Early pregnancy detection has the potential to improve reproductive efficiency of embryo transfer (ET) programs by identifying nonpregnant animals and allowing their prompt reutilisation. Although methods are available to determine pregnancy after ~28 days, recent studies have highlighted the potential of indirect measurements, such as colour Doppler ultrasonography of the corpus luteum (CL), to serve as early pregnancy diagnostic methods. The objective of this study was to evaluate the sensitivity, specificity, and positive (PPV) and negative predictive value (NPV) of multiple indirect methods for pregnancy diagnosis at Day 21 in in vitro-produced ET recipients. Dairy heifers (n=796) were synchronized using a modified 5-day CoSynch and received a fresh in vitro-produced embryo 7±1 day after gonadotropin-releasing hormone. Heifers were examined by transrectal ultrasound on Day 21 after gonadotropin-releasing hormone to determine uterine endometrial thickness (UET), CL volume, and CL blood flow. Blood flow to the CL was determined by colour Doppler ultrasonography (Sonovet Pico, 7.5-MHz linear array probe, PRF 1kHz). Blood samples were collected on Day 21 to determine serum progesterone (P4) concentrations. Cutoff points for UET, CL volume, and P4 were evaluated using receiver operating characteristic curves and cutoffs for pregnancy were determined to be <8.75mm, >1800mm3, and >2.65 ng/mL for UET, CL volume, and serum P4, respectively. Heifers were classified as pregnant by CL colour Doppler ultrasound if they exhibited colour pixels in more than 10% of the periphery or exhibited colour pixels internally. Heifers were evaluated by transrectal ultrasonography on Day 28 to determine true pregnancy status. Differences between measurements were determined by generalized estimating equations. Pregnancies per ET on Day 28 was 50.1% (399/796). Sensitivity was lowest for UET and was different (P<0.01) from the other three tests (Table 1). Specificity was greatest (P<0.01) for serum P4, lowest for UET, and intermediate for CL volume and colour Doppler (Table 1). Serum P4, CL volume, and CL colour Doppler showed excellent NPV, indicating that each of these values accurately predicted nonpregnant heifers, the main emphasis of this study. However, PPV was moderate, indicating the potential for false positive results either due to pregnancy loss between Day 21 and 28 or, alternatively, inaccuracy of the method. Thus, on Day 21 of pregnancy, an accurate nonpregnancy diagnosis can be performed in ET recipients by using CL volume with or without CL colour Doppler, allowing for improved reproductive efficiency.
Table 1.Sensitivity, specificity, and positive (PPV) and negative predictive value (NPV) for various indirect methods to determine pregnancy on Day 21 in embryo transfer (ET) recipient heifers
Association of endometrial blood flow as determined by a modified colour Doppler technique with subsequent outcome of in-vitro fertilization
