Rozprawy doktorskie na temat „Placental hypoxia”

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

Introdução: o timo é importante órgão linfoide do sistema imunológico. Estudos mostraram que, durante o período fetal, a atrofia desse órgão faz parte da resposta adaptativa do feto ao ambiente intrauterino adverso, como a desnutrição crônica causada pela insuficiência placentária. Essa situação pode explicar a associação entre restrição de crescimento intrauterino e as alterações no sistema imunológico após o nascimento, na infância e na adolescência. Objetivos: analisar as dimensões do timo fetal pela ultrassonografia em gestações com insuficiência placentária, comparando com gestações de alto risco sem insuficiência placentária e gestações de baixo risco. Métodos: estudo prospectivo com 30 gestações com insuficiência placentária (Doppler de artéria umbilical com índice de pulsatilidade > p95) comparadas com 30 de alto risco e 30 de baixo risco (grupo controle). Os critérios de inclusão foram: idade gestacional entre 26 e 37 semanas, feto único e vivo, ausência de malformações fetais, membranas íntegras, ausência de sinais de trabalho de parto, ausência de infecção materna ou fetal e não realização de corticoterapia antes da avaliação ultrassonográfica fetal. O timo fetal foi identificado na interface com os pulmões, na altura dos três vasos da base do coração, no corte do tórax fetal. Foram realizadas três medidas do diâmetro transverso (DT) e do perímetro (P) do timo, e as médias foram utilizadas para análise, transformadas em escores zeta, de acordo com a idade gestacional em que se efetuou a medida. Foram realizadas as medidas ultrassonográficas da circunferência cefálica (CC) e do comprimento do fêmur (CF) fetal, com as quais se calculou as relações DT/CF, DT/CC, P/CF e P/CC. Resultados: o grupo com insuficiência placentária apresentou mediana significativamente maior do escore zeta do IP da artéria umbilical quando comparado ao grupo de alto risco e controle (4,6 vs. -0,5 vs. -0,2, p < 0,001). As medidas do timo fetal no grupo com insuficiência placentária [escore zeta do DT (média=-0,69; DP=0,83) e escore zeta do P (média=-0,73; DP=0,68)] foram significativamente (p < 0,001) menores quando comparadas aos grupos de alto risco [escore zeta do DT (média=0,49; DP=1,13) e escore zeta do P (média=0,45; DP=0,96)] e controle [escore zeta do DT (média=0,83; DP=0,85) e escore zeta do P (média=0,26; DP=0,89)]. Nas relações estudadas, houve diferença significativa (p < 0,05) na média dos grupos: insuficiência placentária (DT/CC=0,10, P/CF=1,32 e P/CC=0,26); alto risco (DT/CC=0,11, P/CF=1,40 e P/CC=0,30) e controle (DT/CC=0,11, P/CF=1,45 e P/CC=0,31). Conclusão: em gestações complicadas pela insuficiência placentária, ocorre redução das dimensões do timo fetal sugerindo que pode ser decorrente da adaptação fetal ao ambiente intrauterino adverso
Introduction: thymus gland is an important lymphoid organ involved in immune response. Studies have shown that during fetal life, thymus atrophy is part of an adaptive response to a compromised intrauterine environment, like chronic malnutrition due to placental insufficiency. This may explain the association between intrauterine growth restriction and later altered immune function. Objective: to evaluate fetal thymus by ultrasonography in pregnancies with placental insufficiency and compare to high risk pregnancies without placental insufficiency and low risk pregnancies. Methods: a prospective study with 30 pregnancies with placental insufficiency (umbilical artery Doppler with pulsatility index > p95), compared to 30 high risk pregnancies and 30 low risk pregnancies (control group). The inclusion criteria were: gestational age ranging from 26 to 37 weeks, singleton pregnancies, absence of fetal malformations, intact membranes, not in labor, no signs of maternal or fetal infection, and no corticotherapy before the ultrasound evaluation. Fetal thymus was identified in its interface with the lungs, at the level of the tree-vessel view of the fetal thorax. Three measures of thymus transverse diameter (TD) and perimeter (P) were made, and the media were converted into zeta score according to the gestational age. Head circumference (HC) and femur length (F) were also measured and used in the calculation of the relations TD/F, TD/HC, P/F, P/HC. Results: the group with placental insufficiency presented median of umbilical artery PI elevated, when compared to high risk pregnancies and low risk pregnancies (4.6 vs. -0.5 vs. -0.2, p < 0.001). Fetal thymus measurements were significantly (p < 0.001) lower in pregnancies with placental insufficiency [TD zeta score (media=-0.69; SD=0.83) and P zeta score (media=-0.73; SD=0.68)] when compared to high risk pregnancies [TD zeta score (media=0.49; SD=1.13) and P zeta score (media=0.45; DP=0.96)] and control group [TD zeta score (media=0.83; SD=0.85) and P zeta score (media=0.26; SD=0.89)]. There was significant difference (p < 0,05) in the relations studied among the groups: pregnancies with placental insufficiency (TD/HC=0.10, P/F=1.32 e P/HC=0.26), high risk pregnancies (TD/HC=0.11, P/F=1.40, P/HC=0.30) and control group (DT/HC=0.11, P/F=1.45, P/HC=0.31). Conclusion: fetal thymus measurements are reduced in pregnancies with placental insufficiency, suggesting that it is a fetal adaptive response for adverse environment

O presente estudo analisou a hipótese de que as alterações hemodinâmicas fetais estão associadas à disfunção miocárdica fetal em gestações complicadas pela insuficiência placentária. Os objetivos do estudo foram correlacionar os valores do peptídeo natriurético cardíaco do tipo B (BNP) em sangue de cordão umbilical no nascimento com os parâmetros da dopplervelocimetria fetal, bem como com o pH no nascimento. Métodos: estudo prospectivo e transversal, com os seguintes critérios de inclusão: mulheres com gestação com feto único e vivo, insuficiência placentária caracterizada pelo aumento do índice de pulsatilidade (IP) da artéria umbilical (AU), membranas íntegras e ausência de anomalias fetais. Foram excluídos da pesquisa os casos com diagnóstico pós-natal de anomalia do recém-nascido e aqueles nos quais não foi realizada a análise do sangue no nascimento. Os seguintes parâmetros da dopplervelocimetria foram analisados: índice de pulsatilidade (IP) da artéria umbilical (AU) e da artéria cerebral média (ACM), relação cerebroplacentária (RCP) e IP para veias (IPV) do ducto venoso (DV). Os parâmetros de Doppler foram transformados em escore zeta. Amostras de sangue do cordão umbilical foram obtidas imediatamente após o parto para a mensuração do pH de artéria umbilical e do BNP. Resultados: Foram incluídas 32 gestações com diagnóstico de insuficiência placentária 21 (65%) com fluxo diastólico positivo e 11 (35%) com diástole zero ou reversa nas AU. A concentração do BNP apresentou correlação significativa com: escore zeta do IP da AU (rho=0,43; P=0,016); escore zeta da RCP (rho=-0,35; P=0,048); escore zeta do IPV do DV (rho=0,61; P<0,001), pH no nascimento (rho=- 0,39; P=0,031) e idade gestacional (rho=-0,51; P=0,003). Na regressão múltipla foram incluídos os parâmetros antenatais e o escore zeta do IPV do DV (P=0,008) demonstrou ser o único fator independente correlacionado com o BNP no nascimento. A correlação entre o BNP e o escore zeta do IPV do DV é representado pela equação de regressão Log[BNP]=2,34+0,13*DV (F=18,8, P<0,001). A correlação entre o BNP e o pH no nascimento é representado pela equação de regressão Log[BNP]=21,36-2,62*pH (F=7,69, P=0,01). Conclusão: os resultados sugerem que a disfunção cardíaca fetal identificada pelas concentrações de BNP no nascimento correlaciona-se de forma independente com as alterações no IPV do DV, além de correlacionarem-se negativamente com os valores do pH no nascimento
This study examined the hypothesis that fetal hemodynamic changes are associated with fetal myocardial dysfunction in pregnancies complicated by placental insufficiency. The objective was to study the correlation between the concentrations of cardiac natriuretic peptide type B (BNP) in umbilical cord blood at birth and fetal Doppler parameters, as well as the pH at birth. Methods: A prospective crosssectional study with the following inclusion criteria: pregnant women with a single fetus, placental insufficiency characterized by increased pulsatility index (PI) of the umbilical artery (UA), intact membranes and absence of fetal abnormalities. There were excluded from the study