Gotowa bibliografia na temat „Placental hypoxia”

Abstract Context.—Two relatively unknown and recently described placental membrane hypoxic lesions (laminar necrosis and microscopic chorionic pseudocysts) have never been compared with time-honored, focal (infarction), and diffuse hypoxic lesions of placental parenchyma. Objective.—To compare the effect on placental diagnosis of the above placental membrane hypoxic lesions and chorionic disc hypoxic lesions (infarctions and global hypoxic pattern of placental injury). Design.—Twenty-three clinical (maternal and fetal) and 32 gross and microscopic placental features were retrospectively compared in 4590 placentas from a placental database built during a 13-year period: 168 placentas with at least one hypoxic disc lesion (infarct or global hypoxia) and at least one membrane lesion (microscopic chorionic pseudocysts or laminar necrosis (group 1), 750 placentas with at least one hypoxic villous lesion but no membrane lesion (group 2), 480 placentas with at least one membrane lesion but no villous lesion (group 3), and 3192 placentas with no hypoxic villous or membrane lesions (group 4). Results.—Several clinical and fetal conditions and placental features known to be associated with in utero hypoxia had a statistically significant correlation with the index hypoxic placental lesions, both villous and membranous. Of placentas from patients associated with clinical conditions at risk for hypoxia, 15% featured only hypoxic membrane lesions without a chorionic disc hypoxic lesion. Conclusions.—Recognizing placental membrane hypoxic lesions increases the sensitivity of placental examination in diagnosing placental hypoxia by at least 15%. The risk of in utero hypoxia is increased when microscopic chorionic pseudocysts and laminar necrosis occur in conjunction with villous hypoxic lesions.

Vascularity is increased in placentas from high- compared with low-altitude pregnancies. An angiogenic response to hypoxia may protect an organ from further hypoxic insult by increasing blood flow and oxygen delivery to the tissue. We hypothesized that increased placental vascularity is sufficient to adapt to high altitude. Therefore, indexes of hypoxic stress would not be present in placentas from successful high-altitude pregnancies. Full-thickness placental biopsies were 1) collected and frozen in liquid nitrogen within 5 min of placental delivery and 2) fixed in formalin for stereologic analyses at high (3,100 m, n = 10) and low (1,600 m, n = 10) altitude. Hypoxia-inducible transcription factor (HIF-1) activity was analyzed by ELISA. Western blot analyses were used to evaluate HIF-1α, HIF-1β, HIF-2α, von Hippel-Lindau protein, VEGF, Flt-1, enolase, and GAPDH. Magnetic resonance spectroscopy was used to evaluate endogenous metabolism. The ratio of placental capillary surface density to villous surface density was 70% greater at high compared with low altitude. HIF-1 activity and HIF-1-associated proteins were unchanged in placentas from high- vs. low-altitude pregnancies. Placental expression of HIF-1-mediated proteins VEGF, Flt-1, enolase, and GAPDH were unchanged at high vs. low altitude. Succinate, GSH, phosphomonoesters, and ADP were elevated in placenta from high compared with low altitude. Placentas from uncomplicated high-altitude pregnancies have greater vascularity and no indication of significant hypoxic stress at term compared with placentas from low altitude.

Apoptosis (programmed cell death) in the human placenta is likely to play a major role in determining the structure and function of that organ. Fetal growth restriction (FGR) has been shown to be associated with increased levels of placental apoptosis. Altered regulation of apoptosis may play an important pathophysiological role in FGR. As reduced placental perfusion and reduced oxygenation are features of FGR, one aim of this study was to determine the effects of hypoxia on apoptotic activity, as assessed by DNA laddering, of placental tissue in vitro. In addition, levels of placental apoptosis may be affected by pharmacological agents routinely used in obstetric patient management. Thus an additional aim of this study was to determine the effects of several relevant pharmacological agents on the levels of DNA laddering during in vitro incubation of human placentae under hypoxic conditions. Incubation of normal placental explant tissue at 37 °C for 1–2 h under hypoxic conditions significantly increased placental DNA laddering compared with that in non-incubated tissue, whereas levels of DNA laddering during incubation for up to 2 h under normoxic conditions were not significantly higher than those in non-incubated tissue. The DNA laddering activity of placental explants after 2 h of incubation under hypoxic conditions was significantly increased with increased concentrations of magnesium, but remained unchanged by the inclusion of pethidine, aspirin, nifedipine, dexamethasone, heparin or indomethacin in the incubation mixture. These results suggest that hypoxia may stimulate apoptotic activity in cultured human placental tissues, and that hypoxia-stimulated placental apoptosis may be further increased by increasing the extracellular magnesium concentration.

