Differential transferrin expression in placentae from normal and abnormal pregnancies: a pilot study

Abstract

Background: The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.

Methods: In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.

Results: Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).

Conclusion: These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.

Figure 1

Placenta lysate stained with CBB after 2D-SDS PAGE (A) and immunodetection with TRO-tf-01 antibody (B). A 13 cm Immobiline DryStrip of pH range 4–7 was used for the separation of the total protein lysate according to pI, in the second dimension, 10% polyacrylamide slab gel (SDS) was used. After immunoblotting with MoAb TRO-tf-01, only one isoform (arrow) of transferrins (underlined) was labeled (B). Detected antigen was identified by MALDI-TOF analysis as human transferrin (TF, sequence coverage of 31%, Z-score of 2.31). Molecular weight is shown on the left.

Figure 2

Western blot analysis of chorionic villi of the placentae from normal and selected abnormal pregnancies with antibody TRO-tf-01. Actin is used as a control for protein loading; lysates (NP-40) were loaded on 10% acrylamide gel (SDS, reducing conditions). Quantitative evaluation of TRO-tf-01 expression (O.D.) in the placentae is placed at the bottom. Placentae of mothers with 1 – drug abuse during pregnancy (DrA), 2 – pregnancy-induced hypertension together with gestational diabetes (PIH+GD), 3 – gestational diabetes (GD), 4 – normal pregnancy, 5 – pregnancy-induced hypertension (PIH).

Figure 3

Immunoreactivity of TRO-tf-01 in chorionic villi of placentae from normal and selected abnormal pregnancies. A) Fetal membranes show very strong expression of transferrin by amniotic epithelium (full arrowhead) and on extravillous cytotrophoblast cells (arrowhead). B) Control experiment without primary antibody shows no transferrin staining in the syncytium of normal term placenta. C) Very low expression of transferrin in the syncytium of normal term placenta. D) Placenta of mother with gestational diabetes shows strong villous expression of transferrin. E) Placenta of mother with pregnancy-induced hypertension shows strong expression of transferrin in villous syncytium. F) Placenta of drug-abuse mother during pregnancy; the transferrin expression is very strong. Hematoxylin counterstained the nuclei.

Figure 4

Quantitative evaluation of TRO-tf-01 immunoreactivity in placental samples. Each column represents mean (± SE) from fifteen net optical densities (Net O.D.) measured in three different placentae. NP, normal pregnancies; GD, gestational diabetes during pregnancy; PIH, pregnancy-induced hypertension during pregnancy; DrA, drug-abusing mothers during pregnancy. Statistical analysis has shown highly significant differences (p < 0.001) between normal (a) and each of the selected abnormal pregnancy types (b).