RU2614692C1 - Method for prediction of fetal growth retardation for pregnant women with placental insufficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method comprises placental protein PP13 level determination in the venous blood of women in the I trimester of pregnancy. If its level is reduced down to 5.34 pg/ml or less, fetal growth retardation at the background of placental insufficiency at later stages of pregnancy is predicted.

EFFECT: improved accuracy of fetal growth retardation diagnosis, which allows to assign a timely adequate therapy in an earlier period.

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Description

The invention relates to medicine, namely to obstetrics and perinatology, and will find wide application in the development of obstetric tactics for managing patients with fetal growth retardation (PSC).

Of particular interest from obstetrician-gynecologists is manifested in fetal growth retardation associated with high perinatal morbidity (17-36%) and mortality (8-24%) of newborns (WHO). Reliable morphological criteria of placental insufficiency are signs of delay (insufficiency) of the first (at 6-8 weeks) and most importantly the second (at 14-16 weeks) waves of cytotrophoblast invasion into the walls of the uteroplacental arteries (Milovanov A.P., 1999), but the clinical signs of the disease do not appear in either I or II trimesters if there is no ectragenital or neuroendocrine pathology. These facts make the prediction of severe forms of fetal growth retardation absolutely necessary, and priority should be given to determining the development of this complication in the first trimester of pregnancy. Usually, the right treatment allows you to avoid the development of severe forms of PSA.

In this regard, it seems relevant to clarify the early diagnostic markers of fetal growth retardation (PFR) in order to conduct timely treatment measures.

The analysis of patent and medical literature has allowed us to analyze the existence of existing methods for diagnosing fetal growth retardation. A more modern method of diagnosing PSC is the use of several fetometric indicators, but they are informative only in the third trimester of pregnancy - at 26-38 weeks (A.N. Strizhakov, A.N. Bunin, M.V. Medvedev // Obstetrics and gynecology. 1988. No. 6. S. 32-35). Ultrasound diagnosis of PSA is based on a comparison of fetometric indicators obtained as a result of the study, with normative indicators for a given gestational age. One of its most informative parameters is the biparietal size of the fetal head. However, measuring only this indicator cannot be used to detect fetal growth retardation.

Recently, Dopplerometric determination of volumetric blood flow in the fetal aorta, umbilical vein, or internal carotid artery with the subsequent analysis of blood flow velocity curves has been used to identify the syndrome of VAR. Given that a qualitative assessment of blood flow using the systolodiastolic ratio (C / D) and the resistance index reflects the same patterns, in widespread clinical practice for the diagnosis of PSA is also used to determine the pulsation index (PI), an increase which occurs with PSA in the umbilical vein and a sharp decrease in the internal carotid artery. However, all these studies are most reliable in the period from 17-20 weeks of pregnancy. With placental insufficiency and fetal growth retardation, changes in dopplerograms occur, but they are non-specific and in a large percentage of cases occur in the second trimester of gestation. The diagnostic value of one-time dynamic scintigraphy with fetal growth retardation is 75-79% (EM Vikhlyaeva, ZS Hodzhaeva // Obstetrics and gynecology. 1984. No. 6. P. 18-24).

A known method for diagnosing the threatening condition of the fetus according to patent No. 2076624 (1997), which makes it possible to simultaneously register the electrical activity of the heart of the mother and the fetus, and in the absence of a correlative relationship, the threatening condition of the fetus or the degree of violation of the fetus. Regarding this analogue, it can be emphasized that its use in clinical practice allows us to assess the presence of abnormalities in the development of the fetus at a much later stage of pregnancy.

A known method for diagnosing intrauterine growth retardation described in patent No. 2086981 (1997), according to which the absolute content of B-lymphocytes (Ba) and the relative content of neutrophils having receptors for the C3 complement component (EAC-RON) are determined in a woman’s venous blood , and with Ba values greater than or equal to 0.3 × 109 μg / L, and EAC-RON greater than or equal to 16%, the fetal condition is assessed as inappropriate for physiological development and intrauterine growth retardation is diagnosed.

This method is not specific, since changes in the content of B-lymphocytes, as well as neutrophils can be detected in a variety of diseases of the mother, not associated with PSA, and the accuracy of the method is only 80%.

