Abstract
To maintain a healthy pregnancy, the exchange of substances between mother and fetus is vital. All transport of these substances takes place through the placenta, which is a temporary organ that serves its purpose from the implantation of the blastula to the birth of the term fetus, supplying nutrients, gas and waste transport between the maternal blood and the developing fetus and maintaining pregnancy by producing hormones. The placenta consists of cells of both maternal and fetal origin and forms a complex barrier between the maternal and fetal blood that allows for passage of different molecules, either by passive or facilitated diffusion or active transport systems. This makes placental transport studies interesting when investigating fetal exposure to foreign or innate substances.
The aim of this thesis is to investigate the transport of selected substances across the human placenta using the human dually perfused recirculating placenta perfusion model. Perfusion models are very useful in studying effects and actions of various live tissues, in order to extrapolate the findings to a real life exposure situation. Simplified models make validated inter-laboratory comparison and extrapolation to the in vivo situation critical.
In my PhD study I have focused on validation and studies with placental perfusion of substances with a high molecular weight, which require transport or carrier molecules to be transported from the maternal to the fetal side, and longer perfusion time demanding more specialized media. Control substances ensure sufficient overlap between maternal and fetal circulations, and a tight fetal system. The water soluble antipyrine which has a low molecular weight is used as a positive control, as it diffuses passively across the placenta and reaches equilibrium within two hours of perfusion with a fetal flow rate of 3 mL/min. Negative controls are added to ensure that substance transfer is not due to leakage, e.g. high molecular weight substances that only pass the placental barrier with bulk flow through a leakage in the fetal system. Dextran (40kD) can be used as a negative control when adding a small amount to the fetal reservoir. To be able to detect any trace of dextran in the maternal reservoir in case of a leakage, the dextran is labeled with FITC and analyzed by fluorescence measurement (Paper I). Inter-laboratory comparisons have confirmed the reproducibility of the system1.
The first high molecular substance perfused was Benzo[a]pyrene (BaP), a highly carcinogenic substance produced by everyday combustion. The transport kinetics of this substance is interesting, as it is influenced by the concentration and species-origin of albumin in the medium. BaP showed a higher trans-placental transport when perfused with medium containing physiological concentrations of human serum albumin as opposed to lower concentrations of bovine serum albumin. Albumin functions as a transport molecule for BaP by keeping it suspended in the medium and loading BaP into the placental barrier cell-layer closest to the maternal blood (the syncytiotrophoblast) (Paper II).
The placental transport of three polybrominated diphenyl ethers: BDE-47, BDE-99 and BDE-209 were studied in collaboration with researchers from the National Environmental Research Institute, who performed the chemical analysis. The perfusions showed decreased transfer across placenta with increased bromination of the molecule2.
In collaboration with Dr Margaret Saunders’ group at BIRCH, Biophysics Research Unit, St Michael’s Hospital, Bristol, the placental transport of two non-dioxin-like polychlorinated biphenyls (PCBs) (PCB52 and 180) were studied in the Copenhagen placental perfusion model and in the BeWo b30 clone monolayer transfer model in Bristol. The transfer of PCB 180 was more rapid in both systems, and PCB 52 adhered more to the placental tissue3.
As the method was developed, the transport of different recombinant human antibodies across the placenta could be investigated. Recombinant antibodies of different isotypes and with known mutations were developed at the Department of Clinical Immunology, Rigshospitalet. To measure the properties of the mutated versions we developed a perfusion model where an antibody with a normal structure is used as an internal calibrator concomitantly with the mutated version. Thus the transfer of mutated antibodies can be compared with the transfer of normal antibodies. This will contribute to the knowledge on treating pregnant women or fetuses directly using antibody therapy. Placental perfusion studies of antibodies were performed with two isotypes (IgG1 and IgG3) and two mutated versions of IgG3 (HM5 and HM5R435H), with antigen affinity for either malaria (+RAM1) or the RhD molecule on the red blood cells (+GAN). The antibody specificity did not affect transport. Preliminary data suggest a similar transfer-rate for IgG1 and a mutated IgG3 with two mutations –one in the hinge region and one in the Fc-region. The mutated versions with a single mutation in the hinge region had the lowest transport rate. (IgG1= HM5R435H >>HM5) (Paper III).
To investigate the placental cell layer facing the maternal blood (the syncytiotrophoblast) a cell assay using a human trophoblast cell line (BeWo clone b30 monolayer) has been established in our group. The transport through this system of two of the monoclonal antibodies mentioned above was also investigated, and they showed comparability with placental transport although with a slower transport rate. In the BeWo monolayer transport model, cell death happened ½-3 hours after addition of BaP (1 µM) in a toluene solution (0.14%).
The morphology of perfused placental tissue has only been briefly described in the literature and to our knowledge a prospective detailed examination of a series of perfused placentas has not previously been published. In collaboration with two pathologists trained in examining placental tissue, a study was made on perfused placentas, both from successful and unsuccessful perfusions, describing the morphology and condition of both perfused and non-perfused tissue after up to six hours perfusion time. The success-criterion of fetal leak below 3 mL/hours perfusion was significantly inversely correlated with the histological finding “trophoblastic vacuolisation”. The study also proposes a standard method for examining perfused placental tissue (Paper IV).
