Tereza Cindrova-davies

ABSTRACT High-altitude pregnancy is a natural model for the effects of chronic mild hypobaric hypoxia on placental and fetal growth. Hypoxia is an important cause of complications of pregnancy e.g. intrauterine growth restriction.(1) Hypoxia increases cytotrophoblast (CTB) proliferation and apoptosis and decreases fusion in vitro . However, controversy exists over its effects in vivo.(2,3) For the first time, this study investigated the effect of high-altitude on cell proliferation, apoptosis and fusion in normal term placentae from non-native women living at sea-level (London) and high-altitude (Leadville, Colorado; 3100 m).

Background: Preeclampsia continues to be a prevalent pregnancy complication and underlying mechanisms remain controversial. A common feature of preeclampsia is utero-placenta hypoxia. In contrast to the impact of hypoxia on the placenta and fetus, comparatively little is known about the maternal physiology. Methods: We adopted an integrative approach to investigate the inter-relationship between chronic hypoxia during pregnancy with maternal, placental, and fetal outcomes, common in preeclampsia. We exploited a novel technique using isobaric hypoxic chambers and in vivo continuous cardiovascular recording technology for measurement of blood pressure in sheep and studied the placental stress in response to hypoxia at cellular and subcellular levels. Results: Chronic hypoxia in ovine pregnancy promoted fetal growth restriction (FGR) with evidence of fetal brain-sparing, increased placental hypoxia-mediated oxidative damage, and activated placental stress response pathways. These chang...

This depository contains all microscopy images presented in ‘Menstrual flow as a non-invasive source of endometrial organoids’ paper accepted at Communications Biology; Tereza Cindrova-Davies et al. present a method for deriving endometrial organoids using menstrual flow collected from women. The approach shows promise as a personalized and non-invasive way to investigate gynecological conditions such as endometriosis and failed implantation following IVF. Fig. 1b-d images present time line images detailing propagation of organoids from menstrual flow and scratch. Fig. 1b: The images represent the growth of menstrual organoids directly derived from menstrual blood. Fig. 1c-d: The images illustrate the growth of organoids from frozen menstrual (Fig. 1c) and scratch (Fig. 1d) digests of the same patient. Fig. 1e: Images depict organoids derived from an endometrial scratch and menstrual flow of the same patient; they are indistinguishable morphologically. Fig. 1f depicts single cell assay images to compare the growth of paired menstrual vs. scratch organoids derived from 4 patients. Images show organoids seeded from single cells (5,000 cells per 20 μl drop Matrigel) after 9d of growth. Fig. 1h shows representative images of immunostaining against the marker of proliferation, Ki67, in menstrual and scratch organoids of one patient. Fig. 3a images depict menstrual organoids seeded and grown for 4 d, followed by treatment with culture medium alone, or with hormones, which consisted of pre-treatment with β-estradiol (10 nM) for 2 d, followed by treatment with β-estradiol, progesterone (1 μM) and cAMP (1 μM) (EPC), EPC plus prolactin (20 ng/ml), or EPC plus prolactin (20 ng/ml), hPL (20 ng/ml) and hCG (100 ng/ml) for 4 d. At the end of each experiment, organoids were removed from Matrigel using Cell Recovery solution and processed for immunohistochemistry. The images show immunohistochemical upregulation of PAEP/GdA, acetylated tubulin, progesterone receptor (P4-R) and prolactin receptor (PRL-R) in response to hormones. Fig. 3e shows representative images of haematoxylin and eosin (H&E) staining of hormonally-treated menstrual and scratch organoids from one patient, showing morphological differences between untreated vs. hormonally treated organoids. Fig. 3f depics Bandeiraea simplicifolia -II (BSA-II) lectin staining of semi-thin resin sections of menstrual and scratch organoids treated with hormones. Suppl. Fig. 1: Derivation and characterisation of menstrual flow organoids. Time line images detailing propagation of organoids from menstrual flow and scratch. The top panel represents the growth of menstrual organoids directly derived from menstrual blood. The middle and bottom panels illustrate the growth of organoids from frozen menstrual and scratch digests of the same patient. Suppl. Fig. 2: Arachis hypogaea agglutinin (AHA) lectin staining of semi-thin resin sections of menstrual and scratch organoids treated with hormones. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Semi-thin resin sections were stained with AHA lectin. Suppl. Fig. 3: Periodic Acid Schiff (PAS) staining of menstrual and scratch organoids treated with hormones. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Paraffin-embedded sections were stained with PAS reagents to detect glycogen (purple). Arrows indicate purple glycogen staining, which is also secreted into the lumen of organoids treated with hormones. Suppl. Fig. 4: Haematoxylin and eosin (H&E) staining of hormonally-treated menstrual and scratch organoids from three patients. Menstrual and scratch organoids were grown for 4 d, and treated with culture medium alone or β-estradiol for 2 d followed by β-estradiol, progesterone and cAMP (EPC), EPC plus prolactin, or EPC plus prolactin, hPL and hCG for 4 d. Paraffin-embedded sections were stained with H&E reagents. The images show morphological differences between untreated vs. hormonally treated organoids from 3 patients. There is evidence of a columnar epithelial morphology with increased vacuole formation analogous to the hypersecretory phenotype of early pregnancy.Centre for Trophoblast Researc

