Two- and three-dimensional high-frequency real-time ultrasonography for estimating gestational age and assessing...

Rats are commonly used model animals in reproductive system and embryonic development research, and accurate pregnancy staging and gestational age (GA) assessment are crucial for experimental studies. Traditional methods (such as vaginal plug examination and abdominal palpation) have limitations and cannot precisely determine the time of fertilization or assess embryonic developmental features. As a noninvasive and convenient real-time imaging technique, ultrasonography has been widely used in human fetal assessment but has been less intensively applied in rat pregnancy research. This study aimed to use commercial high-frequency two-dimensional (2D) and three-dimensional (3D) ultrasonography to establish and validate quantitative GA estimation formulae for rats, display the 3D structure of the gestational sac (GS) and fetal rat, and preliminarily examine the blood flow Doppler changes in pregnant rats and fetal rats.

A total of 23 pregnant Wistar rats were included in the study and divided into an observation group (12 rats with detected vaginal plugs) and a validation group (11 rats without detected plugs but with ultrasonography-confirmed pregnancy). A Voluson E10 ultrasound diagnostic instrument (GE HealthCare), equipped with a 2D linear array probe (4-10 MHz) and a 3D volumetric probe (6-18 MHz), was used to examine the pregnant rats. Parameters such as the maximum diameter of the GS, crown-rump length (CRL), biparietal diameter (BPD), and abdominal circumference (AC) were measured, and linear and multiple regression models were constructed with GA serving as the dependent variable. The normality of data was verified via the Shapiro-Wilk test, and intraobserver reproducibility was assessed via intraclass correlation coefficients (ICCs). Doppler flow spectra of the maternal uterine artery (UtA) and fetal ductus venosus and umbilical artery were also analyzed.

The GS was first visualized on embryonic day 7 (E7). CRL demonstrated a strong linear correlation with GA from E9 to E15 (R²=0.95; P<0.001), while the combination of BPD and AC provided a more reliable estimation from E16 onward (R²=0.90; P<0.001). With higher GA, peak systolic velocity and end-diastolic velocity of the maternal UtA and fetal ductus venosus gradually increased, whereas the systolic:diastolic ratio and resistance index progressively decreased. All Doppler parameters showed excellent intraobserver reproducibility (ICC ≥0.85). In the prospective validation cohort (n=11), the GA prediction model achieved an overall accuracy of 81.82% within 1 day and 100% within 2 days. 3D ultrasonography clearly depicted the beaded distribution of GS and the spatial structure of fetal rats.

Commercial high-frequency ultrasound facilitates the reliable and quantitative assessment of rat pregnancy and GA with good reproducibility. Serial Doppler imaging can reveal characteristic maternal-fetal hemodynamic changes throughout gestation. 3D ultrasonography allows for the noninvasive visualization of the spatial arrangement of GS and fetal morphology. These findings provide a standardized imaging protocol for future studies in rat reproductive toxicology, pharmacology, and pregnancy-related disease models.