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Presence of cell-free fetal DNA in plasma of women with ectopic pregnancies.

Ectopic pregnancy (EP) is a major cause of maternal morbidity and mortality, accounting for ~6% to 13% of first-trimester pregnancy-related deaths (1-3). The availability of highly sensitive assays for serum (3-human chorionic gonadotropin (hCG) and an increase in the sensitivity of transvaginal sonography have dramatically improved the diagnosis of EP (4). In clinical practice, the usefulness of a single [beta]-hCG measurement to confirm the absence of an EP has been questioned (5), and the measurement of serial [beta]-hCG (5, 6) and progesterone (7) concentrations has been proposed. However, measuring serial [beta]-hCG concentrations is not a practical method when the patient presents for an emergency evaluation.

New markers for preoperative detection of EP could decrease the time to diagnosis and reduce the possibility of tubal rupture. To find an early single marker for EP, several studies have measured placental markers (pregnancy-associated plasma protein A, serum-specific protein-1, human placental lactogen, and leukemia-inhibiting factor) and nonplacental proteins (glycodelin and vascular endothelial growth factor) and compared results for women with an EP with those for women with an intrauterine pregnancy (IUP) (8-10).

The first reports regarding the presence of cell-free fetal DNA in maternal plasma (11) were followed by several studies focusing on the detection and possible consequences of this cell-free DNA in other body fluids, such as urine, cerebrospinal fluid, and peritoneal fluid (12-14). Other studies focused on the analysis of cell-free DNA quantity and quality in different fetal disorders and pathologic pregnancies (15-17). The aim of our study was to measure cell-free fetal DNA in plasma from EP patients and to compare the results with IUP cases and investigate a possible relationship between measured 0-hCG and cell-free DNA concentrations.

A total of 58 pregnant women (5-11 weeks of amenorrhea, based on menstrual dating, and a positive urine pregnancy test) with pelvic pain and pelvic sonography suggestive of possible EP, were enrolled in the study at the 1st Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary. Fourteen of these women had normal IUPs, 10 had spontaneous abortions, and 36 had EPs. The EP patients were treated with laparoscopic surgery, and salpingectomy was performed in all of these cases. Another 45 women with confirmed IUPs, who had been referred to the department for termination of pregnancy, were also enrolled. All patients gave informed consent for participation in the study.

We collected blood samples for [beta]-hCG determinations and DNA isolation. All of the women subsequently underwent pelvic sonography. The diagnosis of EP was always confirmed by histologic examination. Blood was collected into sterile EDTA tubes, and plasma was rapidly separated by centrifugation at 8408. Plasma was immediately stored at -80[degrees]C until isolation. To extract DNA from 200 [micro]L of plasma, we used the silica adsorption method with a High Pure PCR Template Preparation Kit (Roche GmbH) according to the manufacturer's instructions. DNA was eluted with 100 [micro]L of elution buffer. To determine the presence of cell-free fetal DNA, quantitative real-time PCR was performed on all samples, with sex-determining region Y (SRY) gene-specific primers (forward, 5'-GGC AAC GTC CAG GAT AGA GTG A-3'; reverse, 5'-TGC TGA TCT CTG AGT TTC GCA TT-3'; Roche GmbH). Each PCR reaction consisted of 1 [micro]L of DNA Master SYBR Green I mixture (LightCycler FastStart DNA Master SYBR Green I, containing Taq polymerase, deoxynucleotide triphosphates, Mg[Cl.sub.2], and SYBR Green I dye; Roche), 1 [micro]L of DNA, and 2.5 pmol of primers. The amplification program included an initial denaturation at 95[degrees]C with an 8-min hold, followed by 45 cycles with denaturation at 95[degrees]C with a 10-s hold, annealing at 58-65[degrees]C with a 5-s hold, and extension at 72[degrees]C with a 10-s hold. One positive control containing male DNA and 2 negative controls, one without DNA template and the other with a DNA sample obtained from a nulligravid woman, were run simultaneously with every assay. Calibration curves were obtained by serial dilution of the [beta]-globin gene (DNA Control; Roche) according to the supplier's instructions. Using LightCycler software (Roche), we calculated the concentration of each gene product with the kinetic approach. Immunoassays were used to measure serum [beta]-hCG concentrations. The detection limit was 1 [micro]g/L, and the within- and between-run CVs were 7.3% and 10%, respectively. We used SPSS 11.0 for Windows software (SPSS Inc.) for statistical analysis of the data.

