Introduction
Preeclampsia affects ~5–8% of all pregnancies and it is the leading cause of maternal and fetal morbidity and mortality in the developing countries.Reference Vlkova, Szemes and Minarik1 It is usually closely linked with intrauterine growth restriction, abnormal fetal oxygenation and preterm delivery. Hypertension, proteinuria and edema are the leading clinical symptoms of pregnant women suffering from preeclampsia. The etiology is unknown, but it is hypothesized that particles of abnormal invasive fetal throphoblasts are responsible for the onset of this disease.Reference Kestlerova, Feyereisl and Frisova2 It is assumed that the feto-maternal interface is poorly regulated in the early stage of pregnancy and causes lean placentation which leads to the symptoms.
In 1997 Lo et al. showed that cell free fetal DNA is present in the plasma and serum of pregnant women.Reference Lo, Corbetta and Chamberlain3 Since then this discovery has opened new possibilities for noninvasive prenatal diagnosis.Reference Lo and Chiu4 The analysis of the fetal DNA is also used in a connection with the research of preeclampsia.Reference Scharfe-Nugent, Corr and Carpenter5 It is not clear if the fetal trophoblasts together with other co-factors are the starting cause of other pathologic mechanisms in preeclampsia. However, it is known that during preeclampsia, the cells of trophoblasts disintegrate more than during normotensive pregnancy.
In numerous studies it was shown that fetal DNA is higher in preeclamptic patients. The fetal nucleic acids are released into circulation in the form of nucleosome bodies, which represent a mixture of DNA and proteins and are recognized as foreign by the immune system of the mother. Therefore, toll-like pathogen recognition receptors might mediate immune reactions, which could result in spontaneous preterm birth.Reference Scharfe-Nugent, Corr and Carpenter5
Various animal models of preeclampsia are used for the research of its pathogenesis. The most common models use mice or rats. Preeclampsia is induced by nitric oxide inhibition, lipopolysaccharide administration or manipulation of uterine circulation.Reference Yan, Cui and Huang6, Reference Yan, Liu and Cui7 It was even proved that some symptoms of preeclampsia can be induced by administration of fetal DNA which triggers an inflammatory reaction.Reference Yan, Cui and Huang6 In that experiment human fetal DNA was used for pregnant mice. It is unclear whether this has any relevance to the observed results. The aim of the present study was to prove whether injected rat fetal DNA induces preeclampsia-like symptoms in pregnant rats.
Materials and methods
Animals
Fourteen female Wistar rats (Charles River, Prague, Czech Republic) were housed with free access to food, consisting of a standard laboratory diet and tap water. They were maintained on a constant 12-h light-dark cycle at the room temperature 21±2°C. The female rats were caged overnight with fertile males for mating. The day 0 of pregnancy was defined as the day when sperms were found in a vaginal smear. All procedures were approved by the Ethics Committee of Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University.
Materials
Fetal DNA was isolated using a commercial kit (Puregene Blood Core Kit, Qiagen, Hilden, Germany) from a rat fetus. A solution of xylazine (Ecuphar N.V., Oostkamp, Belgium) and ketamine (Richterpharma, Wels, Austria) was used for anesthesia. The commercial kits QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), RNeasy Mini Kit (Qiagen, Hilden, Germany) and TRIzol (Sigma Aldrich Chemie GmbH, Steinheim, Germany) were used for DNA and RNA isolation.
Experimental protocol
The rats were randomly assigned to one of two treatment groups – fetal DNA (n=7) and control (n=7) on gestation day 14. The rats in the fetal DNA group were injected i.p. daily with 400 μg of purified rat fetal DNA dissolved in millipore water starting on the day 14 until the day 18 of pregnancy. Control rats received an equivalent volume of millipore water. From the day 14 to 18 animals were also weighed. Animals were put in metabolic cages on the 18th day of pregnancy for 24 h urine collection. They were anesthetized with an i.p. injected combination of xylazine (10 mg/kg body weight) and ketamine (100 mg/kg body weight). A sample of aortic blood was collected into collection tubes containing ethylenediaminetetraacetic acid. Plasma was separated from whole blood samples by centrifugation at 2000 g for 5 min and stored at −20°C for further analysis. Kidneys, placentas and fetuses were removed and weighed. One random sample of placenta from each animal was collected, immediately shock-frozen in liquid nitrogen as well as a sample of the cortex of left kidney and then stored at −80°C for further assessment. Urine samples were collected in metabolic cages during 24 h and stored at −20°C for further analysis.
