Human Reproduction Vol.17, No.2 pp. 490–492, 2002
Serum vascular endothelial growth factor as a possible marker for early ectopic pregnancy Afaf Felemban, Aref Sammour and Togas Tulandi1 Department of Obstetrics and Gynecology, McGill University, 687 Pine Avenue West, Montreal, Quebec, Canada 1To
whom correspondence should be addressed. E-mail:
[email protected]
BACKGROUND: This study evaluated serum vascular endothelial growth factor (VEGF) concentrations in women with normal intrauterine pregnancy (IUP), arrested IUP and ectopic pregnancy (EP). METHOD: This was a prospective, case–control study evaluating serum VEGF concentrations among 45 early pregnant women who subsequently were found to have an EP, a normal IUP or an arrested IUP (15 women in each group). Patients were stratified according to serum VEGF concentrations above and below 200 pg/ml. RESULTS: There was a significant difference in VEGF concentrations among women with EP, arrested IUP and normal IUP (306.1 ⍨ 26.5, 169.7 ⍨ 16.6 and 27.0 ⍨ 4.4 pg/ml respectively, P < 0.001). With a cut-off concentration of 200 pg/ml, serum VEGF could distinguish normal IUP from EP with a sensitivity of 88%, a specificity of 100% and a positive predictive value of 100%. Between EP and arrested IUP, the sensitivity was 87.5%, specificity 75% and positive predictive value of 77.8%. CONCLUSIONS: VEGF is a potential marker for EP. Its concentrations in women with EP are higher than in those with normal and arrested IUP. Key words: arrested pregnancy/ectopic pregnancy/pregnancy/VEGF
Introduction Management of ectopic pregnancy (EP) has changed dramatically over the years and a conservative approach now predominates (Yao and Tulandi, 1997; Tulandi and Sammour, 2000). In order to allow a conservative treatment, an early diagnosis is important. Transvaginal ultrasound and serum β-human chorionic gonadotrophin (HCG) determinations are the two most widely used methods. However, transvaginal ultrasound examination can be helpful only when an intrauterine gestation or an adnexal mass is seen (Cacciatore et al., 1994; Tulandi and Sammour, 2000). Similarly, serial determinations of serum β-HCG can distinguish normal intrauterine pregnancy (IUP) from a non-viable pregnancy, but it cannot separate an arrested IUP from an EP (Kadar et al., 1982; Tulandi and Sammour, 2000). Another serum marker is progesterone (McCord et al., 1996; Mol. et al., 1998; Perkins et al., 2000). Its concentrations are higher in women with normal IUP, but its use to distinguish between EP and arrested IUP is also not reliable. Accordingly, investigators are looking for other markers for EP. One of the promising markers is vascular endothelial growth factor (VEGF; Daniel et al., 1999). VEGF is a potent angiogenic factor and angiogenesis is needed for successful reproduction. Its levels in women with polycystic ovarian syndrome are higher than in those with normal ovaries (Tulandi et al., 2000) and it may play a role in the pathophysiology of ovarian hyperstimulation syndrome (Lee et al., 1997). 490
The purpose of our study was to evaluate serum VEGF concentrations in women with normal and arrested IUP, and EP.
Materials and methods From June 1999 until June 2000, all patients attending the McGill Reproductive Center with early diagnosis of pregnancy underwent blood sampling at 5 weeks gestation for possible serum VEGF measurements. These patients were asymptomatic. A total 12 ml of blood was withdrawn; 2 ml of blood for β-HCG and another 10 ml for growth factor determinations. Blood samples for growth factors were collected in siliconized tubes containing a protease inhibitor cocktail. Samples were centrifuged (400 g for 10 min) and the supernatants were stored at –80°C until assayed. The Research and Ethics Board of the Royal Victoria Hospital approved the study. VEGF concentrations were measured collectively from the first 45 samples from women who subsequently were diagnosed to have an EP, a normal IUP and an arrested IUP (15 women in each category). The diagnosis was made by serial transvaginal ultrasound examinations. Following our standard practice, serum β-HCG concentrations were also measured. All blood samples were from singleton pregnancies at 5 weeks gestation. An arrested IUP was defined as an intrauterine sac without fetal tissue or fetal cardiac activity. Serum VEGF was measured in duplicate by commercial ELISA (human VEGF165; RandD system, Minneapolis, MN, USA) specific for the human molecule. The sensitivity of the assay was ⬍5 pg/ml. The inter-assay coefficient of variation for the VEGF assays ranged from 5.0 to 8.8%. © European Society of Human Reproduction and Embryology
VEGF and ectopic pregnancy
Table I. Serum β-HCG and VEGF concentrations in women with ectopic pregnancy, arrested intrauterine pregnancy and normal pregnancy. Values are mean ⫾ SEM Type of pregnancy
β-HCG (mIU/ml)
VEGF (pg/ml)*
Ectopic Arrested intrauterine Normal intrauterine
384.0 ⫾ 51.3 769.9 ⫾ 185.3 1847.4 ⫾ 535.5†
306.1 ⫾ 26.5 169.7 ⫾ 16.6 27.0 ⫾ 4.4
*P ⬍ 0.0001 between the three groups. †P ⬍ 0.01 between normal intrauterine pregnancy and the other two groups.
