BACKGROUND: The placenta is a multifunctional organ whose primary functions are to nourish and protect the fetus throughout gestation. The immune response of the placenta plays an important part in gestational outcome. Microbial infection during pregnancy can be detrimental to both maternal health and fetal development, increasing the risk for miscarriage, preterm birth, and congenital abnormalities. However, evaluating immunological response has been an on-going challenge for scientists and clinicians due to the complexity of the maternal-fetal interface. Research has been done to understand the mechanisms by which pathogens activate placental immune response, but our understanding is still lacking in many areas due to the dynamic changes that occur in immunology over the gestational timeline. The primary challenge faced by researchers is the availability of placental tissue, which is limited by donors and their finite viability in culture once harvested. Additionally, legal restrictions placed on fetal-tissue research have severely limited advancement in the field. Human induced pluripotent stem cells (hiPSCs) present a unique tool to study the differentiation of trophoblasts and maternal-placental immunology without the need of fetal tissue. OBJECTIVE: The goal of this project is to develop an in vitro model for studying placental immunology and pathogenesis using human induced pluripotent stem cell (hiPSC)-derived trophoblasts. Our aim is to report a robust protocol for producing hiPSC-derived trophoblasts and to characterize them against primary trophoblasts using both gene and protein expression detection techniques. Successful modeling of human trophoblasts would allow us the unique opportunity to investigate the cellular interface between the maternal and fetal systems without needing to isolate primary human trophoblasts. Once we produce and fully characterize several hiPSC cell clones from multiple normal individuals, we will demonstrate the use of these cells as a model for infections at the maternal-fetal interface by exposing them to viral pathogens known to target the placenta. METHODS: Earlier publications have reported the differentiation of embryonic stem cells into trophoblasts in culture by using bone morphogenetic protein-4 in conjunction with inhibitors of activin A and FGF2-signaling (BMP4/A83-01/PD173074; BAP-treatment). We applied this approach to hiPSC lines from two different lineage origins and characterized the outcome against known trophoblast markers. We also developed a novel approach to maintain proliferative trophoblast stem cells in culture long term. Two viral pathogens, a recombinant vesicular stomatitis virus strain engineered to express a green fluorescent protein (rVSV-GFP) and a strain of Zika virus (ZIKV-PRVABC59), were used to determine if it is possible to infect hiPSC-derived trophoblasts in culture. RESULTS: Using this approach, hiPSC readily differentiate into trophoblasts by day 8 of culture. These cells demonstrate formation of multinuclear syncytium, invasive capacities, and secretion of placental hormones. Further characterization using quantitative real-time PCR and immunofluorescent staining indicates that these cells express a number of trophoblast markers at levels comparable to those expressed by primary first-trimester trophoblasts. We were also able to maintain a putative CT population which retains the capacity to double and give rise to terminal cell types. HiPSC-derived trophoblasts infected with rVSV-GFP and ZIKV-PRVABC59 tested positive for viral infection by 72 hours post-infection (HPI), demonstrating the use of these cells as an in vitro model for studying placental pathogens at the maternal-fetal interface. / 2022-06-08T00:00:00Z

Publicación a texto completo no autorizada por el autor / El documento digital no refiere asesor / Determina los factores asociados al Desprendimiento prematuro de placenta en gestantes atendidas en el Hospital Docente Madre Niño San Bartolomé durante el periodo 2008-2012. Estudio de tipo observacional, analítico comparativo, retrospectivo y transversal. Se estudió a 39 pacientes gestantes con desprendimiento prematuro de placenta (DPP) y 21 gestantes como grupo comparativo que no tenían DPP. Para determinar las diferencias de medias de las variables cuantitativas se utilizó las pruebas t de student y para determinar la relación entre variables y los factores asociados se usó las pruebas de chi-cuadrado y Odds ratio. La edad promedio de las pacientes con sospecha de desprendimiento prematuro de placenta es de 23.1±5.3 años, teniendo en su mayoría entre 19 a 35 años (75%), la mayoría son sólo convivientes (71.7%) de educación secundaria (96.7%). El grupo etario de las pacientes no está relacionado significativamente con el diagnóstico de desprendimiento prematuro de placenta (p=0.562), de la misma forma el estado civil de las pacientes no está relacionado significativamente con el DPP (p=0.067), tampoco lo está el grado de instrucción de las pacientes (p=0.291). De los factores médicos obstétricos se observó que existe diferencia entre los peso y talla promedios entre gestantes con y sin DPP (p<=0.001) y (p=0.038) respectivamente. No se ha encontrado asociación significativa de las gestantes con DPP con la paridad (p=0.49), contro prenatal (p=0.075), tipo de gestación (0.495), índice de masa corporal (0.131). Con respecto a los factores maternos conductuales no se detectó ningún caso con hábitos nocivos. Referente a los otros factores estudiados sólo la ausencia de la desproporción céfalo pélvica tiene relación significativa (p=0.002) con la presencia del DPP. La prevalencia del desprendimiento prematuro de placenta en el periodo 2008 a 2012 es de 39 / 60 casos que cumplieron los criterios de inclusión y exclusión. No hay factores socio-demográficos y médicos obstétricos asociados al desprendimiento prematuro de placenta en gestantes atendidas en el Hospital Docente Madre Niño San Bartolomé durante el periodo 2008-2012, sólo la ausencia de la desproporción céfalo pélvica tiene relación significativa (p=0.002) con la presencia del DPP en el grupo de estudio. / Trabajo de investigación

