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

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

Paraoxonase 2 protein is spatially expressed in the human placenta and selectively reduced in labour

Humans parturition involves interaction of hormonal, neurological, mechanical stretch and inflammatory pathways and the placenta plays a crucial role. The paraoxonases (PONs 1–3) protect against oxidative damage and lipid peroxidation, modulation of endoplasmic reticulum stress and regulation of apoptosis. Nothing is known about the role of PON2 in the placenta and labour. Since PON2 plays a role in oxidative stress and inflammation, both features of labour, we hypothesised that placental PON2 expression would alter during labour. PON2 was examined in placentas obtained from women who delivered by cesarean section and were not in labour and compared to the equivalent zone of placentas obtained from women who delivered vaginally following an uncomplicated labour. Samples were obtained from 12 sites within each placenta: 4 equally spaced apart pieces were sampled from the inner, middle and outer placental regions. PON2 expression was investigated by Western blotting and real time PCR. Two PON2 forms, one at 62 kDa and one at 43 kDa were found in all samples. No difference in protein expression of either isoform was found between the three sites in either the labour or non-labour group. At the middle site there was a highly significant decrease in PON2 expression in the labour group when compared to the non-labour group for both the 62 kDa form (p = 0.02) and the 43 kDa form (p = 0.006). No spatial differences were found within placentas at the mRNA level in either labour or non-labour. There was, paradoxically, an increase in PON2 mRNA in the labour group at the middle site only. This is the first report to describe changes in PON2 in the placenta in labour. The physiological and pathological significance of these remains to be elucidated but since PON2 is anti-inflammatory further studies are warranted to understand its role

Arsenic-Associated Oxidative Stress, Inflammation, and Immune Disruption in Human Placenta and Cord Blood

BACKGROUND: Arsenic (As) exposure during pregnancy induces oxidative stress and increases the risk of fetal loss and low birth weight. OBJECTIVES: In this study we aimed to elucidate the effects of As exposure on immune markers in the placenta and cord blood, and the involvement of oxidative stress. METHODS: Pregnant women were enrolled around gestational week (GW) 8 in our longitudinal, population-based, mother-child cohort in Matlab, an area in rural Bangladesh with large variations in As concentrations in well water. Women (n = 130) delivering at local clinics were included in the present study. We collected maternal urine twice during pregnancy (GW8 and GW30) for measurements of As, and placenta and cord blood at delivery for assessment of immune and inflammatory markers. Placental markers were measured by immunohistochemistry, and cord blood cytokines by multiplex cytokine assay. RESULTS: In multivariable adjusted models, maternal urinary As (U-As) exposure both at GW8 and at GW30 was significantly positively associated with placental markers of 8-oxoguanine (8-oxoG) and interleukin-1β (IL-1β); U-As at GW8, with tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); and U-As at GW30, with leptin; U-As at GW8 was inversely associated with CD3+ T cells in the placenta. Cord blood cytokines (IL-1β, IL-8, IFNγ, TNFα) showed a U-shaped association with U-As at GW30. Placental 8-oxoG was significantly positively associated with placental proinflammatory cytokines. Multivariable adjusted analyses suggested that enhanced placental cytokine expression (TNFα and IFNγ) was primarily influenced by oxidative stress, whereas leptin expression appeared to be mostly mediated by As, and IL-1β appeared to be influenced by both oxidative stress and As. CONCLUSION: As exposure during pregnancy appeared to enhance placental inflammatory responses (in part by increasing oxidative stress), reduce placental T cells, and alter cord blood cytokines. These findings suggest that effects of As on immune function may contribute to impaired fetal and infant health

Hepcidin and iron species distribution inside the first-trimester human gestational sac