Patent No. 2172958 (2001) proposes a method for diagnosing PSA, which allows diagnosis in the early stages of fetal growth retardation by analyzing peripheral blood, including DNA isolation, polymerase chain reaction and determination of the PL-AII allele of the glycoprotein IIIA gene.

It should be noted that this method has a major drawback, primarily the limited application, due to the complexity of its implementation, expressed in multi-stage and the duration of its execution.

Another method for early prediction of fetal growth retardation syndrome is to determine the content of HLA-DR + monocytes in the peripheral venous blood (patent 2265224, 2004), and when its value is equal to or less than 57.9%, the development of the VAR syndrome is predicted.

It should be noted that this method is also limited in application due to the complexity and duration of its implementation.

RF patent No. 2331365 (2007) proposes a method for diagnosing fetal developmental retardation syndrome by determining the allantoin content in the blood of a pregnant woman, and diagnosing fetal developmental retardation syndrome with a concentration of 2.9 to 5.8 μmol / L.

This method is not specific, since a pronounced decrease in this indicator is observed not only with PSC, but also with non-developing pregnancy and early spontaneous abortions.

A known method for the diagnosis of fetal growth retardation (RF patent No. 2 425 379, 2011) by examining the biological fluid of a pregnant woman, characterized in that in a pregnant woman in terms of 16-19 weeks in the amniotic fluid, the content of arginine and proline is determined by capillary electrophoresis, their coefficient is calculated relations and with a magnitude of a coefficient equal to 3.2 and higher, a fetal growth retardation is diagnosed.

The disadvantages of this method include the occurrence of a threat of termination of pregnancy due to the invasiveness of the method during amniocentesis, as well as the diagnostic rather than prognostic orientation of the method.

A known method for predicting intrauterine growth retardation (patent No. 2526178, 2014), characterized in that from early pregnancy determine the relative content of CD3 + CD16 + 56 + lymphocytes, the level of the C3 component of complement and soluble tumor necrosis factor receptor (sTNF-R ) in the venous blood of a woman, the prognostic index (PI) is calculated by the formula:

P1 = -0.8911X1-0.0321X2-2.3669X3 + 11.0779,

where X1 is the relative content of CD3 + CD16 + CD56 + - lymphocytes,%;

X2 is the concentration of the C3 component of complement, mg / dl;

X3 - concentration of sTNF-R, ng / ml

and with PI less than 0, intrauterine growth retardation in the second half of pregnancy is predicted, and with PI more than 0, a conclusion is made about the low risk of developing this pathological condition.

The disadvantages of this method include its complexity in connection with the need to determine a large number of indicators and a long lead time.

The prototype of the claimed invention selected a method for predicting intrauterine growth retardation (patent No. 2246733, 2005) by examining a pregnant woman’s blood serum, characterized in that the content of insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF) are determined, their ratio is calculated and with a coefficient value of 28.5 or lower, a fetal developmental delay is diagnosed.

This method has several disadvantages:

1. The method is not specific enough - a change in the level of growth factors is observed with various complications of pregnancy - gestosis, an undeveloped pregnancy, the threat of abortion, premature birth (A.V. Orlov et al. // Ross, Bulletin of obstetrician and gyne. - 2005. - No. 3. S. 7-9).

2. The method is time-consuming and time-consuming to complete.

3. The implementation of the method requires expensive equipment and imported sets of reagents for enzyme-linked immunosorbent assay, which complicates its widespread use in clinical practice.

These disadvantages are eliminated in the claimed invention.

The objective of the invention: improving the accuracy and specificity by developing a specific method for the diagnosis of fetal growth retardation (PSA).

The problem is solved by the fact that the level of placental protein PP 13 is determined in the venous blood of women in the first trimester of pregnancy and, with a decrease in its level to 5.34 pg / ml or less, the development of placental insufficiency with fetal growth retardation at subsequent stages of pregnancy is predicted.

The technical result obtained during the use of the invention consists in increasing the accuracy of diagnosis of fetal growth retardation, which allows prescribing timely adequate therapy in an earlier period.

The novelty of the proposed method lies in the fact that for the first time it is proposed to diagnose PCA in pregnant women in the early period by examining the level of placental protein PP13 in venous blood.