opyright a 2013 American Society for Inve ublished by Elsevier Inc. All rights reserved ttp://dx.doi.org/10.1016/j.ajpath.2013.01.001 Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine b-synthase and cystathionine g-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms comparedwith preeclamptic placentas with normal waveforms and controls. These findingswere confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Dopp...

Preeclampsia is a complex multifactorial disease. Placental oxidative stress, a result of deficient spiral artery remodeling, plays an important role in the pathophysiology of preeclampsia. Antiangiogenic factors secreted from malperfused placenta are instrumental in mediating maternal endothelial dysfunction and consequent symptoms of preeclampsia; the mechanism is likely to involve increased ET-1 secretion and reduced NO bioavailability. Therapeutic interventions so far remain only experimental and there is no established remedy for the treatment of preeclampsia. This review concentrates on the evidence for the therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins. These compounds display pleitropic antioxidant, anti-inflammatory, and pro-angiogenic effects in animal and in vitro studies. Although clinical trials on the use of antioxidant vitamins in pregnancy proved largely unsuccessful, the scope for their use still exists given the beneficial card...

Introduction: Intrauterine growth restriction (IUGR) is a leading cause of perinatal morbidity and mortality, and is often associated with abnormal Doppler umbilical artery waveforms indicative of increased vascular resistance. The increased resistance is thought to involve stem villus arteries (SVAs), but how it develops remains unknown. Oxidative stress following deficient maternal spiral artery conversion is believed to be the primary insult in unexplained cases of IUGR and pre-eclampsia. Cystathionine-g-lyase (CSE) has been reported to be downregulated in SVAs during IUGR and pre-eclampsia1. Methods: SVA explants from uncomplicated human pregnancies delivered by elective Caesarean section were subjected to oxidative stress in vitro by cycles of hypoxia-reoxygenation (HR), or to normoxia in the presence or absence of CSE inhibitor propargylglycine (10 mM) for 1 or 3 days. Results: HR induced oxidative stress, and vascular smooth muscle (SM) dedifferentiation, with reduced expression of the contractile proteins and differentiation markers SM Myosin Heavy Chain and SM a-actin. SM dedifferentiation was accompanied by acquisition of a synthetic phenotype characterised by increased SM proliferation, and marked ECM remodelling, as evidenced by changes in collagen deposition. HR also reduced CSE expression in SVAs and produced a decline in H2S production of similar magnitude. CSE inhibition under normoxic conditions increased the level of oxidative stress in SVAs and produced changes in SM marker expression similar to those induced by HR. Under oxidative stress, changes in Hsp90 and catalase levels were paradoxically correlated with changes in CSE expression. Conclusions: Our results show that oxidative stress can trigger, via CSE downregulation and reduced H2S signalling, a positive feedback loop of signalling that drives SM dedifferentiation into a synthetic phenotype. This could result in vascular remodelling, leading to compromised SVA distensibility, and hence increased SVA and placental resistance in pathological pregnancies. 1. Cindrova-Davies et al. 2013 Am J Pathol 182,1448-1458.

