This study was conducted to document the size of primigravid women and their infants and placentas born at term in the Coloured community of Cape Town. It also explored the relationship between maternal, infant and placental size at birth. One thousand nine hundred and fifty seven firstborn infants delivered at term to Coloured women by the Peninsula Maternity Service during 1975 and 1976 were examined. The birth weight, crown-heel length and head circumference of each infant were measured, the gestational age assessed and the ponderal index of weight to length calculated. In addition the standing height, delivery weight and postdelivery weight of 395 of their mothers were measured and the Quetelet index of weight to height determined. The trimmed weight and chorionic plate area of 992 of the study infants' placentas were also measured and the placental thickness calculated.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Barbora Umanová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Expression and function of placental drug transporters in health and disease There are many physiological changes during pregnancy. Placenta is a crucial organ which mediates exchange of nutrients, metabolites and respiratory gases, provides endocrine functions and fetal protection. A pregnant woman and her fetus may be exposed to various potentially harmful substances during pregnancy, including drugs that may endanger fetal health. Protection of the fetus from xenobiotics is enabled by drug transporters. Drug transporters are membrane proteins expressed in most tissues of the human body. In the placenta, they are localized in the placental syncytiotrophoblast and occur also in the endothelial cells of the fetal vessels. They belong into two large superfamilies of transporter proteins: ATB-binding cassette (ABC) and solute carrier (SLC). While ABC transporters mediate exclusively efflux of their substrates, SLC are predominantly influx transporters. Therefore, these transport proteins play a key role in the disposition of drugs, some of which facilitate drugs entry into a fetus, and others actively...

The plant cell wall is a vitally important interface connecting plant cells to their outside environment and neighboring cells. Acting as a hub for defense, signaling, and physiological processes, the plant cell wall was a crucial innovation in plant evolution. Current cell wall models are largely based on what has been observed in plants like Arabidopsis, Pisum sativum, Nicotiana tabacum, and Phaseolus vulgaris. These models are unable to consider the variety of polymers in a given wall, the mechanical and functional properties such polymers impart, and the complexity of interactions among polymeric cell wall constituents. This work deepened the understanding of wall composition of specialized walls that fall outside of the scope of current plant cell wall models. A detailed survey of cell wall polymer distribution in the transfer cell walls in three key bryophyte species the model moss Physcomitrium patens, hornwort Phaeoceros carolinianus, and liverwort Marchantia polymorpha was done utilizing histochemical techniques in the light and florescent microscopes coupled with immunocytochemical localization with monoclonal antibodies (MAbs) in the transmission electron microscope (TEM). This work demonstrated that the occurrence, abundance, and types of polymers differ among taxa and between the two generations, are more influenced by developmental and life history needs than the similar function of the cells in individual taxa. A notable difference between generations was seen in M. polymorpha with the LM2 and JIM13 MAbs targeting AGP epitopes. However, findings in P patens appear to lack the differential labeling observed in both M. polymorpha and P. carolinianus. Using these same techniques, the walls and matrices involved in the process of spermatogenesis were examined in the moss P. patens and noted differences in abundance and location of cell wall polymers during sperm cell differentiation. Another notable finding of this work was that high concentrations of arabinose as components of AGP and pectins are important in the walls of P. patens during the process of spermatogenesis. The final study focused on utilizing herbarium specimens to explore the application of immunogold localization on dried collections of the moss Polytrichum up to 100 years old. The studies compiled in this dissertation demonstrate that the major cell wall components, cellulose, pectins, hemicelluloses, and callose, are constituents of special walls in three bryophytes, but they are differentially expressed within cell types and across these plants. Taken together, these works contribute significant new data on the composition of plant cell walls by focusing on bryophytes and the unique cell walls vital to the life history processes of spermatogenesis and placental function. These findings also show that both field-collected and herbarium samples are successfully labeled with MAbs at the TEM level, unlocking the potential for further studies across time and taxa using plant collections.

