Intrauterine crowding and placental insufficiency are main reasons for prenatal losses in swine. Two studies were conducted to examine: 1) conceptus development and in vitro steroidogenic capability of three regions of the placenta (middle, inner, polar) at d 30, 50, 70, and 90 of gestation; and 2) the effects of intrauterine position and fetal sex on conceptus development and in vitro steroidogenic activity of the placenta and endometrium at d 40, 60, 80, and 100 of gestation. All variables were examined in gilts that were uterine intact before hysterectomy (n=19) and in gilts unilaterally hysterectomized-ovariectomized (UHOX) before breeding (n=17) to induce intrauterine crowding. Placentas were combined according to the sex of the fetus associated with the placental unit (except at d 30). Placentas were sectioned into middle, inner and polar regions. Placental tissues were incubated, and release of progesterone (P₄) and estrone (E₁) was determined. Fetal survival rate was greater (P< .01, .05, .001 at d 50, 70 and 90, respectively) from intact versus UHOX gilts. Placental length and weight, fetal length and weight, and allantoic fluid volume were greater in intact compared to UHOX gilts. The polar region of the placenta released less P₄ than the other regions at d 50, 70 and 90. Uterine status (P< .005) affected P₄ release only at d 90. Sex of the fetus did not affect placental P₄ release. Region of the placenta affected E₁, release at d 30 (P< .01) and d 50 (P< .06). Uterine status did not affect E₁ release. Sex of the fetus affected (P< .001) E₁ release only at d 50. In the second study, a total of 45 gilts was used. Placentas and endometrium were combined based upon the intrauterine position of the associated fetus. Placental and endometrial tissues were incubated and release of P₄ and E₁ was determined. Uterine status (intact or UHOX) did not affect the variables measured. Intrauterine position affected fetal and placental weights (P< .02 and .01, respectively) at d 40 of gestation. No significant effect of intrauterine position was detected on placental and endometrial P₄ release. At d 100 of gestation, placentas associated with fetuses bordered in utero by fetuses of the same sex released more (P< .01) E₁ compared to placentas associated with fetuses bordered by fetuses of the opposite sex. Estrone release by the endometrium was not significantly affected by intrauterine position. Only trace amounts of testosterone and dehydroepiandrosterone sulfate were measured in the fetal fluids at all days of gestation. Intrauterine position had no effect on P₄, E₁ or androstenedione concentrations in fetal fluids. Collectively, the results indicated a) a differential release of P₄ and E₁ by the three regions of the placenta at certain days of gestation, b) no compensatory increase in placental steroidogenic activity per unit of tissue when total placental mass was reduced, and c) a limited effect of intrauterine position on placental and endometrial steroidogenesis at various days of gestation. / Ph. D.

La grossesse est une période de majoration du risque vasculaire, participant à une morbi-mortalité maternelle et fœtale pouvant justifier des mesures de prévention primaire et secondaire. Notre travail évalue l'impact de certains déterminants et l'apport de nouveaux acteurs moléculaires impliqués dans la dysfonction vasculo-placentaire. Le but ultime étant d'optimiser les prises en charge et de développer de nouvelles stratégies thérapeutiques. Nous avons étudié les complications vasculaires placentaires associées à des marqueurs biologiques connus : mutation du facteur V Leiden, mutation du gène de la prothrombine et marqueurs conventionnels du syndrome des anticorps anti-phospholipides (SAPL). Nos résultats montrent que les femmes à antécédents de fausses couches précoces répétitives et porteuses, soit du polymorphisme du facteur V, soit du polymorphisme du facteur II, soit d'un SAPL (traité par héparine et aspirine faible dose), ont un risque élevé de fausse couche tardive lors d'une nouvelle grossesse. Les femmes à antécédent de fausse couche tardive et porteuses des mêmes particularités biologiques, traitées pendant leur grossesse selon les recommandations (héparine pour l'anomalie du facteur V ou II, héparine plus aspirine faible dose pour le SAPL), ont un risque diminué de récidive de perte fœtale tardive mais demeurent, dans le groupe SAPL, fréquemment exposées aux complications tardives de la grossesse malgré la prophylaxie antithrombotique. Nous avons évalué l'apport de nouveaux marqueurs de la dysfonction vasculaire placentaire. Nous montrons que le polymorphisme Ile89Leu du gène de la phosphatase alcaline placentaire (PLAP), enzyme