INTRODUCTION
The main
objective of obstetrics is to reduce maternal and child morbidity and
mortality. Undoubtedly, timely and optimal delivery plays an important role in
solving this problem [9, 13]. The most important medical and biological
problem of modern obstetrics is premature birth [3, 10, 16].
According to statistics, premature infants account for 58.8-68.5% of early
neonatal mortality and 66.1-74.9% of infant mortality. Stillbirth in premature
births is observed 8-13 times more often than in timely ones. Perinatal
mortality in premature newborns is more than 26 times, and higher than in
full-term infants, i.e. its spontaneous interruption at the time from
conception to 37 weeks [4, 12, 18].
According to
literature data, the F13A1 gene encodes factor XIII, etc. fibrin-stabilizing
factor (fibrinase), which is involved in the
formation of insoluble fibrin, which is the basis of a blood clot, or thrombus [8, 11, 15]. At the same time, blood clots formed in the
presence of fibrinase undergo lysis
very slowly. An increase in the activity of factor XIII is accompanied by an
increase in the adhesion and aggregation of blood plates. In patients with
thromboembolic complications, fibrinase activity is
increased [2, 6, 14].
The aim of our research is to assess the detectability of
polymorphism of genotypes G103T of the F13 gene in pregnant women with
premature birth.
MATERIAL AND METHODS
We examined 90 women aged 21 to 36 years. Among them, patients with PB were 52 persons aged 19
to 42 years and 38 patients with physiological pregnancy without PB, who made
up the control group. General clinical,
instrumental, functional (ultrasound, Doppler), and ELISA studies were
performed in all pregnant women. Pregnant women were consulted by related
specialists (therapist, neurologist, infectious disease specialist,
dermatologist, endocrinologist, etc.). By informed consent, the molecular genetic study of the
G103T gene of the F13A1 gene was performed by polymerase chain reaction in real
time. Molecular genetic examination of biomaterials (DNA) was performed on the
basis of the clinical laboratory of Genotechnologies
LLC. DNA/RNA isolation from all biological blood samples was performed
using the Ribot-prep kit (Interlabservice,
Russia).
To identify the
polymorphism of the genotype consisting of alleles G>A of the F2 gene,
allele-specific primers from the manufacturer were selected from DNA samples.
200 DNA samples were examined for genotyping DNA samples by polymerase chain
reaction (PCR). To do this, the 96-cell automated amplifier "Applied Biosystems Veriti" was
optimized according to the following program: initial denaturation once at 180
seconds 94 ° C, 94 ° C - 10 seconds, 64 ° C - 10 seconds, 72 ° C - 20 seconds
in the program, we performed these specified actions 40 times to occur
polymerase chain reaction. Statistical analysis of the results was carried out
using the statistical software package "OpenEpi
2009, Version 2.3".
THE RESULTS OF THE STUDY
The
results of molecular genetic studies are presented in Table 1.
In the
studies of Malyshkin A.I., Fetisova
I.N., Zholobov Y.N. et al. (2018) found that an
increased risk of bleeding in patients with threatening preterm birth compared
to women with a physiological course of pregnancy was evidenced by the results
of an analysis of the distribution of gene and genotypic frequencies at the
F13A1 0103T locus [1, 5, 17]. At the same time, the frequency of occurrence of
the mutant allele T of the F13A1 gene in the control group was significantly
higher than in patients with threatening PB (38,36% и 25,24%, respectively; р=0,015; OR=0,54 [0,34-0,86]) and in patients with realized
PB (38,36% и 23,00%, respectively; р=0,015, OR=0,48 [0,27-0,85]).
It is
noteworthy that the heterozygous carriage of the F13A1 T variant in the
examined groups was similar, however, the homozygous
carriage of the F13A1 T allele in women of the control group was almost 4 times
higher than that in patients with threatening PB. The F13A1 gene encodes a
fibrin-stabilizing factor protein responsible for the final step in the blood
coagulation cascade.
In our
cases, unfavorable allele variants were most often detected in women with PB,
which was 1.9 times higher than in control women with a physiological course of
pregnancy (Р <0,05). According to A.D. Makatsaria (2011) with the F13A1 T/T genotype, the ability
of the protein to “crosslink” fibrin monomers changes, as a result of which
fibrin clots become thinner and more unstable, which increases the risk of
bleeding [7, 19]. It
should be noted that the reliable determinability of carriage of T/T mutant
genotypes in the F13A1 gene in pregnant women with the threat of preterm birth
allows us to regard this fact as a genetic component of an increased risk of
placental circulation disorders, bleeding and early termination of pregnancy.
The data obtained on
the frequency of occurrence of genotypic variants may indicate that the
polymorphism of the F13A1 gene is of pathogenetic
significance in the mechanism of development of preterm labor with a complex
sentence.
Considering the fact that
both the mutant allele and the mutant genotype carrying one "T"
allele were significantly less frequently detected in patients with PB against
the background of complex suggestions that the T/T genotype may also have a
protective effect. However, the proof
of this hypothesis requires a significant increase in the sample of patients
due to the rare frequency of occurrence of a polymorphic genotype with two
mutant alleles.