El Desprendimiento Prematuro de Placenta (DPP) es una de las Hemorragias del Tercer Trimestre que condiciona morbimortalidad materna y perinatal (15); la morbimortalidad materna se debe fundamentalmente a la magnitud de la hemorragia y a las complicaciones derivadas de ella, así también los procedimientos quirúrgicos destinados a su control (15, 27, 39) , en tanto la morbimortalidad perinatal es dependiente de la prematurez y de la asfixia del recién nacido. El Desprendimiento Prematuro de Placenta (DPP) constituye el 31% de las Hemorragias del Tercer Trimestre(16) presentando una incidencia muy variable que oscila de 0.12% a 3% (12,13,15,16,19) a nivel mundial, cifras encontradas en numerosos estudios; a nivel nacional estas cifras varían de 0.2% a 2%(1,5,13,28) lo cual concuerda con datos estadísticos obtenidos en el Hospital Docente Madre Niño San Bartolomé que arroja una incidencia de 0.45%(14,53) y en el Instituto Especializado Materno Perinatal la incidencia es del 1%(7,17,23) ; algunas literaturas refieren que los Desprendimiento Prematuro de Placenta suficientemente extensos como para matar al feto se dan en el 0.11% - 1.29%(22,28) mientras que las mujeres que han tenido previamente esta complicación la tasa de recidiva es del 10%(22,25) y en caso de presentarse en dos embarazos consecutivos con Desprendimiento Prematuro de Placenta la incidencia aumenta al 25%(1,14,22,28)por lo que muchas veces se recomienda terminar prematuramente el embarazo debido al riesgo de recidiva pero no hay suficiente evidencia que apoye esto. La tasa de mortalidad materna en el Desprendimiento Prematuro de Placenta se encuentra entre 0.5% y 5% (15) dependiendo de la precocidad con que se haga el diagnostico, la mortalidad fetal oscila entre 50% a 80%, la perinatal entre 20% y el 40% y los que sobreviven tienen un 40% a 50% de enfermedad neonatal. / Tesis

ABSTRACT Preeclampsia (PE) is a one of the more common pregnancy complications that affects 5-8% of pregnancies worldwide and produces significant morbidity and mortality for mother and fetus. Shallow trophoblast invasion and insufficient maternal spiral artery remodeling early in gestation is believed to lead to a relatively hypoxic placenta with inflammatory and trophoblast endoplasmic reticulum (ER) stress. These stresses cause an imbalance in trophoblast expression of angiogenic/anti-angiogenic molecules with decreased placental growth factor (PGF) and increased soluble fms like tyrosine kinase-1 receptor (sFlt-1) production. The decrease in trophoblast PGF seems to be mediated at the transcriptional level while increased expression of sFlt-1 is mediated by alternative splicing. Two variants known to be elevated in PE are the sFlt-1i13 and sFlt-1e15a isoforms. Both share the first 13 exons of Flt-1. A read through into intron 13 and utilization of an alternative poly (A) signal sequence produces the sFlt-1i13 variants protein, while sFlt-1e15a results from alternative splicing of exon 14 to exon 15a, rather than exon 15, and utilization of an alternative poly (A) signal sequence. This angiogenic imbalance contributes to the clinical manifestations of PE later in pregnancy including maternal hypertension and proteinuria. Currently, there are no pharmacological options available for the prevention of PE and the only way to reverse PE symptoms is by delivery. The overall goal of my project was (1) to investigate potential therapeutic mechanisms that could be used to relieve the maternal symptoms of PE by correcting the angiogenic imbalance, and (2) to gain a better understanding of the alternative splicing mechanisms responsible for producing sFlt-1 gene expression in human trophoblast. PE shares some similar pathophysiology and risk factors with cardiovascular diseases. This has prompted use of statins as a potential therapy for PE. However, existing preclinical investigations for statin use has been mostly restricted to PE animal models without elucidating the cell types