INTRODUCTION
A life threatening disorder during pregnancy and postpartum period is preeclampsia. It is a triad of oedema, hypertension and proteinuria occurring primarily after the 20th gestational week and most frequently near term (Marbie et al., 1994). Intrauterine growth retardation (IUGR), pre-term delivery, low birth weight, fetal death and neo-natal death due to complications of pre-term delivery are common perinatal outcomes associated with preeclampsia (Ware-Jauregui et al. 1999). Preeclampsia affects between 0.4% and 2.8% of all pregnancies in developed countries and many more in developing countries, leading to as many as 8370000 cases worldwide per year (Villar et al., 2003). In developing nations, the incidence of the disease is reported to be 4-18%, (Villar 2006).
Oxidative stress of the placenta is considered to be a key intermediary step in the pathogenesis of preeclampsia. Patients with preeclampsia have been shown to be under increased oxidative stress (Hosen et al., 2015; Harma et al., 2005) and oxygen free radicals created by such stress induce several types of DNA damage (Mert et al., 2012). Several other studies also suggested that Reactive Oxygen Species (ROS) attack the integrity of DNA in the nucleus and causes nucleotide modifications, DNA strand breaks and chromatin cross-linking (Sakkas et al., 1999; Said et al., 2005). Though the association of oxidative stress is well documented, the DNA damage rate in preeclampsia is poorly understood. Several methods have been employed to monitor genetic damage to mononuclear leukocytes as an indicator of general genetic damages. These include the micronucleus (MN) test, analysis of chromosomal aberrations, the sister-chromatid exchange (SCE) test, gene mutation tests, and the comet assay (Deng et al., 2005). Of these tests, the comet assay (single-cell gel electrophoresis) is a well-established genotoxicity test that is simple, rapid and sensitive; the test has been used to assess the extent of endogenous DNA damage (Deng et al., 2005; Kocyigit et al., 2005).
Preeclampsia is also a common problem in Bangladesh. Though we have showed the association of oxidative stress (Howlader et al., 2007), and maternal hypothyroidism (Hosen et al., 2014) with preeclampsia in our previous studies, the association of DNA damage with preeclampsia is not clear. So far in my knowledge, there are no other studies conducted to explain the rate of DNA damage in Bangladeshi PE patients. We hypothesized that patients with preeclampsia may also suffer from other complications after delivery because of DNA damage. If untreated various complications may develop in patients. The objective of this study was to measure the DNA damage in PE patients.
MATERIALS AND METHODS
Study Subjects
The study was conducted on 52 subjects (27 preeclamptic pregnant women and 25 healthy uncomplicated pregnant women) matched by age. Preeclamptic pregnant women were recruited from Dhaka Medical College Hospital and uncomplicated pregnant women were recruited from Azimpur Maternity Hospital, Dhaka, Bangladesh.
Subjects were selected based on following criteria:
- Systolic blood pressure greater than 140 mmHg or a raise of at least 30 mmHg.
- Diastolic blood pressure greater than 90 mmHg or a raise of at least 15 mmHg.
- Proteinurea of 300 mg in a 24 hours urine collection.
- Antepartum and postpartum Preeclampsia.
Subjects with uncomplicated pregnancies were normotensive throughout gestation and had no proteinurea.
Sample Collection
About 5.0 mL of peripheral blood was drawn from each individual with the help of an expert. Then the blood was transferred to a sterile EDTA containing glass tube without any disturbance. Blood samples were kept in an ice chamber following collection and during transportation to the laboratory. The whole blood was kept at -20° C until analysis.