Noninvasive blood pressure measurement
Systolic blood pressure was measured noninvasively in conscious animals on day 19 of pregnancy. Animals were pre-warmed in a heating chamber maintained at 35°C for 5–20 min. The systolic blood pressure was measured by a pneumatic tail cuff device and LabChart6 (ADInstruments GmbH, Spechbach, Germany). Measurement was performed three to five times for each rat, the mean value was used for the analysis.
Biochemical analysis
Proteinuria
Proteinuria was measured with a pyrogallol red-molybdate complex according to the method modified by Watanabe N. et al. in urine samples.Reference Watanabe, Kamei and Ohkubo8 A microplate-based protocol was used. Absorbances were read at 600 nm and the blank was subtracted from all values. Concentrations were subsequently calculated according to a standard curve.
Creatinine
Creatinine was measured by the method according to Jaffé, both, in urine and in plasma. Frozen samples of plasma and urine samples were thawed and centrifuged to pellet particulate matter. Each plate was run using appropriate standards and blanks. Plates were read at 495 nm using the software Kim Imunochemical Processing (Daniel Kittrich Software Production, Praha, Czech Republic). Exported values were calculated according to a standard curve. Creatinine clearance was calculated using both measured creatinine concentrations and diuresis.
DNase activity
DNase activity was measured in plasma samples using a commercial ELISA kit (Orgentec Diagnostika GmbH, Mainz, Germany). After all the procedures according to kit, the intensity of the yellow color is measured photometrically at 450 nm. The amount of the color is inversely proportional to the DNase activity.
Statistical analysis
Statistical analysis was performed using unpaired Student t-test. Data were analyzed with GraphPad software (version 6.0a for Mac, Graphpad Software Inc., La Jolla, California, USA). Statistical significance (P) was defined as a probability value of <0.05. Data are presented as mean±standard deviation (s.d.).
Results
Pregnancy outcome
The litter size and weights of fetuses were not different between control and fetal DNA group (11.5±0.7 v. 12.3±1.128; P>0.05; 2.1±0.3 v. 2.3±0.5; P=ns) respectively (Fig. 1). The mean diameters of placentas were also comparable (1.3±0.02 cm for the control group v.1.3±0.03 cm for the fetal DNA group; P=ns). No significant difference was found between the groups in placental weight (0.4±0.03 g for the control group v. 0.4±0.03 g for the Fetal DNA group; P=ns) (Fig. 1).
The litter size was similar in the control group and fetal DNA group (a). There was no significant difference in fetal weight between groups (b). Diameter of each placenta was comparable between the groups (c). No significant difference was found between groups in placental weight (d).
Blood pressure
There was no significant difference in the systolic blood pressure between the control group and the fetal DNA group on the day 19 of pregnancy (113.8±3.2 v. 109.1±4.5 mmHg; P=ns) (Fig. 2).
Blood pressure was measured on the day 19 of pregnancy. control group did not differ from fetal DNA group (a). Proteinuria was comparable in control and fetal DNA groups (b). The albumin/creatinine ratio did not differ between the groups (c). DNase activity was measured in plasma. There was no difference between control group and fetal DNA group (d).
Biochemical outcomes
Proteinuria of pregnant rats was comparable between the control and fetal DNA group (4.7±0.7 v. 3.6±1.03 mg/24hod; P=ns). Albumin to creatinine ratio in the urine as another measure of proteinuria was similar in both analyzed groups (0.04±0.006 v. 0.04±0.01 mg/mmol; P=ns) (Fig. 2). Similarly, no correlation was found for creatinine clearance. DNase activity was similar in control pregnant rats and pregnant rats injected with rat fetal DNA (15,351±114.8 v. 15,009±509.7 uKuU/ml; P=ns) (Fig. 2).
Creatinine was also measured in plasma using commercial Arbor kit. However there was found no difference between groups (2054±347.4 v. 2234±351 mmol/l; P=ns) (Fig. 2).
