ASSOCIATION BETWEEN INHERITED THROMBOPHILIA IN PREGNANCY AND MICRONUCLEUS FREQUENCY IN PERIPHERAL BLOOD LYMPHOCYTES
Šošić GM, Jović N2, Rakić B, Dimitrijević A, Varjačić M
*Corresponding Author: Gordana M. Šošić, B.Sc., Department of Cytogenetic Diagnosis, Obstetrics and Gynecology Clinic, Clinical Center “Kragujevac,” 30 Zmaj Jovina Street, 3400 Kragujevac, Serbia. Tel: +381-63-835-66-24. Fax: +381-34-37-00-73. Email: gordana.sosic.2011.02@gmail.com
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DISCUSSION

Micronucleus presence is an indicator of genomic instability and accumulated damages that appeared during the lymphocyte’s life cycle and can be detected in vitro [3]. Every process that affects the DNA damage and/or chromosomes and spindle apparatus directly or indirectly increases the MN frequency. The most important processes are oxidative processes in the cell [2], gene polymorphisms and mutations that affect genomic instability [3]. Micronuclei are present in humans, as well as spontaneously formed in the range of 0-12 MN/1000BN cells [7]. In our study, the individual variation of the MN frequencies in the group of pregnant women was within a wide range of MN frequency variations (1-25 MN/1000BN). Wide varia tions of the MN frequency in the same group may be the result of different factors that influence chromosomal damage. Kopjar et al. [3] reported mean frequency of 6.9 ± 3.32 MN/1000BN cells in their study performed on healthy male and female subjects aged 20 to 61 in Croatia. The authors concluded that due to the differences in the organization of the research and the size of the population examined, the mean MN frequency was often higher or lower than the one determined in their research [3]. Our research has shown that the mean MN frequency in the studied population of pregnant women aged 20 to 33 is 6.09 ± 4.78 MN/1000BN cells. The results of the previous studies have shown that chromosomal aberrations could be found in the karyotype of 3.0-6.0% of couples with recurrent miscarriages [24]. Fenech [20] wrote about MN frequency increase as one of the factors that could be connected with recurrent miscarriages, and Furness et al. [19] showed that in the high risk group [patients who had recurrent pregnancy loss (RPL), preeclamsia/eclampsia, intrauterine growth retardation (IUGR), placental abruption or preterm delivery] there was a statistically higher MN frequency comparing to low risk group. Toljic et al. [25] showed that in gestational diabetes (GDM) and pregnancy-induced hypertension (PIH), there were elevated levels of oxidative stress, as well as damage to DNA and chromosomal aberration, which could be detected owing to elevated values of MNi in PBL of pregnant women. Upon the performed examination, we deduced that the case group (>4MN/1000BN) contained more statistically significant previous miscarriages and a higher number of miscarriages. The results presented in this study show that previous miscarriages and number of miscarriages are separate independent variables and represent significant predictors in the case group (>4MN/1000BN), while the multivariate analysis allocated only thrombophilia as an important predictor of an increased MN frequency in the case group (>4MN/1000BN). Studies analyzing the effects of smoking on MN frequency have shown contradictory findings. Studies performed by Kopjar et al. [3] as well as the one conducted by Nefic et al. [26] showed that smokers have statistically significant higher MN frequency than non smokers. In most of the research studies, the link between smoking and MN frequency was not found. Fenech et al. [27] concluded that radical MN frequency increase can be found only in smokers who smoke more than 30 cigarettes a day and who are not professionally exposed to genotoxic agents. In our study, the maximal number of cigarettes was between 1 and 9 and this study was in accordance with some of the previous findings that showed no link between smoking status and an increased MN frequency. Alcohol is a proven teratogenic agent that influences normal embryo and fetal development and it is implicated in the pathogenesis of the fetal alcohol syndrome. It is not known if any amount of alcohol is safe in pregnancy, but there are speculations that even a small amount of alcohol may harm the fetus. It is well-known that ethanol can easily cross the feto-placental barrier in both directions and concentrations in fetal and maternal circulation thus equalize [28]. Studies on animal models have shown that ethanol may cause disorders on epigenetic level and that it may also disturb the coordinated process of cellular differentiation [29]. Alcohol toxicity can be seen not only as a direct effect of the ethanol, but also as an indirect effect through its metabolic products and reactive oxygen species (ROS) that appear during alcohol biotransformation [30]. Studies on animal models have shown that alcohol may induce higher ROS production, cause oxidative stress and react with proteins, lipid and DNA causing their damage or complete degradation [31]. Different studies have confirmed the genotoxic effect of ethanol by various cytogenetic and molecular tests [32,33]. In the study by Santovito et al. [34], significant differences in MN frequency between alcoholics and controls were not found, but they did find significant differences in frequency of sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). The results of the studies by Benassi-Evans and Fenech [35] support the hypothesis that chronic exposure to alcohol induces formation of MN in two human B lymphoblastoid cell lines: WIL2-NS and GM13705. We have shown that in the group of pregnant women with frequency of >4 MN/1000BN there were significantly more women who consumed alcohol and the amount of alcohol consumed by the pregnant women was higher, but we did not find that these variables represented significant predictors of an increased MN frequency in the group of cases (>4MN/1000BN). A statistically significant difference in the mean values of the MN frequency in women who consumed and did not consume alcohol during pregnancy was not found. This can be explained by the fact that the minority of women in our study consumed alcohol (<10.0%) and no pregnant women chronically consumed alcohol. During pregnancy hypercoagulability and hypofibrinolysis are present and together with inherited and acquired thrombophilia disorders they can lead to early complications of pregnancy (RPL) and late complications of pregnancy [PE, IUGR, placental abruption, premature birth and intrauterine fetal death (IUFD)] [24]. Prothrombophilic genetic variants Factor V Leiden and Prothrombin A20210G were significantly associated with a higher prevalence of RPL [24,36]. Šošić et al. [37] found that in the group of women with inherited thrombophilia from Šumadija District, Serbia, previous miscarriages were most commonly noticed in double owners of gene variants plasminogen activator inhibitor-1 (PAI-1) 5G/4G and MTHFR, C677T. Karsli et al. [38] found that there was an impact of inherited thrombophilia on the first trimester combined aneuploidy screening test parameters. They determined that PAPP-A levels were significantly higher, whereas fbHCG levels and fetal NT measurements were lower in women with inherited thrombophilia. In a similar study, carried out by Cıkman et al. [39], the effect of inherited thrombophilia on second trimester combined aneuploidy screening test markers was investigated. They found that unconjugated estriol multiple of median (MoM) levels were significantly lower in women with inherited thrombophilia [39]. In our study, the results of the first trimester combined aneuploidy screening test were consistent with previous research of the PAPP-A levels. Micronucleus forming in human cells is connected to many medical conditions. Pristov et al. [21] came up with the result that in pregnant women with thrombophilia, placental tissue is exposed to H2O2-mediated oxidative stress, which can originate from the mother’s blood and the endothelium. The occurrence of H2O2 can be initiated by prothrombotic state present in mother, indicating a potential relation between a pregnancy complicated with thrombophilia and oxidative stress [21]. In our study, thrombophilia in pregnancy has a significant partial contribution to the occurrence of frequencies >4MN/1000BN. Pregnant women with thrombophilia are 26.69-times more likely to have a frequency of >4MN/ 1000BN compared to pregnant women without thrombophilia. In the population of pregnant women, thrombophilia accounts for around one-third of variance in MN frequency.



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