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 changes were linked with dilation of the placental endoplasmic reticulum (ER) cisternae and increased placental expression of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin), combined with a shift towards an angiogenic imbalance in the maternal circulation. Chronic hypoxia further led to an increase in uteroplacental vascular resistance and the fall in maternal blood pressure with advancing gestation measured in normoxic pregnancy did not occur in hypoxic pregnancy. Conclusions: Therefore, we show in an ovine model of sea-level adverse pregnancy that chronic hypoxia recapitulates physiological and molecular features of preeclampsia in the mother, placenta, and offspring.

Relevance. An in-depth study of the state of feto-placental complex, the characteristics of its reactions in response to hypoxia on the background of juvenile immaturity of minors can help to identify the main directions of the prevention of certain complications of pregnancy and childbirth among minors in the first place — placental insufficiency, preeclampsia, fetal hypoxia. Purpose of the study was to explore features of the histological structure of placentas and expression of HIF-1α and VEGF-A markers in placentas in minors.Materials and methods. 74 placentas from minors were investigated by histological methods. The comparison group consisted of 25 placentas from healthy women the average reproductive age (20-30 years). The immunohistochemical study was performed in 35 placentas from minors who were divided into groups according to age and the presence or absence of chronic placental insufficiency. To study vascular status of villous tree of placentas were used the monoclonal antibodies of the vascular endothelial growth factor VEGF-A (Clone UG1; 1 : 50, Dako), to assess hypoxia in placenta were used the monoclonal antibodies of hypoxia — inducible factor HIF-1α (H1 alpha 67; 1 : 100, GRTP (Abcam).Results. It was revealed that the placenta in minors is characterized by hypertrophy (559,5 ± 10,5 g vs. 478,7 ± 12,9 g), it is pathologically immature (33,8% vs. 7,0%), there is significantly greater frequency of chronic placental insufficiency in minors (33,8% versus 4,0%) with significantly lower severity of compensatory-adaptive reactions (86,5% vs. 100%). It is shown increased expression of hypoxia — inducible factor (HIF-1) and vascular endothelial growth factor-A (VEGF-A) in placentas of minor women with chronic placental insufficiency, compared with placentas of minor women without chronic placental insufficiency.Conclusion. Chronic placental insufficiency at the time of birth, detectable more frequently in minor patients can make a difference in violation of adaptive mechanisms and quality of life of children in the postnatal period. Increased expression of HIF-1α and VEGF-A in the placentas of minor women with chronic placental insufficiency reflects activation of angiogenesis in response to intrauterine fetal hypoxia in chronic placental insufficiency.

Context.—In utero hypoxia is an important cause of perinatal morbidity and mortality and can be evaluated retrospectively to explain perinatal outcomes, to assess recurrence risk in subsequent pregnancies, and to investigate for medicolegal purposes by identification of many hypoxic placental lesions. Definitions of some placental hypoxic lesions have been applied relatively liberally, and many of them are frequently underreported. Objectives.—To present a comprehensive assessment of the criteria for diagnosing acute and chronic histologic features, patterns, and lesions of placental and fetal hypoxia and to discuss clinicopathologic associations and limitations of the use thereof. The significance of lesions that have been described relatively recently and are not yet widely used, such as laminar necrosis; excessive, extravillous trophoblasts; decidual multinucleate extravillous trophoblasts; and, most important, the patterns of diffuse chronic hypoxic preuterine, uterine, and postuterine placental injury and placental maturation defect, will be discussed. Data Sources.—Literature review. Conclusions.—The placenta does not respond in a single way to hypoxia, and various placental hypoxic features should be explained within a clinical context. Because the placenta has a large reserve capacity, hypoxic lesions may not result in poor fetal condition or outcome. On the other hand, very acute, in utero, hypoxic events, followed by prompt delivery, may not be associated with placental pathology, and many poor perinatal outcomes can be explained by an etiology other than hypoxia. Nevertheless, assessment of placental hypoxic lesions is helpful for retrospective explanations of complications in pregnancy and in medicolegal investigation.

Local tissue oxygenation profoundly influences placental development. To elucidate the impact of hypoxia on cellular and molecular adaptation in vivo, pregnant mice at embryonic days 7.5–11.5 were exposed to reduced environmental oxygen (6–7% O2) for various periods of time. Hypoxia-inducible factor (HIF)-1α mRNA was highly expressed in the placenta, whereas HIF-2α was predominantly found in the decidua, indicating that HIF-1 is a relevant oxygen-dependent factor involved in placental development. During severe hypoxia, HIF-1α protein was strongly induced in the periphery but, however, not in the labyrinth layer of the placenta. Accordingly, no indication for tissue hypoxia in this central area was detected with 2-(2-nitro-1 H-imidazol-1-yl)- N-(2,2,3,3,3-pentafluoropropyl)acetamide staining and VEGF expression as hypoxic markers. The absence of significant tissue hypoxia was reflected by preserved placental architecture and trophoblast differentiation. In the search for mechanisms preventing local hypoxia, we found upregulation of endothelial nitric oxide synthase (NOS) expression in the labyrinth layer. Inhibition of NOS activity by Nω-nitro-l-arginine methyl ester application resulted in ubiquitous placental tissue hypoxia. Our results show that placental oxygenation is preserved even during severe systemic hypoxia and imply that NOS-mediated mechanisms are involved to protect the placenta from maternal hypoxia.

