22 VASCULAR ENDOTHELIAL GROWTH FACTOR mRNA EXPRESSION IN FETAL MEMBRANES DURING EARLY PREGNANCY AS A BIOMARKER FOR DEVELOPMENT OF HYDROPS PLACENTA LATER IN PREGNANCY IN BOVINE EMBRYOS CREATED THROUGH SOMATIC CELL NUCLEAR TRANSFER (SCNT)

Abstract

Pregnancies obtained via somatic cell nuclear transfer (SCNT) show an increased incidence of pregnancy complications, including poor placental vascularization, hydrops placenta, and abnormal placental morphology. In a preliminary study of hydrops placentas from SCNT clones v. control cow placentas close to term, we observed a thickening of the endometrial epithelium, which appeared pseudostratified; in addition, the subepithelial endometrial vascular bed consisted of large, rare arterioles with thick basement membranes compared with the normal capillary bed observed in control animals. The fetal membranes of hydrops placenta from bovine SCNT clones also exhibited macroscopic morphological differences, which included edema, increased membrane thickness, and a gelatinous appearance. All of these morphological differences were similar to those we have reported for sheep SCNT clones (Palmieri et al. 2007 Placenta 28, 577–584). In the search for biomarkers that may indicate abnormal placental development early in pregnancy, placental tissues were collected on day 40 from pregnancies established after transfer of embryos created through SCNT (n = 17) or IVF (n = 6). Nuclear donor cells used to obtain SCNT embryos were those we have previously found to be highly susceptible to hydrops (from 5 to 19%), whereas the incidence of hydrops in IVF and controls in our laboratories did not exceed 0.5%. A sample of fetal membranes was fixed in 10% buffered formalin for histological analysis; another sample was snap-frozen in liquid nitrogen for analysis of mRNA levels for vascular endothelial growth factor (VEGF) and its receptors (KDR and FLT) by quantitative, real-time RT-PCR. Histological analysis of cell proliferation rate was performed using immunohistochemical localization of proliferating cell nuclear antigen and image analysis software (Image-Pro Plus). There were no differences (P = 0.39) in the rate of cell proliferation of the trophoblast in SCNT v. IVF (29.4 ± 4.9% v. 22.1 ± 3%). However, VEGF mRNA expression was less (P < 0.04) in the fetal membranes of SCNT than IVF on day 40 of pregnancy (0.30 ± 0.04 v. 0.53 ± 0.12, normalized to α-actin mRNA levels). The mRNA expression for KDR and FLT were similar in SCNT and IVF. These data suggest that the lower expression of VEGF, a major placental angiogenic factor, in fetal membranes of early pregnant SCNT fetuses may be an early biomarker for the development of hydrops placenta later in pregnancy.

NDAES Proj. ND01727.