RU2552931C1 - Method for prediction of preeclampsia in second trimester of pregnancy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a method for the prediction of preeclampsia in the second trimester of pregnancy by blood examination, differing by the fact that the activity of acid and neutral proteinases is measured in blood serum of the women 7-8 weeks pregnant; if the activity of acid proteinases is more than 5.6 mcmole/l, while the activity of neutral proteinases is more than 3.9 mcmole/l, the preeclampsia progression in the second trimester of pregnancy is predicted.

EFFECT: higher accuracy and specificity of the method for the prediction of gestational toxicosis.

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Description

The invention relates to medicine, namely to obstetrics, and will be widely used for objectively predicting the development of preeclampsia (preeclampsia) in the second trimester of pregnancy.

The previously used term "gestosis" is currently in accordance with the standards and protocols approved by the Ministry of Health of the Scientific Center for Obstetrics, Gynecology and Perinatology, it is recommended to replace the term "preeclampsia" since 2012. Since the difference between the concepts of gestosis and preeclampsia is only terminological, and the replacement of the term is relatively recent, in some of the analogues to which we refer, the term gestosis is used.

Despite the undoubted successes in the treatment of preeclampsia (preeclampsia), it remains one of the frequent and formidable complications of pregnancy and childbirth, leading to a violation of the health of the mother, fetus and newborn. In the structure of maternal mortality in Russia, this pathology takes third place, its specific gravity is 11.8-14.8% (Torchinov AM, Tsakhilova SG, Sarakhova D.Kh. et al. The relevance of preeclampsia (preeclampsia) in modern obstetrics Problems and solutions (literature review) // Probl. Repro. - 2010. - No. 3. - 87-91). Pathogenetic (pathochemical and pathophysiological) mechanisms are established already in the early stages of pregnancy and are caused by impaired trophoblast invasion and gestational transformation of the uterine spiral arteries with subsequent formation of deep metabolic damage in the mother-placenta-fetus system.

To date, issues of early prediction of the development of preeclampsia (gestosis) at different periods of gestation remain completely unresolved, which complicates the timely implementation of preventive therapy for this obstetric pathology.

Among the known methods for predicting gestosis known in the patent and medical literature are the following.

A known method for predicting gestosis (RF patent No. 22429212, March 27, 2005) by examining a pregnant woman’s blood plasma, characterized in that in the early stages (from 5 weeks of gestation) the level of carbonyl derivatives of proteins is determined and at a value equal to 51.7 units. optical density / g protein and higher predict the development of gestosis.

The increase in the number of carbonyl derivatives is associated with the destruction of proteins due to the development of oxidative stress, which also occurs with fetal growth retardation (Mert I., Oruc AS, Yuksel S. et al. Role of oxidative stress in preeclampsia and intrauterine growth restriction // J. Obstet Gynaecol. Res. - 2012. - Vol.38, N 4. - P. 658-664), which indicates its lack of specificity.

A known method for predicting mild gestosis from early pregnancy by examining the peripheral venous blood of women, characterized in that in the period from 6 to 12 weeks of gestation, the relative content of CD3 + CD16 + lymphocytes is examined and with its values equal to or more than 5.4%, predict the development of mild gestosis (RF patent No. 2265221, 11/27/2005).

The disadvantage of this method is the limited ability to predict only mild severity of gestosis, which is less dangerous for the mother than more pronounced degrees.

According to the patent of the Russian Federation No. 2287823 (11/20/2006), the occurrence of gestosis is predicted by examining the blood serum of a pregnant woman, characterized in that the concentration of antibodies to the neurospecific enolase and gliofibrillar-acid protein is determined in the blood serum and when the level of anti-NDE antibodies is more than 0.6 μg / ml or anti-GFAP antibodies greater than 0.9 μg / ml predict the development of gestosis.

RF patent No. 2304783 (08/20/2007) proposes a method for predicting the development of gestosis by examining the peripheral blood of a pregnant woman, characterized in that, starting from 22 weeks of pregnancy, the concentration of neurospecific enolase and gliofibrillar acid protein is determined in blood serum and with a neurospecific enolase concentration higher than 12.4 ng / ml and gliofibrillar acid protein above 4 ng / ml predict the development of gestosis.

These methods are difficult to perform in an obstetric hospital, because require special imported equipment and expensive reagents. In addition, the study is conducted only from the end of the second trimester of pregnancy, which makes it impossible to earlier predict gestosis.

A known method for predicting the occurrence of gestosis in pregnant women (RF Patent No. 2478960.10.04.2013), including a blood serum enzyme-linked immunosorbent assay in terms of 16-18 weeks of gestation, characterized in that the content of neopterin is determined and the occurrence of gestosis is predicted at a neopterin level above 8.1 nmol / L.