Prenatal exposure to sex hormones has profound effects on neurodevelopment with lifelong implications for mental health. Fetal exposure to aberrant levels of sex hormones alters sexual dimorphism (i.e. degree of feminization or masculinization; sex differences in brain and behavior) and may contribute to the differential susceptibility of males and females to psychiatric risk and neurodevelopmental disorders, including autism. During fetal development, the in-utero environment is regulated by the placenta, a maternal-fetal endocrine structure that serves as a “gate-keeper” between the maternal and fetal circulatory systems. The placenta expresses high levels of aromatase, an enzyme that converts testosterone to estrogen, and it has been proposed that placental aromatase precludes the transfer of maternal testosterone to the fetus. However, this view does not account for individual differences in placental aromatase expression or maternal hormone levels that may account for altered neurobehavioral outcomes. Retinoic acid-related orphan receptor-alpha (RORA) has been identified as a transcription factor that regulates aromatase and as an autism candidate gene – yet the role of RORA in the placenta as a regulator of the prenatal hormonal environment has yet to be determined. The research presented in this thesis aimed to evaluate 1) the relationship between maternal and neonatal hormone concentrations, 2) whether placental aromatase/RORA influences the relationship between maternal and neonatal hormones concentrations, 3) the relationship between maternal sex hormones during pregnancy and neurobehavioral outcomes in the offspring, and 4) the relationship between placental aromatase/RORA and neurobehavioral outcomes in the offspring. Chapters 1 and 2 of this thesis provide a review of the literature pertaining to the sources of and neurodevelopmental consequences of fetal exposure to hormones and the methods used to address our research questions. Chapters 3, 4, and 5 of this thesis used in vivo and in vitro methods to investigate the role of placental aromatase and RORA in regulating fetal exposure to maternal hormones. Results from these studies indicate that maternal testosterone is a strong predictor of neonatal testosterone levels at birth and that aromatase and RORA expression in the human placenta subtly influence the relationship between maternal and neonatal testosterone and estradiol in a sex-dependent manner. Results from our studies using an in vitro approach call into question the widely proposed role of placental aromatase in converting maternal testosterone intro estradiol. Chapters 6 and 7 of this thesis aimed to determine whether variability in maternal testosterone and placental aromatase/RORA expression was associated with increased neurodevelopmental risk as a result of elevated fetal hormone exposure. For the first time in the literature, we report a direct association between elevated maternal testosterone and poorer childhood neurodevelopmental outcome in a sex-dependent manner. We also report that the effects of placental aromatase and RORA expression on childhood outcomes vary depending on the neurobehavioral domain being assessed. Taken together, these studies support the notion that fetal exposure to sex hormones, especially those of maternal origin, can affect neonatal hormone production as well as long-term child neurobehavior. The specific mechanisms by which the placenta regulates fetal exposure require further investigation.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Lenka Ťupová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of doctoral thesis: Interactions of selected antiretroviral drugs and methylmercury with placental membrane transporters. Pregnant women are especially in developed countries exposed to high amount of various xenobiotic including environmental pollutants and drugs. Antiretroviral therapy (ART) is administered to HIV positive pregnant women for the purpose of prevention of HIV mother- to-child-transmission. Pharmacokinetics of many antiretrovirals is limited or enhanced by activity of ATP-binding cassette (ABC) or Solute carrier's transporters, of which many are expressed also in placental tissue. ART therapy usually consists of combination of 3 - 4 antiretroviral drugs, thereby leading to higher risk for development of drug-drug interactions on ABC and SLC transporters. In this study we described influence of non-nucleoside reverse transcriptase inhibitors etravirin and rilpivirin on BCRP- and MDR1-mediated transport of tenofovir disoproxil fumarate (TDF) and/or abacavir. Etravirin showed potent inhibition of BCRP transporter significantly changing transport of both, TDF and abacavir, across monolayers of MDCKII-BCRP...

