RU2718725C1 - Method for early prediction of fetal growth retardation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to obstetrics and pediatrics, and can be used for early prediction of fetal growth retardation. That is ensured by measuring the level of carbonyl derivatives in the peripheral venous blood of women at 6–9 weeks of gestation, and if the value is more than 1.9 mcmol/l, the fetus growth retardation is predicted.

EFFECT: method provides higher accuracy and specificity of early prediction of fetal growth retardation and timely pathogenetic therapy.

1 cl, 2 ex

Description

The invention relates to medicine, namely to obstetrics and perinatology, and can be widely used for early (at 6–9 weeks of gestation) prediction of fetal growth retardation (PSC).

PSR is a serious complication of pregnancy, leading to high perinatal morbidity and mortality. The frequency of this obstetric pathology has not been reduced to the present time and ranges from 5 to 40% in different countries (Strizhakov A.N., Makatsaria A.D., Timokhina E.V., Baimuradova S.M., Kozlova W.A. Clinical the importance of acquired and inherited forms of thrombophilia in the pathogenesis of fetal growth retardation syndrome // Questions of Gynecology, Obstetrics and Perinatology. 2009. T. 8, No. 2. P. 16-21; Dessi A., Ottonello G., Fanos V., J. Physiopathology of intrauterine growth retardation: from classic data to metabolomics // Matern. Fetal Neonatal. Med. 2012. Vol. 25 (5). P. 13-18; Sharma D., Shastri S., Sharma P. Intrauterine Growth Restriction: Antenatal and Postnatal Aspects // Clin. Med. Insights Pediatr., 2016. Vol. 10. P. 67-83; Kady SM, Gardosi J. Perinatal mortality and fetal growth restriction // Best Pract. Res. Clin. Obstet. Gynaecol. 2004. Vol. 18. P. 397-410; Leitner Y., Harel S., Geva R., Eshel R., Yaffo A., Many A. Neurodevelopmental outcome of children with intrauterine growth retardation: a longitudinal, 10-year prospective study // J. Child. Neurol. 2007. Vol. 22 (5). P. 580-587). Currently, despite numerous studies and certain successes, there remains a difficulty in early and objective forecasting of PSA, which is necessary to improve the treatment tactics of this pathology. The above determines the relevance of the development of this method.

Among the methods for diagnosing PSA known in the medical and patent literature, ultrasound studies of a number of fetometric indicators can be noted in comparison with normal indicators for a certain gestational period, but they are informative only in the third trimester.

Dopplerometric mapping of the uterine vessels and determination of the state of their blood supply based on the pulsation index are used to detect PWR (Bunin A.T., Fedorova M.V. Fetal development retardation syndrome: pathogenesis, clinical features, diagnosis and treatment // Obstetrics and Gynecology. 1988. No. 7, pp. 74-78.). The determination of volumetric blood flow in the fetal aorta, umbilical vein, or internal carotid artery is also known, followed by analysis of the blood flow velocity curves (systolic-diastolic ratio and resistance index). However, these indicators are nonspecific and can equally change under different pathological conditions, in particular with preeclampsia (Sidorova I.S., Nikitina N.A. A modern look at the problem of preeclampsia: arguments and facts // Obstetrics and Gynecology. 2013. No. 5 . S. 10-16). Moreover, the predictor value of all these methods for predicting PMS in the second half of pregnancy is low, since there is little time to prevent the development of this pathology.

A known method for diagnosing fetal growth retardation (RF patent No. 2172958, 2001) by analyzing peripheral blood, including DNA extraction, polymerase chain reaction and determination of the PL-All allele of the glycoprotein IIIa gene, in the presence of which the intrauterine growth retardation is diagnosed. The disadvantage of this method is:

1. The complexity, the impossibility of conducting such studies in conventional obstetric hospitals that do not have special equipment necessary for polymerase chain reactions;

2. The inability to carry out a forecast of the development of a missile defense system, which makes it difficult to conduct timely treatment and preventive measures.

3. The lack of information on the accuracy of the method.

RF patent No. 2425379 (2011) proposes a method for diagnosing fetal growth retardation by determining in amniotic fluid at 16-19 weeks of gestation by capillary electrophoresis of arginine and proline content, calculating their ratio and with a coefficient value of 3.2 and higher, diagnosing fetal growth retardation.

This method:

1. Invasive, amniotic fluid taken in the second trimester of pregnancy is used, which can only be carried out by a highly qualified specialist who has permission to carry out this procedure.

