The results of combining these trees into a three-marker test (VEGF, progesterone, and inhibin A) and four-marker test (VEGF, progesterone, inhibin A, and activin A) are shown in Table 3. The three-marker test diagnosed 45% of patients with 97% accuracy, misdiagnosing three participants: one with an ectopic pregnancy and two with intrauterine pregnancies. The four-marker test diagnosed fewer participants but with higher accuracy (99%), misdiagnosing only one participant with an ectopic pregnancy. Validation by bootstrapping demonstrated consistent results as shown in Table 3.
We also evaluated the performance of the tests for the subgroups of patients with hCG levels less than and 1,500 milli-international units/mL or greater (Table 3). The multiple marker profile resulted in perfect discrimination in women who presented with hCG values less than 1,500 milli-international units/mL at the time of presentation for care. Furthermore, more than two thirds of participants with ectopic pregnancy were classified in the low hCG group (70%) in the three-marker test compared with only 24% in the higher hCG group (P=.005).
We assessed the correlation of each marker with hCG. Among the four markers, hCG was the most closely associated with inhibin A in both groups (intrauterine pregnancy: ρ=0.64, P<.001 and ectopic pregnancy: ρ=0.54, P<.001). A weaker but significant association was also noted with activin in the intrauterine pregnancy group (0.42, P<.001), progesterone in the ectopic pregnancy group (ρ=0.52, P<.001), and VEGF in both groups (ρ=−0.48, P<.001 in the intrauterine pregnancy group and ρ=−0.34, P=.005 in the ectopic pregnancy group).
Given the clinical challenge of diagnosing ectopic pregnancy at an early gestational age, the identification of a biomarker to simplify and improve the diagnosis of ectopic pregnancy is a research priority.1,5 There are two general strategies to identify a biomarker, testing putative markers and discovery-driven screening of the proteome. In this study, we concomitantly examined a large set of biomarkers already demonstrated to have discriminative ability but not yet validated. We demonstrated that as single markers, none were adequate to discriminate ectopic pregnancy from intrauterine pregnancy. However, markers with limited ability when used together can often improve prediction,7,8 especially if they reflect different pathways. We developed a three-marker test combining progesterone, inhibin A, and VEGF that could diagnose almost half of the sample with 97% accuracy and the majority of women with a low hCG value with 100% accuracy. A four-marker test incorporating activin A diagnosed slightly fewer patients but with 99% accuracy.
Each of our markers is biologically plausible. Progesterone is secreted by the corpus luteum in early pregnancy before placental production and is a critical hormone for the establishment of normal pregnancy.9 A systematic review found that a single value of serum progesterone had insufficient capacity to diagnose ectopic pregnancy, although it was capable of identifying those at risk.10 In combination with other markers in our study, progesterone played a role in both optimizing sensitivity as well as specificity, likely as a result of its reflection of the viability of the pregnancy.
Inhibin A is also a major peptide product of the corpus luteum and also appears to have a fetoplacental source in early pregnancy.11,12 It is secreted by cytotrophoblasts13 and is involved in the fetomaternal communication necessary to maintain pregnancy.14,15 Previous studies have shown elevated levels of inhibin A to be a good predictor of intrauterine pregnancies but with varying discrimination of ectopic pregnancy among groups with abnormal intrauterine pregnancies.16,17
Vascular endothelial growth factor was the only marker in our diagnostic tree that was elevated in ectopic pregnancies. VEGF rises during the first trimester of pregnancy.18 Vascular endothelial growth factor may also be involved in the implantation of pregnancy in the fallopian tube. There is increased expression of VEGF and its receptor at the implantation site of an ectopic pregnancy compared with the nonimplantation site of the same oviduct.19 Our values are similar to other studies showing statistically significant elevations in serum VEGF levels in ectopic pregnancies compared with normal intrauterine pregnancy levels.20–22
Activin A was a component of our four-marker test. Activin A is localized in placental trophoblast and Hofbauer cells in early pregnancy,23,24 is secreted by cytotrophoblasts, and promotes their invasion in pregnancies to 10 weeks of gestation in vitro.13,25 Activin A levels begin at prepregnancy levels in the first trimester and progressively rise throughout pregnancy.17,26 There are conflicting data on the use of diagnostic value of a single serum value of activin A at presentation with both poor (AUC 0.58)17 and excellent (AUC 1.0)27 discrimination of a low value in predicting ectopic pregnancy. In our study, it enhanced specificity in the four-marker test, although it was not needed for perfect discrimination when we examined the low hCG level group.