The placenta of the anthocerote, Dendroceros tubercularis Hatt., consists of long and branched haustorial cells, that arise from the foot and gametophyte transfer cells. Both cell types contain electron‐dense vacuolar deposits that were digested by pronase and therefore are assumed to be protein. These deposits were negative to the PATAg test for carbohydrates. Protein bodies were also found in the parenchyma cells of the foot and younger meristematic cells at the base of the capsule. Vacuolar deposits of osmiophilic material in the gametophyte cells external to the placenta were stained non‐specifically with PATAg method and were not affected by pronase. The haustorial cells have pleomorphic plastids lacking starch and a thylakoid system, whereas the transfer cells have well developed chloroplasts. No pronase‐sensitive material was detected in the apo plastic space separating gametophyte and sporophyte cells. These results suggest that protein is synthesized in the haustorial cells, perhaps from precursors provided by transfer cells, and is then transferred, via plasmodesmata, to the parenchyma cells of the foot and eventually to the cells of the growing capsule.

Evolution of the terrestrial egg of amniotes (reptiles, birds, and mammals) is often considered to be one of the most significant events in vertebrate history. Presence of an eggshell, fetal membranes, and a sizeable yolk allowed this egg to develop on land and hatch out well-developed, terrestrial offspring. For centuries, morphologically-based studies have provided valuable information about the eggs of amniotes and the embryos that develop from them. This review explores the history of such investigations, as a contribution to this special issue of Journal of Morphology, titled Developmental Morphology and Evolution of Amniote Eggs and Embryos. Anatomically-based investigations are surveyed from the ancient Greeks through the Scientific Revolution, followed by the 19th and early 20th centuries, with a focus on major findings of historical figures who have contributed significantly to our knowledge. Recent research on various aspects of amniote eggs is summarized, including gastrulation, egg shape and eggshell morphology, eggs of Mesozoic dinosaurs, sauropsid yolk sacs, squamate placentation, embryogenesis, and the phylotypic phase of embryonic development. As documented in this review, studies on amniote eggs and embryos have relied heavily on morphological approaches in order to answer functional and evolutionary questions.

Background: In the pregnant population, being physical active and meeting gestational weight gain (GWG) guidelines have numerous health benefits for both mother and infant. Markers of lipid metabolism are known to be influenced by these two variables in the non-pregnant population. However, the relationship between physical activity (PA) and GWG on lipid markers has yet to be assessed during pregnancy. My thesis aims to address this gap in the literature. Methods: The first objective of my thesis was to examine the relationship between maternal PA and GWG on gross measurements of fetal and placental development (n=40). Specifically, three markers of placental efficiency (Pl-E) were examined (birthweight [BW], BW-to-placenta weight ratio, and residual BW). The second objective of my thesis was to analyze maternal serum lipid and glucose markers (n=40), in mid (24-28 weeks) and late (34-38 weeks) gestation as well as from the umbilical cord (UC) as they relate to both PA and GWG. The third objective of my thesis was to explore how PA level and GWG status affect markers of lipid metabolism in term placenta (n=31). Markers of placental lipid transport (FATP1, FABP4, FAT/CD36) were assessed at the protein level, and enzymatic activity of placental lipoprotein lipase was also measured. Lastly, placental lipid storage was assessed by examining triglyceride content, paired with lipid droplet staining. Results: There was no relationship between PA independently or in combination with GWG on any Pl-E markers. A significant association was found between GWG and BW in women who gained weight excessively compared to insufficiently. Neither PA nor GWG categorization was associated with maternal lipid and glucose markers. Total cholesterol levels measured in UC serum were significantly lower in women categorized as active throughout pregnancy (p<0.0001) or whose activity dropped in late gestation (p<0.0001) compared to those who were inactive v throughout gestation. Glucose levels were lower in UC blood of women who gained weight appropriately in mid-gestation compared to those who gained insufficient (p=0.040) or excessive (p=0.021) weight. In terms of placental fatty acid transport, there was a significant interaction between PA status and GWG categorization and placental FATP1 protein expression (F=14.62, p<0.0001). Finally, while no differences were found in placental lipid droplet staining, the droplets were more likely to be clustered within the syncytiotrophoblast border. Conclusion: In conclusion, maternal PA had no association with Pl-E, while GWG was only associated with BW. My thesis work found that while maternal serum lipid markers were not associated with PA and GWG, both maternal PA and GWG status were related to changes in UC and placental lipid markers throughout pregnancy. In combination with previous research from our lab, it is suggested that women who are physically active during pregnancy, and gain weight appropriately may be transporting fewer nutrients (i.e. fatty acid, glucose, cholesterol) to the placenta than those who are inactive, yet simultaneously increasing metabolization. Future research should further investigate these findings by performing functional experiments.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18860号 / 医博第3971号 / 新制||医||1008(附属図書館) / 31811 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 平岡 眞寛, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

Over the approximate nine months of its intrauterine existence, the development of the fetus is supported by the human placenta. This transient organ is central to pregnancy success as it facilitates maternal-fetal exchange, immunological tolerance, and hormone production. Villous trophoblasts mediate placental formation by engaging in a continuous turnover process of proliferation, differentiation, fusion, and apoptosis. In doing so, cytotrophoblasts and syncytiotrophoblasts maintain the integrity of the outer placental lining known as the syncytium. Exposure to drugs, however, can compromise placental establishment, which can in turn adversely impact pregnancy and fetal health. Specifically, cannabis is widely used by women of reproductive age and during pregnancy. While maternal cannabis use is linked to poor outcomes such as preterm birth and neurodevelopmental delays in exposed children, the underlying mechanisms are not well-defined. First, we characterized a functionally relevant cell line to model differentiation and fusion. In a comparison of the BeWo and BeWo b30 cell lines, our findings demonstrated that both models similarly undergo fusion. We then explored the implications of exposure to delta-9- tetrahydrocannabinol (∆9-THC) on the immunological roles of villous trophoblasts. We observed that cytotrophoblast differentiation and fusion were associated with localized inflammation due to elevated interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) but inhibited interleukin-4 (IL-4) and interleukin-10 (IL-10) production. ∆9-THC exposure impaired this T helper 1/2 cytokine balance through decreased IL-2 and TNF-α as well as increased IL-4 and IL-10 levels. Subsequently, we investigated the effects of ∆9-THC in TNF-α- and IL-10-dominant environments, to represent inflammatory and immunomodulatory microenvironments, respectively. Coincident with inflammation, ∆9-THC attenuated trophoblast fusion and the biosynthesis of steroid hormones, progesterone and cortisol, through perturbed cytochrome P450 regulation. This thesis ultimately lays a foundation for understanding how cannabis use during pregnancy may compromise the fusogenic, immune and endocrine functions of villous trophoblasts in the placenta. / Thesis / Master of Science (MSc) / The human placenta is a pregnancy-specific organ that supports the health of the mother- to-be and fetus. Stem cells known as cytotrophoblasts undergo differentiation and fusion to support the establishment of the syncytium, which creates a boundary that separates the maternal and fetal circulations. In the case of cannabis consumption during pregnancy, its biologically active components can travel to the placenta, cross the syncytium, and enter fetal blood. Our primary objective was to determine how cannabis exposure can impact the formation and maintenance of the syncytium. While maternal use has been linked to short- and long-term consequences for child health, existing research lacks a complete understanding of the underlying mechanisms. We demonstrate that cannabis exposure alters the production of important immune and hormonal factors during cytotrophoblast fusion, which may play a role in mediating poor placental development. Ultimately, it is critical to explore the implications of cannabis use for female reproductive health due to a rising trend in its use.