Study population
A prospective clinical cohort study of 290 female patients was conducted within the clinical units of the FSBEI HE ASMU of the Ministry of Health of the Russian Federation from 2012 to 2018. Two groups were singled out: the study group of 140 patients with the GA genotype (average age 31.2±4.7 years) and the control group of 150 women with the «wild» GG genotype (average age 32.3±3.9 years).
A distinctive feature of our study was that we analyzed all pregnancy cases of the registered patients, not a single episode of gestation. So, in our opinion, it is of fundamental importance, since often a woman's reproductive plans are not limited to the birth of one child, and it is important to understand the combination of which factors can lead to obstetric complications in carriers of the prothrombin G20210A genotype both in the first and subsequent pregnancies. Considering the above, during the observation period, the course and outcome of 814 pregnancies were analyzed: 363 in the group of the prothrombin G21210A mutation carriers and 451 in the control group. The association with the carriage of the prothrombin G20210A genotype was studied only in the case of planned pregnancies; artificial abortions were excluded.
Under complications of pregnancy we meant the following conditions: reproductive losses before 12 weeks of gestation, development of PE, FGR, and antenatal fetal death. Under non-developing pregnancy we understood anembryonic pregnancy - the absence of an embryo in a fetal egg or the death of an embryo. Preeclampsia and fetal growth retardation were diagnosed according to the criteria of international consensus [23, 24].
Inclusion criteria
Criteria for the inclusion in the study group: prothrombin G20210A mutation, 18 – 45 years of age; singleton progressive pregnancy occurred without hormone stimulation; no abnormalities in the development of internal genital organs; no decompensated extragenital diseases; informed consent of the woman to be the subject of additional research methods. Criteria for the inclusion in the control group were the same as for the study group; however, the patients were not prothrombin G20210A mutation carriers.
Exclusion criteria
Factor V Leiden mutation [F5L (1691)GA]; decreased functional activity of antithrombin III and proteins C or S; genital malformations; multifetal pregnancy; pregnancy resulting from assisted reproductive technologies; decompensated extragenital diseases; autoimmune diseases, including antiphospholipid syndrome; chromosomal aberrations in spouses.
The clinical characteristics of the comparison groups according to traditional risk factors for the development of placental dysfunction are presented in Table 1.
Table 1 Clinical characteristics of patients admitted to the study
Variable
|
The study group, GA genotype n = 140
|
The control group GG genotype n = 150
|
Statistical value
|
absolute number
|
(%)
|
absolute number
|
(%)
|
OR
|
95%CI
|
p
|
Age
|
18-35 years old
|
101
|
72.1
|
112
|
74.7
|
0.9
|
0.521-1.480
|
0.6268
|
>35 years old
|
39
|
27.9
|
38
|
25.3
|
1.1
|
0.675-1.917
|
0.6268
|
Caucasian race
|
130
|
92.9
|
138
|
92.0
|
1.1
|
0.472-2.705
|
0.7831
|
BMI (kg/m2)
|
<18.5
|
3
|
2.1
|
2
|
1.3
|
1.6
|
0.266-9.844
|
0.6
|
18.5-25
|
98
|
70.0
|
112
|
74.7
|
0.8
|
0.472-1.32
|
0.3747
|
≥25
|
25
|
17.9
|
30
|
20.0
|
0.6
|
0.482-1.567
|
0.8696
|
≥30
|
8
|
5.7
|
4
|
2.7
|
2.1
|
0.651-7.516
|
0.2033
|
≥35
|
6
|
4.3
|
2
|
1.3
|
3.3
|
0.657-16.698
|
0.1466
|
Hypertensive disease
|
34
|
24.3
|
13
|
8.7
|
3.4
|
1.699-6.723
|
0.0005
|
The groups were comparable by age (p> 0.05) and ethnicity: 93.2% of the study group and 91.9% of the control group were represented by Caucasians (p> 0.05). The body mass index of the patients also did not have significant differences. It should be noted that before the reproductive function was performed, the number of patients suffering from essential hypertension was comparable [OR = 1.8, 95% CI: 0.8926-3.8746; p = 0.0976]. By the end of the observation, the number of hypertension cases in the group of G20210A genotype was registered 3.4 times more often than in the control group. Upon that, in 13 women hypertension was the result of PE.
The study was approved by the local ethics committee of the FSBEI HE ASMU of the Ministry of Health of the Russian Federation (protocol No. 5 of June 25, 2012).
Preeclampsia and fetal growth retardation were diagnosed according to international consensus criteria 23, 24.
Laboratory assays
All patients were diagnosed with prothrombin G20210A mutation and prothrombin activity, none of them receiving anticoagulants. Eight time points were selected to evaluate prothrombin activity, based on trophoblast invasion waves and major pregnancy periods: 7–8 weeks, 12–13 weeks, 18–19 weeks, 22–23 weeks, 27–28 weeks, 32–33 weeks, 36–37 weeks and 2-3 days after delivery 25,26.
Prothrombin G20210A mutation was diagnosed by means of the polymerase chain reaction (PCR) method using reagents from Litekh SPA (Russia). Material for the study was human genomic DNA isolated from peripheral blood leukocytes. The analysis was based on the Real-Time PCR method using competing TagMan probes complementary to the polymorphic DNA sequence. In all patients, prothrombin activity was measured using Factor II deficient plasma (Siemens) on an automatic coagulometer (Siemens BCS XP) according to the previously described method 27.
Statistics
Statistical data processing was performed using the MedCalc Version 17.9.7 statistical software package (license BU556-P12YT-BBS55-YAH5M-UBE51). Variation series were checked for normal distribution using the Shapiro-Wilk W-test. Laboratory values are presented as scatter plots with box plots (box-and-whisker plot). The box plot represents a median (Me) - middle of the sample, shown as a marker on the inside line of each box; interquartile range - interval between the 25th and 75th percentiles containing the central 50% of the sample’s observation, shown as a box; 95% confidence interval (95% CI) for the median shown as straight lines (whiskers) coming out of the box.
To compare the levels of prothrombin activity in two independent samples, the Mann-Whitney nonparametric statistical U-test was used. To determine the prognostic value of prothrombin activity index for the development of pregnancy complications in prothrombin G20210A mutation carriers, the ROC curve was used, with subsequent AUC calculation.
For qualitative features, the total and relative values were given in percentage; verification of statistical hypotheses on the coincidence of the observed and expected frequencies was performed using the χ2 criterion and Fisher's exact test. For binary features, the relative risk (RR) and 95% confidence interval (95% CI) were calculated. The critical significance level of discrepancies (p) was defined as p<0.05.