Preeclampsia is a pregnancy-specific disease characterised by de novo development of concurrent hypertension, proteinuria and oxidative stress in the placenta. In the placenta, intervillous blood flow increases after 10 weeks of gestation and results in exposure of trophoblast cells to oxygen. Hypoxia occurs during the development of placenta in the first trimester and is implicated in trophoblast differentiation. Ca2+ is a universal intracellular second messenger involved in many processes such as signal transduction, hormone secretion and programmed cell death. Human placental primary cell cultures were established from first-trimester human placentas (at 7 to 12 weeks of gestation). In this study, calcium-related proteins (CRPs; TRPV6, PMCA1, NCKX3 and CaBP-28k) were investigated at normoxia (5% CO2 in 95% air) or hypoxia (2% O2/93% N2/5%CO2) for 12 h in human placental cell line (BeWo) and human placental primary cell (hPC). We confirmed mRNA expression by real-time PCR and protein expression by Western blot analysis. The data were 2 or 3 individual experiments with triplicate samples and analysed by one-way ANOVA using Tukey's multiple comparison test. In hypoxia, the level of TRPV6 mRNA and protein was not changed, however, calcium transporters' (NCKX3, CaBP-28k) mRNA and protein expressions were significantly increased in hypoxic BeWo cell compared with control (normoxia). In addition, expression of PMCA1 mRNA and protein was decreased in hypoxic BeWo cells. In hPC, CRPs (TRPV6, PMCA1, NCKX3 and CaBP-28k) mRNA and protein expressions were significantly induced by hypoxic stress compared with control. These results, taken together, indicate that alterations of calcium transporters in hypoxic stress may be involved in calcium transport in the placenta and protection of the placental trophoblasts from the oxidative stress during the pregnancy.

BACKGROUND Prematurity refers to live births before 37 weeks of gestation, wherein the baby is born before the body and its organ systems achieve perfect maturity, and this disorder is still a global problem. The high incidence of prematurity is a problem in developing and also in developed countries. Certain conditions accompanying pregnancies like preeclampsia, infection, and placental insufficiency, may trigger uterine hypoxia, causing premature birth. The placental condition is related to the intra-uterine fetal condition. In prolonged placental hypoxia, there occurs a compensatory mechanism, i.e. an increase in placental angiogenesis. This study aimed to evaluate the effect of hypoxia on fetal blood vessel count as compensatory mechanism for tissue hypoxia. METHODS An observational-analytical cross-sectional design using paraffin blocks of conserved premature placentas, comprising 31 samples of hypoxic premature placentas and 28 samples of non-hypoxic premature placentas, selected using non-random consecutive sampling. The samples were made into slides and stained with hematoxylin-eosin for assessment of histological structure, including fetal blood vessel count and integrity, villus conditions, syncytiotrophoblastic nuclear changes, and syncytiotrophoblastic nuclear aggregation. Mann-Whitney test was used to compare the difference of blood vessel count between groups. RESULTS Assessment of histological structure showed a significant increase in fetal blood vessel count in the hypoxic group [8.00 (5-15)] as compared with the non-hypoxic group [7.50 (3-15)]. CONCLUSION The hypoxia in premature placentas caused an increase in the number of fetal blood vessels as a form of compensation for disturbed oxygen homeostasis.

Prematurity refers to live births before 37 weeks of gestation and associated with infant morbidity/mortality. Activation of HIF during the final pregnancy phase is believed to play a critical role in the pathogenesis of premature birth and other pregnancy disorders. This study aimed to analyze the relationship between hypoxicstatus and the intensity of HIF-1α expression in a premature placenta.Stored biological materials premature placenta (paraffin blocks) was used in this study. Thirtyone samples of placental hypoxia (H) and 28 samples of premature placental non-hypoxia (N) as controls, were selected non-random consecutively. Subsequently, immunohistochemistry was performed to analyze HIF-1α expression. TheChi-square testwas used to analyze the data and a p-value <0.05 was considered statistically significant.Moderate to strong intensity of HIF-1α expressionwas observed in 58% of hypoxic placenta samples, whereas most of non-hypoxic placental samples(86%) did not expressed or expressed weaklyHIF-1α.There was a significant correlation between the intensity of HIF-1α expression and placental hypoxia (p <0.05) and Odds Ratio (OR) value was 8.31 with a 95% confidence interval (2.32-29.77). The conclusion shows that hypoxic status is associated with intensity of hypoxia inducible factor (HIF)-1α expression in a premature placenta.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietaty selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