exprimée par les cellules du syncytiotrophoblaste -polymorphisme associé à une augmentation de l'activité PLAP-, exerce un effet protecteur sur l'échec d'implantation et la survenue d'une fausse couche primaire. Un facteur angiogénique (brevet en cours) a également été étudié (génétique, dosage plasmatique, fécondation in vitro) et nous montrons une association de ce marqueur avec les échecs d'implantation et les fausses couches idiopathiques. L'ensemble de ces travaux suggère que ces nouveaux marqueurs moléculaires pourraient contribuer au diagnostic des complications vasculaires de la grossesse et fournir des biomarqueurs d'implantation embryonnaire et/ou de développement placentaire. Ils pourraient suggérer de nouvelles cibles et stratégies thérapeutiques, répondant aux limites des traitements disponibles. / Vascular risk increases during pregnancy, contributing to maternal and foetal morbidity and mortality, and potentially justifying primary and secondary preventive measures. Our work evaluates the impact of some determinants and the contribution of new molecular actors implicated in placental vascular dysfunction. The ultimate aim is to optimize management and to develop new therapeutic strategies. We studied the placental vascular complications associated with known biological markers: the factor V Leiden or prothrombin polymorphisms, and conventional markers of the antiphospholipid antibody syndrome (APS). Women with previous recurrent abortions carrying polymorphisms of either factor V or factor II, or with APS (treated with heparin and low-dose aspirin), had an increased risk of foetal loss during subsequent pregnancies. Women with a previous foetal loss carrying these biological markers, treated according to recommendations during a new pregnancy (heparin for the polymorphisms, heparin plus low-dose aspirin for APS) had a lower risk of foetal loss, but an excess of late complications was observed in the APS group despite prophylaxis. We evaluated the contribution of new markers of placental vascular dysfunction. The placental alkaline phosphatase enzyme (PLAP) is synthesized and expressed by syncytiotrophoblastic cells. We found that the Ile89Leu polymorphism of the PLAP gene provides protection against implantation failure and primary miscarriage and induces increased PLAP activity. We also studied (genetics, plasma determinations, in vitro fertilisation) an angiogenic factor (patent application underway), which we showed to be associated with idiopathic implantation failure and miscarriage. These findings suggest that these molecular actors are potentially useful for the diagnosis of placenta-mediated pregnancy complications and may be relevant biomarkers of embryo implantation and/or placental development. They may indicate new targets for relevant therapeutic strategies, potentially overcoming the limitations of the currently available treatments.

Le retard de croissance intra-utérin par altérations vasculaires placentaires affecte 4% des grossesses actuelles. Ses mécanismes d’installation encore inconnus ont un impact pédiatrique important responsable d’une mortalité non négligeable. La combinaison d’un retard de croissance intra-utérin et de modifications vasculaires placentaires sur un modèle animal est nécessaire pour comprendre ces mécanismes et envisager une étude thérapeutique ultérieure. Le but de ce projet est de mettre au point dans un premier temps un modèle animal qui associe au retard de croissance intra-utérin les modifications vasculaires placentaires retrouvées dans la physiopathologie humaine. Pour cela, deux modèles animaux murins ont été étudiés : l'hyperthermie en fin de gestation et l’anémie par carence martiale pré et péri-gestationnelle. Une étude qualitative et quantitative par échographie (échographie Doppler et échographie de contraste) associée à une étude anatomopathologique. immuno-histochimiques et spectroscopique des placentas a été réalisée. L’optimisation du modèle animal définitif a requis dans un premier temps la mise au point de repères anatomiques inexistants à l'heure actuelle permettant la reproductivité des mesures échographiques ainsi que la mise en évidence de l’innocuité du produit de contraste échographique utilisé. Une étude placentaire globale nous a permis d’enregistrer des modifications structurelles liées au modèle analysé. L’étude de ces deux modèles animaux nous a permis d’établir un protocole de mesure standardisé et de mettre en évidence l’absence d’effet de l’utilisation du produit de contraste échographique sur la gestation. L’hyperthermie tout comme la carence martiale sont responsables d'un retard de croissance intra-utérin avec un effet dose dépendant. Les