Maternal placental vascularity and capillary endothelial cell proliferation during early pregnancy after transfer of embryos generated through assisted reproductive technology in sheep.

Normal placental angiogenesis is critical for fetal growth anddevelopment because it provides for the metabolic demands ofthe developing conceptus. Abnormal placental vascularizationhas been observed during late pregnancy in fetuses from assistedreproductive technologies (ART), which could explain the highrate of embryonic/fetal death and intrauterine growth restrictionoften seen in these pregnancies. How early in pregnancy thisabnormal placental angiogenesis begins is not known. Both clonedembryos (somatic cell nuclear transfer, or SCNT) and monoparentalembryos (parthogenotes, containing only maternal genes; androgenotes,containing only paternal genes) provide important models forstudying defects in placentation and placental vascular development.We hypothesized that compromised placental vascular developmentbegins during early pregnancy after transfer of embryos generatedthrough ART. Uteri were collected on day 20 of pregnancy: (1)after natural mating (pregnant controls); (2) after transferof biparental embryos produced through in vitro fertilization(IVF), and (3) after transfer of monoparental (parthogenetic),in vitro activated (IVA) embryos (n=5 to 6 ewes/group). In addition,uteri were collected from nonpregnant ewes on day 10 after estrus(n=5; nonpregnant controls). Cross-sections (0.5-cm thick) ofeach uterine horn were immersion-fixed in Carnoy's solutionand embedded in paraffin. To determine the proliferation indexof capillary endothelial cells (EC) and quantitatively measurevascularity in the intracaruncular (ICAR; maternal placental)tissues, cross-sections were immunostained to detect proliferatingcell nuclear antigen and histochemically stained with periodicacid-Schiff's reagent, respectively. Photomicrographs of thestained sections were taken from10 tissue areas per slide, followedby computerized image analysis of endometrial ICAR areas. Thefollowing parameters were determined for each section: EC labelingindex (proportion of of cells that were proliferating), capillaryarea density (CAD, capillary area as a proportion of tissuearea), capillary number density (CND, number of capillariesper tissue area), capillary surface density (CSD, capillarycircumference per tissue area). Compared to nonpregnant controls,capillary labeling index of EC was greater (P<0.01) in thepregnant controls by 15-fold, in the IVF group by 24.5- fold,and in the IVA group by 36.4-fold. CND was similar for the nonpregnantand pregnant control groups and was 2-fold greater (P<0.002)than either the IVF or the IVA group. The other 2 measures ofvascularity, CAD and CSD, were greater (P