cases with postnatal diagnosis of abnormality of the newborn and those in which the blood analysis was not performed. The following Doppler parameters were analyzed: UA PI, middle cerebral artery (MCA) PI, cerebroplacental ratio (CPR) and ductus venosus (DV) PI for veins (PIV). The Doppler parameters were converted into zeta score. Blood samples were obtained from the umbilical cord immediately after delivery to measure the pH of the umbilical artery and the BNP. Results: Thirty two pregnancies with placental insufficiency were included, 21 (65%) with positive diastolic flow and 11 (35%) with absent or reversed end diastolic flow in the UA. The concentration of BNP correlated significantly with: UA-PI z-score (rho = 0.43, P = 0.016); CPR z-score (rho = -0.35, P = 0.048); DV-PIV zscore (rho = 0.61, P <0.001), pH at birth (rho = -0.39, P = 0.031) and gestational age (rho = -0.51, P = 0.003). In the multiple regression analysis, antenatal parameters were included and DV-PIV z-score (P = 0.008) was found to be independent parameter correlated with BNP at birth. The correlation between BNP and DV-PIV zscore is represented by the regression equation Log [BNP] = 2.34+0.13*DV (F=18.8, P <0.001). The correlation between BNP and pH at birth is represented by the regression equation Log [BNP] = 21.36-2.62*pH (F=7.69, P = 0.01). Conclusion: The results suggest that fetal cardiac dysfunction identified by BNP concentrations at birth correlated independently with changes in the DV PIV, and correlated negatively with pH values at birth

Objetivo: Avaliar a relação das alterações de fluxo na artéria cerebral média (ACM) com a ocorrência de acidemia no nascimento, em gestações com insuficiência placentária. Métodos: estudo transversal prospectivo com 91 gestações com diagnóstico de insuficiência placentária pelo Doppler de artéria umbilical (AU) alterado (índice de pulsatilidade [IP] > p95). Os critérios de inclusão foram: gestações únicas com idade gestacional (IG) superior a 26 semanas completas, membranas ovulares íntegras, ausência de anomalias cromossômicas ou congênitas. Os parâmetros da dopplervelocimetria analisados foram: IP da AU, IP da ACM, pico de velocidade sistólica (PVS) da ACM, relação cerebroplacentária (RCP) e índice de pulsatilidade para veias (IPV) do ducto venoso (DV). Foi analisada a última avaliação fetal realizada imediatamente antes do parto ou anterior à corticoterapia. Todos os parâmetros foram analisados por meio do escore zeta ou múltiplos da mediana (MoM), baseados nas médias, desvio-padrão e valores de referência para cada IG. Imediatamente após o parto, uma amostra de sangue da artéria umbilical foi obtida para a medida do pH, e os casos classificados de acordo com a presença (pH < 7,20) ou ausência de acidemia no nascimento. Resultados: Quarenta e sete (51,6%) recémnascidos apresentaram acidemia no nascimento. Os fetos que evoluíram com acidemia apresentaram valor de escore zeta do IP da AU significativamente maior (mediana 2,1 vs 1,7; p=0,014), assim como maior proporção de casos com diástole zero ou reversa (51,0% vs 31,8%; p=0,006). Quanto à ACM, o escore zeta mostrou-se significativamente menor nos casos com pH < 7,20 (mediana -2,7 vs -2,1; p=0,042), porém, em relação ao PVS não foi possível estabelecer diferença significativa entre os grupos (p=0,051). A acidemia no nascimento se associou a menores valores de RCP (mediana 0,5 vs 0,7; p=0,006), porém não ao seu escore zeta (p=0,055). Em relação ao território venoso, maiores valores do escore zeta do IPV do DV associaram-se à acidemia (mediana 2,4 vs 0,6; p=0,015). Na análise de correlação entre os valores de pH no nascimento e os resultados da avaliação da dopplervelocimetria fetal, foi constatada correlação significativa entre o valor do pH no nascimento e o escore zeta do IP da AU (rho=-0,31; p=0,003), IP da ACM (rho=0,26; p=0,012), da RCP (rho 0,25; p=0,015) e IPV do DV (rho=-0,32; p=0,002), e PVS da ACM MoM (rho=-0,21; p=0,042). A regressão logística identificou o escore zeta do IP da AU e escore zeta IP da ACM como variáveis independentes para a predição de acidemia no nascimento, classificando corretamente 67,03% dos casos. Conclusão: em casos de insuficiência placentária, o IP da AU e da ACM são preditores independentes associados com a acidemia no nascimento. Este estudo reforça que o grau de insuficiência placentária e a capacidade de adaptação fetal estão diretamente relacionados com a acidemia no nascimento
