Methods Of Detecting Ectopic Pregnancy

Abstract

A method for determining the likelihood of an ectopic pregnancy and spontaneous miscarriage is described, by measuring the levels of markers, especially hCG and CA-125 which have been found to be characteristic of these conditions. Preferably measuring these biomarker levels at earliest possible presentation of patients with general clinical symptoms and applying cut-off values described determines the likelihood of an ectopic pregnancy and spontaneous miscarriage.

Claims

1. A method of determining the likelihood of an ectopic pregnancy or spontaneous miscarriage comprising measuring the level of one or more markers selected from total hCG(hCGt), free beta subunit of hCG (hCGβ), hyperglycosylated form of hCG (hCGh), CA125 and progesterone in a sample obtained from a pregnant woman.

2. The method of claim 1 wherein the levels of combination of two or more of said markers is measured.

3. The method of claim 1 wherein an ectopic pregnancy can be distinguished from a normal viable intrauterine pregnancy.

4. The method of claim 1 wherein the level of hCGt is measured.

5. The method of claim 1 wherein the levels of hCGt and CA125 are measured.

6. The method of claim 1 wherein the level of hCGh is measured.

7. The method of claim 1 further comprising analysing the results obtained from an ultrasound scan to determine the presence of a foetal sac and optionally a foetal heartbeat.

8. The method of claim 1 wherein a woman likely to suffer a spontaneous miscarriage can be distinguished from a normal viable intrauterine pregnancy.

9. The method of claim 1 wherein a woman likely to suffer a spontaneous miscarriage can be distinguished from an ectopic pregnancy.

10. A method according to claim 1, wherein the sample is from a pregnant woman at between 4 and 10 weeks gestation.

11. The method of claim 1 wherein the sample is a serum sample.

Description

[0046] The application will now be described in the examples below which refer to the following figures:

[0047] FIG. 1 shows Box plots of hCGt, hCG(3, hCGh, progesterone and CA125 in the sera of women with viable pregnancy, spontaneous miscarriage, and ectopic pregnancy at presentation. 1=CA125; 2=TH ratio; 3=TC; 4=hCGh; 5=THBPC; 6=TB; 7=hCGb; 8=hCGt; 9=progesterone. T=hCGt; B=hCGβ; H=hCGh; C=CA125; P=progesterone.

[0048] FIG. 2 shows ROC analyses for (top) inclusion of ectopic pregnancy and exclusion of viable pregnancy; and (bottom) inclusion of ectopic pregnancy and exclusion of spontaneous miscarriage for hCGt, hCGβ, hCGh, progesterone and CA125. Single- and combined markers are in 1, 3 and 2, 4, respectively. T=hCGt; B=hCGβ; H=hCGh; C=CA125; P=progesterone.

EXAMPLE 1

Materials and Methods

Women and Samples

[0049] Serum samples were collected between September 2002 and March 2003 at the accident and emergency department (AE) and early pregnancy assessment unit (EPAU) of Homerton NHS Hospital Trust London, from 441 women at initial presentation with a positive pregnancy test in the first trimester, lower abdominal pain and/or vaginal bleeding. Gestational age was estimated based on date of last menstrual period as is standard practice in EPAU. The final diagnoses were confirmed through follow up on pregnancy outcome, laparoscopy, and histology of products of conception. 7/441 patients were lost to follow up and 58/441 samples were incorrectly stored and therefore disposed of The remaining 376 patients had a final diagnoses of viable pregnancy in 175 women; spontaneous miscarriage (inevitable, incomplete, complete) in 175 women; and ectopic pregnancy in 26 women. 2 patients initially diagnosed as ectopic pregnancies by current assessment practice were confirmed as spontaneous miscarriage upon follow up. Clinical data for the initial and final diagnoses were obtained by blinded personnel and matched for each patient. Samples were stored at −20 ° C. Ethical approval was obtained from the North East Thames Region Health Authority Ethics Committee.

