Investigating the porcine feto-maternal interface throughout gestation: associations with foetuses of different size and sex
MetadataShow full item record
Background Inadequate foetal growth cannot be remedied postnatally, leading to severe consequences for neonatal and adult development. Furthermore, sexual dimorphism in placental development has been suggested in humans although this remains poorly investigated in the pig. Hypotheses Intrauterine Growth Restriction (IUGR) occurs due to aberrant conceptus attachment, which leads to alterations in angiogenesis and vascularity of the feto-maternal interface. Altered gene expression and vascularity will be observed at the feto-maternal interface in male foetuses compared to female foetuses. Increased apoptosis and decreased proliferation will be observed in the feto-maternal interface associated with the lightest foetuses compared to the closest to mean litter weight (CTMLW) foetuses. Aims This thesis aimed to investigate the association between foetal size and sex and: integrin signalling; apoptotic and proliferation pathways; umbilical arterial (UA) blood flow; and angiogenesis and vascularity at the feto-maternal interface. This was performed by the collection of placental and endometrial samples associated with conceptuses or foetuses of different size (lightest and CTMLW) and sex at gestational day (GD) 18, 30, 45, 60 and 90. Results – Integrin Subunits, Secreted Phosphoprotein 1 (SPP1) and Fibronectin (FN) Integrin receptors exist as heterodimers consisting of an α and a β subunit at the porcine feto-maternal interface. They bind to a number of ligands including secreted phosphoprotein 1 (SPP1) and fibronectin (FN), and play a central role in the establishment of pregnancy. The mRNA expression of the integrin subunits (ITG) ITGα2, ITGαV, ITGβ1, ITGβ3, ITGβ5, ITGβ6 and ITGβ8, and the ligands SPP1 and FN was quantified by qPCR in placental and endometrial samples supplying foetuses of different size and sex throughout gestation. Temporal changes in mRNA expression were observed in both tissues. At GD45, placental samples supplying the lightest foetuses had increased ITGα2 expression compared to those supplying the CTMLW foetuses (P=0.07). Endometrial samples supplying the lightest foetuses had decreased expression of ITGβ1 and SPP1 at GD45 and 60 respectively (P≤0.05). Placental samples associated with female foetuses had decreased expression of ITGβ6 and FN (P≤0.05) compared to those supplying male foetuses at GD45 and 90 respectively. FN and ITGβ3expression was increased in endometrial samples supplying female foetuses compared to their male littermates at GD30 and 60 respectively (P≤0.05). In contrast, SPP1 expression was decreased in endometrial samples supplying female foetuses compared to their male littermates at GD60 (P≤0.05). SPP1 protein staining per uterine gland was assessed throughout gestation by immunofluorescence. Whilst temporal changes in staining were observed, with a similar profile to the mRNA expression data, no associations between foetal size or sex, and uterine gland staining were observed. Results – Apoptosis and Proliferation The mRNA expression of candidate genes involved in apoptosis or proliferation (Bax, Bcl2, P53 and Ki67) was quantified by qPCR. Temporal changes were observed in both tissues. Placentas associated with the lightest foetuses had decreased P53 and Ki67 expression compared to the CTMLW foetuses at GD45. However, at GD60, P53 expression was increased in placentas supplying the lightest foetuses compared to CTMLW foetuses. Intriguingly, endometrial P53 expression was increased in samples associated with the lightest foetuses compared to the CTMLW foetuses at GD45. A trend towards females having decreased placental Bax expression was observed at GD45 (P=0.06). At GD60 the expression of Bcl2, P53 and Ki67 was decreased in endometrial samples associated with females compared to their male littermates. At GD30, Bax expression was increased in endometrial samples associated with female foetuses compared to their male littermates. TUNEL staining revealed an increased prevalence of apoptotic cells in placentas at GD60 compared to GD45 however, no association between foetal size or sex, and apoptotic cell number was observed. Results – Doppler Ultrasound Human IUGR is diagnosed prenatally by alterations in UA blood flow, detected by Doppler ultrasound. Doppler ultrasound was used under moderate sedation over a 30-minute period to monitor umbilical arterial (UA) blood flow in the right uterine horn of Large White X Landrace gilts at GD30, 45, 60 and 90. Gilts were scanned prior to euthanasia to examine relationships between litter size, sex ratio and five UA parameters of interest. In GD90 gilts where scans were obtained from all foetuses in the scanned horn, relationships between individual foetal weight and sex were examined. A subset of the gilts were sedated, scanned and recovered (SSR) earlier in gestation to assess the influence of sedation on later foetal development by comparing with control litters that had not been sedated previously. Temporal