Determination of DNA Damage Using the Alkaline Comet Assay
The comet assay, also known as the single-cell gel electrophoresis (SCGE) assay, was performed as described by Singh et al. (1988) and Deng et al. (2005) with the following modifications: 10 mL amounts of fresh mononuclear leukocyte cell suspensions (roughly 20,000 cells) were mixed with 20 mL of 0.7% low melting-point agarose in PBS at 37° C. Next, 20 mL of each mixture was layered onto a slide precoated with a thin layer of 1% normal melting-point agarose (NMA) and immediately covered with a cover slip. Slides were held for 20 min at 4° C to allow the agarose to solidify. After removal of cover slips the slides were layered again with 20 mL of 0.7% low melting-point agarose in PBS. Slides were held for 10 min at 4° C to allow the agarose to solidify. After removal of cover slips the slides were immersed in freshly prepared cold (4° C) lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% Triton X-100, and 10% DMSO [added just before use]; pH 10–10.5) for at least 1 h. Slides were next immersed in freshly prepared alkaline electrophoresis buffer (0.3 M NaOH and 1 mM Na2EDTA; pH > 13) at 4° C to allow DNA to unwind (40 min) and then electrophoresed (25 V/300 mA, 25 min). All manipulations were performed under minimal illumination. After electrophoresis, the slides were neutralized (0.4 M Tris–HCl; pH 7.5) for 5 min.
After neutralization, modified version of silver staining was used as described by Nadin et al. (2001). The slides were fixed for 10 minutes in fixative solution A (15% TCA, 5% ZnSO4.7H2O and 5% Glycerol) and following washing for 5 minutes (twice), slides were dried. Slides were re-hydrated for 5 minutes in deionized water and submerged in a horizontal staining jar containing freshly prepared staining solution (5% Na2CO3, 0.1% NH4NO3, 0.1% AgNO3, 0.25% tungstosilicic acid and 0.15% formaldehyde). Staining was carried out for 30 minutes in dark and gentle shaking condition. Slides were brought out from the staining jar and rinsed the slides thrice (each of 5 minutes) with deionized water. Then slides were immersed in a stopping solution E (1% glacial acetic acid) for 5 minutes and air-dried.
The slides were examined under conventional light microscope [(Motic-BA200)(400× magnification)] equipped with CCD camera (Nikon Cool Pix 99F). Hundred cells per slide were randomly examined. The comet images were captured by the camera and then transferred to computer for analysis. For comparing the extent of DNA damage in different samples, comet images were analyzed using a software named Computer Assay Software Project (CASP, version 1.2.2), developed by Konca et al. (2003). The DNA damage rate was expressed as the percentage (%) of head and tail DNA. The higher percentage of head DNA represented the less DNA damage while the higher percentage of tail DNA represented higher DNA damage.
STATISTICAL ANALYSIS
All the results were expressed as mean ± SEM. The statistical analysis of the data was carried out with Statistical Package of Social Science (SPSS), version 17 and Graph Pad Prism version-5. The comparisons between two groups were tested by unpaired t-test. A 95% confidence interval was used. p values less than 0.05 were considered as statistically significant.
RESULTS
Statistically significant differences among complicated and uncomplicated pregnancy are indicated in Table 1 to 2 and in Fig. 1 and 2 along with their significant values.
Clinical and Laboratory Data
These are shown in Table 1 and in Fig. 1. The maternal age of study subjects was not significantly different. On the other hand, the gestational age was significantly decreased in preeclampsia as compared with normal pregnancy (p<0.001) (Table 1). The fetal weight was also significantly lower in preeclampsia as compared with normal pregnancy (p<0.001) (Table 1).
Table 1: Baseline characteristics of the study subjects.
| Parameters | Mean ± SEM | p value | |
| Control (n=25) | PE Patient (n=27) | ||
| Maternal ages (years) | 26±0.1 | 25.04±0.1 | ns |
| Gest. ages (weeks) | 38.36±0.7 | 34.11±0.5 | <0.001 |
| Birth weight (kg) | 2.9±0.1 | 2.2±0.1 | <0.001 |
Unpaired t-test was done as the test of significant. p<0.05 was taken as level of significant.
Fig. 1: (a) Systolic and (b) Diastolic blood pressure of study subjects. Unpaired t-test was done as the test of significant. p<0.05 was taken as level of significant. ***; p<0.001
As shown in Fig. 1 the systolic and diastolic blood pressure levels were significantly lower in normal pregnancy as compared with preeclampsia (p <0.001, respectively).