The aim of this thesis is to investigate the transport of selected substances across the human placenta using the human dually perfused recirculating placenta perfusion model. Perfusion models are very useful in studying effects and actions of various live tissues, in order to extrapolate the findings to a real life exposure situation. Simplified models make validated inter-laboratory comparison and extrapolation to the in vivo situation critical.
In my PhD study I have focused on validation and studies with placental perfusion of substances with a high molecular weight, which require transport or carrier molecules to be transported from the maternal to the fetal side, and longer perfusion time demanding more specialized media. Control substances ensure sufficient overlap between maternal and fetal circulations, and a tight fetal system. The water soluble antipyrine which has a low molecular weight is used as a positive control, as it diffuses passively across the placenta and reaches equilibrium within two hours of perfusion with a fetal flow rate of 3 mL/min. Negative controls are added to ensure that substance transfer is not due to leakage, e.g. high molecular weight substances that only pass the placental barrier with bulk flow through a leakage in the fetal system. Dextran (40kD) can be used as a negative control when adding a small amount to the fetal reservoir. To be able to detect any trace of dextran in the maternal reservoir in case of a leakage, the dextran is labeled with FITC and analyzed by fluorescence measurement (Paper I). Inter-laboratory comparisons have confirmed the reproducibility of the system1.
The first high molecular substance perfused was Benzo[a]pyrene (BaP), a highly carcinogenic substance produced by everyday combustion. The transport kinetics of this substance is interesting, as it is influenced by the concentration and species-origin of albumin in the medium. BaP showed a higher trans-placental transport when perfused with medium containing physiological concentrations of human serum albumin as opposed to lower concentrations of bovine serum albumin. Albumin functions as a transport molecule for BaP by keeping it suspended in the medium and loading BaP into the placental barrier cell-layer closest to the maternal blood (the syncytiotrophoblast) (Paper II).
The placental transport of three polybrominated diphenyl ethers: BDE-47, BDE-99 and BDE-209 were studied in collaboration with researchers from the National Environmental Research Institute, who performed the chemical analysis. The perfusions showed decreased transfer across placenta with increased bromination of the molecule2.
In collaboration with Dr Margaret Saunders’ group at BIRCH, Biophysics Research Unit, St Michael’s Hospital, Bristol, the placental transport of two non-dioxin-like polychlorinated biphenyls (PCBs) (PCB52 and 180) were studied in the Copenhagen placental perfusion model and in the BeWo b30 clone monolayer transfer model in Bristol. The transfer of PCB 180 was more rapid in both systems, and PCB 52 adhered more to the placental tissue3.
As the method was developed, the transport of different recombinant human antibodies across the placenta could be investigated. Recombinant antibodies of different isotypes and with known mutations were developed at the Department of Clinical Immunology, Rigshospitalet. To measure the properties of the mutated versions we developed a perfusion model where an antibody with a normal structure is used as an internal calibrator concomitantly with the mutated version. Thus the transfer of mutated antibodies can be compared with the transfer of normal antibodies. This will contribute to the knowledge on treating pregnant women or fetuses directly using antibody therapy. Placental perfusion studies of antibodies were performed with two isotypes (IgG1 and IgG3) and two mutated versions of IgG3 (HM5 and HM5R435H), with antigen affinity for either malaria (+RAM1) or the RhD molecule on the red blood cells (+GAN). The antibody specificity did not affect transport. Preliminary data suggest a similar transfer-rate for IgG1 and a mutated IgG3 with two mutations –one in the hinge region and one in the Fc-region. The mutated versions with a single mutation in the hinge region had the lowest transport rate. (IgG1= HM5R435H >>HM5) (Paper III).
To investigate the placental cell layer facing the maternal blood (the syncytiotrophoblast) a cell assay using a human trophoblast cell line (BeWo clone b30 monolayer) has been established in our group. The transport through this system of two of the monoclonal antibodies mentioned above was also investigated, and they showed comparability with placental transport although with a slower transport rate. In the BeWo monolayer transport model, cell death happened ½-3 hours after addition of BaP (1 µM) in a toluene solution (0.14%).
The morphology of perfused placental tissue has only been briefly described in the literature and to our knowledge a prospective detailed examination of a series of perfused placentas has not previously been published. In collaboration with two pathologists trained in examining placental tissue, a study was made on perfused placentas, both from successful and unsuccessful perfusions, describing the morphology and condition of both perfused and non-perfused tissue after up to six hours perfusion time. The success-criterion of fetal leak below 3 mL/hours perfusion was significantly inversely correlated with the histological finding “trophoblastic vacuolisation”. The study also proposes a standard method for examining perfused placental tissue (Paper IV).
| Original language | English |
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| Publication status | Published - 31 Mar 2011 |
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Keywords
- Faculty of Health and Medical Sciences