Placental vascularity and growth factor expression in singleton, twin, and triplet pregnancies in the sheep

For singleton, twin, and triplet pregnancies, uteri were collected on day 140 of pregnancy. For each ewe (n = 18), placentomes were fixed by arterial perfusion supplying the fetal (cotyledon) and maternal placenta (caruncle). Tissue sections were stained for determination of vascularity by image analysis. Further, protein expression for factor VIII, vascular endothelial growth factor (VEGF) and its receptor, VEGFR1, as well as basic fibroblast growth factor (FGF2) and its receptor, FGFR, in tissue sections was determined by immunohistochemistry and image analyses. Cotyledonary and caruncular samples were analyzed for expression of mRNA for Vegf and its two receptors, Vegfr1 and Vegfr2, as well as Fgf2 and Fgfr. Fetal number did not affect placental capillary density or factor VIII expression, whereas increased fetal number reduced total cotyledon and caruncle capillary volume. While expression of Vegf, Vegfr1, Vegfr2, and Fgfr mRNA in cotyledonary but not caruncular tissue was greater in twin pregnancies compared to singleton and triplet pregnancies, protein expression of VEGF in the placentome decreased with increasing numbers of fetuses, VEGFR1 did not change, and FGFR was greater in twin versus singleton and triplet pregnancies. Fetal number did not affect the expression of Fgf2 mRNA in placental tissues, whereas FGF2 protein expression was less in triplet compared to singleton and twin pregnancies. Reduced fetal and placental weights in twins and/or triplet pregnancies are associated with an overall decrease in total placental vascularity, VEGF and FGF2 and/or FGFR protein expression, but not in angiogenic factor mRNA expression or VEGFR1 protein expression in sheep.