We found 15 SRY-positive cases in the EP group and 14 in the IUP group. The SRY-negative cases were considered to be female fetuses and were excluded from further analysis. The mean gestational age, 51 days in the SRY-positive EP group and 59 days in the SRY-positive IUP group, did not differ significantly between the 2 groups. According to an independent sample t-test, however, the mean (SD) plasma concentrations of cell-free DNA differed significantly between the 2 groups [565 (136) genome-equivalents (GE)/mL in the EP group and 72 (19) GE/mL in the IUP group (P <0.005)]. The serum concentration of cell-free DNA was significantly higher (P <0.001) in women with EP than in those with IUP (Fig. 1). The serum concentrations of [beta]-hCG also differed significantly between the EP and IUP groups [4780 (1760) IU/L vs 132 000 (11500) IU/L, respectively (P <0.0001)]. As expected, the serum concentrations of [beta]-hCG were significantly higher (P <0.0001) in women with IUP than in those with EP.

With a serum SRY concentration >80 GE/mL, 2 SDs above the mean, as a cutoff concentration to distinguish an EP from an IUP, the positive predictive value was 78% and the negative predictive value was 83%; the sensitivity of the method was 84%, and the specificity was 76%.

When we applied the Pearson correlation test, we found no significant (P = 0.05) correlation between plasma SRY and serum [beta]-hCG concentrations. The mean (SD) values for the SRY:[beta]-hCG ratio were 2.65 (4.81) and 0.002 (0.003), respectively, in EP and IUP cases.

[FIGURE 1 OMITTED]

Transvaginal sonography in combination with serum [beta]-hCG concentration measurement is a commonly used method for the clinical diagnosis of EP. Most meta-analyses have shown that a single [beta]-hCG concentration measurement is not adequate to differentiate between EP and IUP. In the absence of a single noninvasive test for the detection of EP, diagnosis requires the exclusion of IUP.

The amounts of placental-specific proteins and nonplacental markers in peripheral circulation differ in EP and IUP. At the same time, serum progesterone assays have limited value for differentiating between EP and spontaneous abortion (19). Fetal fibronectin concentrations are lower in women with an EP than in women with an IUP, but this marker is not sensitive or specific enough for the diagnosis of EP (18). Although serum leukemia-inhibiting factor concentrations are lower in EP than in IUP, their diagnostic value is limited. Increased vascular endothelial growth factor concentrations had a sensitivity of 60%, a specificity of 90%, and a positive predictive value of 86% for EP (8-10). The presence of cell-free fetal DNA in the peripheral circulation of pregnant women raises many questions and opens new horizons for noninvasive prenatal diagnosis and immunologic research. The quantity of cell-free DNA increases during pregnancy and decreases rapidly after birth (20). The results of several studies regarding the origin of cell-free DNA have confirmed its placental trophoblast origin (21). The amount of cell-free DNA measured in pathologic pregnancies, such as preeclampsia, is higher than in uncomplicated pregnancies (17). Taking into account that the trophoblast is the source of cell-free DNA, the possible explanation of this phenomenon seems to be the difference in placentation between uncomplicated and pathologic pregnancies. In EP, the development of trophoblastic structures and the invasion of vessels differ from those in IUP and may lead to differences in the amounts of cell-free DNA in maternal plasma.

We cannot draw firm conclusions because the relatively small group sizes in our study may have increased variance around the test characteristic estimate, and there may have been a selection bias related to the severity of symptoms, sonographic findings, pathologic diagnoses, and the use of a sex-dependent marker. Nevertheless, these findings warrant further prospective studies of the association between cell-free fetal DNA concentrations and EP. These results confirm that plasma concentrations of cell-free fetal DNA are significantly higher in women with an EP than in women with an IUP. They also suggest that plasma cell-free DNA measurement may have diagnostic value in EP.