[0050] Hormone Assays

[0051] Sample analyses were carried out by automated analysis on the DPC IMMULITE 2000 platform in accordance with the manufacturer's instructions. Sera of 376 samples were assayed for matched hCGt, hCGβ (referring to the free beta subunit of hCG and not a result of an hCG assay designated as ‘βhCG’ but measuring both free beta subunit and intact hormone hCG (total hCG)), progesterone, and CA125. Sufficient residual sera was available from 149 patient samples for hCGh measurement using the ITA (Invasive Trophoblast Antigen—hCGh) assay performed using the discontinued Nichols Institute Diagnostics Advantage system. Quality controls for the lower-, mid- and upper linear range were run concurrently with test samples. The detection limits were hCGt, 0.4 mIU/mL; hCGO, 0.02 ng/mL; progesterone, 0.2 ng/mL; and CA125, 1 U/mL.

[0052] Data were recorded in Excel 2007 and statistical analyses were performed with Stats-Direct software. Differences between median concentrations were analysed using Kruskal-Wallis (Conover-Inman) tests for single- and combination markers. The assigned value for significance was P<0.05, and optimum cutoff during ROC analyses was based on equal sensitivity: specificity weighting except for CA125 during ectopic pregnancy against spontaneous miscarriage where cutoff was optimised for 100% sensitivity.

[0053] Ultrasound Detection of Intrauterine Pregnancy

[0054] As a matter of routine the EPAU report in patient's notes whether a foetal sac and foetal heart beat are detected upon ultrasound examination of patients referred to the on call ultra-sonographer. Thus we could retrospectively compare biochemical analysis with an ultrasound finding. In subsequent analysis patient notes were examined to see if ultrasound findings would reduce false positive arising from any biochemical screening due to spontaneous miscarriage.

[0055] Results

[0056] The gestational ages were similar for viable pregnancy, spontaneous miscarriage, and ectopic pregnancy with a mean of 7.3, 7.6, and 6.3 weeks, respectively (Table 1). This eliminated bias associated with variability of marker concentration with gestational age.

[0057] The current ultrasound based screening for ectopic pregnancy detected 11 (42%) of the ectopic pregnancy at presentation and 12 (46%) on follow-up scan, but 2 (8%) were only correctly identified upon emergency re-admission, both women presented with hemorrhagic shock.

[0058] The data sets for the concentration of each marker were skewed therefore the medians were compared. Significant differences were seen between all 3 conditions (in all pairwise combinations) in all markers except hCGh, hCGt×hCGh×hCGO×progesterone×CA125 (THBPC), and THB for ectopic pregnancy against spontaneous miscarriage; and CA125 and hCGt/hCGh ratio (T/H) for viable pregnancy against spontaneous miscarriage (Table 2). All markers had significantly lower concentrations in ectopic pregnancy compared to viable pregnancy (Table 2 and FIG. 1). ROC analyses (FIG. 2 and Table 3) for ectopic pregnancy against viable pregnancy revealed that the best performing marker was hCGt×CA125 (TC) with area under curve (AUC) of 0.912. The best performing single marker was hCGt with AUC of 0.878. At a cutoff of −3736 mIU/mL, hCGt had 100% sensitivity, 76% specificity, 39% PPV, and 100% NPV (Table 3). In the category of ectopic pregnancy against spontaneous miscarriage, CA125 and TC were the best markers with AUC of 0.802 and 0.794, respectively (FIG. 2). At a cutoff of −42.029 U/mL, CA125 had 100% sensitivity, 43% specificity, 21% PPV, and 100% NPV; and at a cutoff of 43,276.8, TC had 92% sensitivity, 60% specificity, 26% PPV, and 98% NPV (Table 3). The overall performance of progesterone was poor compared to other markers (Table 3 and FIG. 2).

[0059] In an algorithm in which low level total hCG (<3736 mIU/mL) was used to detect all ectopic hCG (100% sensitivity) but had high false positive due to simultaneous detection of many pregnancies destined to miscarry, miscarriage (Table 4) resulted in a 40.9% false positive rate (defined as any non-ectopic pregnancy). The addition of low CA-125, at a cutoff of 41.98 U/mL, excluded many of the miscarrying intrauterine pregnancies; maintaining a 100% sensitivity and reducing the false positive rate to 24.9%. Although retrospectively, analysis of the ultrasound reports for evidence of a uterine pregnancy indicated that if ultrasound assessment had been added as a third stage test identified, a further 43 false positives (all subsequently identified as miscarriages) would have been excluded. Ultrasound at this stage would maintain a 100% sensitivity for ectopic pregnancy but reduce the false positive rate by a further 12% to 14.4% (see Table 4).

