The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

Transport of large neutral amino acids into BeWo cells.

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into columns and superfused. Unidirectional influx of l -phenylalanine (l -phe) and l -leucine (l -leu) across the microvillous border of the cells was studied using a rapid paired-tracer dilution technique. Influx of l -phe and l -leu comprised both saturable and non-saturable components. K(m)values for l -phe and l -leu were 0.57+/-0.01 m m and 0.05+/-0.01 m m, respectively, with V(max)values of 120.4+/-0.5 nmol/mg/min and 41. 7+/-0.2 nmol/mg/min. Non-saturable uptake components were 29.0+/-0.1 nmol/mg/m m and 37.9+/-0.1 nmol/mg/min/m m respectively. l -leu uptake was found to be sodium-independent. The uptake of l -[(3)H]phe was strongly inhibited (90-100 per cent) by unlabelled l -phe, d -phe, l -leu or 2-aminoendobicyclo-[2,2, 1]-heptane-2-carboxylic acid (BCH) but not by l -arginine (l -arg) or methyl alpha-aminoisobutric acid (Me-AIB). Pre-incubation of Bewo cultures for 24 h in the presence of an additional 1.2 m ml -phe (simulating maternal phenylketonuria) significantly reduced both the K(m)and V(max)components of l -phe influx. l -arg (2 m m) had no effect on l -leu influx whereas 2 m ml -phe completely inhibited saturable l -leu influx. These data suggest that the microvillous border of differentiated BeWo cells transport large neutral amino acids predominantly via system L rather than by B(0) or y(+)L transporters.

Regulation of the choline transport system in superfused microcarrier cultures of BeWo cells.

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into syringe barrels and superfused. The unidirectional choline uptake across the microvillous membrane of the cells was measured by a rapid single-circulation paired-tracer dilution procedure using methyl[3H]choline with D-[14C]mannitol as the extracellular reference molecule. Choline influx was saturable with a K(t) of 214+/-15 microM and a V(max) of 45.29+/-0.94 nmol/min/mg of cell protein. Uptake of labelled choline was partially inhibited by nicotine, strongly inhibited by hemicolinium-3, and was reduced by about 50 per cent in sodium-free perfusates. A range of agents was added to the stirrer flasks 24 h prior to the experiments to determine if intracellular or extracellular levels of choline or its metabolic product, acetylcholine, regulated choline uptake. Pre-incubation with 2 mM choline reduced the choline maximal uptake by half, while pre-incubation with 100 microM alpha-NETA [2-(alpha-naphthoyl)ethyltrimethyl-ammonium] reduced the influx by 77 per cent. Choline influx was also reduced to about half in the presence of 100 microM vesamicol, bethanecol or neostigmine. It is concluded that BeWo cells possess a choline transporter similar to that described in isolated cytotrophoblasts and syncytiotrophoblast microvillous membrane preparations, and that uptake appeared to be regulated by both intracellular and extracellular concentrations of choline and acetylcholine. Therefore, these cells provide a novel model for studying the role of acetylcholine in human placenta.

In vitro modulation of L-arginine transport in trophoblast cells by glucose.

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.

Image analysis of protein profiles from paired microvillous and basal syncytiotrophoblast plasma membranes from term human placenta and characterization of IgG binding to membrane vesicles.

Image analysis of SDS-PAGE profiles of highly purified, paired maternal-facing (microvillous; MVM) and fetal-facing (basal; BM) plasmalemma membrane vesicles from six term human placentae showed that, while individual MVM or BM profiles were extremely reproducible, the two membrane populations were substantially different--although all of the seven major bands of molecular mass, 98.4, 79.4, 71.1, 45.1, 40.9, 39.5 and 34.5 kDa found in MVM were present, albeit in differing amounts, in BM. BM were characterized by the presence of five low molecular weight bands which were not present in MVM. Despite this consistency of the membrane preparations, binding of 125I-IgG or its fragments showed marked variability in both MVM and BM. At pH 7.4, both MVM and BM bound similar amounts of 125I-IgG with Kd values of 5.2 +/- 1.9 x 10(-6) M (s.e., n = 8) and 2.9 +/- 0.4 x 10(-6) M respectively, (P > 0.05). There were 1.2-1.6 x 10(15) binding sites/mg protein. Affinity constants for Fc fragment binding to MVM and BM were similar to those for IgG, although the Bmax value for BM Fc binding was greatly reduced compared to that for IgG (P > 0.001). Fab binding to MVM and BM was also saturable but substantially lower than that of Fc, whereas binding of F(ab')2 was low and linear. Both MVM and BM bound marginally more IgG at pH 6.0 than at pH 7.4. These data provide further evidence for receptor-mediated transcytosis of maternal IgG across the placenta and confirm that the placental IgG transporter differs from classical Fc gamma receptors.