Prostanoids can suppress vascular smooth muscle cell (VSMC) proliferation, but the mechanism through which this is mediated has not been identified. In this study, we show rat aortic VSMCs to express the EP 1 , EP 2 , EP 3 , EP 4 , and IP receptors. The EP 4 receptor–specific agonist, 11-deoxy-PGE 1 , induced a time-dependent phosphorylation of protein kinase C and extracellular signal-regulated kinase (ERK) 1/2 in serum-depleted (0.1%) VSMCs, whereas the EP 2 receptor agonist, butaprost, was without effect. PGI 2 or iloprost at the IP receptor inhibited basal ERK phosphorylation with IC 50 values of ≈10 nmol/L. Iloprost also attenuated the sustained activation of ERK induced by endothelin-1 or basic fibroblast growth factor (bFGF). Endothelin-1 or bFGF significantly increased the number of VSMCs counted 24 hours later compared with basal, and both responses were blocked by the MEK inhibitor, U0126, or iloprost. Under basal conditions, U0126 or iloprost reduced the number of viable ...

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of ‘uterine milk’ proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of comm...

Development of the human placenta takes place in contrasting oxygen concentrations at different stages of gestation, from ~20 mmHg during the first trimester rising to ~60 mmHg at the start of the second trimester before gradually declining to ~40 mmHg at term. In view of these changes, the early placenta has been described as ‘hypoxic’. However, placental metabolism is heavily glycolytic, supported by the rich supply of glucose from the endometrial glands, and there is no evidence of energy compromise. On the contrary, the trophoblast is highly proliferative, with the physiological low-oxygen environment promoting maintenance of stemness in progenitor populations. These conditions favour the formation of the cytotrophoblastic shell that encapsulates the conceptus and interfaces with the endometrium. Extravillous trophoblast cells on the outer surface of the shell undergo an epithelial-mesenchymal transition and acquire invasive potential. Experimental evidence suggests that these c...

The placenta responds to adverse environmental conditions by adapting its capacity for substrate transfer to maintain fetal growth and development. The effects of early-onset hypoxia on placental morphology and activation of the unfolded protein response (UPR) were determined using an established rat model in which fetal growth restriction is minimized. We further established whether maternal treatment with the mitochondria-targeted antioxidant (MitoQ) confers protection during hypoxic pregnancy. Wistar dams were exposed to normoxia (21% O) or hypoxia (13% to 14% O) from days 6 to 20 of pregnancy with and without MitoQ treatment (500 μM in drinking water). On day 20, animals were euthanized and weighed, and the placentae from male fetuses were processed for stereology to assess morphology. Activation of the UPR in additional cohorts of frozen placentae was determined with Western Blotting. Neither hypoxic pregnancy nor MitoQ treatment affected fetal growth. Hypoxia increased placent...

Premature ageing has been implicated in placental dysfunction. Senescence can be activated by oxidative stress, a key intermediary in the pathophysiology of pre-eclampsia. We examined senescence markers across normal gestation, and in pathological and post-mature pregnancies. Inducers of oxidative stress were used to mimic senescence changes in term explants. Placental samples were collected with ethical approval and informed consent: first and second trimester samples from surgical terminations; term and pre-term controls, and early-onset pre-eclampsia samples from caesarean deliveries. Paraffin and EM blocks of post-mature placentas were from an archival collection. Term explants were subjected to hypoxia-reoxygenation (HR) or hydrogen peroxide (HO). p21 was increased significantly in term homogenates compared to first and second trimester samples, and was significantly higher in PE compared to term controls. Immunostaining revealed nuclear localisation of p21 and phosphorylated h...

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 bi...

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...