[0060] Discussion

[0061] The use of a single point predictor biomarker to identify early ectopic pregnancy would aid diagnosis at initial presentation, thereby facilitating medical intervention prior to life-threatening tubal rupture, and thus help reduce maternal mortality. To date, no such single predictor has been found. Clinicians rely on transvaginal ultrasound, which is sensitive if an adequate serum hCG assay is used and levels fall above the discriminatory zone (1000-1500 mIU/mL) and is also dependent on serial 48 hour measurements of hCG to determine the doubling time. However, the discriminatory zone approach is clearly flawed in detecting ectopic pregnancies below the threshold and would thus have missed more than half (15/27 or 56%) of the ectopic pregnancies in our study. The doubling time approach is also hindered by the diagnostic delay time of 1.5 days, and the potential tubal rupture which may occur outside the healthcare setting if the repeat hCG measurement is performed on an out-patient basis. A highly sensitive single-point biomarker algorithm would improve maternal safety by detecting all ectopic pregnancies at presentation, and desirably also possess enough specificity to reduce medical costs from the needless follow up of patients with viable pregnancy. The reduced diagnostic turnaround time would also help alleviate the anxiety of patients. Previously, in a smaller study, it has been found that single point measurements of serum hCG isoforms, particularly hCGβ and hCGt, had better predictive function than corresponding urinary isoforms in distinguishing women with ectopic pregnancy from women with viable pregnancy. However neither marker could distinguish ectopic pregnancy from spontaneous miscarriage. This much larger study, examined similar markers with further inclusion of hCGh, progesterone and CA125. These were in either single or combination algorithms (Table 1).

[0062] We found that all the markers had significantly lower concentrations (P<0.0001) in ectopic pregnancy compared to viable pregnancy, though performances were relatively less so with progesterone (P=0.0004), and hCGt/hCGh ratio (P=0.02) (Table 2). All markers, therefore, appeared to have the potential for discriminating between ectopic and viable pregnancy, thus corroborating the previous study. However, some markers appeared to be better than others, as indicated by ROC analysis (FIG. 2 and Table 3). The best performing marker was hCGt×CA125 (TC) with area under curve (AUC) of 0.912. At a cutoff of ≦80,893.5, serum TC measurement was able to detect women with ectopic pregnancy and exclude those with viable pregnancy with a sensitivity of 100%, specificity of 78%, positive predictive value (PPV) of 40%, and negative predictive value (NPV) of 100%; combining all biomarkers did not improve on this algorithm (FIG. 2). The best performing single marker, and thus potentially the most cost effective marker of choice, was hCGt with AUC of 0.878. At a cutoff of ≦3736 mIU/mL, hCGt had 100% sensitivity, 76% specificity, 39% PPV, and 100% NPV. At a cutoff of ≦7.5 ng/mL, hCGβ had 96% sensitivity, 74% specificity, 36% PPV, and 99% NPV (Table 3).

[0063] Progesterone was significantly different between all pairwise combinations of ectopic pregnancy, viable pregnancy and spontaneous miscarriage (Table 2). However, here serum progesterone levels in ectopic pregnancy were marginally higher than in spontaneous miscarriage (FIG. 1); At a similar cutoff of 20.4 ng/mL (Table 3), we found a sensitivity of only 65% for ectopic pregnancy. Overall, despite the significant all-pairwise Kruskal-Wallis results (Table 2), ROC analyses revealed that the performance of progesterone was poor, compared to the other markers (FIG. 2 and Table 3). This may be attributable to the wide range of overlap for serum progesterone between ectopic pregnancy, spontaneous miscarriage and viable pregnancy (FIG. 1) which was reflected in the wide confidence intervals (Table 3). We found that all the markers except THBPC, THB, and hCGh were discriminatory between ectopic pregnancy and spontaneous miscarriage (Table 2). This included hCGt (P=0.002) and hCGβ (P=0.014), ROC analyses indicated that CA125 and hCGT with CA-125 were the best markers to distinguish ectopic pregnancy from spontaneous miscarriage, with AUC of 0.802 and 0.794, respectively (FIG. 2). At a cutoff of ≦42.029 U/mL, CA125 had 100% sensitivity, 43% specificity, 21% PPV, and 100% NPV; and at a cutoff of ≦43,276.8, TC had 92% sensitivity, 60% specificity, 26% PPV, and 98% NPV (Table 3). CA125 appeared to distinguish ectopic pregnancy, from intrauterine pregnancy (spontaneous miscarriage and viable pregnancy) as a combined group (Table 2, and FIG. 1). This was consistent with previous studies where serum CA125 was found to be lower in ectopic pregnancy compared to viable pregnancy, but not between viable pregnancy and spontaneous miscarriage. The trend with hCGh (P=0.69) was consistent with previous work and appeared overall to discriminate viable pregnancy from non-viable pregnancy (ectopic pregnancy and miscarriage) as a group (Table 2). The non-discrimination of ectopic pregnancy from spontaneous miscarriage found in THBPC and THB may thus have been due to hCGh, which was the marker common to both.

