Discussion - part 2

Current applications for the technique described here include counting early embryos in experimental animals. This practice will allow early confirmation of pregnancy in valuable animals, which can be removed from mating if already pregnant or recycled into mating if not. In addition, our technique can help determine whether fetal wastage has occurred between early pregnancy and delivery. For example, Becker and coworkers treated tumor-bearing pregnant mice with angiogenic inhibitors to determine whether there was any effect on pregnancy. The tumors went away, while pregnancies progressed. The authors were able to show that the number of early pregnancies was the same as the number of animals delivered, thereby demonstrating that there was no fetal wasteage. The system also can be used to document delayed development or growth retardation in mutant or manipulated animals compared with wild-type animals.

Discussion

Discussion
Here we describe the use of a commercially available ultrasound system to provide a simple and accurate method to stage and morphologically assess embryonic mouse development. Using a similar technique, Chang and coworkers found that decidual sac size is a reliable and accurate marker of GA. We used US measurements of CRL, BPD, and sac width to predict GA statistically by using formulas based on best linear fit to the data. CRL and BPD, in particular, are used routinely in the ultrasound staging of human gestational development, and many operators and investigators are likely to be familiar with them.

Musculoskeletal system

Musculoskeletal system. Limb buds were first apparent at 10.5 DPC in 10 of 27 (37%) fetuses, and in the remaining 17 fetal animals, limb buds became apparent the following day (Figure 9). Individual hind- and forelimb digits became discernible at 15.5 DPC; femur length could be measured at this time as well (Figure 10).

Cardiovascular system

Cardiovascular system. Fetal heart activity was detectable by use of gray-scale US at 10.5 DPC in all gestations. Blood flow within the umbilical cord was demonstrated at this time point as well by use of power Doppler color imaging. Power Doppler color imaging of the heart revealed the interventricular septum by 14.5 DPC and the atrial septum at 17.5 DPC. The interventricular septum could be detected by use of gray-scale at 16.5 DPC. By 11.5 DPC, blood flow could be demonstrated within the major thoracoabdominal arteries, and the circulation could be followed within the ascending, descending, and abdominal aorta and into the hypogastric arteries, umbilical cord, and placenta (Figure 6).

Results / Sixteen successful pregnancies yielded 92 fetuses

Results
Sixteen successful pregnancies yielded 92 fetuses.
Biometric measurements. At 9.5 DPC, all gestations appeared as rounded sacs with a diameter (mean + standard error) of 4.4 + 1 mm (Figure 1). Placentas and fetal poles were differentiable by 10.5 DPC (Figure 2). Correlation between CRL and GA was highly significant beyond 9.5 DPC (r = 0.97, P < 0.0001), and a simple regression equation was derived to predict GA: CRL/2 + 9 d (R² = 0.935, P < 0.001; Figures 3 and 4 and Table 1). BPD was a significant predictor of GA beyond 12.5 DPC (R² = 0.845, P < 0.001; Figures 3 and 5 and Table 2).

Study variables

Study variables. The study variables included both biometric measurements and evaluation of morphologic features. The biometric measurements assessed were gestational sac dimensions, crown-rump length (CRL), biparietal diameter (BPD), and thoracoabdominal diameter (TAD). The morphologic features evaluated included: presence of fetal heart activity, the 4 chambers of the heart, great vessels, axial and appendicular skeleton, thorax, abdomen, and brain.

Imaging

Imaging. Transabdominal US was carried out with a 15-MHz transducer (Acuson Sequoia System, 3.5 x 1 cm 15L8 transducer, Siemens Corporation, Mountain View, CA). The system we used costs between $300,000 and $400,000, depending on options and discounts. The transducer probe costs between $17,000 and $18,000.

Materials and Methods / Technique

We mated 36 female mice individually with 12 males in 3 cycles. Females were pretreated with hormonal supplementation with 10 IU pregnant mare serum gonadotrophin in normal
saline administered intraperitoneally 48 h prior to mating and 10 IU human chorionic gonadotrophin diluted in normal saline administered intraperitoneally immediately prior to mating. For each of 3 cycles, 12 different females and the same 12 males were paired overnight and separated the next morning. This practice allowed precise determination of coitus and was used in preference to presence or absence of vaginal plugs. Noon of the first day postcoitus was considered to be 0.5 d post-coitus (DPC), and nonpregnant animals were mated again in subsequent cycles. 

Ultrasound Diagnosis of Mouse Pregnancy and Gestational Staging - part 2

However, like magnetic resonance microscopy, the ultrasound backscatter microscopy system is technically complex and not readily available. In addition, various technical limitations, such as a narrow transducer aperture (5 mm) and only superficial depth penetration capability (7 mm), may limit broader applicability of this system. Although ultrasound backscatter microscopy appears to be highly valuable for rigorous anatomic and physiologic genotype-phenotype analyses, it may be cumbersome, if not impractical, for more simple tasks such as determining gestational age (GA).