Oxidative stress of the placenta is considered a key intermediary step in the pathogenesis of preeclampsia, but the cause for the stress remains unknown. Ischaemia-reperfusion injury, as a result of intermittent placental perfusion secondary to deficient trophoblast invasion of the endometrial arteries, is a possible mechanism. This thesis therefore tests whether hypoxia-reoxygenation (H/R) in vitro can induce placental oxidative stress, and cause increased apoptosis and production of tumour necrosis factor-α as seen in the preeclamptic placenta. The first aim was to examine the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species (ROS) was detected using a fluorescent marker when hypoxic villous samples were reoxygenated. The expression of oxidative stress markers including nitrotyrosine residues, 4-hydroxy-2-nonenal adducts, and inducible heat shock protein 72 was greatly increased in villous samples subjected to H/R compared to the controls maintained under constant hypoxia. Furthermore, preloading villous samples with ROS scavengers such as desferrioxamine and α-phenyl-N-tert-butylnitrone significantly reduced the levels of oxidative stress in H/R. Having demonstrated that in vitro H/R is capable of inducing oxidative stress in a reproducible and manipulable manner, investigations were next carried out to study the effects of resultant oxidative stress on apoptosis within the trophoblast. Compared to hypoxic and normoxic controls, there was a significant increase in the release of cytochrome c from mitochondria, activation of caspase , and cleavage of poly (ADP-ribose) polymerase in villous samples subjected to H/R. These events were associated with an increased number of syncytiotrophoblastic nuclei displaying apoptotic changes and increased lactate dehydrogenase release into the medium. The causal relationship between the generation of ROS and these apoptotic changes was revealed by the fact that pre-administration of desferrioxamine attenuated the insult.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Intrauterine Growth Restriction (IUGR) is a pregnancy-related pathology characterized by a placental insufficiency phenotype and a multifactorial etiology that still needs to be completely clarified. IUGR is associated with increased risk of maternal and neonatal perinatal mortality and morbidity and a tendency to develop cardiovascular and metabolic pathologies in the adulthood. A deeper knowledge of the alterations occurring in IUGR has therefore become essential to find therapeutic tools to prevent fetal, neonatal and future adult complications. A specific placental phenotype has been associated with IUGR, characterized by placentation defects, altered transport of oxygen and nutrients to the fetus, impaired mitochondria content and increased oxidative stress (OxS). Mitochondria (mt) are eukaryotic ubiquitous organelles whose number range from hundreds to thousands of copies per cell. As they are the fuel stations of all cells, more than 95% of ATP is synthesized in these organelles Besides this well-known function, many essential pathways involve mitochondria, such as mt biogenesis. Mt biogenesis is a complex of mechanisms needed to mitochondria ex-novo creation: mt DNA duplication and translation of mt factors controlling the transcription machinery that produce all respiratory chain complexes (RCC). IUGR hypoxic features, and the consequent higher OxS, affect mitochondria as showed by in vivo models increased mt oxygen consumption trigger by hypoxia or in vitro downregulation of mt biogenesis. The aim of this study was to investigate, by ex vivo experiments and in vitro models, different types of placental cells to deeper characterize the placental insufficiency features of IUGR, with specific attention to the consequences of its hypoxic environment. IUGR and physiological placenta bioenergetics were first examined, by analyzing both mitochondrial (mt) content and function in whole placental tissue and in several placental cell types (cytotrophoblast and mesenchymal stromal cells). Mt DNA content resulted higher in IUGR placentas compared to controls, as well as NRF1 (biogenesis activator) mRNA levels. Oppositely, both mtDNA and NRF1 expression levels were significantly lower in cytotrophoblast cells isolated from IUGR placentas compared to controls. The observed divergence between placental tissue and cytotrophoblast cells may suggest that other placental cell types (e.g. syncytiotrophoblast, endothelial cells and mesenchymal stromal cells), that are subjected to different oxygen - and consequently oxidative stress - levels may be responsible for the mt content increase in the whole placental tissue. Moreover, a different exposure to progesterone may also explain this mt content divergence, since progesterone, regulating mt biogenesis, is produced by syncytio but not in cytotrophoblast cells. In IUGR cytotrophoblast cells, respiratory chain complexes (RCC) showed lower, though not significantly, gene expression levels and no differences in their protein expression compared to controls. In contrast, mt bioenergetics - represented by cellular O2 consumption - was higher in IUGR versus controls, especially in more severe IUGR cases. Thus, despite the protein content of RCC was not altered, their activity was significantly increased in IUGR cytotrophoblast cells, possibly due to a more efficient RCC assembly. Finally, as O2 consumption resulted inversely correlated to mtDNA in cytotrophoblast cells, a functional (respiration) compensatory effect to the decreased mitochondrial content might be hypothesized. Estrogen-Related Receptor (ERRγ) is a very interesting transcriptional factor involved both in mt biogenesis and function and in estradiol production (through CYP19 aromatase up-regulation). ERRγ and CYP19 mRNA levels were therefore analyzed, for the first time in human IUGR placentas. In whole placental tissue CYP19 showed higher expression in IUGR compared to controls, progressively increasing with IUGR severity. Higher ERRγ expression in IUGR cases was also found, though not significantly. These data are consistent with mtDNA and NRF1 results, thus confirming altered mt biogenesis and content in IUGR and strengthening the hypothesis of a restore attempt made through the stimulation of mt biogenesis. An additional effect of ERRγ increase is CYP19 upregulation. The observed higher CYP19 expression may indicate a protective mechanism exerted through estradiol against oxidative stress. Opposite to their placental tissue expression, ERRγ levels in cytotrophoblast cells significantly decreased in the IUGR group compared to controls. This is consistent with literature evidences of O2-dependent ERRγ gene expression in trophoblast cells. As