The pregnancy outcome prediction (POP) study: Investigating the relationship between serial prenatal ultrasonography, biomarkers, placental phenotype and adverse pregnancy outcomes

Placental dysfunction is implicated in many major complications of pregnancy associated with adverse maternal and infant outcome, such as preeclampsia, fetal growth restriction and stillbirth. Yet, despite years of intensive research, screening for these complications is still largely based upon clinical grounds rather than ultrasonic and/or biochemical assessment of placental function. One of the few widely employed methods for assessment of risk, low first trimester levels of PAPP-A (Pregnancy Associated Plasma Protein A), was identified through secondary analysis of data collected to identify new methods of screening for Down's syndrome rather than as a purposeful search for screening tests for abnormal placentation. Development of improved methods for population screening requires better mechanistic understanding of the pathways leading to placentally-related complications of human pregnancy. This is in addition to a need for identification of biomarkers which reflect the underlying pathology, while predicting associated disease with high sensitivity and specificity. In this paper, we outline some of the challenges and opportunities in this area. Furthermore, we illustrate how some of these can be addressed in research studies using the example of the Pregnancy Outcome Prediction (POP) study, a prospective cohort study conducted in Cambridge, UK.This study was funded by the NIHR Cambridge Comprehensive Biomedical Research Centre [grant number A019057], the Medical Research Council [grant number MR/K021133/1] and the Stillbirth and Neonatal Death Society (SANDS)

The relationship between human placental morphometry and ultrasonic measurements of utero-placental blood flow and fetal growth.

INTRODUCTION: Ultrasonic fetal biometry and arterial Doppler flow velocimetry are widely used to assess the risk of pregnancy complications. There is an extensive literature on the relationship between pregnancy outcomes and the size and shape of the placenta. However, ultrasonic fetal biometry and arterial Doppler flow velocimetry have not previously been studied in relation to postnatal placental morphometry in detail. METHODS: We conducted a prospective cohort study of nulliparous women in The Rosie Hospital, Cambridge (UK). We studied a group of 2120 women who had complete data on uterine and umbilical Doppler velocimetry and fetal biometry at 20, 28 and 36 weeks' gestational age, digital images of the placenta available, and delivered a liveborn infant at term. Associations were expressed as the difference in the standard deviation (SD) score of the gestational age adjusted ultrasound measurement (z-score) comparing the lowest and highest decile of the given placental morphometric measurement. RESULTS: The lowest decile of placental surface area was associated with 0.87 SD higher uterine artery Doppler mean pulsatility index (PI) at 20 weeks (95% CI: 0.68 to 1.07, P < 0.001). The lowest decile of placental weight was associated with 0.73 SD higher umbilical artery Doppler PI at 36 weeks (95% CI: 0.54 to 0.93, P < 0.001). The lowest decile of both placental weight and placental area were associated with reduced growth velocity of the fetal abdominal circumference between 20 and 36 weeks (both P < 0.001). CONCLUSION: Placental area and weight are associated with uterine and umbilical blood flow, respectively, and both are associated with fetal growth rate.This study was funded by the NIHR Cambridge Comprehensive Biomedical Research Centre (grant number A019057) and Stillbirth and Neonatal Death Society (SANDS). GE donated two ultrasound machines for use in the project.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.placenta.2015.12.00

Influence of speed of sample processing on placental energetics and signalling pathways: implications for tissue collection.

INTRODUCTION: The placenta is metabolically highly active due to extensive endocrine and active transport functions. Hence, placental tissues soon become ischaemic after separation from the maternal blood supply. Ischaemia rapidly depletes intracellular ATP, and leads to activation of stress-response pathways aimed at reducing metabolic demands and conserving energy resources for vital functions. Therefore, this study aimed to elucidate the effects of ischaemia ex vivo as may occur during tissue collection on phosphorylation of placental proteins and kinases involved in growth and cell survival, and on mitochondrial complexes. METHODS: Eight term placentas obtained from normotensive non-laboured elective caesarean sections were kept at room-temperature and sampled at 10, 20, 30 and 45 min after delivery. Samples were analyzed by Western blotting. RESULTS: Between 10 and 45 min the survival signalling pathway intermediates, P-AKT, P-GSK3α and β, P-4E-BP1 and P-p70S6K were reduced by 30-65%. Stress signalling intermediates, P-eIF2α increased almost 3 fold after 45 min. However, other endoplasmic reticulum stress markers and the Heat Shock Proteins, HSP27, HSP70 and HSP90, did not change. Phosphorylation of AMPK, an energy sensor, was elevated 2 fold after 45 min. Contemporaneously, there was an ∼25% reduction in mitochondrial complex IV subunit I. DISCUSSION AND CONCLUSIONS: These results suggest that for placental signalling studies, samples should be taken and processed within 10 min of caesarean delivery to minimize the impact of ischaemia on protein phosphorylation

Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions.This work was supported by the Wellcome Trust (084804/2/08/Z). Funding to pay the Open Access publication charges for this article was provided by theWellcome Trust.This is the final published version. It originally appeared in Molecular Human Reproduction, at http://dx.doi.org/10.1093/molehr/gau105

Computational modeling of the structure-function relationship in human placental terminal villi.