This method is characterized by the disadvantages inherent in the previous method, it should also be noted that the content of neopterin - a marker of cellular immunity activity, an indicator of the development of the inflammatory process, changes with a number of pathological conditions (Sviridov EA, Telegin T.A. Neopterin and its restored forms : biological role and participation in cellular immunity // Successes in Biological Chemistry. - 2005. - T. 45. - P.355-390).

A known method for predicting preeclampsia (RF patent No. 2485894, 06/27/2013), including determining the function of endothelium in early pregnancy, characterized in that in the first half of pregnancy twice at a period of 5-7 weeks and 16-19 weeks, the functional activity of the endothelium is assessed by the results of a cuff compression test on the brachial artery of the mother and calculate the rate of flow-dependent dilatation (BPD) of the brachial artery after the first and second studies according to the formula, and if the IPE decreases or increases by less than 4% in relation to the initial values of the first study, the development of gestosis is predicted, and if the IFE indicator increases by 4% or more compared to the initial values, an uncomplicated pregnancy is predicted without clinical manifestations of gestosis.

The disadvantages of this method include the need for a double examination of a pregnant woman and its nonspecificity, since the rate of flow-dependent dilatation can be changed even with such a complication of gestation as placental insufficiency, the development of which is also associated with a modification of the functional activity of the endothelium (Zaynulina M.S. The role of endothelial dysfunction in pathogenesis of placental insufficiency // Regional blood circulation and microcirculation. 2003. - No. 2. - S. 36-42).

According to the patent of the Russian Federation No. 2481578 (05/10/2013), the development of severe preeclampsia is predicted using a blood test, characterized in that a leukocyte intoxication index is calculated in a pregnant woman in the second trimester and, with a value above 1.6, the development of severe preeclampsia is predicted.

The disadvantage of this method is the late determination of the prognostic indicator only in the second trimester, the increase of which is higher than the standard values in this period is considered as a risk of severe preeclampsia. The final diagnosis: severe preeclampsia is set only in the third trimester. In addition, this method predicts the development of only a severe degree of preeclampsia.

In all patents cited there is no data on the accuracy of the method.

The prototype of the claimed invention selected a method for predicting gestosis (RF patent No. 2263913, 10.11.2005) by examining the peripheral venous blood of women, characterized in that in the period of 6-13 weeks of gestation, the activity of cathepsin D is determined and if its value is equal to or more than 0.021 units. Act. f / h predict the development of gestosis in the 3rd trimester.

This method, like the previous ones, has several disadvantages:

- limited information content of the method: the use of one enzyme only from the group of acidic lysosomal endopeptidases;

- lack of accuracy and specificity of the method;

- the impossibility of predicting an earlier development of gestosis;

- the authors do not indicate which reaction products catalyzed by cathepsin D are detected by them at 280 nm; therefore, they give values in relative rather than absolute units of the substance being determined.

These disadvantages are proposed to eliminate in the present method.

The objective of the invention is to develop a highly informative method that allows for 7-8 weeks of pregnancy to predict the development of preeclampsia (gestosis) in the second trimester.

The problem is solved as follows: in the blood serum of women at 7-8 weeks of pregnancy, the activity of acidic and neutral proteinases is determined and, with an activity of acidic proteinases above 5.6 μmol / L and activity of neutral proteinases above 3.9 μmol / L, the development of preeclampsia is predicted ( preeclampsia) in the second trimester of pregnancy.

The technical result obtained during the use of the invention consists in increasing the accuracy and specificity of the method for predicting gestosis, which will allow timely prescribing appropriate adequate pathogenetic therapy.

The novelty of the proposed method lies in the fact that for the first time it is proposed to predict the development of preeclampsia (preeclampsia) in the second trimester of pregnancy by a single determination in the blood serum of a woman in 7-8 weeks of pregnancy activity of acidic and neutral proteinases.

In a complex chain of disorders leading to the development of preeclampsia (preeclampsia), an imbalance in the balance of protein anabolism and catabolism, which play a key role in all regulatory cellular responses (Whitford D. Proteins: Structure and Function. - West Sussex, England: John Wiley & Sons Ltd., 2005 .-- 542 p.). A shift in this balance may be accompanied by profound biochemical changes in the maternal body, as well as the fetoplacental system (Pogorelova T.N., Linde V.A. Placenta metabolism and molecular mechanisms of its regulation. Clinical and experimental study. - LAP LAMBERT Academic Publishing, 2012. - 200 s.). A significant place in these processes is occupied by proteinases, which play a central role in the processes of biological decay and renewal of protein molecules. Moreover, only the combined action of acidic and neutral endo- and exoproteinases, which function both in lysosomes and in other subcellular fractions: mitochondria, microsomes, nuclei, and the cytosol, fully regulates protein metabolism (Dean R. Cell decomposition processes. - M .: Publishing House -to "Mir", 1981. - 120 p.).