[0064] For the construction of a diagnostic algorithm, we found that either the single marker hCGt, or the combination marker hCGt and CA125 (depending on cost implications), appeared to be most favorable in excluding viable pregnancy from ectopic pregnancy (Table 3). Similarly, CA125, or hCGt×CA125 (TC), appeared most favorable in excluding spontaneous miscarriage from ectopic pregnancy (Table 3), and would potentially further reduce the false positives obtained after excluding viable pregnancy. We thus simulated a combination algorithm for diagnosis of ectopic pregnancy (Table 4), where, a positive hCGt assay (i.e. <3737 mIU/mL) was followed by a CA125 assay (deemed positive when <41.98 U/mL). The combined algorithm produced 100% sensitivity (26/26), 75.1% specificity and 24.9% false positive rate (FPR) for identifying ectopic pregnancy, even at initial presentation. This is in comparison to the current protocol, where follow up was required to identify 57.7% (15/26) of ectopic pregnancies (Table 4). Furthermore, the algorithm would be safe in excluding 75.1% (263/350) of non-ectopic pregnancies compared with 64.9% (227/350) at initial presentation in the current protocol. Significantly, this indicates that 36 additional patients would have been spared the anxiety of a possible ectopic pregnancy representing 10% of our study population. The CA125 assay was useful in reducing the FPR from 40.9% to 24.9% without compromising the 100% sensitivity (Table 4). Since 43 of the residual false positives had a gestation of >5.5 weeks, the FPR could potentially be reduced further to 14.37% with the application of ultrasonography, based on the detection capability for localising pregnancies with gestation of >5.5 weeks (at which stage the yolk sac is visible).

[0065] In conclusion, in early pregnancy, single-point serum measurements of hCGt <3737 mIU/mL together with CA125 <41.98 U/mL, are a promising algorithm for detecting ectopic pregnancy with 100% sensitivity, 75.1% specificity, and 24.9% false positive rate. The addendum of ultrasonography to the algorithm has potential to further reduce the false positive rate significantly, and needs to be explored. This algorithm has the advantage of a single point detection over current protocols which require follow up, and appears safe in excluding non-ectopic pregnancy at presentation. Indeed, the adoption of this biochemical screening would have detected the two women (8%) who were not identified as ectopic pregnancy by EPAU at presentation or follow up and subsequently presented with hemorrhagic shock, however a significant number of spontaneous miscarriage are also detected. Nevertheless this biochemical algorithm enables the identification of women with a high risk of ectopic pregnancy in early pregnancy and allows or intervention antecedent to tubal rupture and a reduction in maternal morbidity.

