Normality of distributions was tested by means of Kol-mogorov–Smirnov test; the parametric data were analyzed using mean and standart deviation. Since they were not parametric, the median and extreme values of each group were used. The Mann–Whitney U test, chi square and t tests were used for the comparison of the control and preec-lamptic groups. A p value less than 0.05 was considered to be statistically signifi cant.
Results
The clinical characteristic of the study groups is shown in table 1.
There was no signifi cant difference in maternal age, parity and the mode of delivery between groups. There was a signifi cant difference in gestational age at delivery (p <
0.0002). The newborns born to mothers with preeclampsia were delivered preterm and have signifi cant lower birth weight (p < 0.001). In the preeclampsia group, there was one stillbirth at 20 weeks of gestation. Intrauterine growth retardation was diagnosed in fi ve women. Four women had HELLP syndrome.
Median concentrations of fetal DNA of the preeclamptic group was signifi cantly higher than of control group (p <
0.000005). Median concentration of fetal DNA in control patients at 20 to 38 weeks of pregnancy were 42.5 cop-ies per millilitre (range 4.8–81.2 copcop-ies/mL) and median concentration of fetal DNA in preeclamptic women were
326.2 copies per millilitre (range: 133.3–1105.2 copies/mL) – table 2.
There were signifi cant differences in systolic blood pres-sure (p < 0.00001), diastolic blood prespres-sure (p < 0.0001), uric acid (p < 0.0001) and proteinuria (p < 0.001) between preeclamptic and control group.
In order to establish the cut off value, sensitivity, spe-cifi city and likelihood ratio the concentration of fetal DNA in preeclamptic women blood samples the ROC analysis was performed.
As shown in table 3. The positive but statistically in-signifi cant correlation between the values of systolic blood pressure, diastolic blood pressure, uric acid concentration, urine protein and fetal DNA concentration in preeclamptic women blood samples was revealed.
The fetal distress usually originating in intensifi cation of preeclampsia symptoms was correlated with statistically signifi cantly higher concentration of fetal DNA in preec-lamptic women plasma.
The higher but statistically insignifi cant concentration of fetal DNA in preeclamptic women blood with additional fetal hypotrophy, HELLP syndrome or imminent eclampsia symptoms was also noted (tab. 4)
ROC analysis shows that the assessment of fetal DNA in pregnant women blood is reliable and clinically useful factor differentiating patients with and without PE. It is proven by high value of AUC – 0.967 and high level of likelihood ratio (LR) – 12.0.
On the basis of ROC analysis the cut off level of fetal DNA concentration was established to be 81.2 copies/mL and in accordance to it the sensitivity, specifi city and like-lihood ratio for occurrence of additional complications of preeclampsia were estimated (fi g. 1).
As shown in table 5 the estimated fetal DNA concentration in pregnant women blood > 81.2 copies/mL is characterised by high sensitiveness in case of occurrence of additional complica-tions of preeclampsia such as intrauterine growth retardation (IUGR), HELLP syndrome, severe PE or fetal distress.
Low values of likelihood ratio (LR < 2.0) also reveal limited clinical use of marking fetal DNA in pregnant women blood as a factor accompanying mentioned above complications of PE.
Discussion
Detection of fetal DNA is a sensitive method of study-ing fetal cells traffi ckstudy-ing in pregnancy [17]. Quantitative real time PCR allows not only the identifi cation but also the quantifi cation of DNA.
Our study with ROC analysis showed that assessment of fetal DNA in pregnant women plasma is reliable and clinically useful factor differentiating patients with and without PE. It is proven by high value of AUC – 0.967 and high level of likelihood ratio LR – 12.0.
FETAL DNA CONCENTRATION IN MATERNAL CIRCULATION AND SEVERITY OF PREECLAMPSIA 23
T a b l e 1. Clinical patients characteristics T a b e l a 1. Charakterystyka pacjentów
Parameters / Parametry
Group / Grupa preeclampsia p
preeklampsja (n = 16)
control kontrolna
(n = 11) Maternal age (years) / Wiek rozrodczy (lata)
SD
31.0 6.04
30.5
3.55 NS*
Gestational age at delivery (weeks) / Długość ciąży w czasie porodu (tygodnie) Median / Mediana (min.–max.)
