RU2850617C1 - Method for predicting risk of preeclampsia in planned pregnancies in women without family history of condition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to clinical laboratory diagnostics, obstetrics and gynaecology, and can be used to predict the risk of developing pre-eclampsia when planning pregnancy in women without a family history of the condition. Before pregnancy, venous blood is taken from a woman during the follicular phase of the ovarian-menstrual cycle to determine progesterone levels. Genotyping of the polymorphic variant T(-786)C of the endothelial NO synthase (NOS3) gene is performed. A progesterone level above 4.6 nmol/L in combination with the presence of the C/C genotype of the T(-786)C variant of the NOS3 gene predicts the risk of developing preeclampsia.

EFFECT: possibility of predicting the risk of preeclampsia by determining progesterone in the follicular phase of the cycle and the C/C genotype of the T(-786)C variant of the NOS3 gene.

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Description

The invention relates to clinical laboratory diagnostics, obstetrics and gynecology, and can be used by doctors to predict the risk of preeclampsia and carry out preventive measures.

Preeclampsia (PE) is a hypertensive complication of the second half of pregnancy associated with the development of multiple organ failure. It is one of the leading preventable causes of adverse maternal and fetal outcomes, affecting 3-5% of pregnancies worldwide.

Delayed complications for the mother include cardiovascular diseases (hypertension, coronary heart disease), cerebrovascular disorders (stroke, cognitive impairment), terminal renal disease, venous thromboembolism, diabetes mellitus and metabolic disorders [Wieteska M., Maj D., Gorecka A., Zaremba B. Preeclampsia - long-term effects on mother and child. J. Educ. HealthSport. 2021;11(8):261-7; Akhmadeev H.P., Fatkullin I.F., Fatkullina L.S. Cardiovascular consequences of major obstetric syndromes. Obstetrics and Gynecology. 2023;4:5-11]. The pathogenesis of these lesions is based on a number of factors, but the leading role is played by persistent endothelial dysfunction, which persists for 5-8 years after PE [Sandvik M.K., Leirgul E., Nygard O., Ueland P.M. et al. Preeclampsia in healthy women and endothelial dysfunction 10 years later. Am. J. Obstet. Gynecol. 2013;209(6):569.el-10].

Genetic predisposition to PE has been confirmed by large cohort studies based on national registry data and patient family histories. Currently, over 100 polymorphic gene variants associated with its development are known. Carrying these polymorphisms does not always lead to the development of PE, as it is also influenced by environmental conditions, lifestyle, and other factors [Ganeeva A.V., Gabidullina R.I., Kapelyushnik P.L., Dmitrieva D.D. The Role of Polymorphisms of Blood Pressure Regulatory Genes in the Pathogenesis of Preeclampsia. Women's Health and Reproduction. 2023. No. 3 (58)].

The leading diagnostic criterion for PE is arterial hypertension (AH). Blood pressure (BP) regulatory genes associated with the development of PE include genes involved in the renin-angiotensin-aldosterone system (RAAS) and endothelial dysfunction.

A patent search has shown that there are ways to predict the risk of developing PE using genetic markers.

Thus, patent RU 2723627 C1 (08.10.2019) “Method for predicting severe preeclampsia in pregnant women carrying a prothrombin gene mutation, genotype F2G20210A” shows a method for predicting a severe form of PE in pregnant women in the presence of a mutation in the prothrombin gene, which is associated with a change in prothrombin activity.

In patent RU 2795660 C1 (16.08.2022) “A method for predicting the risk of developing preeclampsia in women taking into account genetic markers,” the risk of PE was determined only by a combination of polymorphic variants of the HFE and BAG6 genes in the genotype.

A similar method is described in patent RU 2834808 C1 (30.08.2024) “Method for predicting the risk of developing preeclampsia based on genetic testing”, in which the prediction of the risk of PE was based on the analysis of genetic testing of polymorphic loci of the OBFC1, PLCE1, CERS5 genes.

Patents RU 2775433 (17.12.2021) "Method for predicting the risk of developing preeclampsia based on molecular genetic analysis" and RU 2775434 (17.12.2021) "Method for predicting the risk of developing preeclampsia based on genetic testing" also contain a description of a method for predicting the risk of PE based solely on molecular genetic analysis (polymorphic variants of the UGT2B4 gene and the PPP1R12C gene) with the identification of leading haploblocks as risk factors.

In patent RU 2738675 C1 dated July 28, 2020, "A method for predicting the risk of developing preeclampsia in women without an adverse family history, taking into account genetic factors," the polymorphism of the progesterone receptor gene (PGR) also served as a genetic marker, predicting a high risk of developing PE when the T allele of the rs 1042838 polymorphic locus of the PGR gene was detected.

There are methods for predicting the risk of developing PE in pregnant women with a polymorphism of the platelet glycoprotein IIIa ITGB3 gene associated with fetal growth retardation syndrome (patent RU 2754956 C1 dated 06.04.2021 "Method for predicting the risk of developing preeclampsia in pregnant women with fetal growth retardation syndrome").

A similar formula using a polymorphic variant of the F13A1 gene as a marker of PE is proposed in patent RU 2741861 C1 dated August 13, 2020, "A method for predicting the weight of a newborn in pregnant women with preeclampsia combined with fetal growth retardation syndrome."

In the patent literature reviewed, the authors did not find a method for predicting the risk of PE before pregnancy in healthy women of reproductive age based on the determination of sex hormones (estradiol, progesterone, testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), vasoactive endothelial factors (nitric oxide (NO), endothelin-1 (ET-1)) in the blood plasma and molecular genetic testing of the T(-786)C variant of the endothelial NO synthase gene (NOS3).

There are a number of studies in the literature establishing the role of the -786C allele of the T(-786)C variant of the NOS3 gene in women with PE [Fetisova I.N., Panova I.A., Rokotyanskaya E.A. [et al.]. Genetic factors in the development of preeclampsia. Bulletin of the Ivanovo Medical Academy. - 2015. - Vol. 20, No. 3. - P. 13-16]. When studying the relationship between the presence of single nucleotide polymorphisms of the NOS3 gene in the genotype and hypertensive disorders during pregnancy, conflicting results were obtained [Bebyakova N.A. et al. The role of the -786T> C polymorphism of the endothelial NO synthase gene in the formation of risk factors for the development of arterial hypertension // Human Ecology. - 2018. - No. 4. - P. 36-42.; Ragia G., Nikolaidis E. et al. - Endothelial nitric oxide synthase gene polymorphisms -786T> C and 894G> T in coronary artery bypass graft surgery patients // Hum Genomics. - 2010. - Vol. 4 (6). - P. 375-383.]. These studies did not examine sex hormones, which are modifiers of the production of endothelial factors (NO and ET-1), the imbalance of which leads to the development of hypertension.

The aim of the proposed method is to comprehensively assess the risk of preeclampsia taking into account the polymorphic variant T(-786)C (rs2070744) of the endothelial NO synthase (NOS3) gene, the level of sex hormones and vasoactive endothelial factors.

A hypothesis has been put forward according to which PE may be preceded by the presence of a hereditary genetically determined disorder of vascular tone, characterized by an increased risk of vasoconstriction and, as a consequence, the risk of PE against the background of carriage of the T(-786)C variant of the endothelial NO synthase gene (NOS3).

Two population-based studies were conducted at the Department of Medical Biology and Genetics, the Consultative and Diagnostic Outpatient Clinic, and the Central Research Laboratory of the Northern State Medical University of the Russian Ministry of Health in Arkhangelsk. All study participants signed voluntary informed consent, approved by the local ethics committee of the Northern State Medical University of the Russian Ministry of Health.

First study

The first study involved 384 women aged 60 to 74 years, residents of Arkhangelsk. The average age of the participants was 67.2 years. Exclusion criteria included the presence of medical conditions requiring regular psychiatric monitoring, thus eliminating the potential influence of mental disorders on biomarker measurements.

Molecular genetic typing was performed as part of the study. Genomic DNA samples isolated from peripheral blood leukocytes were used as the test material. The blood was stabilized with a 4% EDTA solution, and the sample was frozen and stored at -20°C prior to testing.

For genotyping of the T (-786)C polymorphism of the NOS3 gene, the polymerase chain reaction (PCR) method with a fluorescent product detection scheme in real time was used on a LightCycler-96 amplifier (Roche Switzerland/Germany) at the molecular genetic laboratory of the Central Scientific Research Laboratory of the Saratov State Medical University using reagents from Litekh (Moscow).

The NOS3 gene is localized on the short arm of chromosome 7 at locus 7q36.1 and contains 27 exons, 26 of which are coding. It has a size of 23.5 kb. Several mutations in the NOS3 gene are known, one of them is a single nucleotide substitution in the promoter region of the gene T(-786 C). Variant T(-786)C (rs 2070744) is a point mutation in the promoter region of the NOS3 gene, namely a substitution of thymine for cytosine, leading to a significant decrease (up to almost 50%) in the activity of the NOS3 enzyme promoter and a decrease in NO levels [Doshi A.A., Ziolo M.T. et al. A promoter polymorphism of the endothelial nitric oxide synthase gene is associated with reduced mRNA and protein expression in failing human myocardium // J Card Fail. 2010. Vol.16 (4). P.314-319.]. The “wild” variant of polymorphism is the T/T genotype.

Information was collected from older women regarding their place of birth, lifestyle, and women's health (number of pregnancies and births, onset of menstruation and menopause, and causes of menopause). Participants were also asked about pregnancy complications, such as gestational hypertension and PE. Furthermore, information was provided on whether they had been diagnosed with hypertension and the nature and characteristics of antihypertensive therapy, if any.

Qualitative variables are presented as absolute values and percentages (%). Group comparisons for qualitative characteristics were performed using the Pearson χ2 test. Logistic regression was used to calculate the odds ratio (OR) with a 95% confidence interval (CI), and the results are presented along with the p value for the corresponding association. The critical significance level was p < 0.05. Stata 18.0 (StataCorp, Texas, CollegeStation, USA) was used for statistical analysis.

Molecular genetic testing identified all alleles and genotypes of the T(-786)C variant of the NOS3 gene. The predominant genotypes were those containing the wild-type variant of the allele, namely the T allele, while the rarest variant was the C/C genotype (Table 1, Table 2), which contains two mutant alleles.

When analyzing the distribution of PE cases among women with different genotypes, it was found that PE was more often observed in carriers of the C/C genotype (p = 0.027). The frequency of PE among women with the C/C genotype was 5.88%. For women with the T/T and T/C genotypes, the frequency of PE was recorded at 1.29% and 0.69%, respectively. According to the results of logistic regression analysis, among women aged 60-74 years, carriers of the C/C genotype had 4.8 times higher odds (OR 4.80; 95% CI: 1.04 - 2.09; p = 0.044) of developing PE in the past compared to carriers of the T/T and T/C genotypes. Thus, the T(-786)C variant of the NOS3 gene may be a marker of predisposition to the development of PE.

Second study

To expand our understanding of risk factors for PE, a second study was conducted. This study included 116 healthy young women, average age 19.6, living in the Arkhangelsk region. The study included young women with established, regular, ovulatory (assessed by basal rectal temperature), and normal menstrual cycles. The study was conducted during two phases of the ovarian-menstrual cycle: follicular (days 5-7) and luteal (days 19-21). Inclusion criteria also included the absence of endocrine, reproductive, or cardiovascular diseases.

The study measured vasoactive endothelial factors: endogenous NO from stable metabolites—nitrates and nitrites—using the Griess reagent in biological fluids, biochemically using the Total NO/Nitrite/Nitrate kit (R&D Systems, USA), and ET-1 by enzyme-linked immunosorbent assay (ELISA) using the BIOMEDICAGRUPPE kit (Austria). Additionally, the vasodilator NO/vasoconstrictor ET-1 ratio was calculated based on the concentration of these factors in the blood. This index allows one to assess the balance of vasoactive endothelial factor production.

The assessment of peripheral vascular tone (PVT) was carried out using the tetrapolar rheography method.

Determination of sex hormones (estradiol, progesterone, testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) was performed by ELISA using the Vector-BEST diagnostic kit (Russia). Molecular genetic testing was also conducted using the PCR method, as in elderly women.

To describe quantitative variables, the median (Me) and values corresponding to the first and third quartiles (Q1-Q3) were used. Correlation analysis was used to analyze relationships between variables, including calculating Pearson correlation coefficients and determining the level of statistical significance of the coefficients. Results were considered statistically significant at p < 0.05. The SPSS statistics software package (StatSoft, USA) and the open-access SNPStats program were used for the analysis.

According to the literature, individuals with at least one C allele for the NOS3 gene variant under study have a lower amount of mRNA and a low level of NO in the blood compared to wild-type T/T homozygotes [Miyamoto Y., Saito Y. et al. Replication protein Al reduces transcription of the endothelial nitric oxide synthase gene containing a -786T→C mutation associated with coronary spastic angina//Hum Mol Genet. 2000. Vol. 9(18). P. 2629-2637.]. However, in this study, in young women, the NO concentration in homozygotes for the mutant C allele in the follicular phase of the ovarian-menstrual cycle was the highest compared to heterozygotes and homozygotes for the T allele. It should be noted that the ET-1 level in carriers of the C/C genotype exceeded the reference value (2.52 fmol/ml) by 2.5 times (Q1-Q3: 0.16-4.87), accordingly, the endothelial factor index (NO/ET-1) in this phase of the cycle, despite the high NO level, was shifted towards vasoconstriction. The development of an imbalance of vasoactive endothelial factors in the luteal phase in C/C homozygotes also explains the higher level of IPF, which was above the threshold value (more than 90 conventional units). Correlation analysis revealed the presence of a significant positive correlation between the level of NO and ET-1 in the luteal phase of the cycle (r=0.851; p=0.05).

In young women with the C/C genotype, testosterone, FSH, and LH levels did not exceed reference values and did not differ statistically significantly from those in heterozygotes and homozygotes for the T allele. Estradiol levels also did not exceed the reference range, but were statistically significantly higher in the follicular phase of the cycle in homozygotes for the mutant C allele, compared with other genotypes. Progesterone levels in the follicular phase are noteworthy: 9.23 nmol/L (Q1-Q3: 2.97-21.42), which exceeds the upper limit of the reference range (0.6-4.6 nmol/L) by 2 times. It should be noted that progesterone levels exceeding the reference value were also observed in C/T heterozygotes in this phase of the cycle - 5.31 nmol/l (Q1-Q3: 4.20-6.41).

Correlation analysis revealed a significant positive correlation of moderate strength between NO and progesterone levels in heterozygotes (r=0.298; p=0.05) and homozygotes for the mutant C allele (r=0.290; p=0.05). A significant positive correlation was also found between NO and estradiol levels (r=0.755; p=0.03) in the follicular phase of the ovarian-menstrual cycle.

Thus, the relationship between estradiol and progesterone with the level of NO is manifested only in the presence of the mutant allele C of the T(-786)C variant of the NOS3 gene in the genotype.

Therefore, the -786C allele is associated with an imbalance of vasoactive endothelial factors, as well as high progesterone levels during the follicular phase of the cycle. Thus, the T(-786)C variant of the NOS3 gene may be a marker of predisposition to preeclampsia.

To predict the risk of preeclampsia in women of reproductive age when high levels of progesterone are detected in the follicular phase of the ovarian-menstrual cycle during pregnancy planning, it is necessary to conduct molecular genetic testing for the presence of the T(-786)C polymorphic variant of the NOS3 gene in the genotype to assess the risk of preeclampsia and implement preventive measures to prevent the development of preeclampsia.

As examples of the specific application of the developed method, a voluntary examination of healthy women of Russian nationality of reproductive age was conducted, including an enzyme-linked immunosorbent assay for the determination of sex hormones, as well as a molecular genetic examination of the rs2070744 locus of the NOS3 gene.

Example 1.

A young married couple sought help at a family planning center. The woman, who had no family history of preeclampsia, had venous blood drawn from her cubital vein to determine sex hormone levels during the ovarian-menstrual cycle. High progesterone levels were detected during the follicular phase. Upon further observation, the woman's diagnosis of early severe preeclampsia, which required emergency delivery, was verified at 27 weeks of pregnancy. Subsequently, genotyping of the T-786 C (rs 2070744) polymorphic variant of the NOS3 gene was performed, revealing the -786 C allele in the C/C genotype of the rs 2070744 polymorphic locus of the NOS3 gene.

Example 2.

Woman A., with no family history of preeclampsia, consulted a gynecologist to plan a pregnancy. Venous blood was drawn from the cubital vein for subsequent determination of sex hormones during the ovarian-menstrual cycle. High progesterone levels were detected during the follicular phase. Subsequently, DNA marker genotyping was performed, which revealed the C/C genotype of the rs2070744 polymorphism in the NOS3 gene, placing her at high risk for preeclampsia. Upon further observation, this woman was diagnosed with late-onset moderate preeclampsia at 34 weeks' gestation.

Example 3.

A woman named T., with no family history of preeclampsia, had venous blood drawn from her cubital vein during preconception preparation for subsequent determination of sex hormones during the ovarian-menstrual cycle phases. High progesterone levels were detected during the follicular phase. Subsequently, DNA marker genotyping was performed, revealing the C/C genotype of the rs2070744 polymorphic locus of the NOS3 gene, which placed her in a high-risk group for preeclampsia. Following pregnancy, a series of preventive measures for preeclampsia was administered. This woman showed no clinical or laboratory signs of preeclampsia throughout the entire gestation period. The pregnancy ended with natural delivery at 38-39 weeks. The newborn's Apgar score was 8-9 points.

Thus, the proposed method makes it possible to predict the risk of PE development taking into account the T(-786)C polymorphic variant of the NOS3 gene and the progesterone level exceeding the reference in the follicular phase of the ovarian-menstrual cycle.

Claims (1)

A method for predicting the risk of developing preeclampsia when planning pregnancy in women without an adverse family history, characterized by the fact that before the onset of pregnancy, venous blood is taken from a woman in the follicular phase of the ovarian-menstrual cycle to determine progesterone, genotyping of the polymorphic variant T(-786)C of the endothelial NO synthase gene (NOS3) is carried out, and when determining the level of progesterone in the follicular phase of the cycle above 4.6 nmol/l in combination with the presence of the C/C genotype of the T(-786)C variant of the NOS3 gene, the risk of developing preeclampsia is predicted.