Violation of the production of angiogenic factors (vascular endothelial growth factor, placental growth factor) is a universal pathogenetic process in the formation of the main pathological conditions of pregnancy (Orlov V.I., Orlov A.V., 2006; Botasheva T.L., 2014). In addition to angiogenic factors, placental proteins that can take an active part in the implantation process are of considerable interest. Of particular interest in the genesis of placental insufficiency is the inadequate production of the PP-13 placental protein by syncytrophoblast. During the implantation of the blastocyst into the endometrium, invasion of trophoblast villi and transformation of the maternal spiral arteriole occurs. It was found that PP13 is produced by syncytiotrophoblast, secreted into the intercellular space and participates in the formation of a specific buffer zone that facilitates trophoblast invasion (Kliman HJ, Sammar M., Grimpel YI 2012), PP 13 has several functions: 1) has lysophospholipase activity participates in the release of fatty acids, in particular linoleic to-you, which contributes to normal implantation; 2) binds to sugar residues of extracellular matrix molecules (such as annexinll), which creates a “molecular bridge” for placental implantation in the endometrium; 3) is involved in the production of prostaglandins, in particular, prostacyclin, which plays an important role in the vascular conversion of uterine spiral arteries; 4) binds to beta and gamma actin within the trophoblast, thus facilitating the penetration and formation of the placental layer 5) is associated with immunological aspects and hemostasis, because in the zone where there is a high concentration of PP 13, many T cells, neutrophils, and macrophages were found (Schneuer F.J., Nassar N., Tasevski V., Ashton A.W. 2012).

Basically, placental insufficiency is the result of a complex multifaceted and in some cases maladaptive reaction of the fetus and placenta to various pathological conditions of the mother's body, the study of the mechanisms of formation of which is of considerable interest to this day.

Our studies showed that the absolute level of PP 13 in case of PSA, unlike the physiological course of pregnancy, was statistically significantly lower in the first trimester of gestation (p = 0.0125), because PP 13 is produced by syncytiotrophoblast. Also in our study, it was found that with a deficiency of PP13, an inferior invasion of TP villi occurs, which leads to the development of PN and the formation of severe forms of PSA. Studying the expression of the placental protein PP 13, it was found that with its level of PP 13 less than 5.34 was diagnosed in 97% of cases of PSA. Studies of the level of the placental protein PP13 must be used to predict PSA and the choice of an adequate obstetric tactic, this is due to the fact that we have established a relationship between the outcome of the birth and the level of PSA: in women with PSA with a reduced level of PP13, term birth was observed in 46% of cases, premature birth in 54% of cases (p = 0.0215). Thus, in case of PSA, a decrease in the level of PP 13 below 5.34 in the first trimester of gestation is a prognostically significant sign of the development of PSA in women with placental insufficiency (sensitivity 92%, specificity 93%).

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

The material for the study was taken at the same time of the day, on an empty stomach using modern analysis methods. Ultrasound and Doppler studies in dynamics were carried out on the same apparatus, by the same specialist.

The specified method is carried out by examining the venous blood of women with placental insufficiency in the 1st trimester of pregnancy and determining the level of the placental protein PP 13. Material for the study was taken at the same time of the day, on an empty stomach using modern analysis methods. Ultrasound and Doppler studies in dynamics were carried out on the same apparatus by the same specialist.

The level of PP13 in blood serum was determined by enzyme-linked immunosorbent assay kits from the company R&D System (USA).

Wash buffer, standard solutions, substrate solution were prepared according to the instructions for the set.

100 μl of working buffer were added to all wells of the plate coated with murine polyclonal antibodies to human PP 13. 50 μl of the standard dilution solution was added to the blank well, 50 μl of the prepared standard samples were added to the wells for constructing the standard curve, and 50 μl of the studied samples were added to the remaining wells. The plate was covered with a film and incubated for 2 hours at room temperature with continuous shaking on a ST 3 shaker (Latvia).

At the end of the incubation, the wells of the plate were washed with washing buffer 4 times on an ELISA WASHNER HUMAN microplate washer (USA).

Then, 200 μl of a solution of conjugate-polyclonal antibodies to human PP 13 with horseradish peroxidase were added to all wells. The plate was incubated at room temperature for 2 hours with continuous shaking on a ST 3 shaker (Latvia).