TABLE-US-00001 TABLE 1 Descriptive staustics for concentrations of hCGt, hCGB, hCGh, progesterone and CA125 in the sera of women with viable pregnancy, spontaneous miscarriage, and ectopic pregnancy. Statistics Viable pregnancy Spontaneous miscarriage Ectopic pregnancy Gestation, weeks Mean (SD) 7.3 (2.4) 7.6 (2.3) 6.3 (2.9) Range 1-12 2-12 0.7-12 hCGt, mlU/ml. Mean (SD) 47,490 (52,525) 11,702 (25,840) 1152 (1048) Median (IQR) 27,644 (3768-81,237) 2546 (588-10,957) 781 (364-1806) hCGβ, ng/mL Mean (SD) 24 (20) 8 (11) 3 (3) Median (IQR) 22 (6-38) 4 (1-11) 2 (1-4) hCGh, ng/mL Mean (SD) 794 (1729) 154 (369) 31 (30)31 (30) Median (IQR) 439 (118-908) 17 (6-58) 22 (10-45) Progesterone, ng/mL Mean (SD) 48 (19) 21 (22) 31 (22) Median (IQR) 56 (35-64) 15 (5-29) 25 (13-55) CA125, U/mL Mean (SD) 49 (66) 69 (99) 15 (12) Median (IQR) 27 (15-21) 31 (16-70) 10 (6-22) T.sup.a × B.sup.b Mean (SD) 1,855,125 (2,584,570) 309,408 (1,100,852) 5093 (8588) Median (IQR) 578,909 (19,250-2,690,350) 8789 (782-91,162) 1851 (196-6892) T.sup.a × B.sup.b × C Mean (SD) 107,551,463 (314,125,661) 20,371,636 (87,192,559) 70,312 (106,167) Median (IQR) 16,132,936 (615,576-93,587,040) 317,905 (29,178-4,438,013) 10,676 (4221-100,402) T.sup.a/H.sup.d Mean (SD) 314 (1190) 233 (612) 59 (58) Median (IQR) 71 (37-138) 96 (44-146) 38 (27-75) Log  (T.sup.a × H  × B.sup.b) Mean (SD) 8 (2) 5 (2) 4 (1) Median (IQR) 9 (7-9) 5 (4-6) 5 (4-5) Log.sub.10 (T.sup.a × H.sup.d × B.sup.b × Mean (SD) 11 (2) 7 (3) 7 (1) P  × C ) Median (IQR) 12 (10-13) 7 (5-9) 7 (6-8) T.sup.a x C Mean (SD) 2,637,744 (6,480,122) 775,330 (2,211,175) 18,153 (22,418) Median (IQR) 662,847 (99,111-2,660,938) 90,293 (16,558-441,047) 7005 (3530-29,825) .sup.ahCGt. .sup.bhCGβ. .sup.cCA125. .sup.dhCGh. .sup.eProgesterone. indicates data missing or illegible when filed

TABLE-US-00002 TABLE 2 Kruskal-Wallis all pairwise comparisons (Conover- inman) of hCGt, hCGβ, hCGh, progesterone and CA125 in the sera of women with viable pregnancym spontaneous miscarriage, and ectopic pregnancy at presentation. Event Marker P-value Result Ep vs VP T P < 0.0001 Significant TBC P < 0.0001 Significant TB P < 0.0001 Significant THBPC P < 0.0001 Significant THB P < 0.0001 Significant H P < 0.0001 Significant B P < 0.0001 Significant C P < 0.0001 Significant TC P < 0.0001 Significant P P = 0.0004 Significant T/H P = 0.0228 Significant EP vs. MIS TC P < 0.0001 Significant C P < 0.0001 Significant TBC P = 0.0001 Significant T P = 0.0018 Significant TB P = 0.0033 Significant T/H P = 0.004 Significant P P = 0.0046 Significant B P = 0.0135 Significant THB P = 0.2717 Not significant THBPC P = 0.468 Not significant H P = 0.687 Not significant VP vs. MIS T P < 0.0001 Significant TC P < 0.0001 Significant TBC P < 0.0001 Significant P P < 0.0001 Significant THBPC P < 0.0001 Significant THB P < 0.0001 Significant H P < 0.0001 Significant B P < 0.0001 Significant C P = 0.0714 Not significant T/H P = 0.25 Not significant T. hCGt; B, hCGβ; H, hCGh; C, CA125: P, progesterone.