2. Diagnosis in this period does not allow the implementation of preventive measures to prevent this obstetric pathology.

3. Despite the assertion about the specificity of the method, its value is not indicated (in%), a change in the amino acid content in the amniotic fluid at 16-18 weeks of gestation was also detected with preeclampsia (Pogorelova TN, Gunko V.O., Palieva N. V., Alliluyev I.A., Kaushanskaya L.V., Larichkin A.V. Imbalance of free amino acids in amniotic fluid during preeclampsia // Obstetrics and Gynecology. 2019. No. 2. P. 60-67.).

A known method for predicting intrauterine growth retardation of the fetus (RF patent No. 2526178, 2014), characterized in that from early pregnancy determine the relative content of CD3 ⁺ / CD16 ⁺ CD56 ⁺ lymphocytes, the level of the C ₃ component of complement and soluble tumor necrosis factor receptor ( sTNF-R) in the venous blood of women. The prognostic index (PI) is calculated by the formula:

PI = -0.8911X ₁ -0.0321X ₂ -2.3669X ₃ +11.0779,

where X ₁ is the relative content of CD3 ⁺ / CD16 ⁺ CD56 ⁺ lymphocytes,%;

X ₂ is the concentration of the C3 component of complement, mg / dl;

X ₃ is the concentration of sTNF-R, ng / ml.

At PI <0, intrauterine growth retardation in the second half of pregnancy is predicted, and at PI> 0, a conclusion is made about the low risk of developing this pathological condition.

The disadvantages of the method include:

1. Multicomponent, complex calculation, making it difficult to obtain an opinion on the forecast.

2. The lack of information about the accuracy and especially the specificity of the method, since a change in many immunological parameters including the used CD ⁺ lymphocytes also occurs in gestosis (RF patent No. 2265221, 2005).

RF patent No. 2540928, 2015 proposes a method for predicting the development of placental insufficiency with fetal growth retardation syndrome of the 2nd to 3rd degree, according to which coagulation factor genes are analyzed in the peripheral venous blood after DNA extraction. 20210G / A FII, 1691G / A FV and 10976G / A FVII. In the case of the detection of the 10976G FVII allele and the 10976GG FVII genotype, an increased risk of developing placental insufficiency with a fetal growth retardation syndrome of the 2nd or 3rd degree in pregnant women is predicted. In the presence of combinations of genotype 20210GG FII and allele 10976A FVII; alleles 20210G FII, 10976A FVII with genotype 1691GG FV or alleles 20210G FII and 10976A FVII predict a low risk of developing placental insufficiency with fetal growth retardation syndrome of the 2nd-3rd degree.

The disadvantages of this method include the following:

1. Methodological complexity: DNA extraction and analysis of several coagulation factor genes. Their study requires a modern genetic laboratory.

2. Similar analytical studies have not been carried out in women with placental insufficiency without the formation of a syndrome of RPS. At the same time, such a complication of gestation is observed quite often. In this case, it is necessary to exclude the detection of the 10976GFVII allele and 10976GGFVII genotype in order to prove the role of PSA in these reactions.

3. The lack of information on the accuracy of the method.

There is a method for predicting the degree of risk of developing fetal growth retardation syndrome (patent No. 2578386, 2016), which includes collecting analytical, anthropometric data, the age of the mother upon the occurrence of a real pregnancy, determining numerous clinical indicators and echographic signs, perform a ball assessment of the identified indicators. With a score of 1-30, a low risk of developing fetal growth retardation syndrome is determined. With a score of 55-70 - a high degree of risk.

The disadvantage of this method, as well as some of the already mentioned methods, is:

1. Late determination of prognostic indicators (only in the II and III trimesters), the increase of which (in points) is considered as a risk of the development of the PSC syndrome, although in the III trimester this obstetric pathology, especially with a high score, may already take place.

2. The extreme multiplicity of prognostic parameters.

3. The lack of information on the accuracy and specificity of the method.

4. The use of only relative parameters reduces the sensitivity of the method.

The prototype of the claimed invention selected a method for early prediction of fetal growth retardation syndrome (RF patent No. 2295132, 03/10/2007) by examining the peripheral venous blood of a pregnant woman, characterized in that at 6-12 weeks of gestation an indicator of the content of vasculoendothelial growth factor (VEGF) is determined and its values equal to or less than 20 pg / ml, the development of the syndrome of fetal growth retardation is predicted.