Other markers of pregnancy development (pregnancy-specific β-1-glycoprotein, pregnancy-associated plasma protein-A, human placental lactogen, and glycodelin) were less discriminatory. Levels of human placental lactogen and glycodelin were low in both the ectopic pregnancy and intrauterine pregnancy groups. Pregnancy-specific β-1-glycoprotein and pregnancy-associated plasma protein-A were both significantly higher in intrauterine pregnancies than ectopic pregnancies with AUCs of 0.76 and 0.67, respectively, but did not add to discrimination beyond that of the markers in the model. Pregnancy-specific β-1-glycoprotein22,28 and pregnancy-associated plasma protein-A22,29–31 are elevated in normal pregnancies, but their discriminatory ability increases as the pregnancy advances.23,28–30 Other recently examined markers potentially involved in the progression of ectopic pregnancy such as inflammatory cytokines IL-6, IL-8, and TNF-α31,32 and the smooth muscle enzyme CK33–38 demonstrated poor discrimination with AUCs between 0.5 and 0.6.
The higher level of hCG in the intrauterine pregnancy group over the ectopic pregnancy group was expected. To aid in the diagnosis of women with an ectopic pregnancy, new markers have to improve discrimination above that of hCG alone. Although our identified markers do have some correlation with hCG, it is only modest and in some cases only noted in intrauterine pregnancy or ectopic pregnancy (and not the other). Of interest was that our new models demonstrated perfect discrimination with more than two thirds of ectopic pregnancies definitively classified in the subgroup with low hCG (less than 1,500 milli-international units/mL). Determination of pregnancy type in this subgroup would be impossible by current diagnostic methods and the improved results, both in terms of diagnostic ability and number diagnosed, in this subgroup are encouraging.
Others have proposed a multiple marker for the diagnosis of ectopic pregnancy. One group in Switzerland developed a multiple marker test, the “triple marker analysis” comparing sera from 43 patients with ectopic pregnancy and 79 normal intrauterine pregnancy from an antenatal clinic in Switzerland.23 Their test had a sensitivity of 97.7% with a specificity of 92.4% in diagnosing ectopic pregnancy.23 We were able to achieve a sensitivity of 69% and specificity of 84% using their algorithm. Pregnancy-associated plasma protein-A did discriminate between the groups in our study, but not as well as inhibin A or activin A. The combination of VEGF and progesterone with these two markers may prove to be a more robust test.
Strengths of this study include the large sample size, the number of biomarkers studied concurrently, a study population including only symptomatic women at risk for ectopic pregnancy, and the combining of markers to create a test with superior accuracy. A limitation of this study is that results from Classification and Regression tree are “data-driven” with a potential for overstating positive results, although validation with bootstrapping was reassuring. Finally, we have not yet evaluated these markers in women with a failed intrauterine gestation to confirm that we are discriminating the location and not solely viability of an early gestation. However, a test to discriminate viability of an early gestation would still be of clinical value. These finding are considered preliminary, and many of the assays are not routinely used in clinical practice. Future planned external validation will be performed in a separate set of samples, collected over a shorter time span, and among patients with failed intrauterine pregnancies in addition to intrauterine pregnancies and ectopic pregnancies.
In summary, many of the markers previously examined in smaller groups of women were found to be poor discriminators although differences between the groups may be statistically significant. We have examined a large number of markers concurrently in women at risk for ectopic pregnancy and have combined them to create a test that diagnoses ectopic pregnancy with excellent accuracy.
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