value of 77.8%. Among women with normal IUP, there was no correlation between serum β-HCG concentrations and VEGF concentrations (r ⫽ –0.16, NS).
Figure 1. Serum VEGF concentrations among women with normal intrauterine pregnancy, arrested pregnancy and ectopic pregnancy. The solid lines represent median concentrations. The broken line represents the serum cut-off level used for the calculation of sensitivity, specificity, and positive predictive value.
The data were analysed using analysis of variance with the Bonferroni post hoc test. P ⬍ 0.05 was considered statistically significant (two-tailed).
Results The mean (⫾SD) age of the women with EP, arrested IUP and normal IUP was 32.0 ⫾ 5.2, 33.2 ⫾ 3.7 and 34.8 ⫾ 4 years respectively. Serum β-HCG concentrations were significantly higher in women with normal IUP than the other two groups, but no difference was found between the concentrations in women with EP and arrested IUP (Table I). The serum concentrations of VEGF among women with EP were significantly higher than in those with normal IUP and arrested IUP. Furthermore, the levels in women with arrested IUP were higher than those seen in women with normal IUP (Figure 1). When cut-off concentrations of 200 pg/ml for VEGF were used, normal IUP could be distinguished from EP with a sensitivity of 88%, a specificity of 100% and a positive predictive value of 100%. Between EPs and arrested IUPs, the sensitivity was 87.5%, specificity 75% and positive predictive
Discussion One of the important aspects of successful implantation is the establishment of angiogenesis and this can be mediated by a number of growth factors. Because the implantation milieu in the Fallopian tube is different from that in the endometrium, the production and secretion of these growth factors could be affected. VEGF is a potent angiogenic factor and its secretion depends on local conditions including hypoxia (Torry and Torry, 1997; Evans et al., 1998). In contrast to HCG and progesterone, which are trophoblast dependent, VEGF is produced by both trophoblast and endometrium (Evans et al., 1998). Daniel et al. first reported that there is a difference in serum VEGF concentrations in women with IUP, women with EP and women with abnormal IUP (Daniel et al., 1999). They postulated that this could be due to increased production of VEGF originating from the hypoxic condition of the tube and/ or from altered production of VEGF binding protein in abnormal pregnancies. Measuring pO2 in utero-placental tissue, Jauniaux et al. found that the tissue concentration of oxygen in early pregnancy is unusually low (Jauniaux et al., 2001). Whether the level is further reduced in ectopic tissue is unknown. In agreement with a previous study (Daniel et al., 1999), it was found that the serum VEGF concentrations in women with EP are higher than in those with normal IUP and arrested IUP. The finding that VEGF concentrations in women with EP are different from those in arrested IUP is important. Unlike progesterone and HCG concentratons, it seems that by using serum VEGF concentrations, it might be possible to distinguish between EP and arrested IUP. All serum concentrations of VEGF in women with normal IUP were ⬍200 pg/ml. With this cut-off point, a normal IUP could be distinguished from an EP with a sensitivity of 88%, a specificity of 100% and a positive predictive value of 100%. Combining serum VEGF and progesterone has been shown to increase the specificity and positive predictive value of the test (Daniel et al., 1999). In our study, we did not measure progesterone concentrations. If our results can be confirmed in a larger study, VEGF may be useful in detecting an early EP. This will allow early and successful medical treatment. Indeed, the most important 491
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factor associated with the failure of medical treatment with methotrexate is a high serum HCG concentration (Tulandi and Sammour, 2000). We conclude that there is a difference in the concentrations of serum VEGF in women with EP, arrested IUP and normal IUP. The potential use of VEGF as a marker for EP should be investigated further in a larger trial.
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