The disadvantages of this method include:

1. The need for equipment for enzyme-linked immunosorbent assay, which is often absent in obstetric hospitals of medium-sized cities.

2. Insufficient accuracy (81.25%) and specificity (87.5%).

3. A change in the indicator of this important growth factor associated with endothelial dysfunction in the first trimester of gestation also occurs with other pregnancy complications, including the threat of abortion, gestosis, and placental insufficiency, which are also characterized by endothelial dysfunction (A. Orlov. Screening markers of physiological and complicated pregnancy // Abstract of thesis ... doctor of medical sciences Rostov-on-Don, 2006.48 s; Zenkina Z.V., Linde V.A., Drukker N.A., Nekrasova M .G., Shkotova E.O., Ganikovskaya Yu.V. Imbalance of biologically active eschestv amniotic fluid and its importance in the pathogenesis of late preterm births // Proceedings of the XV All-Russian Scientific Forum "Mother and Child". Moscow, 2014. pp 61-62.).

In this regard, the specificity of the forecast method based on the determination of only this factor is small.

These disadvantages are proposed to eliminate in the present method.

The objective of the invention is to develop a highly informative method that allows in 6-9 weeks to predict fetal growth retardation.

The problem is solved as follows: in the peripheral venous blood of a woman, the level of carbonyl derivatives is determined and, with its value above 1.9 μmol / l, fetal growth retardation is predicted with an accuracy of 98%.

The technical result obtained during the use of the invention consists in increasing the accuracy and specificity of predicting fetal growth retardation, which allows timely preventive therapy of this pathology before the onset of clinical signs.

The novelty of the proposed method lies in the fact that for the first time it is proposed to predict the development of fetal growth retardation by a single determination in the peripheral venous blood of women at 6-9 weeks of pregnancy the content of carbonyl derivatives.

In a complex chain of disorders leading to the development of fetal growth retardation, molecular-biochemical changes in the mother-placenta-fetus system occurring as a result of various external or internal causes, including as a result of intrauterine hypoxia, which according to the literature with PSA (Pogorelova T.N., Gunko V.O., Avrutskaya V.V., Kaushanskaya L.V., Durnitsyna O.A. Disruption of placental metabolism of amino acids with fetal growth retardation // Biomedical Chemistry. 2017. T. 63 , No. 3. S. 266-271). In many ways, metabolic damage during a complicated pregnancy is determined by a violation of the state of proteins that perform numerous regulatory functions and are the “molecular machine” that implements the cell information program. Damage to the structure of proteins under hypoxia is manifested in their oxidative modification, oxidized forms of proteins serve as early markers of cell damage. One of the main reactions of protein oxidation is carbonylation (Dubinina E.E. Products of oxygen metabolism in the functional activity of cells (life and death, creation and destruction). Physiological and clinical-biochemical aspects. St. Petersburg: Medical Press, 2006 . 400 s). Since various proteins with enzymatic and hormonal activity, as well as performing trophic functions, are capable of undergoing carbonylation, the spectrum of reactions regulated by them is quite wide.

Therefore, a change in the degree of oxidative carbonylation of proteins, manifested in an increase in the level of carbonyl derivatives, may be one of the pathogenetic mechanisms of the formation and further development of fetal growth retardation.

We have established prognostic parameters for the development of this obstetric pathology of pregnancy at 6–9 weeks according to the determination of the level of carbonyl derivatives in peripheral venous blood and with its value above 1.9 μmol / L, the development of fetal growth retardation is predicted.

A detailed description of the method and examples of its clinical use.

The method is as follows.