Wash the plate 4 times with wash buffer as described above.

200 μl of a freshly prepared substrate solution was added to all wells and incubated for 30 minutes, protecting from direct sunlight.

The color reaction was stopped by adding 50 μl of stop reagent to each well.

The optical density was measured at 450 nm and the results were calculated using a MULTILABEL COUNTER 1420 photometer (Finland).

Numerical values of PP 13 concentrations in standard solutions were introduced into the computer software of the device, and after measurement, the concentration of PP 13 in experimental samples in pg / ml was obtained.

With a decrease in the level of placental protein PP 13 to 5.34 pg / ml or less, the development of placental insufficiency with fetal growth retardation in the subsequent stages of pregnancy is predicted.

The efficiency of this method is confirmed by the following clinical examples.

Example 1

First pregnant, I-va, 25 years old. Date of examination 02.11.2014

An ultrasound scan was performed. Diagnosis: Pregnancy 10 weeks.

Venous blood was taken. The level of PP 13 in the blood serum was 6.83 pg / ml, which indicates a favorable prognosis of pregnancy.

In a period of 28 weeks, a second ultrasound and Dopplerometric examination was performed. Ultrasound data: BPR -71 mm for 28 weeks. 5 days; Exhaust gas - 260 mm for 28 weeks. 3 days; Coolant - 243 mm for 28 weeks. 4 days; DB - 54 mm for 28 weeks. 5 days. Placenta I Art. maturity, thickness 28 mm. Diagnosis: Pregnancy 28 weeks. Violations of uterine and fetoplacental hemodynamics were not detected.

Example 2

First pregnant, N-va, 28 years old. Date of examination 02.15.2014

An ultrasound scan was performed. Diagnosis: Pregnancy 9 weeks.

Venous blood was taken. The level of PP 13 in the blood serum was 5.34 pg / ml, which indicates a high likelihood of developing placental insufficiency and fetal growth retardation in the 2-3 trimester.

In the period of 32 weeks, repeated ultrasound and Dopplerometric studies were performed. Ultrasound data: BDP - 72 mm for 29 weeks; Exhaust gas - 267 mm for 29 weeks; Coolant - 250 mm for 29 weeks. 2 days; DB - 55 mm for 29 weeks. 3 days. Placenta II-III Art. maturity, thickness 36 mm. Diagnosis: Pregnancy 32 weeks. Symmetric form of fetal growth retardation. Ultrasound signs of placental insufficiency. Violation of uterine and fetoplacental hemodynamics 2 degrees.

The claimed method examined 115 women whose venous blood in the first trimester of pregnancy determined the level of placental protein PP13 in 6-9 weeks. pregnancy. In 65 patients with a PP13 level lower than 5.34 in the II and III trimesters, the development of placental insufficiency, aggravated by fetal growth retardation, was observed. 50 women whose PP13 level was higher than 5.34, pregnancy proceeded without fetal growth retardation. When conducting a statistical analysis of empirical data, the capabilities of the Excell 2003 table processor and application software packages (Megastat and Statistica 6.0) were used.

Thus, a method is proposed for predicting fetal growth retardation in placental insufficiency in the first trimester of pregnancy based on the determination of such an informative indicator as the level of placental protein PP 13, which allows one to effectively assess the possibility of obstetric complications, ensure the timely delivery of obstetric care, and thereby reduce perinatal losses .

The above confirms the diagnostic value of the proposed method. Thus, the claimed method is more efficient compared to the known and has several advantages:

1. The method is specific (determination of PSA by the level of PP13 is a specific method).

2. It has high accuracy. Reliability is 100%.

3. The method is highly sensitive, makes it possible to determine the PSA at the preclinical stage.

Testing of the claimed invention was carried out on a sufficient volume of clinical material, which confirmed the efficiency of the method and the possibility of its use in clinical practice.

Claims (1)

A method for predicting fetal growth retardation in women with placental insufficiency by examining the biological fluid of a pregnant woman, characterized in that the level of placental protein PP13 is determined in the venous blood of women in the first trimester of pregnancy and, when its level is reduced to 5.34 pg / ml or less, development is predicted fetal growth retardation due to placental insufficiency in subsequent stages of pregnancy.