modifications vasculaires placentaires retrouvées dans le modèle hyperthermie à type d'ischémie-hémorragique n’apparaissent pas consécutives à des modifications vasculaires installées mais séquellaires de cet incident aigu. Les modifications hémodynamiques enregistrées dans le cadre de la carence martiale sont plutôt en faveur de modifications vasculaires en accord avec la pathologie humaine. L’étude spectroscopique ne met pas en évidence de changement métabolomique particulier. Ces deux modèles permettent donc l’installation d’un retard de croissance intra-utérin corrélé positivement à l’intensité du protocole. Le modèle anémie tend à se rapprocher au mieux de la pathologie humaine ; son étude reste à approfondir sur des effectifs plus conséquents. / The intra-uterin growth restriction by placental vascular modifications affects 4% of the current pregnancies. lt’s still unknown installation mechanism has an important pediatric impact with a significant mortality. The association of intra-uterin growth restriction and placental vascular defects on an animal model is necessary to understand this mechanism and envisage a therapeutic study later. The aim of Ibis project is to establish at first an animal model which associates intra-uterin growth restriction and vascular placental modifications found in the human physiopathology For that purpose, two murine models were studied : hyperthermia at the end of the gestation and anaemia by iron deficiency before and during the gestation. A qualitative and quantitative study by ultrasonography (Doppler ultrasonography and contrast ultrasonography) associated with anatomopathological, immune-histochemical and spectroscopic studies of the placentas was realized. The optimization of the definitive animal model required at first anatomical marks development, allowing reproduction of the ultrasonographic measures as well as revelation of the ultrasound contrast product harmlessness. A global placental study allowed us to note structural modifications connected to the analyzed model. The study of these two animals models allowed us to establish a standard measuring protocol and show that ultrasonographic contrast product use had no effect on the gestation. The hyperthermia, as the iron deficiency, is responsible of an intra-uterin growth restriction with a positive effect dose related. The vascular placental modifications found in the hyperthermia model as hemorrhage and ischaemia areas do not appear as the result of vascular modifications but after-effects of this acute incident. The hemodynamical modifications registered within the framework of the iron deficiency are rather in favour of vascular modifications in agreement with the human pathology. The spectroscopic study does not show metabolomic modifications. These two models allow the installation of an intra-uterin growth restriction positively correlated with the protocol intensity. The anaemia model gets closer to the human pathology; its study remains b he continued with more consequent numbers.

Objetivo: A finalidade deste estudo foi avaliar o Doppler venoso em gestações gemelares monocoriônicas (MC) com insuficiência placentária e a relação do fluxo sanguíneo venoso com a acidemia no nascimento ou óbito fetal. Método: Estudo prospectivo que incluiu 18 gestações gemelares MC com insuficiência placentária. Os critérios de inclusão foram: gestação gemelar MC e diamniótica, dopplervelocimetria da artéria umbilical (AU) alterada, membranas integras e ausência de defeitos congênitos fetais. Casos que apresentassem a síndrome de transfusão feto- fetal foram excluídos. Os seguintes parâmetros de Doppler foram avaliados: índice de pulsatilidade (IP) da AU, índice de pulsatilidade para veias (IPV) do ducto venoso (DV), IP e velocidade sistólica máxima (Vmax) da artéria cerebral média (ACM), a média da velocidade máxima (TAMxV) da veia umbilical (VU) e a TAMxV da veia portal esquerda (VPE). Os parâmetros dopplervelocimétricos foram transformados em escore zeta (desvios padrão da média) ou múltiplos da mediana (MoM), de acordo com os valores de referência. Amostras de sangue do cordão umbilical foram obtidas imediatamente após o parto para a mensuração do pH da artéria umbilical no nascimento. Resultado: O pH < 7,20 ocorreu em nove recém nascidos (25%), pH< 7,15 em quatro (11,1%) e em quatro (11,1%) casos houve óbito intrauterino. Os escores zeta da TAMxV da VU e da VPE foram significativamente menores no grupo com pH < 7,2 ou óbito intrauterino (respectivamente: -1,79 vs. - 1,22, p=0,006; -2,26 vs. -1,13, p=0,04). Nos casos com pH< 7,15 ou óbito intrauterino a pulsação da VU foi mais frequente (50% vs. 10,7%, p=0,03) e a TAMxV da VU foi significativamente