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Drug transporter expression during in vitro differentiation of first-trimester and term human trophoblasts
A48
Abstracts / Placenta 35 (2014) A1eA112
P1.120-N. PLACENTAL FTO GENE EXPRESSION IS CORRELATED WITH MATERNAL FATTY ACID CONCENTRATION IN BLOOD AT EARLY AND LATE PREGNANCY Mildrey Mosquera a, b, Jane K. Cleal a, Sheila J. Barton a, John W. Holloway a, Charlene Sibbons c, a, Hazel M. Inskip a, Philip C. Calder a, Cyrus Cooper a, Keith M. Godfrey a, Rohan M. Lewis a, SWS study Group a a University of Southampton, Southampton, UK; b Universidad del Valle, Cali, Colombia; c University of Surrey, Surrey, UK Introduction: The fat mass and obesity related (FTO) gene is an RNA demethylase. Genetic polymorphisms in the FTO gene are associated with obesity and placental FTO mRNA expression has been associated with birth weight. We investigated the relationship between FTO and prenatal growth in a new cohort and determined whether maternal plasma phospholipid fatty acid concentrations in early and late pregnancy correlate with FTO gene expression. Methods: We used data and samples from the Southampton Women’s Survey, a mother-offspring cohort study with information collected from the mothers before conception. 102 tissue samples used for this analysis were selected from 300 collected in total, based on availability of placental transport and neonatal dual-energy X-ray absorptiometry body composition data. FTO mRNA expression was measured by quantitative rtPCR. Pearson’s correlation coefficient (rp) was used to determine partial correlations adjusted for sex and gestational age between mRNA levels, neonatal body composition and maternal plasma phospholipid fatty acid concentrations at 11 (n¼41) and 32 (n¼90) weeks gestation. Results: FTO gene expression was correlated with birth weight (r¼0.21, p¼0.033), neonatal head circumference (r¼0.30, p¼0.002) and neonatal fat mass (r¼0.23, p¼0.020). Higher placental FTO expression was associated with lower maternal 11 week plasma phospholipid concentrations of 20:1n-9 (r¼0.31, p¼0.049), 20:3n-6 (r¼-0.36, p¼0.023), 20:4n-3 (r¼-0.34, p¼0.033) and 24:1n-9 (r¼-0.31, p¼0.045), and with higher maternal 32 week plasma concentrations of 22:5n-3 (r¼0.26, p¼0.012) and 22:6n-3 (r¼0.26, p¼0.015). Conclusion: This study shows relationships between maternal plasma phospholipid fatty acid levels and placental FTO expression. This suggests potential mechanisms by which placental FTO may be regulated and perhaps mediate effects on placental function and fetal growth.
P1.121. EXPRESSION OF MIDKINE IN THE HUMAN PLACENTA: A POSSIBLE ROLE FOR EARLY PLACENTAL DEVELOPMENT Atsuko Togo, Yumiko Goto, Kanako Mitsuzuka, Hidetoshi Kanno, Atsuya Narita, Satoshi Asai, Masaki Miyazawa, Hitoshi Ishimoto Dept. of OBGYN, Tokai University School of Medicine, Isehara, Kanagawa, Japan Objectives: Midkine (MK) is a heparin-binding growth factor with various bioactivities including cell proliferation, migration, differentiation and angiogenesis, most of which are essential components of organogenesis. MK expression is temporally regulated; MK is highly expressed during midgestation. Strong MK expression is found in tissues where epithelialmesenchymal interactions or remodeling are taking place. Although public databases and previous studies show minimal or low expression of MK in the term placenta of mice and humans, its placental expression profile has not yet been well-documented. In this study, we investigated placental mRNA expression and localization of MK throughout gestation, and possible regulatory effects of MK on gene expression of syncytins in BeWo cells. Methods: Human tissues were obtained under informed consent and an institutional review board-approved protocol. Murine samples were from commercial sources. The following methods were used: 1) TaqMan realtime quantitative RT-PCR (qPCR) for mRNA expression, 2) immunohistochemistry for MK localization, and 3) qPCR for mRNA levels of syncytins in BeWo cells treated with recombinant human MK (rhMK). Results: Murine placental MK mRNA remained at constant levels from E10 to E16, and increased by 40-50% at E18. In contrast, in the human
placenta, MK mRNA showed the highest level in the first trimester and decreased dramatically thereafter to a scant level at term. Human MK was localized mainly to syncytiotrophoblasts, and the extent and intensity of immunoreactive MK followed a gestational pattern similar to that of MK mRNA. In BeWo cells, rhMK treatment significantly increased syncytin-2 mRNA levels in a dose-dependent manner (24 hrs: by 2.4-fold at 10 ng/ml). However, rhMK did not significantly change syncytin-1 mRNA. Conclusion: The temporal and spatial expression pattern of MK and its regulation of syncytin-2 expression suggest a role for MK in early stages of human placental development.
