevention and treatment.