The simultaneous determination of acidic and neutral proteinases in the blood serum increases the accuracy and specificity of the method and makes it possible to more objectively predict the development of preeclampsia even at the preclinical stage. The ability to predict the development of this obstetric pathology in the second trimester is of undoubted practical value (compared with the forecast for its development in the third trimester), since the frequency of complications in the development of preeclampsia in this period is 1.5-3.4 times higher than with its late manifestation (Savelyeva G.M., Krasnopolsky V.I., Strizhakov A.N. et al. What classification of gestosis (preeclampsia) should a clinical practice doctor adhere to? // Questions of gynecology, obstetrics and perinatology. - 2013. - No. 2. - S. 5-8).

We have established prognostic parameters for the development of this obstetric pathology in the second trimester, as determined in the blood serum at 7-8 weeks of pregnancy, the activity of acidic and neutral proteinases and with the value of acidic proteinases above 5.6 μmol / L and the activity of neutral proteinases above 3.9 μmol / L predict the development of preeclampsia.

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

The method is as follows.

In a pregnant woman, in order to predict the possible development of complicated gestation, including preeclampsia, in the morning on an empty stomach they take blood from the cubital vein.

0.2 ml of serum is added to 0.2 ml of blood serum as a substrate protein in 0.1 M Tris-HCl buffer (pH 7.8) and incubated for 30 minutes at 38 ° C. The reaction is stopped by the addition of 0.2 ml of a 5% trichloroacetic acid solution, after which the samples are centrifuged at 3000 rpm. In the control samples, the protein substrate is added after stopping the reaction. In centrifuges, the amount of tyrosine formed during proteolysis is determined by the classical Lowry method using Folin's reagent. Samples are colorimetric in the visible spectrum at 750 nm. The activity of neutral proteinases is defined as the difference in optical density between the experimental and control samples and is expressed in μmol / L. The determination of acidic proteinases and the calculation of their activity are carried out in a similar way, using as a reaction substrate a 1% solution of hemoglobin in sodium acetate buffer (pH 3.5). The calculation of tyrosine content is carried out according to calibration graphs compiled using standard amino acid solutions (ICN, Belgium).

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

Example 1. Patient B-eva, 24 years old. She was examined on an outpatient basis in the consultative clinic of RNIIAP. The first pregnant. Pregnancy 7 weeks., Suspicion of an ectopic pregnancy. With ultrasound examination and analysis of hCG in the blood, this diagnosis was excluded. The woman was also determined in the blood serum activity of acidic and neutral proteinases. The activity of acidic proteinases was 5.7 μmol / L, neutral - 4.0 μmol / L, which, according to the claimed method, testified to the possibility of preeclampsia. The pregnant woman refused hospitalization for preventive therapeutic measures. At 20 weeks of gestation, the woman was admitted to the pathology department of pregnant women. Diagnosis: preeclampsia of moderate severity (edema, blood pressure 140/90). Inpatient treatment of preeclampsia was performed. At the end of the course of therapy, she was discharged in satisfactory condition. Pregnancy ended in childbirth on time. Thus, the diagnosis made by the present method was confirmed.

Example 2. Patient K-ova, 23 years old. She was examined on an outpatient basis in the consultative clinic of RNIIAP. Re-pregnant. Diagnosis: 8 weeks pregnancy. Anemia. After a generally accepted examination, an additional determination was made of the activity of proteinases in the blood serum. The activity of acidic proteinases was 5.6 μmol / L, the activity of neutral 3.9 μmol / L. These activity values indicate the absence of biochemical signs of the possibility of preeclampsia.

Subsequent monitoring of the woman until the end of pregnancy, which ended in the birth of a full-term baby, confirmed the absence of preeclampsia.

According to the claimed method, 95 women were examined in whom the activity of acidic and neutral proteinases was determined in the blood serum at 7-8 weeks of gestation. Of the examined patients, in 34 the activity of acidic proteinases was 5.6 μmol / L and lower, the activity of neutral 3.9 μmol / L and lower. Observation in this group of pregnant women did not reveal the development of preeclampsia. In 61 patients, these values were respectively above 5.6 μmol / L (neutral proteinases) and above 3.9 μmol / L (acidic proteinases). In 60 of 61 women with the indicated activity of proteinases, preeclampsia was diagnosed in the second trimester of pregnancy, and only one of them did not develop preeclampsia. The analysis of the obtained values showed that a truly positive result occurred in 60 cases, a false positive in one case, a truly negative result in 34 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 proposed method. Thus, the inventive method is more efficient compared to the known and has several advantages.

1. High accuracy - 98%, specificity - 98%.

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

3. The one-time study.

4. The possibility of early prediction of the development of preeclampsia. The inventive method for predicting the development of preeclampsia in the second trimester 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 predicting preeclampsia in the second trimester of pregnancy by a blood test, characterized in that the activity of acidic and neutral proteinases is determined in the blood serum of women at 7-8 weeks of pregnancy and when the activity of acidic proteinases is higher than 5.6 μmol / L and the activity of neutral proteinases is higher than 3 , 9 μmol / L predict the development of preeclampsia in the second trimester of pregnancy.