Discussion
Fetal DNA in maternal plasma is higher in preeclampsia than in healthy pregnant women.Reference Lo, Corbetta and Chamberlain3 It is, however, not clear whether it is one of the inducing factors, which initiates preeclampsia, or whether it increases due to preeclampsia. Other diseases, such as systemic lupus erythematosus (SLE), increases the percentage of the risk of preeclampsia complication from 8% up to 30%.Reference Malatyalioglu, Kurtoglu and Kokcu9 Increased lupus activity during pregnancy may result in elevated risk for poor pregnancy outcome, such as preterm birth, low birth weight or pregnancy loss. Complications are usually associated with infection within the uterus. Placenta studies, however, do not show spread rates for infection during SLE. Several experimental markers for preeclampsia, including soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PIGF) have been found to correspond to preeclampsia in lupus patients as they do in women with SLE.Reference Malatyalioglu, Kurtoglu and Kokcu9 Given the observational and associative character of the currently known facts about preeclampsia, the only way, how to prove these possibilities is an interventional experiment.
The main finding of this study is that injected rat fetal DNA does not affect the rat gestation. Neither the blood pressure, nor renal functions of the pregnant rat, nor the fetuses and placentas were affected by rat fetal DNA injections during the last third of gestation. This is in contrast to the previously published results showing that fetal DNA might be a trigger of preeclampsia.Reference Scharfe-Nugent, Corr and Carpenter5 There are, however, several important differences between these two experiments. In the present study rat fetal DNA was applied to pregnant rats, while in the published experiment human fetal DNA was injected to pregnant mice. Even more important, the Irish group did not measure systolic blood pressure or proteinuria, the major hallmarks of preeclampsia. Correspondingly there was a review recently written by Martin et al.,Reference Martin, Krishna and Martina10 who considered several publications focused on preeclamptic human patients. Their research approved again that it is still not well-known if the fetal DNA starts off the preeclampsia pathogenesis. Some of the publication did not even found the increased levels of fetal DNA among pregnant women who paradoxically demonstrated other symptoms of preeclampsia. The reason for this may have been the different week of gestation of women whose fetal DNA was measured or the the different body mass index of patients.Reference Martin, Krishna and Martina10
Another option to model preeclampsia is to apply the nitric oxide synthase inhibitor L-NAME.Reference Yan, Cui and Huang6 It increases blood pressure and proteinuria in the pregnant animals. In a preliminary experiment we were able to reproduce the effects of L-NAME. Thus, the methods used are sensitive, but were not able to prove the effects of fetal DNA. One of the limitation of the present study is the lack of inflammation assessment. Neither proinflammatory cytokines, nor molecular intracellular markers of immune system activation were analyzed. On the other hand, the chosen parameters measured are more closely related to preeclampsia.
An animal experiment completed by McDonnold et al.Reference Martin, Krishna and Martina10 made an experiment in which mice were injected either with adenovirus carrying fms-like tyrosine kinase 1 or the murine immunoglobulin G2α Fc fragment. The injection of fms-like tyrosine kinase 1 induced preeclampsia and proved that preeclampsia alters weight of the fetus and postnatal growth.Reference McDonnold, Tamayo and Kechichian11 Results of Lin et al.Reference Lin, Liu and Shan12 showed that the activation of toll-like receptor 9 could be triggered by cytosine-guanine (CpG)-DNA motifs, a synthetic mimic of bacterial DNA, and induced a strong immune response resulting in embryo loss or preterm birth, as it happens during preeclampsia.Reference Lin, Liu and Shan12 Neither of these two groups measured fetal DNA level.
One potential explanation for the lack of effects in this experiment is the fact that rat fetal DNA was isolated and purified from proteins. This DNA is prone to be quickly degraded by DNases. A difference in DNase activity was not found between groups, but this does not mean that injected fetal DNA was not degraded. Fetal DNA in pregnant women is usually bound to protective proteins such as histones. Fetal DNA circulating in maternal plasma bound to proteins is very likely protected from the effect of DNases and might, thus, induce inflammatory processes.Reference Orozco, Jorgez and Horne13 Further experiments are needed to prove this hypothesis, especially experiments with modified DNA protected from DNases.
In conclusion, according to our knowledge, this is the first study analyzing the effects of rat fetal DNA on rat pregnancy. No effects could be observed and, thus, further follow-up studies should be conducted to prove whether fetal DNA has any role in the etiology or pathogenesis of preeclampsia.
Acknowledgments
None.
Financial Support
The authors alone are responsible for the content and writing of the paper. This work was supported by the Slovak research and development agency through contract APVV-0754-10.
Conflicts of Interest
None.
Ethical Standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guides on the care and use of laboratory animals (Regulation of Government of Slovakia, no. 377/2012) and has been approved by the institutional committee (Ethical Committee, Department of Pathological Physiology, Faculty of Medicine, Comenius Univesity).