Thus, the data of our study showed the
connection of the "T" allele and the heterozygous genotype of the
polymorphism of the F13A1 gene with the development of premature birth in women
of the Uzbek population. At the same time, the risk of pathology formation with
the carrier of the "T" allele and the G/T genotype increases by 2.1
(OR=2.12) and 1.5 (OR=3.8) times, respectively. The presence of wild allele and
genotype of polymorphism of the F13A1 gene in patients plays a protective role
in relation to the formation of premature birth. The obtained result also
indicates that the variant allele and heterozygous genotype of the polymorphism
of the F13A1 gene predicts the risk of premature birth on a complex sentence.
To assess the frequency of occurrence
of various genotypes of the polymorphic gene F13 and the potential influence of
a number of dynamic factors determining the genetic structure of the
population, as well as to assess the population risk of the development of PB,
we analyzed the expected and observed frequency of the genotypes of the
polymorphism under study and the correspondence of the frequency distribution
to the Hardy-Weinberg equilibrium.
Analysis of the expected frequencies of
genotypes of the F13A1 gene in the control group showed that the observed
frequency of genotypes of functional genotypes G/G was 84.2%, then the expected frequency was 60.6%, which was 1.4 times
lower than the observed indicators. While the observed frequency of the
heterozygous variant of G/T was – 15.7, the expected increased 2.2 times and
was - 34.5%. The study showed mutant genotypes of the F13A1 gene in the control
group. Therefore, at the observed frequency – 0, the expected frequency was –
13.3, which indicates an increase in the definiteness of the carrier
polymorphism of the association of mutant genotypes.
The analysis of the obtained results indicates that the distribution of
all F13A1 polymorphism genotypes in the main and control groups corresponds to
the Hardy-Weinberg equilibrium, indicating that there is no influence of
systematic or random factors that can change the genetic structure of
populations. The study of the genetic structure of this marker
revealed a relatively high level of expected heterozygosity
and homozygous variants of mutant alleles in the main and control groups of
pregnant women (36,14% and 34,5%; 5,6% and 13,3% accordingly). In both groups,
the indicator D is to the left of 0, that is, it is negative (D<0). The
revealed fact indicates higher frequencies of expected heterozygotes and
homozygotes, rather than actually calculated frequencies of genotypes.
When analyzing the frequency distribution of alleles
and genotypes of this polymorphism in the group of pregnant women with fetal
growth restriction syndrome, significant differences were found compared with
the control group. The functionally unfavorable allele of the T gene F13A1 was
1.9 times statistically not significantly predominant in the studied alleles in
pregnant women with PB compared with pregnant women without PB (χ2=2.30;
p<0.13; OR=2.12; 95%CI 0.79 – 5.71).
The distribution of genotype frequencies of this
polymorphism also revealed significant differences between the pregnant group
and the comparison group in the general sample (P<0.05). Associations of
"functionally unfavorable" genotypes of G/T (χ2=2.15; p<0.14; OR=1.60; 95%
CI 0.54 – 4.73) and T/T (χ2=2.15;
p<0.14; OR=3.81; 95%CI 0.18 – 81.73) with the development of preterm labor
were revealed.
Analyzing the results of molecular genetic studies, we
can say that the T allele and the heterozygous genotypes of the F13A1
polymorphism are one of the markers of an increased risk of thrombophilia in
pregnant women with a complex sentence, and the functional G allele and the
functionally favorable G/G genotype are functional markers for the development
of pathology (χ2=2.15; p<0.14; OR=3.81; 95%CI 0.18 – 81.73).
The connection with the development
of the studied problems of the functionally unfavorable T/T genotype requires
additional research.
Thus, the results of our own studies published in
the literature and conducted by us indicate that the F13A1 hemostasis gene
plays an important role in the development of an increased risk of placental
circulatory disorders, the development of bleeding and early termination of
pregnancy.
CONCLUSIONS:
1.
The analysis of the detectability of the genotypes of
the F13A1 gene showed that the homozygous variant of the functional genotypes
G/G in the control group of pregnant women without PB was detected in 84.2% of
cases (32/38), and in the main group – 73.1% (38/52), which was 1.5times lower
than the control group (χ2=2.15; p<0.14; OR=0.51; 95%CI 0.18 – 1.48).
Whereas the heterozygous variant of the G/T gene F13 in the control group was
determined in 15.7% of cases (6/38). And in the main group of patients with PB,
the heterozygous variant of the G/T gene F13A1 was determined in 23.07% of
cases (12/52), respectively, which was 1.5 times higher than the indicators of
control individuals (χ2=2.15; p<0.14; OR=1.60; 95% CI 0.54 – 4.73).
2.
The mutant genotype of the T/T gene F13A1 in our cases
was determined in 3.8% of cases (2/52) in the main group. (χ2=2.15; p<0.14; OR=3.81; 95%CI 0.18 – 81.73). In
the control group of patients, this genotype was not determined.
3.
Based on the Hardy-Weinberg equation, in
the main group, the heterozygous variant G/T of the observed frequency of the
F13A1 gene was 23.08%, and the theoretically expected frequency was 36.14%,
respectively, which indicates an increase in this indicator by 1.6 times (P
<0.05). the frequency of the mutant homozygous variant of the T/T gene F13
was 3.8%, and the expected one was 5.6%, which was 1.5 times higher than the
indicators observed (Р<0,05).
4.
Analyzing the results of molecular genetic studies, it
can be said that the T allele and heterozygous genotypes of F13A1 polymorphism
are one of the markers of increased risk of thrombophilia in pregnant women
with PB (χ2=2.15; p<0.14; OR=3.81; 95%CI 0.18 – 81.73).
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