Superfused microcarrier cultures of choriocarcinoma cells: a dynamic model for studying transport of glucose and amino acids.

We here describe a novel procedure for culturing BeWo and JAr choriocarcinoma cells on solid microcarrier beads. The regime developed to stir the beads resulted, after about 10 days, in small aggregates of two to six beads covered with a layer of differentiated cells. Bead aggregates were packed into small columns and superfused, providing a dynamic in vitro system for studying the uptake mechanisms for sugars and amino acids. The rapid, unidirectional uptake of tritiated L-phenylalanine, L-serine, L-arginine and D-glucose was determined, relative to an extracellular reference tracer, in cells superfused in 0.5 ml (range 0.3-0.8 ml) columns. Several sequential measurements could be made in the same column. Twenty-four hour pre-incubation with dexamethasone (0.25 microM) was found to increase the transport of D-glucose. Uptake of D-glucose was reduced by over 80 per cent following 20 min perfusion of the cells with 1 mM phloretin. Pre-incubation with growth hormone (0.2 microgram/ml) decreased the transport of serine, whereas nicotine (0.5 microgram/ml) decreased both serine and phenylalanine uptake. Atropine (1 mM) or 5-oxoproline (0.5 mM) had no short-term effects on amino acid uptake. Insulin (12.5 mIU/ml) had no effect on the transport of the amino acids but caused a small but significant increase in glucose transport (P < 0.05). This model allows characterization of human trophoblast function without the complications resulting from the presence of other cell types in placental slices or fragments.

Superfused Microcarrier Cultures of BeWo Choriocarcinoma Cells : A Dynamic Model for Studies of Human Trophoblast Function.

The human placenta is a unique and still very poorly understood organ During gestation, it is responsible for performing most synthetic and transport functions that are necessary to ensure the normal growth and development of the fetus in utero. Subsequently, at birth it is discarded. Current understanding of the role of the placenta in maintaining fetal homeostasis is based primarily on studies using either artificially perfused term placental lobules, placental slices or explants, or isolated plasma membrane fractions (1-3). Studies with placental tissue are complicated by the presence of serial tissue layers in the maternofetal barrier, i e., syncytiotrophoblast, cytotrophoblast, and fetal endothelium, as well as by the presence of other cell types within the placental tissue, such as the Hofbauer cells or placental macrophages.

Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels.

The microvessels of the human placenta resemble those of skeletal muscle, both in endothelial cell junctional organization and in the single passage extraction of radiolabeled tracers. Addition of histamine (100 microM) to the fetal perfusate in an isolated term lobule resulted in a rapid and sustained rise (40-80%) over a 30-min perfusion period in the single circulation extraction values of 57Co-labeled cyanocobalamin and of 51Cr-labeled EDTA, but not of 22Na. Extraction values for 125I-albumin were not increased in histamine-perfused vessels nor was any focal leakage of label observed in serial cryostat sections of lobules perfused with rhodamine-conjugated albumin. There was no electron microscopic evidence of transendothelial channels in the microvascular bed; no gaps were seen at the paracellular cleft regions or in neighboring cytoplasm in any microvessels. Tilting of sections on a goniometric stage showed a significant increase in the separation between adjoining endothelial membrane leaflets at tight junctional regions (from 4.1 to 6.1 nm) although the dimensions of the wide zones remained unchanged. Placental microvessels contain the endothelial adhesion molecules PECAM-1 and VE-cadherin in the wide regions of paracellular clefts: PECAM-1 is also localized on the luminal membrane. Histamine-stimulated microvessels showed an altered staining pattern for both of these molecules, at the expense of the cleft regions. These changes in adhesion molecule distribution and tight junctional membrane separation may be parts of a series of events which leads to increased permeability during inflammatory events.

Sequential preparation of highly purified microvillous and basal syncytiotrophoblast membranes in substantial yield from a single term human placenta: inhibition of microvillous alkaline phosphatase activity by EDTA.

The human placental syncytiotrophoblast is a highly polarised epithelial layer responsible for regulating materno-fetal exchange. We here describe a novel procedure for isolating paired fractions of the maternal-facing and fetal-facing plasma membranes from this syncytium, from a single placenta, without the need for homogenisation procedures. This reduces the potential for contamination of these membrane fractions by intracellular membranes, or from plasma membranes from other cell types within the placenta. Microvillous membrane vesicles (MVM) were obtained by gentle stirring of dispersed villous tissue. The tissue sedimented at the end of this procedure was subjected to sequential ultrasonication to release the basal membrane (BM). Crude MVM was subsequently purified on a discontinuous sucrose gradient. Crude BM was further purified using either discontinuous Ficoll or sucrose gradients. The Ficoll procedure, while producing a BM fraction extremely enriched in marker enzyme, resulted in unacceptably low protein recoveries and hence the sucrose gradient procedure was also adopted for BM. Yields for MVM and BM produced on sucrose density gradients approached 30 mg/100 g tissue. The MVM fraction was composed of vesicles of 232 +/- 9 (S.E.) nm diameter of which nearly 90% were 'right side out'. These membranes were 37-fold enriched in the marker enzyme alkaline phosphatase. Purified BM vesicles were 317 +/- 14 nm in diameter, also approximately 90% 'right side out' and over 40-fold enriched in dihydroalprenolol binding. Cross-contamination or contamination from intracellular membranes was negligible. MVM alkaline phosphatase activity was shown to be inhibitable in a dose- and time-dependent manner by EDTA present in the storage buffer.

