Transfer and Metabolism of Cortisol by the Isolated Perfused Human Placenta

Context: Fetal overexposure to glucocorticoids in utero is associated with fetal growth restriction and is postulated to be a key mechanism linking suboptimal fetal growth with cardiovascular disease in later life. Objective: To develop a model to predict maternal-fetal glucocorticoid transfer. We hypothesized placental 11-β-hydroxysteroid dehydrogenase-type 2 (11β-HSD2) would be the major rate-limiting step in maternal cortisol transfer to the fetus. Design: We used a deuterated cortisol tracer in the ex vivo placental perfusion model, in combination with computational modeling, to investigate the role of interconversion of cortisol and its inactive metabolite cortisone on transfer of cortisol from mother to fetus. Participants: Term placentas were collected from five women with uncomplicated pregnancies, at elective caesarean delivery. Intervention: Maternal artery of the isolated perfused placenta was perfused with D4-cortisol. Main Outcome Measures: D4-cortisol, D3-cortisone, and D3-cortisol were measured in maternal and fetal venous outflows. Results: D4-cortisol, D3-cortisone, and D3-cortisol were detected and increased in maternal and fetal veins as the concentration of D4-cortisol perfusion increased. D3-cortisone synthesis was inhibited when 11-β-hydroxysteroid dehydrogenase (11β-HSD) activity was inhibited. At the highest inlet concentration, only 3.0% of the maternal cortisol was transferred to the fetal circulation, whereas 26.5% was metabolized and 70.5% exited via the maternal vein. Inhibiting 11β-HSD activity increased the transfer to the fetus to 7.3% of the maternal input, whereas 92.7% exited via the maternal vein. Conclusions: Our findings challenge the concept that maternal cortisol diffuses freely across the placenta and confirm that 11β-HSD2 acts as a major “barrier” to cortisol transfer to the fetus.


Model equations
where , and are the concentrations (mol/L) of solute which can be either D4-cortisol (D4F), D3-cortisone (D3E) or D3-cortisol (D3F) in the maternal "m", syncytiotrophoblast "s" and fetal "f" compartment respectively. Similarly, the volumes (L) of the different compartments are indicated with subscripts using the same notation. and (L/min) are the fluid flow rates in the maternal and fetal circulation.
, is the maternal inlet concentration, which is zero for all solute species except D4cortisol. Note that the fetal inlet concentration is zero for all species and therefore has not been included. and denote the effective overall permeability constants (L/min) for the microvillous membrane (MVM) and basal membrane (BM) including surface area. These diffusive permeability constants were assumed to be the same for

Model parameters
The total cotyledon volume was based on the average cotyledon weight from the experiments (30.8 × 10 -3 kg, n = 5), which was directly equated to the volume in L. The volume fractions of the maternal, syncytiotrophoblast and fetal compartments distinguished in the model were set to 34%, 15% and 7.4% respectively, as in our previous work. [14,22] The flow rates in the maternal and fetal circulations = 14 × 10 -3 L/min and = 6 × 10 -3 L/min were directly based on the experimental settings. To account for any discrepancies between nominal and actual values, the D4-cortisol input concentrations used in the model were calculated based on the combined maternal and fetal steady state output during the blocking phase. The Michaelis-Menten constant was set to 44 × 10 -9 mol/L, based on the value for the enzyme 11β-HSD2 for cortisol.
[23] In first instance the same value was adopted for both metabolic conversion steps in Equations 4-6.

Parameter estimation
The remaining parameters in the model were determined by fitting the experimental data. The following error criterion was defined for a certain species and compartment in general: The D3-cortisol concentrations measured experimentally were 300 times smaller compared to D4-cortisol and did not contribute significantly to the overall mass balance. Therefore the conversion to D3-cortisol was neglected in the parameter estimation by setting 3 →3 to zero. In addition, the measured D3-cortisone values could not be directly related to concentration. Therefore D3-cortisone was not fitted, but instead the experimental values for D3-cortisone were scaled to allow comparison of the relative changes predicted by the model. Thus, only the D4-cortisol values in the maternal and fetal compartments (averaged over 5 placentas) were fitted according to the following overall error criterion: In total 3 parameters were fitted, the membrane permeability constants and and the maximum rate of conversion from cortisol to cortisone 4 →3 . Time integration of Equations 1-3 was performed in Matlab (R2016a) using the ode45 function (Runge-Kutta (4, 5) method). Parameter estimation by minimising Eq. 8 was implemented using the fminsearch function (Nelder-Mead method). Initial parameter estimates were varied to verify that the algorithm converged to a unique solution.

Supplementary Tables
Supplementary Table 1 Mass spectral conditions for analysis of analytes and internal standards by positive ion electrospray ionisation blank samples were diluted in 500 µL of water and processed using the same extraction method and analysis conditions as perfusate samples. Standard curves were plotted by calculating the peak area (analyte peak area / internal standard peak area). Weighting of 1/x and was applied to form standard curves of best fit with a regression coefficient above 0.99. The ion ratio (quantitative ion/qualitative ion) of the analytes was calculated using MultiQuant software and results were not considered acceptable if the ratio was greater than 20% of the ratio of the standards. Inter-assay fourteen point standard curve validation (n=6 different day respectively) was used to assess the limits of quantification of accuracy and precision for each analyte. Precision was based on the percentage relative standard deviation (%RSD), which was calculated using peak area ratios. Tissue sample* is intra-assay (amount, ng for tissue replicates (n=6). Inter-assay was not performed for tissue samples, as all tissue samples were analysed on the same day. Low values are the limit of quantification for