Placental oxygen transport takes place at the final branches of the villous tree and is dictated by the relative arrangement of the maternal and fetal circulations. Modeling techniques have failed to accurately assess the structure-function relationship in the terminal villi due to the geometrical complexity. Three-dimensional blood flow and oxygen transport was modeled in four terminal villi reconstructed from confocal image stacks. The blood flow was analyzed along the center lines of capillary segments and the effect of the variability in capillary diameter, tortuosity and branching was investigated. Additionally, a validation study was performed to corroborate the simulation results. The results show how capillary variations impact motion of the fetal blood, and how their bends and dilatations can decelerate the flow by up to 80%. Vortical flow is also demonstrated not to develop in the fetal capillaries. The different geometries are shown to dictate the transport of gases with differences of over 100% in the oxygen flux between samples. Capillary variations are key for efficient oxygen uptake by the fetus; they allow the blood to decelerate where the villous membrane is thinnest allowing for a better oxygenation, but also by reducing the vessel diameter they carry the oxygenated blood away fast. The methodology employed herein could become a platform to simulate complicated in-vivo and in-vitro scenarios of pregnancy complications.The first author gratefully acknowledges the generous support of Centre for Trophoblast Research, University of Cambridge, UK, which funded this work

RNA-seq reveals conservation of function among the yolk sacs of human, mouse, and chicken

The yolk sac is phylogenetically the oldest of the extraembryonic membranes. The human embryo retains a yolk sac, which goes through primary and secondary phases of development, but its importance is controversial. Although it is known to synthesize proteins, its transport functions are widely considered vestigial. Here, we report RNA-sequencing (RNA-seq) data for the human and murine yolk sacs and compare those data with data for the chicken. We also relate the human RNA-seq data to proteomic data for the coelomic fluid bathing the yolk sac. Conservation of transcriptomes across the species indicates that the human secondary yolk sac likely performs key functions early in development, particularly uptake and processing of macro- and micronutrients, many of which are found in coelomic fluid. More generally, our findings shed light on evolutionary mechanisms that give rise to complex structures such as the placenta. We identify genetic modules that are conserved across mammals and birds, suggesting these modules are part of the core amniote genetic repertoire and are the building blocks for both oviparous and viviparous reproductive modes. We propose that although a choriovitelline placenta is never established physically in the human, the placental villi, the exocoelomic cavity, and the secondary yolk sac function together as a physiological equivalent.M.G.E. is the recipient of a Research Fellowship from St. John’s College, University of Cambridge. This study was supported by Medical Research Council Grant MR/L020041/1

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment

Evolutionary history of endogenous Human Herpesvirus 6 reflects human migration out of Africa

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or “reactivation” of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa

Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with “protein glycosylation” and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders

Placental Streptococcus agalactiae DNA is associated with neonatal unit admission and foetal pro-inflammatory cytokines in term infants.

Streptococcus agalactiae (Group B Streptococcus; GBS) is a common cause of sepsis in neonates. Previous work detected GBS DNA in the placenta in ~5% of women before the onset of labour, but the clinical significance of this finding is unknown. Here we re-analysed this dataset as a case control study of neonatal unit (NNU) admission. Of 436 infants born at term (≥37 weeks of gestation), 7/30 with placental GBS and 34/406 without placental GBS were admitted to the NNU (odds ratio (OR) 3.3, 95% confidence interval (CI) 1.3-7.8). We then performed a validation study using non-overlapping subjects from the same cohort. This included a further 239 cases of term NNU admission and 686 term controls: 16/36 with placental GBS and 223/889 without GBS were admitted to the NNU (OR 2.4, 95% CI 1.2-4.6). Of the 36 infants with placental GBS, 10 were admitted to the NNU with evidence of probable but culture-negative sepsis (OR 4.8, 95% CI 2.2-10.3), 2 were admitted with proven GBS sepsis (OR 66.6, 95% CI 7.3-963.7), 6 were admitted and had chorioamnionitis (inflammation of the foetal membranes) (OR 5.3, 95% CI 2.0-13.4), and 5 were admitted and had funisitis (inflammation of the umbilical cord) (OR 6.7, 95% CI 12.5-17.7). Foetal cytokine storm (two or more pro-inflammatory cytokines >10 times median control levels in umbilical cord blood) was present in 36% of infants with placental GBS DNA and 4% of cases where the placenta was negative (OR 14.2, 95% CI 3.6-60.8). Overall, ~1 in 200 term births had GBS detected in the placenta, which was associated with infant NNU admission and morbidity