Objectives: To evaluate the relationship between middle cerebral artery (MCA) parameters and acidemia at birth, in pregnancies complicated by placental insufficiency. Methods: The study was performed as a prospective cross-sectional analysis of Doppler measurements in 91 patients with the diagnosis of placental dysfunction by abnormal umbilical artery (UA) Doppler (pulsatility index [PI] > p95). Inclusion criteria were: singleton pregnancy, intact membranes, abscence of fetal congenital or chromosomal abnormalities. The Doppler parameters analyzed were: UA PI, MCA PI, MCA peak systolic velocity (PSV), cerebroplacental ratio (CPR) and pulsatilility index for veins (PIV) of ductus venosus (DV). It was analyzed the last assessment obtained right before birth or the antenatal steroids. Umbical artey blood samples were collected at birth, and acidemia was defined as pH below 7.20. Results: Forty seven (51.6%) newborns had acidemia at birth. Those who developed acidemia showed a UA PI z-score significantly higher (median 2.1 vs 1.7, p = 0.014), as well as a higher proportion of cases with absent or reverse end diastolic flow (51.0% vs 31.8%, p = 0.006). Regarding the MAC, the PI z-score was significantly lower in cases with pH < 7.20 (median -2.7 vs. -2.1, p = 0.042), but concerning PSV z-score, no significant relation between the groups could be established (p = 0.051).The acidemia at birth was associated with lower values of CPR (median 0.5 vs 0.7, p = 0.006), but not with its z-score (p = 0.055). In relation to the venous territory, greater values of DV PIV z-score were associated with acidemia (median 2.4 vs 0.6, p = 0.015).The correlation analysis between the pH values at birth and the Doppler measurements, a significant correlation was observed between the pH at birth and UA PI z-score (rho = -0.31, p = 0.003 ), MCA PI z-score (rho = 0.26, p = 0.012), CPR z-score (rho 0.25, p = 0.015), PIV DV zscore (rho = -0.32, p = 0.002), and PSV MCA MoM (rho = -0.21, p = 0.042). Logistic regression identified the UA PI z-score and the MCA PI z-score as independent predictors for acidemia at birth, correctly classifying 67.03% of cases. Conclusion: In pregnancies with placental insufficiency, the UA PI and the MCA PI are independent predictors associated with acidemia at birth. This study reinforces that the degree of placental insufficiency and the fetal adaptation capacity are directly related to acidemia at birth

An essential event during early pregnancy is the invasion of trophoblasts into the maternal decidua, which is necessary for proper implantation and establishment of maternal-fetal interface and ultimately allows for proper nutrient exchange and immunological tolerance of the growing fetus. For this invasion to occur, cells originating from the trophectoderm undergo an epithelial to mesenchymal transition to become invasive extravillous trophoblasts and begin invading the uterine decidual tissue. Through the secretion of matrix metalloproteinases and through interactions with many cytokines and cell-adhesion molecules, this well-orchestrated process of trophoblast invasion results in extensive remodeling of the maternal spiral vasculature by the extravillous trophoblasts. Ultimately, the spiral arteries are transformed from high resistance, low flow vessels to low resistance, high flow vessels to allow for adequate perfusion of the placenta and developing fetus. Preeclampsia is a leading cause of maternal morbidity worldwide and is associated with the onset of hypertension and proteinuria, typically after 20 weeks of gestation. While the hypertension typically resolves following delivery of the fetus and placenta, both the mother and growing child are faced with long-term adverse health effects such as the development of cardiovascular disease and metabolic disorders. Preeclampsia is characterized by widespread maternal inflammation and endothelial dysfunction triggered by the secretion of soluble factors from the placenta into the maternal circulation. It is thought that the onset of these adverse