well as for mt DNA and NRF1 levels, other cell types could be responsible for ERRγ increase in the whole placental tissue. CYP19 expression was not significantly different between IUGR and controls in cytotrophoblast cells, though it positively correlates with ERRγ levels, but low CYP19 levels are reported for cytotrophoblast cells, and this might complicate the detection of any difference. Interestingly, a significant positive correlation linked maternal BMI and expression of both ERRγ and CYP19 genes (in whole placental tissue: positive trend/cytotrophoblast cells: negative trend). An estradiol-dependent regulation of leptin production through ER (Estrogen Receptor) – ERR is known. Leptin, an anti-obesity hormone produced also by placenta, increase during. The future measure of plasmatic levels of both leptin and 17β estradiol in maternal blood will verify this speculation. Then in vitro experiments were performed to assess possible biomolecular mechanisms regulating mithocondrial content in Intrauterine Growth Restriction, by culturing primary placental cells under normal oxygen conditions and hypoxia, a typical feature of IUGR. Fluctuations in placental oxygen concentration may generate oxidative stress (OxS), that is enhanced in Intrauterine Growth Restriction condition. As mitochondria are the major producers of intracellular reactive oxygen (O2) species through free radicals generated by the mt oxidative phosphorylation, altered intrauterine O2 conditions might affect mt DNA content and function, leading to increased oxidative stress in IUGR placental cells. Using trophoblast primary cell lines could help to understand O2 conditions that placentas may be exposed to in IUGR pregnancy. Exposure of trophoblast cultures to hypoxia is an in vitro model commonly used in the last few years. Preliminary data from performed experiments show that the oxygen lack in cytotrophoblast cells leads to increased mt DNA levels. The evidence that O2 levels may regulate mt biogenesis in cytotrophoblast cells highlights their deep sensitivity to O2 conditions. However, further data are needed to confirm these preliminary results, also considering the implied difficulties in adapting the primary cytotrophoblast cultures, very sensitive to O2 concentration, to an in vitro model. A future goal will be reproduce particularly hypoxia/re-oxygenation intervals characterizing placental insufficiency and generating OxS and measuring cell apoptosis levels and autophagy markers (e.g. TNF-α, p53, caspases). Finally, in vitro experiments were performed to isolate and characterized p-MSCs from physiological and affected by IUGR placentas. p-MSCs have never been investigated before in IUGR pregnancies, but their role have been recently studied in preeclamptic placentas. PE p-MSCs show pro-inflammatory and anti-angiogenic features, that may result in abnormal placental development. In the performed p-MSCs cultures, mesenchymal markers enrichment and multipotent differentiation abilities confirm the successful isolation and selection of a mesenchymal stromal cell from placental membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the p-MSC population is earlier selected in IUGR placentas: this faster selection might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment. During placenta development, the lower proliferation rate characterizing IUGR pMSCs could impair the primary villi formation and consequently trophoblast development, since MSCs both serve as structural of trophoblast cells. Moreover, IUGR p-MSCs population display lower endothelial and higher adipogenic differentiation potentials compared to controls. During pregnancy, pMSCs usually contribute to both vasculogenesis and angiogenesis Interestingly, several studies report some alterations in maternal and fetal endothelial progenitor or in the angiogenic capacity of IUGR placental cells. Opposite to endothelial differentiation ability, the adipogenic potential in pMSCs from IUGR is increased compared to controls: as these changes are evident early in life, the predisposition to obesity may be programmed in utero. To further characterize IUGR pMSCs, their mitochondrial (mt) content was investigated by measuring NRF1 and Respiratory Chain UQCRC1 and COX4I1 gene expression levels. Mesenchymal stem cell metabolism is known to be mainly anaerobic, with a shift towards an aerobic mitochondrial metabolism reported during differentiation. Interestingly, p-MSCs cultured with no differentiating medium present a trend towards higher NRF1, UQCRC1 and COX4I1 expression levels in IUGR basal disc samples compared to controls and higher COX4I1 levels in IUGR placental membranes; these differences are not statistically significant likely because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR p-MSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells. The different gestational age between cases and controls, typical of all IUGR versus term-placentas studies, is a possible limit that associate all the performed experiments. However, any significant correlation between gestational age (ge) and the O2 consumption of CIV (which presents the highest significance between IUGR and controls), ge and mt DNA levels, ge and ERRy/CYP19 expression, ge and p-MSCs. CYP19 gene expression have been analyzed assuming that it may represent an index of aromatase content in placental tissue. However, post-translational modifications (glycosylation and phosphorylation) may occur, affecting its functional activity. Finally, a potential limitation of placental mesenchymal stromal cells is that the analysis was performed on IUGR placentas at delivery, whereas placental abnormal development of IUGR pathology is supposed to start already at the beginning of placentation. Taken together, reported data highlight mitochondrial alterations occurring in placentas of Intrauterine Growth Restricted pregnancies, through ex vivo and in vitro approaches. These results shed genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. Mitochondrial content is higher in IUGR total placental tissue compared with normal pregnancies at term. This difference is reversed in cytotrophoblast cells of IUGR fetuses, which instead present higher mitochondrial functionality. These findings suggest different mitochondrial features depending on the placental cell lineage. Indeed, our results on placental Mesenchymal Stromal Cells, showed higher levels of genes accounting for mitohcondrial content and function. The increased placental O2 consumption by placental tissue may represent a limiting step in fetal growth restriction, preventing adequate O2 delivery to the fetus. This limitation has potential consequences on fetal O2 consumption both in animal models and in human IUGR.