changes were observed in all UA parameters (P≤0.001). At GD60 and 90 foetal heart rate decreased with increasing duration of sedation (P≤0.001). Sex ratio and foetal weight were associated with UA blood flow whereas litter size and foetal sex were not. SSR at GD30 and 45 was associated with decreased foetal weight at GD60 (P≤0.001) and 90 (P=0.06), respectively, when compared to controls. These results suggest that maternal sedation during gestation has a significant effect on foetal development, the mechanisms of which warrant further investigation. Results – Angiogenesis and Vascularity Angiogenesis is essential for foetal and placental development and has been shown to be inadequately regulated in instances of complicated pregnancies. Immunohistochemistry on placental (GD45, 60 and 90) and endometrial (GD18, 30, 45, 60 and 90) samples for the endothelial cell marker Platelet and Endothelial Cell Adhesion Molecule 1 (CD31) was performed to assess placental and endometrial vascularity. At GD60, the percentage staining in the chorioallantoic membrane (CAM) of placentas supplying the lightest foetuses was increased compared to those supplying the CTMLW foetuses (P=0.06). The direction of this relationship switched at GD90, with placentas supplying the lightest foetuses having decreased percentage CD31 staining in the CAM compared to those supplying the CTMLW foetuses (P≤0.05). No association between foetal sex and placental vascularity was observed. At GD60, a trend for both the total (P=0.07) and mean number of blood vessels (BV) (P=0.07) to be decreased in endometrial samples supplying the lightest foetuses compared to the CTMLW foetuses was observed. Similarly, a trend towards a decrease in the mean number of uterine glands in endometrial samples supplying the lightest compared to the CTMLW foetuses was observed at GD90 (P=0.08). The total number of uterine glands (P≤0.05), total number of BV (P≤0.05) and mean number of BV (P≤0.05) were increased in endometrial samples supplying female foetuses compared to male foetuses at GD45. The mRNA expression of candidate genes which are quantitative trait loci (QTL) associated or have a central role in angiogenesis and foetal development, including: Uteroferrin (ACP5), CD31, Hypoxia Inducible Factor 1 Alpha Subunit (HIF1A), Heparanase (HPSE), Prostaglandin F2α Receptor (PTGFR) and Vascular Endothelial Growth Factor A (VEGFA) were investigated by qPCR. Temporal changes in expression were observed in both tissues. ACP5 expression was increased (GD60), whilst HIF1A (GD90) were decreased in placentas supplying the lightest foetuses compared to the CTMLW foetuses. CD31 expression was decreased at GD45, and increased at GD60, in placental samples supplying the lightest foetuses compared to the CTMLW foetuses. Decreased expression of CD31 (GD60), HPSE and VEGFA (GD90), alongside increased HIF1A (GD45) expression were observed in endometrial samples supplying the lightest foetuses compared to the CTMLW foetuses. Decreased expression of CD31, PTGFR and VEGFA was observed in endometrial samples associated with male compared to female foetuses at GD30. At GD60, the direction of these differences in endometrial expression was reversed, with increased expression of ACP5, CD31 and VEGFA in endometrial samples associated with male foetuses compared to their female littermates. Results – In vitro Angiogenic Potential of Placental and Endometrial Samples Matrigel branching assays were used to assess the angiogenic potential of GD45 and 60 placental and endometrial conditioned media on endothelial cell branching in vitro. Conditioned media from GD45 placental and endometrial samples increased endothelial cell branching in vitro compared to treatment with media from GD60 samples. Endothelial cell branching was impaired in response to treatment with conditioned media from placental and endometrial samples associated with the lightest foetuses compared to the CTMLW foetuses at both GD. At GD60, conditioned media from female placentas had increased endothelial cell branching compared to those supplying males. Interestingly, the inverse of this was observed in the endometrium, with samples supplying females having a decreased ability to induce endothelial cell branching compared with males. Conclusion This thesis has presented novel findings of associations between foetal size and sex, and placental and endometrial integrin signalling, apoptosis and proliferation, and angiogenesis and vascularity. Currently, this is the first suggestion in the literature that foetal size, and more intriguingly foetal sex, may have a strong influence on the activity of the endometrium. The mechanisms behind these findings warrant further investigation. Switches in the direction of differences at the feto-maternal interface between foetuses of different size were observed throughout gestation, notably between GD45 and 60, highlighting the dynamic nature of the feto-maternal interface and suggesting this as a potential window that could be manipulated by the industry to attempt to rescue the postnatal phenotype of IUGR piglets.