We have investigated factors affecting iron distribution in the first-trimester gestational sac, by the measurement of transferrin, non-transferrin-bound iron (NTBI) and pro-hepcidin (Hep) in maternal serum, coelomic fluid (CF) and amniotic fluid (AF) and by immunostaining for Hep in villous and secondary yolk sac biopsies. These samples were obtained from 15 first-trimester pregnancies at 8–11 weeks gestation. Transferrin concentrations were significantly lower in fetal (0.56 mg/ml) than maternal serum (1.71 mg/ml), with very low concentrations in CF and AF (0.09 mg/ml). In contrast, transferrin saturations were significantly higher in fetal (77%) than maternal serum (33%). NTBI was present in fetal serum, CF and AF, presumably as a consequence of low transferrin concentrations in these compartments. Pro-Hep was present at lower levels in fetal (140.0 ± 11.1) than maternal serum (206.2 ± 9.2) and at low concentrations in CF (19.4 ± 3.1) and AF (21.8 ± 5.2). Immunostaining with Hep antibody was found in the syncytiotrophoblast of first-trimester placenta as well as in mesothelial and endodermal layers of the secondary yolk sac at 10 weeks. The presence of Hep in syncytiotrophoblast cells of first-trimester placenta as well as in mesothelial and endodermal layers of the secondary yolk sac suggest a key regulatory role for this protein in iron transfer to the first-trimester fetus. The low transferrin concentrations and the presence of NTBI in CF and AF suggest that transferrin-independent iron transfer is important in early gestation

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.This work was supported by the Medical Research Council (MR/L020041/1), the Centre for Trophoblast Research, University of Cambridge and the Wellcome Trust (RG60992). M.Y.T. has received funding from the E.U. 7th Framework Programme for research, technological development and demonstration under grant agreement no PIEF-GA-2013-629785. J.H. was supported by a Wellcome Trust vacation scholarship. B.-K.K. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (101241/Z/13/Z) and receives a core support grant from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute

The role of oxidative stress and antioxidant supplementation in pregnancy disorders

Oxidative stress is widely implicated in failed reproductive performance including infertility, miscarriage, diabetes-related congenital malformations and preeclampsia. Maternal obesity is a strong risk factor for preeclampsia, and recently, in an animal model of maternal obesity we have reported evidence of oxidative stress in the oocytes of obese animals prior to pregnancy as well as in early stage embryos. This adds to the growing evidence for a greater focus on the pre-conceptual period in prevention of pregnancy disorders including those related to oxidative stress. Our research has also focussed on the role of free radicals and antioxidant capacity in preeclampsia. Assessment by measurement of markers of lipid peroxidation or of antioxidant capacity has provided unequivocal evidence for oxidative stress in this disorder. Partial failure of the process of placentation has been implicated, with recent evidence proposing that ischaemia-reperfusion in the placenta may contribute to oxidative stress in trophoblast. Endoplasmic reticulum stress in the placenta may also play a role. We and others have performed randomised controlled trials to determine whether early supplementation with vitamins C and E in women at risk of preeclampsia may be beneficial but these studies have shown no evidence for prevention of preeclampsia. Whether this represents an inappropriate antioxidant strategy or whether supplementation has been too late in gestation to be beneficial is not known. Other potential approaches to prevention of preeclampsia through amelioration of oxidative stress include provision of supplements in the pre-conceptual period, selenium supplements, anti-peroxynitrite strategies and statins

Differential Effects of Concomitant Use of Vitamins C and E on Trophoblast Apoptosis and Autophagy between Normoxia and Hypoxia-Reoxygenation

Concomitant supplementation of vitamins C and E during pregnancy has been reportedly associated with low birth weight, the premature rupture of membranes and fetal loss or perinatal death in women at risk for preeclampsia; however, the cause is unknown. We surmise that hypoxia-reoxygenation (HR) within the intervillous space due to abnormal placentation is the mechanism and hypothesize that concomitant administration of aforementioned vitamin antioxidants detrimentally affects trophoblast cells during HR.Using villous explants, concomitant administration of 50 microM of vitamins C and E was observed to reduce apoptotic and autophagic changes in the trophoblast layer at normoxia (8% oxygen) but to cause more prominent apoptosis and autophagy during HR. Furthermore, increased levels of Bcl-2 and Bcl-xL in association with a decrease in the autophagy-related protein LC3-II were noted in cytotrophoblastic cells treated with vitamins C and E under standard culture conditions. In contrast, vitamin treatment decreased Bcl-2 and Bcl-xL as well as increased mitochondrial Bak and cytosolic LC3-II in cytotrophoblasts subjected to HR.Our results indicate that concomitant administration of vitamins C and E has differential effects on the changes of apoptosis, autophagy and the expression of Bcl-2 family of proteins in the trophoblasts between normoxia and HR. These changes may probably lead to the impairment of placental function and suboptimal growth of the fetus