13 ULTRASOUND EVALUATION OF FETAL AND PLACENTAL DEVELOPMENT IN SOMATIC CELL NUCLEAR TRANSFER AND ARTIFICIAL INSEMINATION BOVINE PREGNANCIES

Abstract

Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) is still doomed by a high incidence of pregnancy failure. In cattle, most gestation losses initially take place during the peri-implantation period. Then, abnormal placentation associated with fetal overgrowth (abnormal offspring syndrome) occurs in late pregnancy. As a consequence, IETS recommendations include regular ultrasound evaluation of SCNT pregnancies, with qualitative ultrasound evaluation of the placentomes, fetal movements and fluids being proposed. The objective of this study was to evaluate the use of new, quantitative parameters for the assessment of feto-placental development in AI and SCNT bovine pregnancies. Twenty-two heifers of 4 different breeds were used as SCNT recipients and 11 Holstein heifers were used as AI controls (C). All SCNT fetuses were produced as previously published in the laboratory, using the same fibroblast donor. Animals were scanned every 2 weeks from Day 150 using a Voluson-i (GE Medical Systems) with a transabdominal multifrequency probe (2.2 to 6.5 MHz), allowing automatic 3D volume and Doppler acquisition. Each time, 7 placentomes were scored from 0 to 3 according to echogenicity, general appearance and degree of oedema and their 2D surface was measured. Fetal intercostal width (ICW) was measured in a coronal view just behind the heart. Doppler velocimetry indices, pulsatility index (PI) and resistance index (RI) of one of the two fetal umbilical arteries were obtained and the diameters of the umbilical vessels were measured (UD). The 3D power Doppler was performed to obtain vascular index (VI), flow index (FI) and vascular flow index (VFI). Data were analysed by ANOVA with GraphPad Prism^®. So far, 27 heifers have either delivered (n = 10 SCNT and 7 C) or pregnancies have been terminated because of abnormal offspring syndrome (n = 10 SCNT). One thousand five hundred and 197 placentomes have been analysed for 2D and 3D analyses, respectively. The mean placentome score is significantly higher (less normal) in SCNT compared with C (P < 0.0001) but does not vary according to gestational age. The placentome surface is also significantly larger in SCNT vs C (P < 0.0001) but also in SCNT that did not go to term vs those that were alive at birth (P < 0.002). Fetal ICW and UD are consistently larger in SCNT vs C (P < 0.0001) at all stages of pregnancy and there is a significant correlation (r² = 0.81) between ICW at 15 days before term and birth weight. The PI and RI were not different between SCNT and C. Intra-operator reproducibility of 3D analyses was very high (intra-class correlation coefficient: 80 to 95% for a 95% confidence interval). There was no significant difference for VI, FI, or VFI between SCNT and C. Ongoing work taking into account pregnancy outcomes indicates that placentome scores are useful indicators of pregnancy outcome in SCNT pregnancies. In contrast, the abnormal vascularization observed by histology in SCNT placentomes does not appear to be accompanied by abnormal placental blood flow when analysed using quantitative 3D Doppler.

This project received financial support from ANR (ref. PCS-09-GENM-022).