Objetivo: Avaliar a relação entre os parâmetros da cardiotocografia computadorizada (cCTG) e da dopplervelocimetria com a lesão miocárdica fetal e com a ocorrência de acidemia no nascimento, em gestações com insuficiência placentária. Métodos: Estudo prospectivo com 49 gestações complicadas pela insuficiência placentária (Doppler de artéria umbilical anormal - índice de pulsatilidade [IP] > p95) diagnosticada entre 26 e 34 semanas. Todas as pacientes foram avaliadas pelo Doppler de artéria umbilical, artéria cerebral média e ducto venoso e pela cCTG (Sonicaid FetalCare, versão 2.2, por 30 minutos). Foi analisada a última avaliação fetal até 48h antes do parto e anterior à corticoterapia. Foi analisado o sangue de cordão umbilical no parto, para detectar a acidemia no nascimento (pH < 7,20) e a lesão miocárdica fetal (Troponina T cardíaca [cTnT] >= 0,09 ng/mL). A cTnT foi obtida em 38 casos e o pH em 46 casos. Resultados: Quinze (39,5%) recém-nascidos apresentaram cTnT >= 0,09 ng/ml e 20 (43,5%) pH < 7,20. Os fetos que evoluíram com acidemia apresentaram menor número de movimentos por hora na cCTG (mediana 2 vs. 15, p=0,019). Houve correlação positiva entre o pH e o número de movimentos fetais por hora (rho=0,35; P=0,019) e com a frequência cardíaca fetal basal (rho 0,37, P=0,011), e correlação negativa entre o pH e o escore zeta do IP para veias (IPV) do ducto venoso (rho= -0,31, P=0,036). A regressão logística identificou o escore-zeta do IPV do ducto venoso (P=0,023) e a frequência cardíaca fetal basal (P=0,040) como variáveis independentes associadas com a acidemia no nascimento. A ocorrência de lesão miocárdica fetal, quando comparada ao grupo com cTnT normal, apresentou associação significativa com o escore zeta do IP da artéria umbilical (mediana 8,8 vs. 4,0; P=0,003), IPV do ducto venoso (mediana 2,6 vs. -1,4; P= 0,007), frequência cardíaca fetal basal (mediana 146 vs. 139 bpm; P=0,033), número de acelerações entre 10-15 bpm (mediana 0 vs. 1; P=0,013), duração dos episódios de baixa variação (mediana 21 vs. 10 min; P=0,038) e a variação de curto prazo (short-term variation-STV) (mediana 3,7 vs. 6,1 ms; P=0,003). Observou-se correlação positiva entre o valor da cTnT no cordão umbilical e a frequência cardíaca fetal basal (rho=0,33; P=0,042), e correlação negativa entre a cTnT e a STV (rho= -0,37; P=0,021). A regressão logística identificou a STV como fator preditor independente para o dano miocárdico fetal (P=0,01), sendo a STV <= 4,3 ms o melhor ponto de corte para predição do evento (sensibilidade de 66,7% e especificidade de 91,3%). Conclusão: Em gestações com insuficiência placentária detectada antes da 34ª semana gestacional, o IPV do ducto venoso e a frequência cardíaca fetal basal analisada pela cCTG são os preditores independentes associados com a acidemia no nascimento; e o valor da STV avaliada pela cCTG é a variável que melhor prediz a lesão miocárdica fetal. A cCTG é ferramenta importante no manejo de fetos com insuficiência placentária, principalmente quando associada a outros métodos propedêuticos como a dopplervelocimetria
Objective: To evaluate the reliability of fetal heart rate parameters analyzed by computerized cardiotocography (cCTG) and fetal Doppler to predict myocardial damage and acidemia at birth in pregnancies complicated by placental insufficiency. Methods: Forty nine patients with placental insufficiency (abnormal umbilical artery Doppler - pulsatility index [PI] > p95) diagnosed between 26-34 weeks of gestation were prospectively studied. All patients were submitted to Dopplervelocimetry of umbilical artery, middle cerebral artery and ductus venosus and to the cCTG (Sonicaid Fetal Care, version 2.2; 30 minutes of duration). We analyzed the last fetal assessment 48h before delivery and prior to steroid therapy.Umbilical cord blood samples were collected at birth to detect acidemia (pH < 7.20) and myocardial damage (cTnT >= 0.09 ng/ml). The results of cTnT were available in 38 cases and in 46 cases we had the pH values. Results: Fifteen (39.5%) newborns had cTnT >= 0.09 ng/ml and 20 (43.5%) had a pH < 7.20. Fetuses who developed acidemia had fewer fetal movements per hour in cCTG (median 2 vs. 15, P=0.019). There was a positive correlation between pH and the number of fetal movements per hour (rho 0.35, P=0.019) and basal fetal heart rate (rho 0.37, P=0.011), and a negative correlation between pH and the zscore of pulsatility index for veins (PIV) of ductus venosus (rho= -0.31, P=0.036). The logistic regression analysis identified the z-score of PIV of ductus venosus (P=0.023) and basal fetal heart rate (P=0.040) as independent variables associated with acidemia at birth. The occurrence of fetal myocardial injury was significantly associated with z-score of PI of umbilical artery (median 8.8 vs. 4.0, P=0.003), PIV of ductus venosus (median 2.6 vs. -1.4, P=0.007), basal fetal heart rate (median 146 vs. 139 bpm, P=0.033), number of accelerations between 10-15 bpm (median 0 vs. 1, P=0.013), duration of episodes of low variation (median 21 vs. 10 min, P=0.038) and short-term variation (STV) (median 3.7 vs. 6.1 ms, P=0.003). We observed a positive correlation between the value of cTnT in the umbilical cord and basal fetal heart rate (rho=0.33, P=0.042), and a negative correlation between cTnT and STV (rho=-0.37, P=0.021). Logistic regression identified the STV as an independent predictor for myocardial damage (P=0.01), and STV <= 4.3 ms was the best cutoff to predict the event (sensitivity 66.7% and specificity of 91.3%). Conclusion: In pregnancies with placental insufficiency detected before the 34th week of gestation, the PIV of ductus venosus and basal fetal heart rate analyzed by cCTG are independent variables associated with acidemia at birth; and the STV is the parameter that best predicts fetal myocardial injury. The cCTG is an important tool in the management of fetuses with placental insufficiency, especially when associated with other diagnostic methods such as Doppler