P1.122. DRUG TRANSPORTER EXPRESSION DURING IN DIFFERENTIATION OF FIRST-TRIMESTER AND TERM TROPHOBLASTS
VITRO HUMAN
verine Degrelle, Nadine Segond, Audrey Chissey, Danie le Paul Berveiller, Se Evain-Brion, Sophie Gil UMR-S 1139, INSERM, University of Paris Descartes, Paris, France Background: Drug transporters interfere with drug disposition during pregnancy by actively transporting drugs from mother to fetus, and vice versa. Data on their placental expression are scarce, especially during the first trimester. Morphological modifications of trophoblast layers throughout pregnancy might be associated with a distinct functional role of the cytotrophoblasts, reflected by a different expression of drug transporters. Methods: Here we examined drug transporter expression during in vitro differentiation of cytotrophoblastic cells into syncytiotrophoblast. Cells were isolated from first-trimester and term placentas. We used a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)-based drug transporter array to examine mRNA expression of 84 pharmaceutically relevant transporters (ABC and SLC transporters). Results: We report the first data on the expression kinetics of human drug transporters genes during in vitro differentiation of purified human cytotrophoblastic cells isolated from first-trimester and term placentas. This study reveals that some transporters are differentially expressed during pregnancy and during human trophoblastic cells differentiation. Conclusion: This expression panel of placental drug transporters should help to understand transplacental drug transfer and to ensure more rational drug use during pregnancy.
P1.123. PILOT STUDY: YOLK SAC VASCULAR DEVELOPMENT AND VEGF EXPRESSION DURING EARLY GESTATION IN BOVINES DERIVED FROM NUCLEAR TRANSFER Andrea Mess a, Ana Claudia Carreira b, Paula Fratini a, Phelipe Favaron a, Flavio Meirelles c, Maria Angelica Miglino a a Department of ~o Paulo, Sao Paulo, Surgery, Faculty of Veterinary Medicine, University of Sa ~o Paulo, Sao Paulo, SP, Brazil; b Department of Chemistry, University of Sa SP, Brazil; c Faculty of Animal Sciences and Food Engineering, University of ~o Paulo, Sao Paulo, SP, Brazil Sa The bovine yolk sac is vulnerable to morphological alterations in embryos and fetuses derived from reproductive techniques, especially in clones. In particular the vasculature seems to be affected, reflecting the importance of the yolk sac as early source of haematopoiesis, vasculogenesis and angiogenesis. Objectives: In order to better understand such processes, we investigated the early differentiation of the yolk sac in cloned bovine. Methods: Yolk sacs from bovines derived from in vivo breeds (n ¼ 5) and nuclear transfer (n ¼ 5) were analyzed by fine structure and vasculature as well as the expression of VEGF genes. Results: Accordingly, the yolk sac structure was weird in clones with only few vessels that had a very thin, partly interrupted endothelium and
Abstracts / Placenta 35 (2014) A1eA112
P1.120-N. PLACENTAL FTO GENE EXPRESSION IS CORRELATED WITH MATERNAL FATTY ACID CONCENTRATION IN BLOOD AT EARLY AND LATE PREGNANCY Mildrey Mosquera a, b, Jane K. Cleal a, Sheila J. Barton a, John W. Holloway a, Charlene Sibbons c, a, Hazel M. Inskip a, Philip C. Calder a, Cyrus Cooper a, Keith M. Godfrey a, Rohan M. Lewis a, SWS study Group a a University of Southampton, Southampton, UK; b Universidad del Valle, Cali, Colombia; c University of Surrey, Surrey, UK Introduction: The fat mass and obesity related (FTO) gene is an RNA demethylase. Genetic polymorphisms in the FTO gene are associated with obesity and placental FTO mRNA expression has been associated with birth weight. We investigated the relationship between FTO and prenatal growth in a new cohort and determined whether maternal plasma phospholipid fatty acid concentrations in early and late pregnancy correlate with FTO gene expression. Methods: We used data and samples from the Southampton Women’s Survey, a mother-offspring cohort study with information collected from the mothers before conception. 102 tissue samples used for this analysis were selected from 300 collected in total, based on availability of placental transport and neonatal dual-energy X-ray absorptiometry body composition data. FTO mRNA expression was measured by quantitative rtPCR. Pearson’s correlation coefficient (rp) was used to determine partial correlations adjusted for sex and gestational age between mRNA levels, neonatal body composition and maternal plasma phospholipid fatty acid concentrations at 11 (n¼41) and 32 (n¼90) weeks gestation. Results: FTO gene expression was correlated with birth weight (r¼0.21, p¼0.033), neonatal head circumference (r¼0.30, p¼0.002) and neonatal fat mass (r¼0.23, p¼0.020). Higher placental FTO expression was associated with lower maternal 11 week plasma phospholipid concentrations of 20:1n-9 (r¼0.31, p¼0.049), 20:3n-6 (r¼-0.36, p¼0.023), 20:4n-3 (r¼-0.34, p¼0.033) and 24:1n-9 (r¼-0.31, p¼0.045), and with higher maternal 32 week plasma concentrations of 22:5n-3 (r¼0.26, p¼0.012) and 22:6n-3 (r¼0.26, p¼0.015). Conclusion: This study shows relationships between maternal plasma phospholipid fatty acid levels and placental FTO expression. This suggests potential mechanisms by which placental FTO may be regulated and perhaps mediate effects on placental function and fetal growth.