References

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(6.) Kadar N. Serial 0-hCG measurements in the early detection of ectopic pregnancy. Obstet Gynecol 1990;76:475-7.

(7.) McCord ML, Muram D, Buster JE, Arheart KL, Stovall TG, Carson SA. Single serum progesterone as a screen for ectopic pregnancy: exchanging specificity and sensitivity to obtain optimal test performance. Fertil Steril 1996; 66:513-6.

(8.) Daponte A, Pournaras S, Zintzaras E, Kallitsaris A, Lialios G, Maniatis AN, et al. The value of a single combined measurement of VEGF, glycodelin, progesterone, PAPP-A, HPL, and LIF for differentiating between ectopic and abnormal intrauterine pregnancy. Hum Reprod 2005;20:3163-6.

(9.) Wegner NT, Mershon JL. Evaluation of leukemia inhibitory factor as a marker of ectopic pregnancy. Am J Obstet Gynecol 2001;184:1074-6.

(10.) Daniel Y, Geva E, Lerner-Geva L, Eshed-Englender T, Gamzu R, Lessing JB, et al. Levels of vascular endothelial growth factor are elevated in patients with ectopic pregnancy: is this a novel marker? Fertil Steril 1999;72: 1013-7.

(11.) Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350: 485-7.

(12.) Zhong XY, Hahn D, Troeger C, Klemm A, Stein G, Thomson P, et al. Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis? Ann N Y Acad Sci 2001;945:250-7.

(13.) Tong YK, Lo YM. Diagnostic developments involving cell-free (circulating) nucleic acids. Clin Chim Acta 2006;363:187-96.

(14.) Angert RM, Leshane ES, Yarnell RW, Johnson KL, Bianchi DW. Cell-free fetal DNA in the cerebrospinal fluid of women during the peripartum period. Am J Obstet Gynecol 2004;190:1087-90.

(15.) Spencer K, de Kok JB, Swinkels DW. Increased total cell-free DNA in the serum of pregnant women carrying a fetus affected by trisomy 21. Prenat Diagn 2003;23:580-3.

(16.) Sekizawa A, Jimbo M, Saito H, Iwasaki M, Matsuoka R, Okai T, et al. Cell-free fetal DNA in the plasma of pregnant women with severe fetal growth restriction. Am J Obstet Gynecol 2003;188:480-4.

(17.) Hahn S, Holzgreve. W Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 2002;8:501-8.

(18.) Nowacek GE, Meyer WR, McMahon MJ, Thorp JR, Wells SR. Diagnostic value of cervical fetal fibronectin in detecting extrauterine pregnancy. Fertil Steril 1999;72:302-4.

(19.) Dart R, Ramanujam P, Dart L. Progesterone as a predictor of ectopic pregnancy when the ultrasound is indeterminate. Am J Emerg Med 2002; 20:575-9.

(20.) Ariga H, Ohto H, Busch MP, Imamura S, Watson R, Reed W, et al. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion 2001;41:1524-30.

(21.) Bianchi DW. Circulating fetal DNA: its origin and diagnostic potential. Placenta 2004;25:93-101. Previously published online at DOI: 10.1373/clinchem.2006.067587

Levente Lazar,* Balint Nagy, Zoltan Ban, Gyula R Nagy, and Zoltan Papp (I. Department of Obstetrics and Gynecology, Semmelweis University, 27 Baross Street, Budapest, Hungary; * author for correspondence: fax 36-1-317-6174, e-mail lazar_lvente@hotmail.com)
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Title Annotation:Technical Briefs
Author:Lazar, Levente; Nagy, Balint; Ban, Zoltan; Nagy, Gyula R.; Papp, Zoltan
Publication:Clinical Chemistry
Date:Aug 1, 2006
Words:2033
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