mais baixa (-1,89 vs. -1,26, p= 0,003). A análise de regressão logística demonstrou que o escore zeta da TAMxV da VU prediz significativamente acidemia com pH< 7,20 ou óbito intrauterino (p=0,019). O parâmetro de Doppler que prediz significativamente pH< 7,15 ou óbito intrauterino foi a pulsação da VU (p=0,023). Conclusão: Os parâmetros de Doppler da VU podem predizer a acidemia no nascimento ou o óbito fetal em gestações gemelares MC complicadas por insuficiência placentária / Objectives: The aim of this study was to investigate fetal venous Doppler in monochorionic (MC) twin pregnancies complicated by placental insufficiency and the relationship between fetal venous flow and acidemia at birth or intrauterine fetal death. Methods: This was a prospective study of 18 MC twin pregnancies with placental insufficiency. Inclusion criteria were MC diamniotic twin pregnancies, abnormal umbilical artery (UA) Doppler, intact membranes, and absence of fetal congenital abnormalities. The twin-to-twin transfusion syndrome cases were excluded. The following Doppler measurements were studied: UA pulsatility index (PI), ductus venosus (DV) pulsatility index for veins (PIV), middle cerebral artery (MCA) PI and peak systolic velocity (PSV), intra-abdominal umbilical vein (UV) timeaveraged maximum velocity (TAMxV), and left portal vein (LPV) TAMxV. Doppler parameters were transformed into z-scores (SD values from the mean) or multiples of median (MoM) according to normative references. Blood samples were obtained from the umbilical cord immediately after delivery to measure the pH of the umbilical artery at birth. Results: pH<7.20 occurred in 9 newborns (25%), pH<7.15 in 4 (11.1%), and intrauterine fetal death in 4 (11.1%). The UV-TAMxV and the LPVTAMxV z-scores were significantly lower in the group presenting pH <7.20 or intrauterine fetal death (respectively: -1.79 vs. -1.22, p=0.006; -2.26 vs. -1.13, p=0.04). In cases with pH <7.15 or intrauterine fetal death, UV pulsations were more frequent (50% vs. 10.7%, p=0.03) and the UV-TAMxV z-score was significantly lower (-1.89 vs. -1.26, p=0.003). Logistic regression demonstrated that the UVTAMxV z-score significantly predicted pH at birth <7.20 or intrauterine fetal death (p=0.019). The Doppler parameter which independently predicted pH < 7.15 or intrauterine fetal death was the presence of pulsation in the umbilical vein (p=0.023). Conclusion: UV Doppler parameters may predict acidemia at birth or intrauterine fetal death in MC twins complicated by placental insufficiency

Objetivo: A finalidade deste estudo foi avaliar o Doppler venoso em gestações gemelares monocoriônicas (MC) com insuficiência placentária e a relação do fluxo sanguíneo venoso com a acidemia no nascimento ou óbito fetal. Método: Estudo prospectivo que incluiu 18 gestações gemelares MC com insuficiência placentária. Os critérios de inclusão foram: gestação gemelar MC e diamniótica, dopplervelocimetria da artéria umbilical (AU) alterada, membranas integras e ausência de defeitos congênitos fetais. Casos que apresentassem a síndrome de transfusão feto- fetal foram excluídos. Os seguintes parâmetros de Doppler foram avaliados: índice de pulsatilidade (IP) da AU, índice de pulsatilidade para veias (IPV) do ducto venoso (DV), IP e velocidade sistólica máxima (Vmax) da artéria cerebral média (ACM), a média da velocidade máxima (TAMxV) da veia umbilical (VU) e a TAMxV da veia portal esquerda (VPE). Os parâmetros dopplervelocimétricos foram transformados em escore zeta (desvios padrão da média) ou múltiplos da mediana (MoM), de acordo com os valores de referência. Amostras de sangue do cordão umbilical foram obtidas imediatamente após o parto para a mensuração do pH da artéria umbilical no nascimento. Resultado: O pH < 7,20 ocorreu em nove recém nascidos (25%), pH< 7,15 em quatro (11,1%) e em quatro (11,1%) casos houve óbito intrauterino. Os escores zeta da TAMxV da VU e da VPE foram significativamente menores no grupo com pH < 7,2 ou óbito intrauterino (respectivamente: -1,79 vs. - 1,22, p=0,006; -2,26 vs. -1,13, p=0,04). Nos casos com pH< 7,15 ou óbito intrauterino a pulsação da VU foi mais frequente (50% vs. 10,7%, p=0,03) e a TAMxV da VU foi significativamente mais baixa (-1,89 vs. -1,26, p= 0,003). A análise de regressão logística demonstrou que o escore zeta da TAMxV da VU prediz significativamente acidemia com pH< 7,20 ou óbito intrauterino (p=0,019). O parâmetro de Doppler que prediz significativamente pH< 7,15 ou óbito intrauterino foi a pulsação da VU (p=0,023). Conclusão: Os parâmetros de Doppler da VU podem predizer a acidemia no nascimento ou o óbito fetal em gestações gemelares MC complicadas por insuficiência placentária / Objectives: The aim of this study was to investigate fetal venous Doppler in monochorionic (MC) twin pregnancies complicated by placental insufficiency and the relationship between fetal venous flow and acidemia at birth or intrauterine fetal death. Methods: This was a prospective study of 18 MC twin pregnancies with placental insufficiency. Inclusion criteria were MC diamniotic twin pregnancies, abnormal umbilical artery (UA) Doppler, intact membranes, and absence of fetal congenital abnormalities. The twin-to-twin transfusion syndrome cases were excluded. The following Doppler measurements were studied: UA pulsatility index (PI), ductus venosus (DV) pulsatility index for veins (PIV), middle cerebral artery (MCA) PI and peak systolic velocity (PSV), intra-abdominal umbilical vein (UV) timeaveraged maximum velocity (TAMxV), and left portal vein (LPV) TAMxV. Doppler parameters were transformed into z-scores (SD values from the mean) or multiples of median (MoM) according to normative references. Blood samples were obtained from the umbilical cord immediately after delivery to measure the pH of the umbilical artery at birth. Results: pH<7.20 occurred in 9 newborns (25%), pH<7.15 in 4 (11.1%), and intrauterine fetal death in 4 (11.1%). The UV-TAMxV and the LPVTAMxV z-scores were significantly lower in the group presenting pH <7.20 or intrauterine fetal death (respectively: -1.79 vs. -1.22, p=0.006; -2.26 vs. -1.13, p=0.04). In cases with pH <7.15 or intrauterine fetal death, UV pulsations were more frequent (50% vs. 10.7%, p=0.03) and the UV-TAMxV z-score was significantly lower (-1.89 vs. -1.26, p=0.003). Logistic regression demonstrated that the UVTAMxV z-score significantly predicted pH at birth <7.20 or intrauterine fetal death (p=0.019). The Doppler parameter which independently predicted pH < 7.15 or intrauterine fetal death was the presence of pulsation in the umbilical vein (p=0.023). Conclusion: UV Doppler parameters may predict acidemia at birth or intrauterine fetal death in MC twins complicated by placental insufficiency

Le dépistage et le diagnostic de l’insuffisance placentaire (IP), qu’il s’agisse du retard de croissance intra-utérin (RCIU) ou de la prééclampsie (PE), sont des enjeux majeurs de santé publique. En pratique clinique, les propriétés mécaniques du placenta ne sont pas explorées, pourtant des modifications de son architecture tissulaire pourraient engendrer des variations d’élasticité. Parmi les méthodes d’élastographie ultrasonore (US), l’élastographie transitoire paraît adaptée pour une telle application. Cette technique consiste à calculer la vitesse de l’onde de cisaillement (Cs) générée par une vibration externe se propageant dans le milieu considéré. Les valeurs d’élasticité obtenues à partir des méthodes US actuelles ne sont calculées qu’à une fréquence unique. Une modification structurelle du tissu pouvant correspondre à une loi de puissance particulière de la dispersion fréquentielle de Cs, nous avons évalué l’intérêt d’une approche multifréquentielle pour distinguer l’élasticité des placentas normaux et celle de placentas présentant des signes d’IP au troisième trimestre de la grossesse. Nous avons développé un dispositif préliminaire en onde plane (pour l’exploration ex vivo) permettant de valider le principe de la méthode proposée, puis un dispositif d’élastographie transitoire 2D (exploration ex vivo et in vivo). Les données sont ajustées au moyen d’un modèle rhéologique fractionnaire dans lequel le comportement en fréquence est modélisé par une loi de puissance (exposant n du modèle). Nous avons montré que les placentas RCIU présentent des valeurs de Cs et n inférieures à celles des placentas normaux ou des PE. Cette diminution de n pourrait s’expliquer par les lésions anatomopathologiques du RCIU et la diminution de Cs est cohérente avec l’étude d’un modèle murin de RCIU par ligature utérine. Enfin, l’analyse de la dispersion fréquentielle est faisable chez la femme enceinte. La valeur ajoutée de la méthode développée devrait désormais être testée lors d’une large étude clinique. / Screening and diagnosis of placental insufficiency (PI), whether intrauterine growth restriction (IUGR) or preeclampsia (PE) are major public health issues. In clinical practice, the mechanical properties of the placenta are not explored; however changes in its tissue architecture could cause variations in elasticity. Among the ultrasound (US) elastography methods, transient elastography seemed suitable for such an application. This technique consists in calculating the shear wave speed (Cs) generated by an external vibration propagating in the medium under consideration. Elasticity values obtained from current US