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The cellular localisation of C/T antigens were investigated as explained previously in this chapter (for 5-HT receptors). The aggregate scoring for C/T antigens are given in Table 4.3 below.

Table 4.3: Summary of staining intensity of C/T antigens

FT Placentae C/T antigens

Region Cell type CAGE1 FATE1 GAGE1 GAGEE1 MAGEA1 MAGEA4 PASD1

Villous CTB 2±0.5 3±0.4 2±0.6 3±0.0 3±0.0 2±0.7 2±0.6 STB 2±0.5 3±0.5 2±0.6 2±0.9 3±0.4 2±0.5 2±0.6 Mesenchymal cells 0±0.5 2±0.7 0±0.4 0±0.9 0±0.4 0 0±0.5 Decidua Uterine epithelium 2±0.6 2±0.6 2±0.5 3±0.0 3±0.0 3±1.1 3±0.0 Decidual stromal cell 0±0.6 2±0.6 1±0.6 2±0.6 0±1.2 1±0.6 2±0.6

Spiral arteries 1±0.5 1±0.6 2±0.0 3±0.0 N/A 1±0.0 1±0.6

Staining intensity: 0-negative; 1-low; 2-moderate; 3-high (Mode±SD). N/A- not available

 CAGE1

Cytoplasmic regions of the STB (thin arrow) and CTB (bold arrow) in the placental villi were moderately (score 2) stained with CAGE1 (Figure 4.6, Panel F1-5; Table 4.3). The staining in the stromal mesenchymal cells was negligible. The epithelial lining the uterine glands (enclosed in box) and the lining of spiral arteries (arrow heads) of the decidua were moderate to low in CAGE1 staining (Figure 4.6, Panel D1-3). The surrounding stromal cells showed no positive CAGE1 staining.

133 Figure 4.6: Localisation of CAGE1.

CAGE1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Thin arrows- STB; Bold arrows- CTB; Uterine glandular epithelium enclosed in box.

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 FATE1

The trophoblast cells (CTB and STB) of the placental villi were highly stained for FATE1 (See Figure 4.7, Panel F1-5). Although in most of the samples the staining appeared to be mostly cytoplasmic, under higher magnification staining also visible in the nuclear region in some samples (Figure 4.7, Panel F2). The few mesenchymal cells within the stromal layer were also positive to FATE1. The staining in the maternal endometrium was localised around decidualised regions and spiral arteries (Figure 4.7, Panel D1). Moderate staining was also observed in the epithelial linings and in the stromal cells of decidua. The staining was higher in villous than in maternal sections.

135 Figure 4.7: Localisation of FATE1.

FATE1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Thin arrows- STB; Bold arrows- CTB; Arrow head- Spiral arteries; Uterine glandular epithelium enclosed in box. Objective magnification: 20X (Scale bar=100µm); 100X (Scale bar= 20µm).

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 GAGE family

The cellular localisation of two members of the GAGE family, namely GAGE1 and GAGEE1, was investigated. Both the placental villi and maternal decidua showed immuno- positivity for these two antigens (Figure 4.8 and 4.9). The staining intensities of the trophoblast cells (CTB and STB) were moderate (score 2) for GAGE1. However, the GAGEE1 antibody showed high staining intensity in the CTB (score 3) and moderate in STB (score 2) (Figure 4.9, Panel F1). The mesenchymal cells were negative for both antigens. The staining in maternal decidua was concentrated in the inner lining of the spiral arteries and glands. The staining of the stromal cells in the decidua was low for GAGE1 and moderate for GAGEE1. In general, the staining intensity of GAGEE1 was higher than GAGE1 in both placental villi and decidua.

137 Figure 4.8: Localisation of GAGE1.

GAGE1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Thin arrows- STB; Bold arrows- CTB; Arrow head- Spiral arteries; Uterine glandular epithelium enclosed in box. Objective magnification: 20X (Scale bar=100µm); 100X (Scale bar= 20µm).

138 Figure 4.9: Localisation of GAGEE1.

GAGEE1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Bold arrows- CTB; Uterine glandular epithelium enclosed in box. Objective

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 MAGE family

The cellular localisation of the two members of the MAGE family, MAGEA1 and MAGEA4, was investigated.

• MAGEA1

Intense cytoplasmic staining was observed in the STB and CTB layers of the villi (See Figure 4.10, Panel F1-5). The syncytial spout (represented by +) of the mesenchymal villi was also immuno-positive (Panel F4). However, the mesenchymal cells in the stroma were negative for MAGEA1 staining. The epithelial layer lining the glands and vessels in the decidua was stained with the same intensity as trophoblast cells (Figure 4.10, Panel D1-3; Table 4.3). No staining was found on the decidual stromal cells or the lining of the spiral arteries.

• MAGEA4

The cellular localisation of MAGEA4 in the placental villi was similar to MAGEA1 (see Figure 4.11, Panel F1-5). The intensity of staining was only moderate (score 2) when compared to the high (score 3) intensity of MAGEA1 (Table 4.3). In the decidual regions, apart from staining of glandular epithelium, low levels of immuno-reactivity for MAGEA4 were also observed on the lining of the spiral arteries and the surrounding stromal cells (Figure 4.11, Panel D1-3).

140 Figure 4.10: Localisation of MAGEA1.

MAGEA1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Thin arrows- STB; Bold arrows- CTB; + Syncytial knot; Arrow head- Spiral arteries; Uterine glandular epithelium enclosed in box. Objective magnification: 20X (Scale bar=100µm); 100X (Scale bar= 20µm).

141 Figure 4.11: Localisation of MAGEA4.

MAGEA4 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Arrow head- Spiral arteries; Uterine glandular epithelium enclosed in box. Objective

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 PASD1

Moderate levels of staining for PASD1 were observed in the cytoplasmic region of STB and CTB layers of the villous region (Figure 4.12, Panel F1-3 and 5; Table 4.3). However, nuclear positivity for PASD1 was observed in the CTB cells (Figure 4.12, Panel 4). The mesenchymal cells were negative. The uterine glandular epithelium showed high immuno- positivity in the decidua (Figure 4.12, Panel D1-D3). The decidual stromal cells and the inner lining of the spiral arteries showed moderate to low levels of staining.

On the whole, trophoblast (STB and CTB) and epithelial (decidua) cells showed immuno- positivity for most of the C/T antigens investigated. The staining in the mesenchymal cells decidual stromal cells and spiral arteries were antigen-dependent. Although most of the staining for the antigens was observed as granular brown stains in the cytoplasm of the cells, FATE1 and PASD1 showed nuclear staining, especially in certain of the villous CTB cells.

143 Figure 4.12: Localisation of PASD1.

PASD1 localisation is shown on the right (at both 20X and 100X magnification), whilst the negative (no primary Ab) control images are shown in the left hand panels and the positive control (vimentin) images are in the centre. Panel F1-5 show images for placental villous sections from FT. Panel D1-D3 represents the decidual sections from FT as well. Thin arrows- STB; Bold arrows- CTB; Arrow head- Spiral arteries; Uterine glandular epithelium enclosed in box. Objective magnification: 20X (Scale bar=100µm); 100X (Scale bar= 20µm).

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