34.0 (20–38)
38.0
(37–41) 0.0002**
Primiparas / Pierwszy poród 9 2
NS***
Multiparas / Kolejny poród 12 9
Caesarean section / Cięcie cesarskie 13 3
0.01***
Vaginal delivery / Poród przez pochwę 3 8
Birth weight (g) / Masa urodzeniowa (g) Median / Mediana (min.–max.)
1950.0 (210.0–2650.0)
3250.0
(1300.0–3930.0) 0.001**
* t-Student test / test t-Studenta
** Mann–Whitney U test / test U Manna–Whitneya *** chi2 test / test chi2
T a b l e 2. The number of DNA copies and clinical and biochemical characteristics of preeclampsia in evaluated groups T a b e l a 2. Liczba kopii DNA oraz kliniczna i biochemiczna charakterystyka preeklempsji w ocenianej grupie
Parameters / Parametry
Group / Grupa preeclampsia samples p
próbki grupy z preeklampsją (n = 21)
control samples próbki grupy kontrolnej
(n = 11) DNA (copies/mL) / DNA (kopie/mL)
Median / Mediana (min.–max.)
326.2 (133.3–1105.2)
42.5
(4.8–81.2) 0.000005*
Systolic BP (mmHg) / Skurczowe RR (mmHg) SD
167.75 22.09
114.09
13.38 0.00001*
Diastolic BP (mmHg) / Rozkurczowe RR (mmHg) SD
108.7 7.58
69.09
10.2 0.0001*
0.001***
Proteinuria / Białkomocz + 14 0
- 7 11
Uric acid (mg/dL) / Kwas moczowy (mg/dL) Median / Mediana (min.–max.)
6.0 4.5–9.7
4.1
2.8–5.8 0.0001**
* t-Student test / test t-Studenta
** Mann–Whitney U test / test U Manna–Whitneya *** chi2 test / test chi2
T a b l e 3. The correlation of systolic blood pressure, diastolic blood pressure, uric acid concentration, protein concentration in urine and
fetal DNA concentration in pregnant women blood samples in the examined group
T a b e l a 3. Zależność skurczowego ciśnienia krwi, rozkurczowego ciśnienia krwi, stężenia kwasu moczowego, stężenia białka w moczu
i stężenia DNA płodowego w próbkach krwi ciężarnych kobiet w badanej grupie
Parameters / Parametry r p
Systolic blood pressure
Skurczowe ciśnienie krwi 0.12 NS
Diastolic blood pressure
Rozkurczowe ciśnienie krwi 0.54 NS
Uric acid
Kwas moczowy 0.15 NS
Urine protein
Białko w moczu 0.54 NS
Fig. 1. ROC analysis of pregnant women DNA concentration with or without preeclampsia
