ASSOCIATION BETWEEN INHERITED THROMBOPHILIA
IN PREGNANCY AND MICRONUCLEUS FREQUENCY
IN PERIPHERAL BLOOD LYMPHOCYTES
Šošić GM1,*, Jović N2,5, Rakić B3, Dimitrijević A2,4, Varjačić M2,5
*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
page: 11
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MATERIALS AND METHODS
Experimental Design and Subjects. The study
was designed as a case-control study involving pregnant
women admitted to the Department of Obstetrics and Gynecology
of our Clinic in 2015. Having been informed, the
pregnant women signed the agreement to take part in the
study approved by the local Ethics Council (No. 01-12294)
and filled in the questionnaire containing basic medical
history necessary for research in the field of cytogenetics
as well as for evaluations of exposure history. The
study included 74 pregnant women of gestational age 11
to 14 weeks. The excluding criteria were the following:
exposure to environmental and professional mutagens, exposure
to X-ray medical procedures, using oral hormonal
contraceptives in the previous year, the presence of other
chronic diseases (except thrombophilia), the intake of antibiotics
and anti-epileptics during pregnancy and using
narcotics. Blood samples from the pregnant women with
thrombophilia were taken before starting anticoagulant
therapy with LMWH. The pregnant women were grouped
according to the value of the MN in the control group
[≤4MN/ 1000 binucleated (BN) cells] and the group of
cases (>4MN/ 1000BN cells).
Blood samples were drawn following the usual procedure
and they were kept refrigerated for 24 hours. All the
heparinized blood (0.5 mL) was cultured in duplicate in 5 mL
complete medium (Gibco® PB-MAX™ Karyotyping Medium;
Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C.
Cytokinesis-Block Micronucleus Assay. The
CBMN assay was performed 44 hours after the cultivation
began by adding Cytohalazin B™ (Sigma-Aldrich, St.
Louis, MO, USA) to the cultures in final concentration of
4 μg/ mL. After continual incubation of cell cultures for an
additional 28 hours, the cells were treated with cold (4 °C)
hypotonic (0.56% KC1) solution and fixed three times with
fresh Carnoy’s fixative composed of methanol and glacial
acetic acid (ZORKA Pharma-HEMIJA d.o.o, Šabac,
Serbia) in a ratio of 3:1. The cell material was dripped
onto dry and cold microscope slides and the dried slides
were colored by 2.0% Giemsa stain solution (BioGnost®
d.o.o, Zagreb, Croatia). Micronucleus frequencies were
determined by scoring 1000 BN cells per person, according
to the criteria previously defined by Fenech et al. [23].
Statistical Data Processing. The entire statistical analysis
was performed using the Statistical Package for Social
Sciences (SPSS) version 22.0 for Windows software (IBM,
Armonk, NY, USA). The results are shown in the tables.
To present the results of the categorical variables, absolute
values and their percentage distribution were used. The experimental
results of continuous numerical values were presented
as mean ± standard deviation (SD) values. The χ2 test
was applied for determining the differences in the frequency
of categorical variables. The significance of the differences
between the means of studied variables was tested by the
Mann-Whitney and Kruskal-Wallis tests. Binary logistic
regression analysis was applied to identify risk factors and
to assess the impact of independent variables on the case
and control groups. The results are presented as odds ratio
(OR) with 95% confidence interval (95% CI) and the p value.
Univariate analysis was applied to all the parameters, while
multivariate analysis was used for statistically significant
parameters. A p value of <0.05 was considered significant.
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