TABLE-US-00003 TABLE 3 Ranked comparison of ROC performances of serum hCGt, hCGβ, hCGh, progesterone and CA125 for (top), the inclusion of ectopic pregnancy (EP) and exclusion of viable pregnancy (VP); and (bottom), inclusion of EP with exclusion of spontaneous miscarriage (MIS). Marker Sensitivity 95% CI specificity 95% CI PPV NPV Cutoff≦ Area under curve EP vs. VP TC 1.00 0.87-1.00 0.78 0.71-84.sup.  0.40 1.00 80,893.5 0.912 TBC 1.00 0.87-1.00 0.77 0.70-0.83 0.39 1.00 411,745 0.901 THB 1.00 0.83-1.00 0.77 0.67-0.86 0.51 1.00 6.33 0.895 THBPC 1.00 0.87-1.00 0.79 0.69-0.88 0.54 1.00 9.33 0.892 T 1.00 0.87-1.00 0.76 0.69-0.82 0.39 1.00 3736 mIU/mL 0.878 TB 1.00 0.87-1.00 0.74 0.67-0.80 0.37 1.00 38,480 0.877 H 1.00 0.83-100  0.76 0.66-0.85 0.50 1.00 112.2 ng/mL 0.869 B 0.96 0.83-100  0.74 0.61-0.81 0.36 0.99 75 ng/mL 0.857 C 0.58 0.32-0.77 0.85 0.81-0.95 0.36 0.93 11.4 U/mL 0.775 P 0.73 0.52-0.88 0.70 0.62-0.76 0.26 0.95 44 ng/mL 0.704 T/H 0.70 0.46-0.88 0.61 0.51-0.72 0.30 0.90 47.3 0.660 EP vs. MIS C 1.00 0.87-1.00 0.43 0.36-0.51 0.21 1.00 41.98 U/mL 0.802 TC 0.92 0.75-0.99 0.60 0.53-0.68 0.26 0.98 43,276.8 0.794 TCB 0.96 0.80-.99  0.51 0.43-0.59 0.23 0.99 301,999 0.744 T/H 0.75 0.51-0.91 0.67 0.51-0.79 0.50 0.86 61.8 0.730 T 0.89 0.71-0.98 0.54 0.46-0.62 0.23 0.97 2076 mIU/mL 0.694 TB 0.96 0.81-0.99 0.42 0.35-0.49 0.21 0.99 23,378 0.684 P 0.65 0.44-0.83 0.63 0.56-0.71 0.21 0.92 20.4 ng/mL.sup.a 0.667 B 0.85 0.66-0.96 0.47 0.40-0.55 0.20 0.95 3.96 ng/mL 0.639 THB 1.00 0.83-.100 0.24 0.13-0.39 0.37 1.00 6.33 0.572 THBPC 0.95 0.75-.099 0.35 0.21-0.51 0.40 0.94 8.44 0.536 H 0.95 0.83-1.00 0.27 0.09-0.34 0.37 0.92 5.9 ng/mL 0.523 T, hCGt; B, hCGβ; H, hCGh; C, CA125; P, progesterone .sup.a≧

TABLE-US-00004 TABLE 4 Diagnostic representation of the step wise application of a biochemical algorithm for ectopic pregnancy coupled with ultrasound scanning. Current algorithm performance Viable pregnancy Miscarriage Ectopic pregnancy Total Diagnosed at presentation  122 (69.7%) 105 (60%) 11 (42.3%) 238 Diagnosed at follow up  53 (30.3%)  70 (40%) 15 (57.7%) 138 Total  175 175 26 376 Suggested 3 stage analysis 1st stage analysis of serum samples taken at presentation Negative result A. hCGt assay <3736 mlU/mL (non-ectopic pregnancy) Positive result Total Outcome interpretation True negative False positive Sensitivity—true positive Performance  207 (59.1%) 143 (40.9%) 26 (100%) 376 2nd stage analysis of samples scored positive in A B. CA125 assay <41.98 U/mL  56* (16%)  87 (24.9%) 26 (100%) 169 Performance (combined effect of stages A and B 2643 (75.1%)  87 (24.9%) 26 (100%) 376 immunoassay of serum samples taken at presentation) 3rd stage ultrasound of patients scored psoitive in B Potential of an additional third stage True negative False positive Sensitivity—true positive Based on 43/87 false positives identified by serum  306 (87.4%)  44 (14.4%) 26 (100%) 376 immunoassay (gestational age >5.5 weeks) reclassified as non-ectopic following ultrasound scan.