Ryc. 1. Analiza ROC stężenia DNA ciężarnych kobiet z lub bez preeklampsji
24 KARINA ENGEL, TOMASZ PŁONKA, MAREK BILAR ET AL.
T a b l e 4. The number of DNA copies (median, min. and max.) and the presence of fetal distress symptoms and coexisting pregnancy complications in preeclamptic group (examined group)
T a b e l a 4. Liczba kopii DNA (mediana, min., maks.) i obecność symptomów stanu zagrożenia płodu i współistniejących powikłań ciąży w badanej grupie
Parameters / Parametry Mean / Średnia Minimum Maksimum / Maximum p
Fetal distress Stan zagrożenia płodu
+ 503.00 297.60 1105.20 0.004
- 272.15 133.30 1105.20
IUGR +- 321.10418.65 147.10133.30 1105.20761.10 NS
HELLP +- 323.65473.00 133.30147.10 1105.20768.40 NS
Imminent eclampsia Zagrażająca rzucawka
+ 335.60 133.30 768.40
- 256.75 203.70 1105.20 NS
IUGR – intrauterine growth retardation / wewnątrzmaciczne ograniczenie wzrastania płodu + present / obecny
- absent / nieobecny
T a b l e 5. Sensitivity, specifi city and likelihood ratio for estimating the concentration of DNA in additional complications of preeclampsia T a b e l a 5. Stosunek czułości, swoistości i prawdopodobieństwa dla określenia stężenia DNA w dodatkowych powikłaniach preeklampsji
Parameters / Parametry
DNA concentration cut off / Granica stężenia DNA
> 81.2 copies/mL / kopie/mL sensitivity % / czułość
(95% CI)
specifi city % / swoistość
(95% CI) LR
Fetal distress / Stan zagrożenia płodu 100 (69.0–100)
50.0
(28.2–71.8) 2.00
IUGR 100
(54.1–100)
42.3
(23.4–63.1) 1.73
HELLP 100
(40.2–100)
39.3
(21.5–59.4) 1.65
Eclampsia imminent / Zagrażająca rzucawka 100 (58.9–100)
44.0
(24.4–44.1) 1.79
LR – likeihood ratio / wskaźnik wiarygodności
IUGR – intrauterine growth retardation / wewnątrzmaciczne ograniczenie wzrastania płodu
Many studies have demonstrated that there is elevated level of circulating fetal DNA in plasma obtained from women with symptomatic preeclampsia. Levine et al. de-scribed a two-stage elevation of fetal DNA in pregnancies with preeclampsia. They suggested that fi rst stage at 17 to 28 weeks may by due to placental necrosis and apoptosis and the second subsequent elevations may refl ect impaired DNA elimination [13].
Lo et al. found that the median circulating plasma fetal DNA was fi ve-fold increased in 20 women with preeclamp-sia compared to controls [6]. In our presented study, the median concentrations of fetal DNA obtained from women with preeclampsia was also signifi cantly higher then in the control group with uncomplicated pregnancies.
Extracellulary fetal DNA is also increased prior to the onset of preeclampsia. Increased levels of fetal DNA have been reported in the fi rst and second trimesters before the development of the clinical symptoms of preeclampsia [18].
Zhong et al. demonstrated that not only maternal and fetal free DNA was elevated in preeclamptic woman, but they also found that increments in free DNA levels cor-responded to the degree of disease severity [14].
Swinkels et al. in a small study described that HELLP syndrome as a complication of preeclampsia in pregnant
women increases the amount of cell-free fetal and maternal DNA in maternal plasma and serum [19].
In another small study Smid et al. [20] reported that fetal DNA concentration increased in the plasma of 9 pregnant women carrying a growth restricted fetuses but Sekizawa et al. demonstrated that there was no increase of fetal DNA in the plasma of pregnant women with fetal growth restriction [3]. There was no difference in fetal DNA concentration between cases with and without IUGR.
In our study fetal distress, usually because of intensi-fi cation of symptoms of preeclampsia, was associated with statistically signifi cantly higher fetal DNA concentration in preeclamptic women plasma. We also revealed higher, but statistically insignifi cant, concentration of fetal DNA in blood of preeclamptic women who also presented fetal hypotrophy, HELLP syndrome or imminent eclampsia.
On the basis of presented analysis we confi rmed that fetal DNA concentration in pregnant women blood > 81.2 cop-ies/mL is characterised by high sensitiveness in case of complications of pregnancy accompanying preeclampsia such as IUGR, HELLP syndrome, severe PE or fetal dis-tress. However low values of likelihood ratio (LR < 2.0) prove limited clinical use of marking fetal DNA in pregnant women blood as a factor accompanying mentioned above complications of PE.
FETAL DNA CONCENTRATION IN MATERNAL CIRCULATION AND SEVERITY OF PREECLAMPSIA 25
Conclusions
1. Signifi cant increase of fetal DNA concentration in preeclamptic women plasma is associated with severe course of preeclampsia and especially with fetal distress symptoms.
2. The evaluation of number of fetal DNA copies in pregnant women plasma is characterized by high sensitivity but low specifi city of this test in regards to preeclampsia with complications such as HELLP syndrome and intrauterine fetal growth retardation (IUGR).
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A N N A L E S A C A D E M I A E M E D I C A E S T E T I N E N S I S
R O C Z N I K I P O M O R S K I E J A K A D E M I I M E D Y C Z N E J W S Z C Z E C I N I E 2007, 53, 3, 26–33
ALEKSANDRA ZAREK