OBJETIVO: O presente estudo, realizado em gestantes de alto risco com diagnóstico de insuficiência placentária, tem como objetivo avaliar o fluxo sanguíneo fetal na veia portal esquerda (VPE), veia umbilical (VU) e ducto venoso (DV), e estabelecer quais parâmetros associam-se com a acidemia fetal no nascimento. MÉTODO: Pesquisa prospectiva envolvendo 58 gestantes, classificadas segundo a presença ou ausência do diagnóstico de acidemia no nascimento, de acordo com o pH no sangue da artéria umbilical, constituindo-se de: Grupo I: 26 casos (acidemia pH<7,20) e Grupo II: 32 casos (pH normal pH7,20). Foram excluídos da pesquisa os casos com diagnóstico pós-natal de anomalia do RN e aqueles em que não se obteve a mensuração do pH no nascimento. As seguintes variáveis dopplervelocimétricas da VPE e VU foram comparadas entre os grupos: escore-zeta da TAMxV (time averaged maximum velocity) (cm/s), Q/Kg (fluxo sanguíneo por Kg de peso fetal) (ml/min/kg) e presença de pulsatilidade; e o escore-zeta do índice de pulsatilidade para veias (IPV) do DV. RESULTADOS: O escore-zeta da TAMxV (rho=0,392, P=0,002) e o Q/Kg da VPE (rho=0,274, P=0,037), o escore-zeta do IPV do DV (rho=-0,377, P=0,004) e o Q/Kg da VU (rho=0,261, P=0,048) apresentaram correlação significativa com o pH no nascimento. Realizando-se a análise de regressão logística multivariada, as variáveis independentes que restaram no modelo final para a ocorrência de acidemia no nascimento (pH<7,20) foram: escore-zeta da TAMxV da VPE (OR=0,41; IC95% 0,25 a 0,71; P=0,001) e fluxo reverso na VPE (OR=0,004; IC95% 0,00 a 0,15; P=0,003), ambas demonstrando efeito protetor para acidemia. Com o presente modelo, constatou-se que 74,1% dos casos são corretamente classificados para acidemia no nascimento. CONCLUSÕES: pela análise do Doppler venoso fetal na insuficiência placentária constatou-se que a acidemia no nascimento (pH<7,20) está associada de forma independente com o fluxo reverso na VPE e com o escore-zeta da TAMxV da VPE, ambos demonstrando efeito protetor com redução do risco para a acidemia
OBJECTIVE: This study, conducted in high-risk pregnancies with placental insufficiency, aims to avaliate blood flow in the fetal left portal vein (LPV), umbilical vein (UV) and ductus venosus (DV), and establish which parameters are associated with acidemia at birth. METHOD: A prospective research involving 58 pregnant women, classified according to the presence or absence of the diagnosis of fetal acidosis at birth, according to pH in the blood of the umbilical artery, consisting of: Group I: 26 cases (acidemia, pH <7,20) and Group II: 32 cases (normal pH, pH 7,20). Exclusion criteria were patients who had postnatal diagnosis of abnormality of the newborn and those in which the pH measurement was not obtained at birth. The following Doppler variables of LPV and UV were compared between the groups: TAMxV (Time Averaged Maximum Velocity) (cm/s) zeta-score, Q/kg (blood flow per kg of fetal weight) (ml/min/kg) and presence of pulsatility; and DV pulsality index for veins (PIV) zetascore. RESULTS: LPV TAMxV zeta-score (rho=0.392, P=0.002) and Q/kg (rho=0.274, P=0.037), DV PIV zeta-score (rho=-0.377, P=0.004) and UV Q/kg (rho=0.261, P=0.048) showed significant correlation with pH at birth. Performing the multivariate logistic regression analysis, the independent variables that remained in the final model were: TAMxV of LPV zeta-score (OR=0.41; IC95% 0.25 a 0.71; P=0.001) and reverse flow in LPV (OR=0.004; IC95% 0.00 a 0.15; P=0.003), both showing a protective effect to reduce the risk of acidemia. With this model, it was found that 74,1% of cases are correctly classified to birth acidemia. CONCLUSION: by analysis of fetal venous Doppler in placental insufficiency we found that acidemia at birth (pH <7.20) is independently associated with reverse flow in the LPV and LPV TAMxV z-score, both showing a protective effect with reduced risk for the event

