Doppler ultrasonography
Doppler ultrasonography was used to assess the uteroplacental and umbilicoplacental hemodynamic alterations on GD 19.5 based on the method described by Renaud et al.(2011). Briefly, the dams were anaesthetized using 3% isoflurane in air by face mask and hair in abdomen was removed by shaving followed by a chemical depilatory. Maternal breathing and heart rate were monitored during the measurement. For Doppler mode, a high frame rate 22 mHz scanhead (SL3116, Esaote Mylab twice, Italy) was used to obtain the waveform recording for offline analysis. For each dam, measurements were performed on 2-4 implantation sites those were located close to the bladder or ovary. In each implantation site, the Doppler waveforms of 2-3 spiral arteries (SAs), maternal canal and umbilical artery were recorded, and the peak systolic velocity (PSV) was measured. Finally, PSV of umbilical artery and maternal canal from two implantation sites and PSV of spiral arteries from four implantation sites were obtained in each pregnant rat.