methods are calculated at a single frequency. As a structural modification of the tissue may correspond to a particular power law of frequency dispersion of Cs, we evaluated the interest of a multifrequency approach to distinguish the elasticity of healthy placentas and that of placentas with PI signs in the third trimester of pregnancy. We have developed a preliminary plane wave device (for ex vivo exploration) to validate the principle of the proposed method, and then a 2D transient elastography device (ex vivo and in vivo exploration). The data is adjusted using a fractional rheological model where the frequency behavior is modeled by a power law (exponent n of the model). We have shown that IUGR placentas have Cs and n values lower than those of healthy placentas or PE. This decrease in the n value could be explained by histopathological lesions of IUGR. As for the decrease of Cs in cases of IUGR, this result is consistent with the study of an IUGR rat model by uterine ligation. Finally, the frequency dispersion analysis is feasible in pregnant women. The added value of this method should now be tested in a large clinical study.

La distinction entre les fœtus constitutionnellement petits de ceux qui présentent une réelle restriction de croissance liée à une insuffisance placentaire n’est pas aisée avec les mesures échographiques utilisées en pratique courante. Le retard de croissance intra-utérin (RCIU) est responsable d’une part importante de la prématurité induite, et d’une augmentation du risque de mortalité et de morbidité néonatales. C’est pourquoi, l’amélioration de la connaissance de la vascularisation placentaire est indispensable pour mieux identifier et prendre en charge les situations d’hypoxie chroniques foetales associées à l’insuffisance placentaire.Pour quantifier la vascularisation les modalités d’imagerie de perfusion disponibles sont l’échographie et l’IRM. Les études évaluant la quantification de la perfusion placentaire par échographie de contraste sont peu nombreuses. Les avantages et les limites de cet examen ont été évalués sur un modèle murin de RCIU par ligature vasculaire. Ainsi, l’échographie de contraste permettait de quantifier une baisse de la perfusion placentaire sur un modèle de RCIU sans que l’on puisse observer de passage d’agents de contraste ultrasonores au travers la barrière placentaire. Les résultats obtenus ont été comparés aux données obtenues par l’IRM de perfusion. Les paramètres quantitatifs obtenus à partir des courbes de cinétiques du contraste pour chacune des deux modalités d’imagerie étaient comparables sur un modèle identique de RCIU murin. Enfin, une méthode d’étude de l’oxygénation placentaire par imagerie photoacoustique a été évaluée. Cette modalité d’imagerie non invasive permettait d’obtenir en temps réel l’oxygénation placentaire, avec cependant une profondeur limitée d’exploration. Le placenta semblait se comporter comme une réserve en oxygène au cours de l’étude d’une séquence hypoxie – hyperoxygénation maternelle avec une désaturation moins importante que celle observée dans les autres tissus maternels. / To identify fetuses small for their gestational-age who have reached their appropriate growth potential from growth-restricted fetuses due to placental insufficiency is uneasy. Intra Uterine Growth Restriction (IUGR) increases the risk for indicated preterm delivery, neonatal mortality and morbidity. Therefore, improving the knowledge of the placental perfusion is essential to better identify and manage fetal chronic oxygen deprivation associated with placental insufficiency.Contrast Enhanced Ultrasound (CEUS) and MRI are two imaging modalities available to quantify placental perfusion. However, few studies focus on the quantification of placental perfusion with CEUS. First, the advantages and limitations of CEUS were presented in an IUGR rat model by uterine ligation. The placental perfusion observed by CEUS was significantly decreased in the ligated horn. No contrast enhancement was observed in the umbilical vein or the fetus. Then, we compared the CEUS parameters to results obtained by MRI perfusion. Perfusion parameters were obtained from the signal intensity decay curve for the two imaging modalities. Results of such perfusion parameters were comparable in the same IUGR rat model. Finally, we evaluated the response of the placenta to oxygenation by photoacoustic imaging. PA imaging is a real-time, non-invasive method to evaluate placental oxygenation without contrast agents. Our results suggesting that placenta is less affected than maternal tissue by the decline in maternal oxygenation. The placenta may play an important role in protecting the feus against hypoxia.

Background: The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V are located at a single locus on chromosome 17q22-24. Local regulatory (5´ P and 3´ enhancer) sequences and a remote locus control region (LCR) containing a placenta-specific hypersensitive site (HS) IV, have been implicated in the efficient expression of the placental hCS/GH-V genes, in part through gene transfer studies in placental and non-placental tumor cell lines. However, low levels of endogenous expression are reported in placental tumor cells compared to normal term placenta. Thus it was hypothesized that the hCS/GH-V chromatin structure in human choriocarcinoma cells is less accessible to regulatory regions essential for efficient expression due to DNA and/or histone modifications, specifically methylation and acetylation, respectively. Approach: To assess individual hCS-A, hCS-B and hGH-V gene expression in placental and non-placental tumor cells, and assess the effect of increasing “chromatin accessibility” on hCS/GH-V RNA levels by inhibiting DNA methylation and histone deacetylation using 5-aza-2´-deoxycytidine (azadC) and trichostatin A (TSA). Principal Findings: Low levels of hCS-A, hCS-B and hGH-V RNA were detected in placental and non-placental tumor cells compared to term placenta. A significant >5-fold increase in promoter activity was seen in placental but not non-placental cells transfected with hybrid hCS promoter luciferase genes containing 3´-enhancer sequences. Placental JEG-3 cells pretreated with azadC and TSA resulted in a significant >10-fold increase in hCS-A, hCS-B and hGH-V RNA levels compared to TSA treatment alone, however, a modest ~3-fold effect was seen in non-placental MCF-7 cells. By contrast to the effect of pretreatment with azadC, post-treatment with azadC mutes the stimulatory effects of TSA on hCS/GH-V transcripts. The specificity of the response suggests that azadC treatment, and presumably hypomethylation of DNA, results in an increase in response to TSA and histone hyperacetylation at the hGH/CS locus. An assessment of histone H3/H4 hyperacetylation in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure including the hCS 3´-enhancer sequences and LCR. Conclusions: These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3´-enhancer sequences to available placental enhancer transcription factors. / May 2016

The morphological diversity of the external and internal surfaces of the petrosal bone, which contains the structures of the inner ear, across a broad range of therian mammals is documented, and patterns of variation across taxa are identified. One pattern of variation is the result of ontogenetic changes in the ear region, as described for the external surface morphology of a sample of isolated petrosal bones referred to Proboscidea from Pleistocene deposits in central Texas. The morphology of the aquaeductus Fallopii for passage of the greater petrosal branch of the facial nerve supports an ontogenetic explanation for some variation within the proboscidean sample, and a sequence of ossification surrounding the aquaeductus Fallopii is hypothesized. Further ontogenetic patterns are investigated using digital endocasts of the bony labyrinth (preserved on the internal surfaces of the petrosal) constructed from CT data across a growth series of the opossum Monodelphis domestica. Strong correlation between skull length and age is found, but from 27 days after birth onward, there is no correlation with age among most dimensions of the inner ear. Adult dimensions of several of the inner ear structures are achieved before the inner ear is functional in M. domestica. Morphological variation within the inner ear of several eutherian mammals from the Cretaceous of Asia, including zhelestids from the Bissekty Formation of Uzbekistan, is described. The variation within the fossil sample is compared to that observed within extant species of placental mammals, and it is determined that the amount of variation within the Bissekty zhelestid population is within the range of that measured for extant species. Additional evolutionary and physiological patterns preserved within the walls of the bony labyrinth are identified through a high level anatomical comparison of the inner ear cavities across Placentalia as a whole. In particular, features of the inner ear support monophyly of Cetacea, Carnivora, Primatomorpha, and caviomorph Rodentia. The volumetric percentage of the vestibular apparatus (vestibule plus semicircular canals) of aquatic mammals is smaller than that calculated for terrestrial relatives of comparable body size. Thus, aspects of the bony labyrinth are both phylogenetically and physiologically informative. / text

This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.