that respond to statin treatment. Therefore, we sought to determine the effect of statins on angiogenic gene expression in cells that are in direct contact with maternal blood during pregnancy and could contribute to PE: primary trophoblast and endothelial cells. Placental tissue and isolated cells were cultured under hypoxic stress (1% O2) as a model for the hypoxic environment noted in PE. We compared the effectiveness of two types of statins (hydrophilic vs hydrophobic) on angiogenic and anti-angiogenic gene expression from the human tissues and cells. Human placenta villus explants, umbilical vein endothelial cells (HUVECs), and cytotrophoblast were isolated from normal term placentae and cultured under low oxygen tension (1% O2) with serial concentrations of statins. Expression of proangiogenic genes (VEGF and PGF) and the prominent anti-angiogenic sFlt-1 isoforms (sFlt-1i13 & sFlt-1e15a) were analyzed. In villus explants, hypoxia (1% O2) tended to alter angiogenic gene expression in their predicted fashion, by increasing VEGF mRNA (hypoxia marker), decreasing PGF mRNA, and increasing both sFlt-1i13 and sFlt-1e15a mRNA expression. However, the changes in gene expression were quite variable and statistically not significant. Hypoxia significantly increased both sFlt-1i13 and sFlt-1e15a mRNA and protein expression in primary trophoblast but had limited effects on expression in HUVECs. Hypoxia significantly decreased PGF mRNA and protein expression in primary trophoblast, yet significantly increased PGF mRNA and protein expression in HUVECs. Concentrations of pravastatin or simvastatin used had limited effects on altering PGF mRNA and protein expression in any of the cell types. In primary trophoblast, lower concentrations of pravastatin (100/500 µg/ml) had no significant effects on sFlt-1i13 or sFlt-1e15a mRNA expression while higher concentrations (1000 µg/ml) significantly decreased sFlt-1i13 and tended to decrease sFlt-1e15a mRNA expression. Secreted sFlt-1 protein from trophoblast decreased with increasing concentrations of pravastatin. Similarly, simvastatin had limited effects and did not significantly decrease sFlt-1i13 or sFlt-1e15a expression in hypoxic primary trophoblast. Both pravastatin and simvastatin significantly down-regulated sFlt-1i13 and sFlt-1e15a mRNA expression and sFlt-1 protein production in HUVECs. To overcome the effects of statin treatments on sFlt-1 expression, primary HUVECs were treated with farnesyl pyrophosphate ammonium salt (FPP), an intermediate in the cholesterol synthesis pathway. FPP partially restored sFlt-1i13 and sFlt-1e15a mRNA expression. Our data support that the angiogenic imbalance seen in PE can be medicated by hypoxia, and that statin could be a promising medication to limit PE symptoms. The effect of statins may be more evident on endothelial cells than on trophoblast, and the reduction in sFlt-1 expression by statins seems to be partially mediated through the cholesterol synthesis pathway in endothelial cells. The antiangiogenic protein, sFlt-1, plays a central role in the pathophysiology of PE. Excessive amounts of the sFlt-1 receptor in maternal circulation leads to maternal endothelial cell dysfunction and subsequent clinical symptoms of PE. However, the mechanism governing sFlt-1 mRNA expression in trophoblast remains unclear. Jumonji C domain containing gene 6 (JMJD6) has been shown to be involved in splicing of sFlt-1i13 in endothelial cells, although with conflicting outcomes as to whether it increases or decreases alternative splicing of sFlt-1i13. It is unknown if JMJD6 functions to regulate splicing in human primary trophoblast. Therefore, we assessed whether JMJD6 expression is altered in primary trophoblast under hypoxia or ER stress and its ability to regulate alternative splicing of sFlt-1. Human cytotrophoblast were isolated from normal term placentae and were cultured in the presence or absence of ER stress inducer (tunicamycin) or at 1% O2 