Pregnant women examined under the Obstetric Monitoring program, conducted at the outpatient consultative department of the Research Institute of Obstetrics and Pediatrics at the Federal State Medical Institution of the Rostov State Medical University in order to predict possible development of complicated gestation, including fetal growth retardation, perform peripheral venous blood sampling on an empty stomach in the morning on an empty stomach. For analysis using blood serum obtained after centrifugation of whole blood for 5-10 minutes at 1500 rpm (0.1 ml is enough), diluted with saline in a ratio of 1: 5. When analyzing the oxidative modification of proteins, the control and experimental samples contain 0.05 ml of diluted serum and 0.95 ml of 1/15 M phosphate buffer (pH 7.4). After 10 minutes of incubation at 37 ° in a water bath, 1 ml of a 0.01 M solution of 2,4-dinitrophenylhydrazine (2,4-DNPH) is poured into test tubes. Instead of 2,4-DNPH, an equal volume of a 2M HCl solution was added to the control samples. To precipitate proteins, 1 ml of a 20% trichloroacetic acid solution is added to each sample. Samples are incubated at room temperature for 20 minutes, then centrifuged at 3000 rpm for 15 minutes. Next, the precipitate is washed with a mixture of ethyl alcohol and ethyl acetate in a ratio of 1: 1. The resulting precipitate was dissolved in 3 ml of an 8 M urea solution. As a result of oxidative modification of proteins, carbonyl derivatives appear, which in the presence of 2,4-DNPH form 2,4-dinitrophenylhydrazones. The optical density of the resulting compound is recorded spectrophotometrically at a wavelength of 363 nm. The calculation of the level of carbonyl derivatives is carried out using a molar extinction coefficient of 22 × 10 ³ M ^-1 cm ^-1 (Arutyunyan A.V., Dubinina E.E., Zybina N.N. Methods for assessing free radical oxidation and the antioxidant system of the body // Methodical recommendations SPb .: IKF “Foliant”, 2000. 104 p.). The results are expressed in μmol / L. Analysis time is 1.2 hours.

The performance of the proposed method is confirmed by the following clinical examples.

Example 1: patient K-va, 22 years old. She was examined in the outpatient advisory department of NIIAP. She addressed in the first trimester of pregnancy about the threat of miscarriage, manifested by pulling pains in the lower abdomen. Ultrasound examination showed cervical hypertonicity. In a laboratory study of the venous blood of a woman at 6 weeks of gestation, the content of carbonyl derivatives was found to be 2.0 μmol / L, which indicated the possibility of developing fetal growth retardation. In this regard, the patient was assigned to the risk group for the formation of this pregnancy complication and continued to be observed in the outpatient consultation department. In the period of 33 weeks of pregnancy, an ultrasound study revealed a PSA. The woman was hospitalized in the hospital, was monitored and received therapy in accordance with the standard of care. Pregnancy ended with an urgent delivery in the period of 39-40 weeks. and the birth of a full-term baby with a body weight of 2800 g, a height of 50 cm, an Apgar score of 7-8 points. The baby’s diagnosis at birth is the PSA.

Example 2: Patient Ao, 24 years old. She turned to the outpatient consultative department of NIIAP in 8 weeks of pregnancy with a burdened obstetric history: medical abortion, vegetovascular dystonia of the hypotonic type, autoimmune thyroiditis, hypothyroidism. A laboratory blood test revealed that the level of carbonyl derivatives was 1.9 μmol / L, which indicates the absence of a forecast for fetal growth retardation. During the entire period of gestation, the patient was observed in an outpatient consultation department. In the 31st week of pregnancy with ultrasound, the fetal size corresponded to a gestational period. The pregnancy ended with an urgent birth in the period of 39-40 weeks and the birth of a live full-term baby with a body weight of 3410 g, a height of 51 cm, an Apgar score of 8-9 points. Diagnosis at birth: healthy.

According to the claimed method, 64 women were examined in whom the content of carbonyl derivatives was determined in venous blood at 6–9 weeks of pregnancy. Of the examined patients, in 16 the level of carbonyl derivatives was higher than 1.9 μmol / L. Of these, 15 developed PSA and only one development of this pathology did not occur. In 48 pregnant women, the content of carbonyl derivatives was 1.9 µmol / L and lower, and in all cases there was no PSA. The analysis of the obtained values showed that a truly positive result occurred in 15 cases, a false positive in one case, a true negative result in 48 cases, and a false negative was absent.

From the above data it follows that the accuracy and specificity of the method is 98%. The above confirms the prognostic value of the method. Thus, the inventive method is more efficient than known, and has several advantages:

1. High accuracy and specificity - 98%.

2. The availability of the method in wide clinical practice.

3. The speed of obtaining research results.

4. Possibility of early forecasting of air defense systems.

Testing of the claimed invention was carried out on a sufficient volume of clinical material, which confirms the efficiency of the method.

The inventive method for predicting fetal growth retardation allows timely preventive therapy of this obstetric pathology, which ultimately will favorably affect perinatal outcomes and reduce the incidence of newborns.

Claims (1)

A method for early prediction of fetal growth retardation, including the study of peripheral venous blood, characterized in that the level of carbonyl derivatives is determined at 6–9 weeks of gestation and fetal growth retardation is predicted when it is higher than 1.9 μmol / L.