Intrauterine fetal growth restriction (IUGR) is often associated with compromised umbilical arterial flow, indicating increased placental vascular resistance. Oxidative stress is causatively implicated. Hydrogen sulfide maintains differentiated smooth muscle in vascular beds, and its synthetic enzyme cystathionine-γ-lyase (CSE) is down-regulated in growth-restricted placentas. We hypothesized that remodeling of resistance arteries in stem villi contributes to IUGR by compromising umbilical blood flow via oxidative stress, reducing hydrogen sulfide signaling. Stem villus arteries in human IUGR placentas displaying absent or reversed end-diastolic flow contained reduced myosin heavy chain, smooth muscle actin, and desmin, and increased markers of dedifferentiation, cellular retinol-binding protein 1, and matrix metalloproteinase 2, compared to term and preterm controls. Wall thickness/lumen ratio was increased, lumen diameter decreased, but wall thickness remained unchanged in IUGR pl...

Low maternal circulating concentrations of placental growth factor (PlGF) are one of the hallmarks of human pregnancy complications, including fetal growth restriction (FGR) and early-onset pre-eclampsia (PE). Currently, PlGF is used clinically with other biomarkers to screen for high-risk cases, although the mechanisms underlying its regulation are largely unknown. Placental endoplasmic reticulum (ER) stress has recently been found to be elevated in cases of FGR, and to an even greater extent in early-onset PE complicated with FGR. ER stress activates the unfolded protein response (UPR); attenuation of protein translation and a reduction in cell growth and proliferation play crucial roles in the pathophysiology of these complications of pregnancy. In this study, we further identified that ER stress regulates release of PlGF. We first observed that down-regulation of PlGF protein was associated with nuclear localization of ATF4, ATF6α and ATF6β in the syncytiotrophoblast of placenta...

A functioning placental renin angiotensin system (RAS) appears necessary for uncomplicated pregnancy and is present during placentation, which occurs under low oxygen tensions. Placental RAS is increased in pre-eclampsia (PE), characterised by placental dysfunction and elevated oxidative stress. We investigated the effect of high-altitude hypoxia on the RAS and hypoxia-inducible factors (HIFs) by measuring mRNA and protein expression in term placentae from normotensive (NT) and PE women who delivered at sea level or above 3100 m; using an explant model of hypoxia-reoxygenation to assess the impact of acute oxidative stress on the RAS and HIFs. Protein levels of prorenin (P = 0.049), prorenin receptor (PRR; P = 0.0004), and angiotensin receptors (AT1R, P = 0.006) and (AT2R, P = 0.002) were all significantly higher in placentae from NT women at altitude, despite mRNA expression being unaffected. However, mRNA expression of all RAS components was significantly lower in PE at altitude t...

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(+), la...

The maternal circulation to the human placenta is not fully established until 10-12 weeks of pregnancy. During the first trimester the intervillous space is filled by a clear fluid, in part derived from secretions from the endometrial glands via openings in the basal plate. The aim was to determine the activity of the glands throughout the first trimester, and to identify components of the secretions. Samples of human decidua basalis from 5-14 weeks gestational age were examined by transmission electron microscopy and immunohistochemically. An archival collection of placenta-in-situ samples was also reviewed. The thickness of the endometrium beneath the implantation site reduced from approximately 5 mm at 6 weeks to 1 mm at 14 weeks of gestation. The glandular epithelium also transformed from tall columnar cells, packed with secretory organelles, to a low cuboidal layer over this period. The lumens of the glands were always filled with precipitated secretions, and communications wit...

Key points  High‐altitude pregnancy is associated with reduced oxygenation and placental complications, which can affect maternal and fetal outcome. However, most high‐altitude populations are also impoverished and because maternal undernutrition itself is known to promote placental problems, the extent to which complications during high‐altitude pregnancy could be due to maternal oxygen and/or nutrient restriction remains unclear. The aim of the study was to investigate whether reduced placental oxygenation, independent of maternal undernutrition, increases maternal and placental oxidative stress and whether maternal treatment with vitamin C is protective. The study shows that hypoxic pregnancy increased maternal circulating and placental molecular indices of oxidative stress. Maternal vitamin C treatment was protective and increased birth weight. The study offers insight to mechanism and intervention against the effects of high altitude on pregnancy.