Method for prediction of preeclampsia in second trimester of pregnancy

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.

2 ex

 

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

Previously used the term "preeclampsia" is currently in compliance with the approved MoH standards and protocols of the Scientific center of obstetrics, gynecology and Perinatology recommended from 2012 to replace the term "preeclampsia". Because the distinction between preeclampsia and preeclampsia only terminological, and the replacement of the term is relatively recent, in some analogs, to which we refer, uses the term - gestosis.

Despite the undoubted successes in the treatment of preeclampsia (gestosis) she remains a frequent and life-threatening complications of pregnancy and childbirth, leading to health problems in the mother, fetus and newborn. In the structure of maternal mortality in Russia this pathology occupies the third place, its specific weight is 11.8-14.8 per cent (Torchinov A. M., tsakhilov S. G., Arahova D. H. and others the Relevance of pre-eclampsia (preeclampsia) in modern obstetrics. Problems and solutions (literature review) // Probl. Reprod. - 2010. - №3. - 87-91). Pathogenic (pathochemical and pathophysiological mechanisms laid down already in the early stages of pregnancy and due to a violation of invasion of triabl�hundred and gestational transformation of the spiral arteries of the uterus with subsequent formation of deep metabolic damage in the system mother-placenta-fetus.

To date, the early prediction of pre-eclampsia (preeclampsia) during different periods of gestation remain completely unresolved, which makes timely preventative therapy for this obstetric pathology.

Among well-known in the patent and scientific and medical literature methods for the prediction of preeclampsia include the following.

Known method for predicting preeclampsia (patent RF №2249212, 27.03.2005) by examining the blood plasma of the pregnant woman, characterized in that in the early stages (from 5 weeks of gestation) determine the level of carbonyl derivatives of proteins and when the value is equal to 51.7% of the optical density /g of protein and higher predict the development of preeclampsia.

The increase in the number of carbonyl derivatives linked to the degradation of proteins due to the development of oxidative stress, which also occurs when growth retardation (I. Mert, A. Oruc S, 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.

Known method for predicting preeclampsia mild severity from early pregnancy through the study of peripheral venous blood of women, characterized in that in the period from 6 to 12 weeks of gestation examine the relative content of CD3+CD16+ lymphocytes and its values, Rav�s or more than 5.4%, predict the development of preeclampsia mild severity (patent RF №2265221, 27.11.2005).

The disadvantage of this method is the limited possibility to predict only mild preeclampsia less hazardous to the mother, the more pronounced degree.

According to the patent of Russian Federation №2287823 (20.11.2006) predict the occurrence of preeclampsia by analysis of blood serum of a pregnant, characterized in that in the serum to determine the concentration of antibodies to neurospecific enolase and gliofibrillary acidic protein and the level of anti-NDE-antibodies more than 0.6 μg/ml or anti-GFAP antibodies more than 0.9 μg/ml predict the development of preeclampsia.

In the patent of Russian Federation №2304783 (20.08.2007) proposed a method for predicting the development of preeclampsia by examining the peripheral blood of the pregnant woman, characterized in that starting from 22 weeks of pregnancy in serum to determine the concentration of neurospecific enolase and gliofibrillary acidic protein and the magnitude of neurospecific enolase above and 12.4 ng/ml and gliofibrillary acidic protein above 4 ng/ml predict the development of preeclampsia.

These methods are difficult to meet in an obstetric hospital, because require imported special equipment and expensive reagents. In addition, the research is conducted only with the end of the second trimester of his pre�STI, hence the impossibility of an earlier prediction of preeclampsia.

A method of predicting the occurrence of preeclampsia in pregnant women (Patent RF №2478960,10.04.2013), including the study of blood serum by enzyme immunoassay in terms of 16-18 weeks of gestation, characterized in that determine the content of neopterin concentrations and the occurrence of preeclampsia predict when neopterin levels above 8.1 nmol/L.

For this method the characteristic drawbacks of the previous method, it should be noted also that the content of neopterin is a marker activity of cellular immunity, indicator of inflammatory process, changes in a number of pathological conditions (E. A. Sviridov, Telegina T. A. Neopterin and its reduced forms: biological role and involvement in the cellular immune system // advances in biological chemistry. - 2005. - V. 45. - Pp. 355-390).