systemic conditions is initiated by poor placental perfusion and pathologically hypoxic conditions in the placenta. In many cases of preeclampsia, there is evidence of shallow trophoblast invasion which results in incomplete spiral artery transformation, ultimately leading to poor placental perfusion. However, the exact mechanisms underlying the inadequate extravillous trophoblast invasion and remodeling are incompletely understood. LIN28 is an RNA binding protein that is highly expressed in embryonic stem cells, fetal tissues and many cancers, and was discovered as a regulator of the maturation of the Let-7 family of miRNAs. However, as an RNA binding protein, LIN28 has been shown to interact with thousands of mRNA transcripts, leading to both increased and decreased protein expression, and control of many cellular processes such as differentiation, proliferation, migration, invasion, and cellular metabolism. In vertebrates, LIN28 exists as two highly homologous paralogs, LIN28A and LIN28B, however LIN28B is slightly larger and contains a nuclear localization signal not found in LIN28A. While they both function to inhibit Let-7 maturation, there is evidence to suggest they also have independent functions. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to preeclampsia. We found that LIN28B mRNA expression is ~1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in human primary cytotrophoblasts, syncytiotrophoblasts, and decidual cells. We also found that LIN28B mRNA and protein levels are significantly reduced in human placentas from preeclamptic pregnancies compared to placentas from normal pregnancies, while LIN28A expression is unchanged. Upon investigation of human first trimester placenta sections, we found that LIN28B is more highly expressed in the invasive extravillous trophoblasts and syncytial sprouts compared to villous trophoblasts. To support this with in vitro evidence, we found that overexpression of LIN28B in human HTR8/SVneo cells resulted in increased proliferation, migration and invasion, while knockdown of LIN28B in JEG3 cells resulted in decreased proliferation. Furthermore, knockdown of LIN28B in JEG3 cells led to decreased expression of SYN-1, ELABELA, and the chromosome 19 miRNA cluster, with accompanying increases in the pro-inflammatory cytokine TNFα and ITGβ4, an integrin enriched on non-invasive trophoblasts. Moreover, culture of JEG3 and BEWO cells in hypoxia resulted in significantly decreased levels of LIN28B mRNA and protein expression, as well as syncytin-1 and ELABELA mRNA levels, while TNFα was increased. These results provide the first evidence that LIN28B is the predominant paralog expressed in human placenta and decreased LIN28B may play a crucial role in PE pathogenesis by reducing trophoblast invasion, syncytialization and by promoting inflammation.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

Thesis (M.Sc.)--University of Alberta, 2010.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Centre for Neuroscience. Title from pdf file main screen (viewed on January 27, 2010). Includes bibliographical references.

Factor inhibiting HIF (FIH) negatively regulates hypoxia inducible factor-1 (HIF-1) transcriptional activity, selectively controlling certain HIF-1 target genes, such as vascular endothelial growth factor (VEGF) and prolyl hydroxylase domain 3 (PHD3), but not others. PHD3 and VEGF are important for placental development and function and are overexpressed in preeclampsia (PE). The purpose of this study was to examine FIH in both normal and pathological human placentae. I hypothesized that FIH regulates VEGF and PHD3 in the placenta and that this rheostat is altered in PE. Results show that FIH suppresses PHD3 and VEGF in JEG-3 cells; this effect was abrogated by FIH gene silencing. Moreover, my data indicate that seven in absentia homologue-1 (Siah-1) targets FIH for degradation in the placenta; this degradation is enhanced in PE and likely contributes to aberrant VEGF and PHD3 expression. Overall, my data suggest an important role for FIH in the pathogenesis of PE.

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research.
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Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008