P1.121. EXPRESSION OF MIDKINE IN THE HUMAN PLACENTA: A POSSIBLE ROLE FOR EARLY PLACENTAL DEVELOPMENT Atsuko Togo, Yumiko Goto, Kanako Mitsuzuka, Hidetoshi Kanno, Atsuya Narita, Satoshi Asai, Masaki Miyazawa, Hitoshi Ishimoto Dept. of OBGYN, Tokai University School of Medicine, Isehara, Kanagawa, Japan Objectives: Midkine (MK) is a heparin-binding growth factor with various bioactivities including cell proliferation, migration, differentiation and angiogenesis, most of which are essential components of organogenesis. MK expression is temporally regulated; MK is highly expressed during midgestation. Strong MK expression is found in tissues where epithelialmesenchymal interactions or remodeling are taking place. Although public databases and previous studies show minimal or low expression of MK in the term placenta of mice and humans, its placental expression profile has not yet been well-documented. In this study, we investigated placental mRNA expression and localization of MK throughout gestation, and possible regulatory effects of MK on gene expression of syncytins in BeWo cells. Methods: Human tissues were obtained under informed consent and an institutional review board-approved protocol. Murine samples were from commercial sources. The following methods were used: 1) TaqMan realtime quantitative RT-PCR (qPCR) for mRNA expression, 2) immunohistochemistry for MK localization, and 3) qPCR for mRNA levels of syncytins in BeWo cells treated with recombinant human MK (rhMK). Results: Murine placental MK mRNA remained at constant levels from E10 to E16, and increased by 40-50% at E18. In contrast, in the human
placenta, MK mRNA showed the highest level in the first trimester and decreased dramatically thereafter to a scant level at term. Human MK was localized mainly to syncytiotrophoblasts, and the extent and intensity of immunoreactive MK followed a gestational pattern similar to that of MK mRNA. In BeWo cells, rhMK treatment significantly increased syncytin-2 mRNA levels in a dose-dependent manner (24 hrs: by 2.4-fold at 10 ng/ml). However, rhMK did not significantly change syncytin-1 mRNA. Conclusion: The temporal and spatial expression pattern of MK and its regulation of syncytin-2 expression suggest a role for MK in early stages of human placental development.
P1.122. DRUG TRANSPORTER EXPRESSION DURING IN DIFFERENTIATION OF FIRST-TRIMESTER AND TERM TROPHOBLASTS
VITRO HUMAN
verine Degrelle, Nadine Segond, Audrey Chissey, Danie le Paul Berveiller, Se Evain-Brion, Sophie Gil UMR-S 1139, INSERM, University of Paris Descartes, Paris, France Background: Drug transporters interfere with drug disposition during pregnancy by actively transporting drugs from mother to fetus, and vice versa. Data on their placental expression are scarce, especially during the first trimester. Morphological modifications of trophoblast layers throughout pregnancy might be associated with a distinct functional role of the cytotrophoblasts, reflected by a different expression of drug transporters. Methods: Here we examined drug transporter expression during in vitro differentiation of cytotrophoblastic cells into syncytiotrophoblast. Cells were isolated from first-trimester and term placentas. We used a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)-based drug transporter array to examine mRNA expression of 84 pharmaceutically relevant transporters (ABC and SLC transporters). Results: We report the first data on the expression kinetics of human drug transporters genes during in vitro differentiation of purified human cytotrophoblastic cells isolated from first-trimester and term placentas. This study reveals that some transporters are differentially expressed during pregnancy and during human trophoblastic cells differentiation. Conclusion: This expression panel of placental drug transporters should help to understand transplacental drug transfer and to ensure more rational drug use during pregnancy.
P1.123. PILOT STUDY: YOLK SAC VASCULAR DEVELOPMENT AND VEGF EXPRESSION DURING EARLY GESTATION IN BOVINES DERIVED FROM NUCLEAR TRANSFER Andrea Mess a, Ana Claudia Carreira b, Paula Fratini a, Phelipe Favaron a, Flavio Meirelles c, Maria Angelica Miglino a a Department of ~o Paulo, Sao Paulo, Surgery, Faculty of Veterinary Medicine, University of Sa ~o Paulo, Sao Paulo, SP, Brazil; b Department of Chemistry, University of Sa SP, Brazil; c Faculty of Animal Sciences and Food Engineering, University of ~o Paulo, Sao Paulo, SP, Brazil Sa The bovine yolk sac is vulnerable to morphological alterations in embryos and fetuses derived from reproductive techniques, especially in clones. In particular the vasculature seems to be affected, reflecting the importance of the yolk sac as early source of haematopoiesis, vasculogenesis and angiogenesis. Objectives: In order to better understand such processes, we investigated the early differentiation of the yolk sac in cloned bovine. Methods: Yolk sacs from bovines derived from in vivo breeds (n ¼ 5) and nuclear transfer (n ¼ 5) were analyzed by fine structure and vasculature as well as the expression of VEGF genes. Results: Accordingly, the yolk sac structure was weird in clones with only few vessels that had a very thin, partly interrupted endothelium and