to simulate trophoblast stressors during PE. Expression of JMJD6, C/EBP homologous protein (CHOP), and sFlt-1 (sFlt-1i13, and sFlt-1e15a) variants were analyzed. Hypoxic stress significantly increases JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression. ER stress also tended to increase JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression in primary trophoblast. Collectively, our results show that low oxygen tension (1% O2) or ER stress increase JMJD6 mRNA expression which may contribute to increased sFlt-1i13 and sFlt-1e15a variant expression in primary trophoblast. Similarly, JMJD6 knock down with siRNA tends to slightly decrease sFlt-1i13 and sFlt-1e15a mRNA expression in primary trophoblast. JMJD6 overexpression in HTR-8 cells (choriocarcinoma) tended to increase sFlt-1i13 and sFlt-1e15a mRNA expression; however, results using HTR-8 were inconsistent due to extremely low expression of endogenous Flt-1 mRNA. To overcome this, a Flt-1 minigene plasmid was transfected into HTR-8 cell line. Under 1%O2 these cells increased expression of the sFlt-1i13 isoform. To more directly confirm effects of JMJD6 and hypoxia on sFlt-1 expression, HEK293 and JEG3 stable clones harboring the Flt-1 minigene were generated. Preliminary results from selected single colony isolates show that several stable clones express the Flt-1 minigene products. HEK293 and JEG3 stable clones harboring the Flt-1 minigene, HEK293-Flt1#5 and JEG3-Flt1#5 respectively, were cultured at 1%O2 for 48 or 72 hours. Hypoxic stress had no significant on altering sFlt-1 variant production or JMJD6 mRNA expression in HEK293-Flt1#5 cells. However, hypoxic JEG3-Flt1#5 cells significantly increased sFlt-1i13 isoform mRNA expression (˜6 fold) and mFlt-1 mRNA expression (2.5 Fold) and also increased JMJD6 mRNA expression (1.8 Fold). In summary, these data suggest a role for statins as a potential therapeutic approach for the prevention and treatment of PE by decreasing systemic sFlt-1 expression in endothelial cells. This effect seems most significant in endothelial cells. If substantiated by clinical studies, use of statins would offer an affordable and easily accessible therapy to lessen PE symptoms. Moreover, our preliminary data suggest a potential involvement of JMJD6 in splicing process of sFlt-1i13. Confirming of JMJD6 role in splicing of Flt-1 may provide therapeutic strategies to treat Flt-1 associated disorder.

Melatonin is a strong antioxidant that has previously been observed to increase uteroplacental blood flow and increase postnatal calf growth when supplemented during gestation. The objective of the current study was to examine the effects of melatonin implants on uterine blood flow and subsequent offspring growth. Commercial beef heifers and cows were artificially inseminated and assigned to one of two treatment groups supplemented with (MEL) or without (CON) melatonin from days 180 to 240 of gestation. Total uterine artery blood flow was increased in MEL- versus CON-treated cattle. Fetal and birth weight were not different between treatments. However, at castration, body weight was increased in calves from MEL-treated dams compared with CON-treated dams. Further research on placental vascularization and the mechanism in which melatonin impacts angiogenic factors is necessary to understand the relationship between melatonin and compensatory growth that occurs in postnatal offspring.

Prostaglandin E 2 vaginal suppositories are a highly effective method of second-trimester pregnancy termination. Management of a failed prostaglandin abortion must include a search for the cause of the failure. This case report is the first description of a failed prostaglandin abortion associated with placenta accreta.

Two isozymes of triosephosphate isomerase have been isolated to homogeneity from human placenta. Triosephosphate isomerase A and triosephosphate isomerase B were compared in terms of their chemical, and biological properties.

