Trophoblast lineage-specific differentiation and associated alterations in preeclampsia and fetal growth restriction

Abstract

The human placenta is a poorly-understood organ, but one that is critical for proper development and growth of the fetus in-utero. The epithelial cell type that contributes to primary placental functions is called "trophoblast," including two main subtypes, villous and extravillous trophoblast. Cytotrophoblast and syncytiotrophoblast comprise the villous compartment and contribute to gas and nutrient exchange, while extravillous trophoblast invade and remodel the uterine wall and vessels, in order to supply maternal blood to the growing fetus. Abnormal differentiation of trophoblast contributes to placental dysfunction and is associated with complications of pregnancy, including preeclampsia (PE) and fetal growth restriction (FGR). This review describes what is known about the cellular organization of the placenta during both normal development and in the setting of PE/FGR. It also explains known trophoblast lineage-specific markers and pathways regulating their differentiation, and how these are altered in the setting of PE/FGR, focusing on studies which have used human placental tissues. Finally, it also highlights remaining questions and needed resources to advance this field.

Keywords: Cytotrophoblast; Extravillous trophoblast; Placenta; Syncytiotrophoblast; Trophoblast stem cells.

Figure 1.

Sequential sections of an archived day-16 human embryo stained with antibodies to EGFR (A, C) or HLAG (B, D). Formalin-fixed paraffin embedded (FFPE) tissue was sectioned and stained with a rabbit monoclonal antibody against EGFR (clone 5B7, Ventana/Roche) or a mouse monoclonal antibody against HLAG (4H84 clone, Abcam), using a Ventana Discovery Ultra automated immunostainer with standard antigen retrieval and reagents as per the manufacturer’s protocol. Note that EGFR highlights both villous trophoblast surrounding the embryonic sac (ES) and primary chorionic villi (CV), as well as trophoblast giant cells (TGC) embedded within the trophoblastic shell (S), while HLAG predominantly highlights the mononuclear trophoblast within the trophoblastic shell. Immunostaining is shown in brown with blue (hematoxylin) counterstain. C and D represent magnification of the trophoblastic shell area with TGC, bracketed in a rectangular area in A and B, respectively. Magnification of 40x (for A and B) and 80x (for C and D).

Figure 2.

Human placenta at term double-stained with antibodies to EGFR (green) and HLAG (red), counterstained with DAPI (blue). FFPE sections from 12 different term placentas were de-paraffinized and rehydrated, and antigen retrieval was performed using heat and antigen retrieval buffer (Dako) for 20 minutes. Tissues were incubated with the same primary antibodies as used for immunohistochemistry shown in Figure 1, then visualized by Alexa 488- or Alexa 594-conjugated secondary antibodies (Invitrogen) and counter-stained with the nuclear stain, DAPI. A) Chorionic plate (near the fetal surface), showing a layer of EGFR⁺ cytotrophoblast (CTB) immediately below the amniotic mesenchyme (AM), with HLAG⁺ cells emanating from the CTB layer, protruding into the intervillous space (IVS). B) Basal plate (near the maternal surface), showing EGFR⁺ syncytiotrophoblast covering chorionic villi (CV, top right), below which are multiple layers of mature, HLAG⁺ cells. C) An intraplacental trophoblast island within the placental disc, where a chorionic villus remnant (CV) gets surrounded by perivillous fibrin resulting in differentiation of CTB into HLAG⁺ EVT. D) An EGFR/HLAG double-positive multinucleated cell (arrow) at the basal plate, surrounded by HLAG⁺ EVT and unstained decidual cells. Magnification of 80x for A-D.

Figure 3.

Cytotrophoblast heterogeneity in early gestation (6-week gestational age) human placenta. FFPE sections from 5 different placentas were stained, either with antibodies against TP63 (mouse monoclonal antibody specific to p40, BC28 clone, Biocare Medical), EGFR (same antibody as in Figure 1), and CD31 (rabbit monoclonal, ab76533, Abcam) (shown in A), or against CDX2 (rabbit monoclonal, ab76541, Abcam) (shown in B), using a Ventana Discovery Ultra automated immunostainer with standard antigen retrieval and reagents as per the manufacturer’s protocol. A) Section showing triple staining for TP63 (brown), EGFR (magenta), and CD31 (yellow-green), counter-stained with hematoxylin (blue). Note that TP63 is uniformly expressed in the CTB layer (trophoblast layer adjacent to the villous stroma/VS). B) Section showing staining for CDX2 (brown), counter-stained with hematoxylin (blue). Note stretches of CTB that lack CDX2 staining (yellow arrowheads). Magnification of 80x for A-B.