Neonatal emergencies during pregnancy, labour, birth, and the postnatal period are covered. Blood tests during pregnancy and detecting deviations from the norm are included. Fetal emergencies and their management include: in utero transfer, hypoxia, asphyxia, cord presentation, cord prolapse, vasa/placenta praevia, shoulder dystocia, undiagnosed breech, and neonatal resuscitation. Guidelines for admission to a neonatal intensive care unit (NICU) and current neonatal morbidity and mortality data are included. The management of an intrauterine death or stillbirth is included.

Abstract Trophoblast invasion and remodeling of uterine spiral artery is closely related to the healthy pregnancy and normal development of fetus. However, if the trophoblast invasion is inhibited and the spiral artery remodeling process is not sufficiently done, this promotes grave pregnancy diseases such as pre-eclampsia. In this research we focused on one of the major factors that affect trophoblast invasion, oxygen concentration. We designed a microfluidic device fabricated with Polydimethylsiloxane (PDMS) which can mimic the microenvironment of the placenta and control physiological oxygen concentration. Human umbilical vessel cells (HUVEC) and trophoblasts (HTR-8) were cultured inside the microfluidic device to recapitulate the placental microenvironment. HUVECs and trophoblasts were co-cultured under normoxic and hypoxic condition to recapitulated trophoblast invasion. We anticipate this device to be utilized as a research model for improve our understandings on mechanisms of trophoblast invasion and a possible platform for developing treatment methods and medicines for pregnancy disorders.