Physical activity (PA) reduces the risk for deleterious outcomes in both mother and fetus during pregnancy and improves health across the lifespan. How these benefits are bestowed remains poorly understood but may involve the placenta, the critical interface responsible for fetal growth and survival during pregnancy. This thesis first aims to determine whether small extracellular vesicles (sEVs), potential biological mediators of cell-to-cell communication, are released into circulation after acute exercise during pregnancy and how this compares in the non-pregnant state. Pregnant women were found to have greater circulating sEVs levels compared to non-pregnant controls after a moderate-intensity treadmill walk. Since exercise-associated sEVs are proposed to mediate tissue cross talk in response to exercise, exercise-associated sEVs were examined for their ability to influence trophoblasts (specialized placental cells) in vitro using the BeWo choriocarcinoma cell line. Exercise-associated sEVs from pregnant and non-pregnant women interacted with trophoblast-like cells but did not alter their proliferation, gene expression of angiogenic growth factors, or production of the pregnancy hormone, human chorionic gonadotropin. Finally, the relationship between differing intensities of maternal PA and fetoplacental vascular density in a cohort of healthy pregnant women followed prospectively from 24 weeks of gestation until term delivery. Using traditional histopathological point-counting techniques, there was no difference in the fetoplacental vascular density of individuals meeting or exceeding recommended 150 min of moderate-to-vigorous intensity PA. However, the analysis revealed unexpected associations between fetoplacental vascular density and lower intensities of PA, and sedentary time. Together, the work presented in this thesis highlight the potential for exercise-associated sEVs to communicate the benefits of PA to mother and fetus and the need to investigate the effects of varying PA intensities on placental vascular development.

Preeclampsia (PE), a hypertensive disease of pregnancy, occurring at or after gestational week 20. PE can have life threating consequences for both the mother and the baby. PE is a highly heterogenous disease which makes it challenging to identify any effective therapeutic interventions. We previously discovered three molecular subclasses of PE disease. One of these subclasses is characterized by heightened placental inflammation (inflammation-driven PE). Since it is a newly identified form of PE, we currently do not know about the molecular mechanisms driving this inflammation-driven form of PE. Interestingly, we have observed that placentas from this inflammatory PE subclass uniquely express higher levels of NAD+ consuming enzymes- PARPs - and thus exhibit a decrease in NAD+ content. NAD+ is a regulator of cellular energy metabolism and mitochondrial function. Several studies in the non-pregnant populations suggested that pro-inflammatory disease conditions can trigger hyperactivation of NAD+ consuming enzymes causing a depletion in total NAD+ content, leading to mitochondrial dysfunction and organ failure. Thus, we tested the hypothesis that NAD+ depletion causes placental mitochondrial dysfunction in the inflammatory subclass of PE and that boosting NAD+ could prevent development of this form of placental disease. We aimed to profile PARP activity, NAD+ availability, and mitochondrial health in human cases of all three PE subclasses. We examined the causal relationship between inflammation and dysregulated NAD+ signalling in both an in vitro human trophoblast culture model and in a rodent model of inflammation-driven PE. We also evaluated the therapeutic potential of NAD+ booster, nicotinamide riboside (NR) to improve placental health and function in the rodent model of inflammation-driven PE. Our results suggest that along with increased activity of PARP enzymes and decreased NAD+ levels, human inflammatory PE placentas also exhibit decreased levels of mitochondrial proteins and increased oxidative DNA damage. Using an in vitro human placental (HTR8 cell line) inflammation model we showed that increasing NAD+ under an inflammatory condition improved trophoblast mitochondrial and cellular function. Using an in vivo LPS induced rat model of inflammation-driven PE, we demonstrated that NAD+ boosting during pregnancy improved placental mitochondrial function, reduced inflammation and oxidative stress. This subsequently resulted in improved pregnancy outcomes demonstrated by reduced maternal blood pressure, increased placental/fetal weights and increased fetal survival in the LPS model. Overall, this study identifies targeting NAD+ signaling as a promising intervention for PE. NAD+ boosting through NR has been tested in non-pregnant human populations and found to be safe and effective in enhancing NAD+ levels. Thus, findings of this thesis lay the ground to test NAD+ boosting strategies in PE patients in near future.