Permeability of the fetal villous microvasculature in the isolated perfused term human placenta.

1. Capillary permeability-surface area (PS) products for the low molecular weight radioactive tracers, 22Na, 51Cr-EDTA (relative molecular mass 357) and 57Co-cyanocobalamin (relative molecular mass 1353) were measured in the fetal circulation of isolated dually perfused lobules of normal term human placentae using the single circulation, multiple-tracer dilution technique. 2. In lobules perfused with M199 medium, containing dextran and 5 g l-1 bovine albumin, the extractions of all three tracers decreased as the flow was increased over the range of 2-8 ml min-1, and PS products for 51Cr-EDTA and 57Co-cyanocobalamin, but not for 22Na, reached constant values at flows above 0.1 ml min-1 g-1. 3. Flow-independent PS products in the presence of albumin were 0.025 +/- 0.002 ml min-1 g-1 (mean +/- S.E.M., n = 25) for 57Co-cyanocobalamin and 0.057 +/- 0.003 ml min-1 g-1 (n = 25) for 51Cr-EDTA. The ratio of PS values (51Cr-EDTA/57Co-cyanocobalamin) was 2.28, while the ratio of the corresponding free diffusion coefficients was 1.79, indicating substantial restriction to the diffusion of the 57Co-cyanocobalamin. 4. In another series of lobules perfused in the absence of albumin, extraction values for all three test tracers were constant over the same flow range. Values at high flow rates were therefore about twice those measured in the presence of albumin, and PS products for all three tracers failed to reach diffusion-limited values. 6. Lobules perfused with and without albumin were fixed using a glutaraldehyde fixative containing 1% Alcian Blue dye. An ultrastructural examination of the endothelium showed no significant changes in cell or cleft morphology, or in the glycocalyx, in the absence of albumin which might account for the observed permeability change. 7. These data are the first physiological measurements specifically characterizing fetal microvascular permeability in the human placenta. The results suggest that permeability resembles that found in skeletal muscle and, as such, the endothelium presents a significant barrier to the diffusion of large solutes. The observed 'protein effect' indicates that albumin can interact with elements of the solute pathway to increase its restrictiveness.

Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: effects of flow rate, thrombin, and cytochalasin D.

We have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex-3 microcarrier beads, packed into 0.3 ml columns (3 x 10(6) cells) and perfused at varying flow rates (0.3-1.2 ml/min) with HEPES-buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125I]insulin (6 kD) or [125I]albumin (66 kD) was assessed relative to [131I]IgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125I]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 +/- 0.02, n = 28) and insulin (0.51 +/- 0.05, n = 14) in cultures perfused at 0.3-0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 x 10(-5) cm/s) and insulin (4.18 x 10(-5) cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 microgram/ml), whereas permeability to albumin (0.39 x 10(-5) cm/s) remained unchanged. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells.

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of immunoglobulin-G (IgG) in the human placenta.

Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.

Uptake and intracellular routing of peroxidase-conjugated immunoglobulin-G by the perfused human placenta.

Selected lobules of term human placenta were extracorporeally perfused and human immunoglobulin-G complexed to horseradish peroxidase (IgG-HRP) was added to the maternal perfusate. After different durations of perfusion IgG-HRP was visualised by use of diamino-benzidine cytochemistry. Within the first 10 min of perfusion IgG-HRP was found bound to microvilli and coated pits of the syncytiotrophoblast; internalisation into coated vesicles and tubulo-vesicular bodies was also observed. Subsequently, IgG-HRP was found in multivesicular bodies and by 30 min appeared in basal vesicles, the frequency of the latter event increasing with time. No routing of IgG-HRP into Golgi regions or lysosomes could be detected. by 60 min IgG-HRP was found in a few caveolae of fetal endothelium of both terminal and intermediate villi. IgG-HRP was not found in intercellular clefts of the endothelium. The pattern of uptake and routing observed suggests a receptor-mediated transcytosis of IgG-HRP across the syncytiotrophoblast and a transcellular pathway through the endothelium.

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta.

