Associations between fetal size, sex and placental angiogenesis in the pig

Abstract

Inadequate fetal growth cannot be remedied postnatally, leading to severe consequences for neonatal and adult development. It is hypothesized that growth restriction occurs due to inadequate placental vascularization. This study investigated the relationship between porcine fetal size, sex, and placental angiogenesis at multiple gestational days (GD). Placental samples supplying the lightest and closest to mean litter weight (CTMLW), male and female Large White X Landrace fetuses were obtained at GD30, 45, 60, and 90. Immunohistochemistry revealed increased chorioallantoic membrane CD31 staining in placentas supplying the lightest compared to those supplying the CTMLW fetuses at GD60. At GD90, placentas supplying the lightest fetuses had decreased CD31 staining in the chorioallantoic membrane compared to those supplying the CTMLW fetuses. The mRNA expression of six candidate genes with central roles at the feto-maternal interface increased with advancing gestation. At GD60, ACP5 expression was increased in placentas supplying the lightest compared to the CTMLW fetuses. At GD45, CD31 expression was decreased in placentas supplying the lightest compared to the CTMLW fetuses. In contrast, CD31 expression was increased in placentas supplying the lightest compared the CTMLW fetuses at GD60. In vitro endothelial cell branching assays demonstrated that placentas supplying the lightest and male fetuses impaired endothelial cell branching compared to placentas from the CTMLW (GD45 and 60) and female fetuses (GD60), respectively. This study has highlighted that placentas supplying the lightest and male fetuses have impaired angiogenesis. Importantly, the relationship between fetal size, sex, and placental vascularity is dynamic and dependent upon the GD investigated.

Figure 1.

Fetal size is associated with CAM CD31 staining at GD60 and 90. The percentage CD31 staining of the CAM was investigated at GD45, 60, and 90 (A) (N = 20–28 placental samples per group). The association between fetal size (B; N = 5–8 samples per group) and fetal sex (N = 10–14 samples per group) and percentage CD31 staining of the CAM was investigated. Error bars represent SEM. *P ≤ 0.05. ***P ≤ 0.001.

Figure 2.

Fetal size is associated with altered placental ACP5 and CD31 expression. mRNA expression of ACP5 (A), CD31 (B), HIF1A (C), HPSE (D), PTGFR (E), and VEGFA (F) was quantified by qPCR in placental samples associated with the lightest and CTMLW fetuses at GD30, 45, 60, and 90. Mean values presented. N = 4–7 placental samples per group. Error bars represent SEM. *P ≤ 0.05.

Figure 3.

The GD of placental samples influences endothelial cell branching in vitro. Endothelial cells were cultured in conditioned media generated from culture of placental samples at GD45 and 60. The influence of the conditioned media on explant area (A), vessel area (B), vessels as a percentage of area (C), total number of junctions (D), junction density (E), total vessel length (F), average vessel length (G), total number of end points (H), and lacunarity (I) was investigated from 2–10 h post-seeding of the cells. Mean values presented for 6 and 12 GD45 and 60 placental samples, respectively. Error bars represent SEM. *P ≤ 0.05. **P ≤ 0.01.

Figure 4.

Endothelial cell branching is impaired in response to conditioned media from placentas supplying the lightest fetuses at GD45. Endothelial cells were cultured in conditioned media generated from culture of placental samples supplying the lightest and CTMLW fetuses at GD45. The influence of the conditioned media on explant area (A), vessel area (B), vessels as a percentage of area (C), total number of junctions (D), junction density (E), total vessel length (F), average vessel length (G), total number of end points (H), and lacunarity (I) was investigated from 2–10 h post-seeding of the cells. Mean values presented for three placental samples in each group. Error bars represent SEM. *P ≤ 0.05. **P ≤ 0.01.

Figure 5.

Endothelial cell branching impaired in response to conditioned media from placentas supplying the lightest fetuses at GD60. Endothelial cells were cultured in conditioned media generated from culture of placental samples supplying the lightest and CTMLW fetuses at GD60. The influence of the conditioned media on explant area (A), vessel area (B), vessels as a percentage of area (C), total number of junctions (D), junction density (E), total vessel length (F), average vessel length (G), total number of end points (H), and lacunarity (I) was investigated from 2–10 h post-seeding of the cells. Mean values presented for six placental samples in each group. Error bars represent SEM. *P ≤ 0.05. **P ≤ 0.01.

Figure 6.

Fetal sex influences endothelial cell branching in response to placental conditioned media at GD60. Endothelial cells were cultured in conditioned media generated from culture of placental samples supplying male and female fetuses at GD60. The influence of the conditioned media on explant area (A), vessel area (B), vessels as a percentage of area (C), total number of junctions (D), junction density (E), total vessel length (F), average vessel length (G), total number of end points (H), and lacunarity (I) was investigated from 2–10 h post-seeding of the cells. Mean values presented for six placental samples in each group. Error bars represent SEM. *P ≤ 0.05. **P ≤ 0.01.