Known method for predicting preeclampsia (patent RF №2485894, 27.06.2013), including the identification of endothelial function in early pregnancy, characterized in that in the first half of pregnancy twice in a period of 5-7 weeks and 16-19 weeks assess the functional activity of the endothelium on the results of cuff compression test on the brachial artery of the mother and compute the indicator photocatalysis dilatation (PPD) of the brachial artery after the first and second research �about the formula, and if the indicator IFE decreases or increases by less than 4% relative to the initial values in the first study, predict the development of preeclampsia, and if the indicator IFE increased by 4% and more in comparison with initial values, predict uncomplicated pregnancy without clinical manifestations of preeclampsia.

The disadvantages of this method include the need to double examination of the pregnant woman and the nonspecific character, since the index photocatalysis dilation can be changed and at 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 (Zainulina M. S. the Role of endothelial dysfunction in the pathogenesis of placental insufficiency // Regional circulation and microcirculation. 2003. - No. 2. - P. 36-42).

According to the patent of Russian Federation №2481578 (10.05.2013) predict the development of severe preeclampsia with blood, characterized in that a pregnant second trimester count leukocyte index of intoxication and when the value above 1.6 predict the development of severe preeclampsia.

The disadvantage of this method is later than the determination of a prognostic indicator only in the second trimester, the increase above guideline values in this period are considered as risk time�itia severe preeclampsia. Final diagnosis: severe preeclampsia is exposed only in the third trimester. In addition, the present method predicts the development of severe preeclampsia.

In all these patents are no data on the accuracy of the method.

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

This method, like the previous one, has a number of drawbacks:

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

- lack of accuracy and specificity of the method;

- the inability to predict an earlier development of gestosis;

- the authors do not indicate which are the reaction products catalyzed by cathepsin D, they are registered at 280 nm, therefore, lead quantities in relative rather than absolute units of the analyte.

These shortcomings are proposed to be eliminated in the present method.

Object of the invention is to develop a highly informative way to 7-8 weeks of pregnancy to make forecasts.�foresight for the development of preeclampsia (preeclampsia) in the second trimester.

The problem is solved as follows: in the serum of women in 7-8 weeks of pregnancy determine the activity of acid and neutral proteinases and when the value of the activity of acid proteinases above 5.6 mmol/l and the activity of neutral proteinases above to 3.9 mmol/l predict the development of preeclampsia (preeclampsia) in the second trimester of pregnancy.

The technical result obtained during the use of the invention is to improve the accuracy and specificity of the method for predicting preeclampsia that will allow time to appoint adequate pathogenetic therapy.

The novelty of the proposed method lies in the fact that the first proposed to predict the development of preeclampsia (preeclampsia) in the second trimester of pregnancy for a single definition in the serum of women in 7-8 weeks of pregnancy, the activity of acid and neutral proteinases.

In the complex chain of violations, leading to the development of preeclampsia (preeclampsia), the importance of the violation of the balance between anabolism and catabolism of proteins that play a key role in all regulatory cellular reactions (Whitford D. Proteins: Structure and Function. - West Sussex, England: John Wiley & Sons Ltd., 2005. - 542 p.). Shift this balance may be accompanied by profound biochemical changes in the maternal organism, and fetoplacental system (Linear�relova T. N., Linda V. A. Metabolism of the placenta and molecular mechanisms of its regulation. Clinical and experimental study. - LAP LAMBERT Academic Publishing, 2012. - 200 C.). Significant place in these processes is a proteinase that plays a Central role in the biological processes of decay and renewal of protein molecules. And only the combined action of acid and neutral endo - and ectoprotheses, functioning as in the lysosomes and other subcellular fractions: mitochondria, microsomes, nuclei and the cytosol, fully regulates the metabolism of proteins (Dean R. the decay Processes in the cell. - M.: Publishing house "Mir", 1981. - 120 p.).

Simultaneous determination of serum acid and neutral proteinases increases the accuracy and specificity of the method and allows you to more objectively predict the development of preeclampsia is still at a preclinical stage. The ability to predict the development of obstetric pathology in the second trimester has undoubted practical value (compared with the forecast of its development in the third trimester) because the rate of complications with the development of preeclampsia in the period 1.5-3.4 times higher than when it is a late manifestation (Savelyeva G. M., Krasnopolsky, V. I., Strizhakov A. N. etc. What is the classification of gestosis (preeclampsia) should adhere to the doctor's clinical practice? // Questions of gynecology, obstetrics and �perinatologia. - 2013. - No. 2. - P. 5-8).

We have established prognostic parameters of the development of obstetric pathology in the second trimester by definition in the serum at 7-8 weeks of pregnancy, the activity of acid and neutral proteinases and acid proteinases above 5.6 mmol/l and the activity of neutral proteinases above to 3.9 mmol/l predict the development of preeclampsia.