Endogenous immunoglobulin-G was localised in ultrathin frozen sections of human term placenta by use of an indirect immuno electron-histochemical methodology. Immunoreactivity of endogenous IgG to rabbit anti-human immunoglobulin-G antibody was visualised by use of protein-A--colloidal gold complex. Gold marked the syncytiotrophoblast in both coated and uncoated regions of the apical plasmalemma, in vesicles and multivesicular bodies, and in vesicles near the basal plasmalemma. Immunoreactivity was also seen in the interstitial space between the trophoblast and the fetal endothelial layer as well as in various types of vesicles within the endothelial cells. No immunoreactivity was seen in the intercellular clefts of the endothelium. The pattern of localisation observed is consistent with receptor-mediated uptake of immunoglobulin-G into the syncytiotrophoblast of the human placenta followed by release into the interstitial space and then vesicular transport through the endothelium.

Uptake of alpha 2-macroglobulin-trypsin complex by human placenta is mediated by a microvillous membrane receptor.

The fate of native alpha 2-macroglobulin (alpha 2M) or its trypsin complex (alpha 2M-T) was studied in the isolated dually-perfused lobule of term human placenta. [125I]-alpha 2M added to the maternal circuit was unchanged during the course of the perfusion with minimal activity becoming associated with the placental tissue. Transfer of radioactivity into the fetal circulation accounted for only 0.07 per cent of the initial dose after 2 h. In contrast, [125I]-alpha 2M-T was rapidly taken up into the placental tissue (nearly 28 per cent of the initial dose during the 2-h perfusion) and breakdown products were released into both maternal and fetal circulations. At the end of 2 h, radioactivity levels on the fetal side were 13 times higher than those found with the native protein. These indications of a classical receptor-mediated uptake and breakdown pathway were confirmed in experiments in which the acidotrophic agent chloroquine was added to the maternal circuit prior to the alpha 2M-T. In the presence of chloroquine, tissue uptake was inhibited and the subsequent release of radioactive degradation products into the fetal circuit was similar to the levels seen with alpha 2M. Incubation of term trophoblast cells at 37 degrees C with [125I]-alpha 2M-T revealed over three-fold greater cell-associated activity than was found with the native protein. In another series of experiments, a purified microvillous membrane fraction was prepared from term placentae using buffers containing 1 mM iodoacetate. In the presence of this proteolytic enzyme inhibitor, binding studies showed a single class of low affinity receptors for the alpha 2M-T complex capable of binding 4.8 +/- 1.3 (SEM) micrograms of complex per mg of membrane protein. There was no binding of the native protein.

Characterization and specificity of lipoprotein binding to term human placental membranes.

The binding characteristics of very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoprotein fractions to a purified human term placental microvillous membrane preparation were determined. Binding of LDL was saturable with a maximal binding capacity of 270 ng LDL protein per mg of membrane protein. Scatchard analysis revealed the presence of a single population of 3.4 X 10(11) sites per mg of membrane protein and a mean affinity constant of 5.8 X 10(-9) M. Binding of VLDL was also saturable but the maximal capacity was 4.5-times greater than that of LDL. The Scatchard analysis revealed the presence of 2.1 X 10(11) binding sites and an affinity constant nearly one order of magnitude greater than that of LDL. Binding of HDL showed less tendency to saturate. Scatchard analysis showed a similar number of receptor sites to that calculated for VLDL and LDL but the affinity constant for HDL was over 100-fold less than that of VLDL. Self- and cross-inhibition studies of VLDL and LDL binding revealed that VLDL was better at blocking the binding of LDL than was LDL itself. This preferential binding of VLDL suggests that this lipoprotein fraction could be an important source of cholesterol for placental progesterone production.

Uptake and metabolism of epidermal growth factor in the perfused human placenta.

Uptake of 125I-labelled epidermal growth factor into trophoblast, and its subsequent fate, was studied in an isolated dually-perfused lobule of term human placenta. 125I-EGF added into the maternal circulation was rapidly taken up into the placental tissue where a portion was degraded and most of the breakdown products released back into the maternal circuit. At the end of the 2 h perfusion, radioactivity in the tissue accounted for 52% of the initial dose. 12.9% of the radioactivity remaining in the maternal circuit at the end of the perfusion, amounting to only 5.2% of the initial activity, could be identified as intact EGF by immunoaffinity chromatography. About 45 min after the start of the perfusion there was a sustained rise in the 125I activity in the fetal circulation accounting for 4.6% of the initial activity, and a small proportion of this (0.22% of the dose) could be immunologically characterised as EGF. In the presence of the acidotrophic agent chloroquine, there was a similar rapid clearance from the maternal circulation, which was not associated with breakdown. The tissue retention was slightly enhanced and there was very little transfer of activity into the fetal circulation.

Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta.

The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin.