RU2730958C1 - Diagnostic technique for severe preeclampsia in pregnant women - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics, gynaecology, molecular biology, molecular genetics, biochemistry, and can be used for diagnosing severe preeclampsia in pregnant women. Blood plasma of the pregnant woman is examined for concentration of extracellular DNA (ec DNA), number of copies of ribosomal DNA in extracellular DNA (ec rRNA), number of copies of mitochondrial DNA in extracellular DNA (ec mtDNA), nuclease activity of DNase 1. Nuclear leukocyte DNA is used to determine the number of copies of genomic ribosomal DNA (rDNA genome), number of copies of genomic mitochondrial DNA (mtDNA genome). If the ec DNA is more than 1000 ng/ml, ec rDNA more than 1000, ec mtDNA more than 200, the ratio of ec rDNA to the rDNA genome is more than 2, the ratio of ec mdDNA to the mtDNA genome is more than 1.64, the nuclease activity of DNase 1 is more than 10 units/ml, is diagnosed severe preeclampsia.

EFFECT: method provides accurate detection of severe preeclampsia by determining a set of molecular-biological factors - criteria for objective reliable diagnosis of severe asymptomatic forms of preeclampsia.

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Description

The present invention relates to medicine, namely to obstetrics and gynecology, and also, to molecular biology, molecular genetics, biochemistry, can be used to determine the critical (severe) state of pregnant women, including those with atypical preeclampsia for the correct choice of medical tactics ...

Preeclampsia is a complication of pregnancy, which is characterized by profound disorders of vital organs and systems, often accompanied by the development of placental insufficiency and fetal malnutrition. At present, according to the WHO, preeclampsia complicates 2.2% of pregnancies and there is a tendency to an increase in its incidence.

A prospective study of all cases of eclampsia that occurred in the UK in 1992 (383 cases, the incidence of 4.9 per 10,000 births) showed that 38.0% of cases developed seizures before proteinuria and arterial hypertension appeared and were diagnosed [ Douglas K.A., Redman CW / Eclampsia in the United Kingdom // BMJ, 1994; 309: 1395-1400].

Given the fact that all three classic symptoms (arterial hypertension, edema and proteinuria) of preeclampsia are rarely found simultaneously in pregnant women, new names for this complication have been proposed. These include the nomenclature "arterial hypertension", "atypical preeclampsia". To "atypical preeclampsia" include cases with the absence of one or more symptoms, emphasizing the fact of the frequency of development of such options in terms of up to 20 weeks of pregnancy [Sibai V.M. / Diagnosis and management of gestational hypertension and preeclampsia // Obstet Gynecol, 102 (1) (2003), pp. 181-192].

There is a whole range of subjective reasons that make it difficult to assess the severity of preeclampsia. These include the following subjective reasons: lack of accurate knowledge about etiology and pathogenesis, high incidence of atypical preeclampsia, a narrow range of characteristics focused on a small number of criteria that do not belong to specific evidence-based medicine: increased blood pressure, proteinuria, functional diagnostics tests, anthropometry , dopplerometry, etc.

Known methods for predicting severe forms of preeclampsia, based on the determination of molecular biological and genetic factors.

A known method for predicting the risk of developing severe preeclampsia taking into account genetic data (RU 2653765 C1). The method is carried out as follows. DIC is isolated from samples of peripheral venous blood of patients in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris-HCl (pH 7.6) are added to 4 ml of blood. The resulting mixture is stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH 8.0) and 75 mM NaCl are added to the sediment and resuspended. Then add 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) and incubate the sample at 37 ° C for 16 hours.

At the second stage, DNA extraction is carried out sequentially from the obtained lysate with equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is sampled. DNA is precipitated from solution with two volumes of chilled 96% ethanol. Formed DNA is dissolved in bidistilled, deionized water and stored at -20 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers. Get genetic polymorphisms MMP-2 (rs243865), MMP-8 (rs11225395), MMP-8 (rs1320632), MMP-9 (rs17577) and predict an increased risk of developing severe preeclampsia in the presence of a combination of genetic variants G MMP-9) (rs17577) and G MMP-8 (rs1320632), a reduced risk of severe preeclampsia - in the presence of a combination of genetic variants T MMP-2 (rs243865) and A MMP-9 (rs17577). However, the known method does not allow determining the critical state of health of patients with preeclampsia to determine medical tactics - prolongation or termination of pregnancy.

There is also known a method for predicting a high risk of reproductive losses in the first trimester of pregnancy (RU 2611358 C1), which includes the isolation of DNA from the peripheral venous blood of pregnant women and the subsequent amplification of the polymorphic A66G variant of the MTRR methionine synthase reductase gene. The method is carried out as follows: 1000 μl of the patient's whole blood is introduced into an Eppendorf test tube, the sample is centrifuged at a speed of 3000 rpm for 5 minutes. Plasma is removed carefully. Close the tube and freeze it at -20 ° C until the shaped elements are completely frozen for at least 1 hour. Thaw the contents of the tube at room temperature. The reagent "DNA-express-blood" is introduced into the test tube with a volume equal to the volume of formed elements remaining in the test tube. The contents of the tube are thoroughly mixed on a vortex. Place the test tube in a thermostat preheated to 99 ° C and incubate for 15 minutes. At the end, it is cooled to 70 ° C. Centrifuge the tube at 12000 rpm at room temperature for 60 sec. The resulting supernatant is used as a test DNA sample. The isolated DNA is then subjected to polymerase chain reaction using SNP-express reagent kits (Litekh, Russia) according to the manufacturer's instructions. Amplification of allelic variants of proinflammatory cytokine genes - 31C-T (rs1143627) IL-lβ, -174G-C (rs1800795) IL-6 and folate cycle enzymes C677T (rs1801133) MTHFR and when one of four genotypes is detected: -31CT IL-1β / -174GG IL-6 / 677CT MTHFR / 66GG MTRR; -31CT IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR; -31CC IL-1β / -174CC IL-6 / 677CC MTHFR / 66AG MTRR; -31CC IL-lβ / -174GC IL-6 / 677CT MTHFR / 66AG MTRR predict a high risk of reproductive loss in the first trimester of pregnancy. This method also makes it impossible to determine the severity of preeclampsia for the choice of medical tactics - prolongation or termination of pregnancy.

As a prototype, we have chosen the classical method for determining severe preeclampsia in pregnant women according to the Tsangemeister triad (edema, proteinuria, arterial hypertension) for the correct choice of medical tactics [Shalina R.I., Mikhaleva L.M., Simukhina M.A., Konoplyannikov A. G., Shtabnitsky AM / Features of the clinical course of severe forms of preeclampsia in modern conditions // Questions of gynecology, obstetrics and perinatology. 2017.Vol. 16.No.6. S. 16-23].

However, at present, all three classic symptoms of preeclampsia are often absent. So, according to G.M. Savelyeva. et al. [Savelyeva G.M., Shalina R.I., Kurtser M.A. et al. / Eclampsia in modern obstetrics // Midwife. and gynec., 2010, No. 6, S. 4-10], arterial hypertension above 160/100 mm Hg. was determined only in 25.5% of cases of eclampsia, and in the rest it was within 130 / 90-150 / 100 mm Hg and below. Proteinuria was not confirmed in 25.0% of eclampsia cases. Revealed large differences in the severity of the edema syndrome, the nature of subjective symptoms. The presence of all three symptoms was noted only in 58.8% of cases.

We have set the task to develop criteria for an objective reliable diagnosis of severe asymptomatic forms of preeclampsia.

The medical and technical result achieved with the implementation of the invention is to reduce maternal and perinatal mortality and morbidity due to the timely selection of adequate medical tactics, clarification of indications for termination of pregnancy by accurately identifying the severe course of preeclampsia.

The essence of the invention is as follows.

To diagnose severe preeclampsia in pregnant women, the concentration of extracellular DNA (cc DNA), the number of ribosomal DNA copies in cc DNA (cc mtDNA) (cc rDNA), the number of mitochondrial DNA copies in cc DNA (cc mtDNA), nuclease activity are determined in blood plasma. DNase 1. In the nuclear DNA of the pregnant woman's leukocytes, the number of copies of genomic ribosomal DNA (rDNA genome), the number of copies of genomic mitochondrial DNA (mtDNA genome) are determined. When the vk DNA concentration is more than 1000 ng / ml, the vk rDNA is more than 1000, the vk of mtDNA is more than 200, the vk rDNA to rDNA genome ratio is more than 2, the vk mtDNA to mtDNA genome ratio is more than 1.64, the DNase 1 nuclease activity is more than 10 units / ml - diagnosed with severe preeclampsia.

The method is carried out as follows.

Samples of freshly taken (with anticoagulant) peripheral blood of patients were centrifuged at 400g for 10 min, plasma was collected in dry test tubes, then plasma and red precipitates were frozen and stored at -80 ° C.

Molecular biological factors in the blood given in our study:

cc DNA - concentration of DNA in blood plasma in ng / ml;

cc rDNA - the number of copies or repeats of ribosomal DNA in the composition of extracellular DNA;

cc mtDNA is the number of copies or repeats of mitochondrial DNA in the composition of extracellular DNA. In structure, it resembles fetal DNA, has a powerful stimulating effect, can provoke abortion;

DNase 1 - an enzyme that destroys DNA in units / ml;

rDNA genome (genomic rDNA) - the number of copies, repeats of nuclear leukocyte ribosomal DNA in nuclear DNA;

genome of mtDNA (genomic mtDNA) - the number of copies, repeats of nuclear leukocyte mitochondrial DNA in nuclear DNA indicates the death of leukocytes;

cc rDNA / rDNA genome - an integrative indicator indicating the accumulation of ribosomal DNA in the bloodstream;

cc mtDNA / mtDNA genome is an integrative indicator indicating the accumulation of mitochondrial DNA in the bloodstream.

Extracellular and genomic DNA was isolated by the method of extraction with organic solvents, respectively, from blood plasma obtained by centrifugation of whole blood and red sediments (formed elements).

The concentration of genomic DNA was determined spectrophotometrically at a wavelength of 260 nm, taking into account light scattering (320 nm) in a quartz cuvette with a beam path of 1 cm.

The concentration of extracellular DNA was determined fluorimetrically using the intercalating dye Pico Green (Invitrogen).

The content of the number of copies of the studied indicator in the composition of DNA was determined by the method of non-radioactive quantitative dot hybridization. For this, DNA samples of known concentration and a set of calibration samples with a known content of genome repeats were applied in several repeats to nitrocellulose filters, and, after thermal immobilization, incubated with a set of biotinylated oligonucleotide probes, the signal was visualized using streptavidin alkaline phosphatase conjugate (Merck) and colorimetric substrate.

The copy content was calculated from the calibration dependence using computer image analysis, calculating the integral intensity of each stained spot.

The nuclease activity of DNase 1 in blood plasma was investigated using the method of radial diffusion in an agarose gel.

To prove the possibility of implementing the declared purpose and achieving the indicated medical and technical result, we present the following data.

Example 1.

A venous blood was taken from pregnant A. at 32 weeks of gestation. When determining the characteristics of cfDNA in blood plasma, it was found that the concentration of cfDNA was 340 ng / ml, the number of rDNA copies was 638 in cfDNA, the ratio of cc rDNA / rDNA genome was 1.5, the number of mtDNA copies was 180 in cfDNA, and the cc mtDNA / genome ratio was mtDNA - 0.44, DNase nuclease activity 1-5.4 U / ml. During the entire pregnancy, pregnant A. had no symptoms of preeclampsia. She gave birth to a girl on time (weight 3634 g, height 52 cm). The postpartum period was uneventful.

Example 2.

The venous blood was taken from pregnant S. at 36 weeks of pregnancy when she was admitted to the hospital. In blood plasma, the following was found: the concentration of cfDNA was 4890 ng / ml, the number of rDNA copies in cfDNA was 1750, the ratio of cc rDNA / rDNA genome was 5.4, the number of mtDNA copies in cfDNA was 2300, the ratio of cc mtDNA / mtDNA genome was 12. 8, nuclease activity - DNase 1-18 U / ml. Severe preeclampsia was diagnosed.

At the beginning of labor, there were complaints of headache, increased blood pressure, preconvulsive state. Despite the appropriate treatment carried out in the intensive care unit within 2 hours, the patient's condition did not improve, which is why a cesarean section was performed. A boy was born (weight 2100 gr., Height 47 cm). After childbirth, appropriate intensive care was required for 8 days.

Example 3. From pregnant V., venous blood was taken at 39 weeks of pregnancy upon admission to the pathology department with complaints of edema of the lower extremities and an increase in blood pressure up to 150 mm Hg. In blood plasma, the following was found: the concentration of cfDNA was 580 ng / ml, the number of rDNA copies was 780 in cfDNA, the ratio of cc rDNA / rDNA genome was 1.4, the number of mtDNA copies was 198 in cfDNA, and the cc mtDNA / mtDNA genome was 0. 56, nuclease activity - DNase 1-8 U / ml. The patient's condition was regarded as moderate preeclampsia. Symptomatic therapy was carried out, the tactics of managing labor through the vaginal birth canal using epidural anesthesia was chosen. The delivery went without complications, a boy was born weighing 3760 grams, 53 cm long. The postpartum period was uneventful. She was discharged from the hospital on the 5th day in a satisfactory condition.

The proposed method was used to determine severe preeclampsia in 57 pregnant women. In all cases, the diagnosis of severe preeclampsia was confirmed clinically, as a result of the choice of medical tactics based on the proposed method, maternal mortality was not observed, there was no perinatal mortality, the incidence was 61.4% due to the immaturity of the child's lung tissue and respiratory problems.

The use of this diagnostic method will make it possible to identify a critical condition in pregnant women with low-symptom preeclampsia; timely diagnostics and therapy will reduce maternal and perinatal morbidity and mortality, which is of great social and national economic importance.

Claims (1)

A method for diagnosing severe preeclampsia in pregnant women, characterized in that the concentration of extracellular DNA (cc DNA), the number of copies of ribosomal DNA in the composition of extracellular DNA (cc rDNA), the number of copies of mitochondrial DNA in the composition of extracellular DNA (cc mtDNA) are determined in the blood plasma of a pregnant woman, nuclease activity of DNase 1; in the nuclear DNA of leukocytes, the number of copies of genomic ribosomal DNA (rDNA genome), the number of copies of genomic mitochondrial DNA (mtDNA genome) are determined, and when cc DNA is more than 1000 ng / ml, cc rDNA is more than 1000, cc mtDNA is more than 200, the ratio of cc rDNA to rDNA genome more than 2, the ratio of mtDNA to mtDNA genome is more than 1.64, DNase 1 nuclease activity is more than 10 U / ml - severe preeclampsia is diagnosed.