Analysis of DNA Damage in Preeclampsia Women
The DNA damage rates with significant values were presented in Table 2. The head DNA indicated the intact DNA while the tail DNA represented damage DNA. The percentage of head DNA was significantly higher in normal pregnant women compared (p<0.001) to PE women. On the other hand, the percentage of tail DNA was significantly higher in PE women compared (p<0.001) to normal pregnant women. Thus, the DNA damage rate was significantly higher in PE women than the normal pregnant women.
Table 2 DNA damage rate (%) in different study subjects.
| DNA | Mean ± SEM | p value | |
| Control (n=25) | PE Patients (n=27) | ||
| Head DNA (%) | 94.40 ± 0.4 | 87.44 ± 0.9 | <0.001 |
| Tail DNA (%) | 5.6 ± 0.4 | 12.56 ± 0.9 | <0.001 |
Results are expressed as Mean ± SEM. Unpaired t-test was done as the test of Statistical significant. p<0.05 was taken as level of significant.
A correlation between DNA damage rate and gestational ages of PE women were found (Fig. 2). Head DNA (intact DNA) percentage was negatively correlated (r = -0.5, p<0.01) with gestational ages of patients. Thus, higher the gestational age higher the DNA damage rate.
Fig. 2: Correlation between intact DNA and gestational ages. r; correlation coefficient, **; p<0.01
DISCUSSION
Preeclampsia is associated with a high incidence of maternal and fetal mortality (Raoofi et al., 2013). There are no reliable, valid and economic screening tests available for predicting this pregnancy related disease (Cunnigham et al., 2010). In this study, we estimated the effects of DNA damage in preeclampsia during pregnancy. Other related parameters like blood pressure (BP) and birth weight also studied. We found high BP and low birth weight in preeclampsia patients compared to control. In preeclampsia oxidative stress increase and potential free radicals damage the vasospasm which in turn increases the peripheral resistance, hence BP increases (Pankaj Desai, 2013).
Only a few clinical studies have investigated DNA damage in women with pre-eclampsia and the results are controversial. Fujimaki et al. (2011) found a higher extent of oxidative DNA damage in preeclamptic women than in controls. Furness et al. (2010) suggested that such damage was significantly increased in women who developed PE and/or IUGR before clinical signs or symptoms appeared in comparison with women who had normal pregnancies. Wiktor et al. (2004) suggested that the highest level of DNA damage was found in women with preeclamptic pregnancies complicated by IUGR, and not in women with preeclampsia alone. In addition, Mert et al. (2012) found no significant difference in the extent of DNA damage between control and preeclampsia groups.
In general, earlier studies found increased signs of DNA damage in preeclamptic women with IUGR. We found, however, that women with preeclampsia had significantly increased level of DNA damage compared to the women with normal pregnancy. Hilali et al. (2013) also found increased level of DNA damage in mildly preeclamptic women. We also found negative correlation between DNA (intact DNA) and gestational ages in preeclamptic women which indicated that DNA damage rate was higher with the increase of gestational age. Very little study has used the comet assay to explore any possible association between damage to the DNA of mononuclear leukocytes and development of preeclampsia.
Oxidative-stress-induced damage to DNA and macromolecules is associated with the onset and development of many other diseases including cardiovascular disease, neurological degenerations (e.g., Alzheimer’s disease, ischemic stroke), and cancer, as well as the normal ageing processes. Thus the DNA damage in preeclampsia may cause serious problems in mothers even after delivery if untreated. Therefore, evaluation of DNA damage rate by comet assay which is simple, rapid and sensitive can be used as a useful tool for diagnosis and treatment of preeclampsia during pregnancy. This study will help to take preventive care for mothers. So treatment should persist even after delivery of child for few days. Further follow up study is needed for accurate findings.
ACKNOWLEDGEMENT
Thanks to the study subjects for their participation in this study.
CONFLICTS OF INTEREST
No competing financial interests exist.
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