Detailed description of the method and examples of its clinical use.

The method is as follows.

Pregnant women to predict the possible development of complications of pregnancy, including preeclampsia, in the morning on an empty stomach produce blood from the cubital vein.

To 0.2 ml of serum is added as a protein substrate of 0.2 ml of 1% albumin in 0.1 M Tris-HCl buffer (pH 7.8) and incubated for 30 minutes at 38°C. stop the Reaction by adding 0.2 ml of 5% solution of trichloroacetic acid, after which the samples centrifuged at 3000 Rev/min In the control sample protein-substrate is added after stopping the reaction. To determine the number of centrifuges formed by proteolysis of tyrosine classical Lowry method using Folin reagent. Sample colorimetric in the visible region of the spectrum at 750 nm. The activity of neutral proteinases is defined as the difference of optical density between the experimental and control samples and expressed mcmol/L. Determination of acid proteinases and their activity calculation performed in a similar manner, using as substrate the reaction of 1% solution of hemoglobin in the sodium acetate buffer (pH 3.5). Calculation of the content of tyrosine spend on calibration graphs constructed using standard solutions of amino acids (ICN, Belgium).

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

Example 1. Patient B-Eva, 24 years old. Surveyed in the consultative outpatient clinic RNIUP. Perfoermance. Pregnancy 7 weeks, suspected ectopic pregnancy. When ULTRASOUND examination and analysis of HCG in the blood this diagnosis was excluded. The woman has also been conducted on the determination of serum activity of acid and neutral proteinases. The activity of acid proteinases amounted to 5.7 mmol/l, neutral to 4.0 μmol/l, which, according to the claimed method, showed an opportunity for the development of preeclampsia. Pregnant woman refused hospitalization for preventive therapeutic interventions. At 20 weeks gestation, the woman was admitted to the Department of pathology of pregnancy. Diagnosis: preeclampsia moderate severity (edema, BP 140/90). A patient treatment of pre-eclampsia. At the end of therapy was discharged in satisfactory condition. The pregnancy is completed�plastic surgery in full-term childbirth. Thus, the diagnosis of the claimed method was confirmed.

Example 2. The patient-ova, 23 years. Surveyed in the consultative outpatient clinic RNIUP. Avtomobilnaya. Diagnosis: pregnancy 8 weeks. Anemia. After the standard survey, also carried out the determination in the serum activity of proteinases. The activity of acid proteinases was 5.6 mmol/l, the activity of neutral to 3.9 mmol/L. These values of activity indicate the absence of biochemical signs of pre-eclampsia.

Follow-up of a woman until the end of pregnancy, culminating in the full-term birth of a child, confirmed the lack of development of preeclampsia.

According to the claimed method were examined 95 women at 7-8 weeks of gestation in serum determined the activities of acid and neutral proteinases. Of the screened patients in 34 the activity of acid proteinases was 5.6 mmol/l or below, the activity of neutral to 3.9 mmol/l and below. Surveillance in this group of pregnant women for the development of preeclampsia is not revealed. In 61 patients, these figures were respectively above 5.6 mmol/l (neutral proteinase) and up to 3.9 mmol/l (acid proteinase). In 60 of the 61 women with these values is the activity of proteinases in the second trimester of pregnancy was diagnosed W�eclampsia, and only one of them is the development of pre-eclampsia occurred. Analysis of the obtained values showed that the true-positive result occurred in 60 cases, false - positive in one case, a true negative result was in 34 cases, false negative was absent.

From these data it follows that the accuracy and specificity of the method was 98%.

The above confirms the prognostic value of the claimed method. Thus, the claimed method is more effective in comparison with well-known and has several advantages.

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

2. Availability of application of the method in clinical practice.

3. A single study.

4. The possibility of early prediction of pre-eclampsia. The claimed method for predicting the development of preeclampsia in the second trimester makes timely preventive therapy of this obstetric pathology that eventually will have favourable impact on perinatal outcomes and reduce neonatal morbidity.

Method for prediction of preeclampsia in the second trimester of pregnancy by examining blood, characterized in that in the serum of women in 7-8 weeks of pregnancy determine the activity of acid and neutral proteinases and acid activity value PR�Teens above 5.6 mmol/l and the activity of neutral proteinases above to 3.9 mmol/l predict the development of preeclampsia in the second trimester of pregnancy.



 

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