RU2340899C1 - Method of predicting of miscarriage in second trimester of pregnancy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely, to obstetrics, and can be used for prediction of miscarriage in the second trimester of pregnancy. At the pregnant woman define the maintenance of the transforming factor of growth-β2 (TFR-β2) in blood serum. If TFR-β2 is less 1806.8 pg/ml, prognosticate abortion threat. In case TFR-β2 is higher 1806.8 pg/ml, in addition define level of substance P (SP) and, if it more than 0.274 ng/ml, also prognosticate abortion threat in the second trimester.

EFFECT: increase of accuracy, sensitivity and specificity of forecasting of a course of the second trimester of pregnancy.

3 tbl, 3 ex

Description

The invention relates to medicine, namely to obstetrics, and can be used to predict miscarriage in the second trimester of pregnancy.

The task of protecting the health of mothers and children is an important component of healthcare, which is of paramount importance for the formation of a healthy generation from an early period of life (Kulakov V.I., 2005). Realization of this task is possible only when creating optimal conditions for maintaining a woman’s health, resolving issues of rational tactics of pregnancy and childbirth, as well as preventing reproductive losses (Abramchenko V.V., 2004; Sidelnikova V.M., 2005).

In modern obstetrics, the problem of miscarriage does not lose its relevance. According to many authors, the frequency of this pathology, which has no tendency to decrease over the past 20 years, is 10-25% of all pregnancies (Sidelnikova V.M. et al., 2001; Genazzi A.R., 2003). It has now been established that miscarriage is a polyetiological complex, in the development and implementation of which the most important systems of the woman’s body take part. Undoubtedly, the fact that the nervous system has an important regulatory effect on the organs of the immune system. Physiological neuroregulation of the body’s immune response is necessary for its full development (Sotnikova N.Yu., Gromova OA, Novikova EA, 2004).

Therefore, the search for new pathogenetic mechanisms for the development of spontaneous abortion is of great practical interest, because will allow the development of new diagnostic criteria and, as a consequence, management tactics.

To date, a significant amount of work has been devoted to the early diagnosis and prediction of miscarriage. At the same time, two main directions of the developed methods are viewed: biochemical and immunological, on the one hand, and instrumental - on the other. Moreover, ultrasound methods are mainly instrumental.

From the first group of methods, a method is known for predicting the threat of termination of pregnancy in the early stages by determining in peripheral blood the concentration of a factor inhibiting the migration of leukocytes before and after a single immunization with donor leukocytes (RF patent No. 20149898, 1994).

The disadvantages of the method are the technical complexity of the implementation, as well as the inability to dynamically monitor the state of pregnancy using this method.

There is a method of early diagnosis of miscarriage by determining in the peripheral blood the relative content of neutrophils having structures on their surface that react with trophoblastic X-glycoprotein (RF patent No. 2077729, 1997). The disadvantages of the method include its complexity and non-specificity.

A known method for predicting the threat of miscarriage by determining the content of peripheral T-lymphocytes before and after incubation with AMHF (patent No. 2077727, 1997).

A known method for preclinical prediction of miscarriage by determining the plasma content of fibrin-monomer complexes. By their size, termination of pregnancy in I, II or III trimesters is predicted (RF patent No. 2123698, 1998). The disadvantages of the method include its non-specificity.

A known method for predicting the threat of termination of pregnancy by determining the concentration of estriol, placental lactogen and interleukin (IL) - 6 in the blood serum (RF patent No. 2285926, 2006).

This method is accepted by us as a prototype.

The disadvantage of these methods is their complexity.

A known method for predicting miscarriage by ultrasound examination of the pelvic organs and determination of serum progesterone, chorionic gonadotropin and serum alpha-2-microglobulin level (RF application No. 2004135161, 2006). The disadvantage of this method is its complexity and non-specificity.

There is a method for predicting miscarriage by determining the content of cytokines in the peripheral blood and mucus of the cervical canal in women with miscarriage of infectious origin by the level of IFN-γ and IL-8 in peripheral blood and mucus of the cervical canal, the increase of which serves as markers of distress in the early stages of pregnancy ( T.N.Sumedy, L.V. Gankovskaya, E.N.Dolgina, etc. The content of cytokines in the peripheral blood and mucus of the cervical canal in women with miscarriage of infectious origin / / Russian Journal of Immunology, 2005 - vol. 9, - N2. - pp. 93-102). The disadvantage of this method is that it does not allow the diagnosis of the threat of termination of pregnancy in the 2nd trimester.

From the foregoing, it is obvious that today the main disadvantages of methods for predicting miscarriage are nonspecificity and complexity.

The objective of the invention: the development of a specific method for predicting miscarriage in the second trimester of pregnancy in order to prescribe timely and adequate therapeutic measures.

The problem is solved in that in a pregnant woman in the second trimester, the content of transforming growth factor-β2 (TGF-β2) is determined in blood serum, and if TGF-β2 is less than 1806.8 pg / ml, then the threat of termination of pregnancy is predicted if Since TGF-β2 is greater than 1806.8 pg / ml, the level of substance P (SP) is additionally determined and, if it is greater than 0.274 ng / ml, then the threat of termination of pregnancy in the second trimester is also predicted.

Method sensitivity - 89.8%

The specificity of the method is 93.3%

Accuracy - 89%

Reliability (p <0, 001)

A new technical result is that this method has high accuracy, sensitivity and specificity. The proposed research method allows to accurately predict the course of the second trimester of pregnancy, i.e. from 12 to 28 weeks gestation.

Thus, a pathogenetically substantiated prognosis of miscarriage even at the preclinical stage will allow timely identification of a group of pregnant women threatening to develop this pathology, and early preventive treatment, which will significantly reduce the frequency of miscarriage and will positively affect the rates of perinatal morbidity and mortality in the face of an exacerbated demographic situation. .

In the genesis of reproductive losses at any stage of pregnancy, the role of persistent urogenital bacterial-viral infection (Sidelnikova V.M., 2001), endocrine pathology (Sidelnikova V.M., 1986) has been convincingly proven. Given the allogeneity of the fetus, there is no doubt that immune mechanisms play an important role in the initiation of premature termination of pregnancy (Ailamazyan E.K., Pavlov O.V., Selkov S.A., 2004). Maintaining homeostasis in the body is ensured by the interaction between the endocrine and immune systems through neurotransmitters, which include neuropeptides.

Substance P (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) is one of the most famous peptides discovered in 1931. This neuropeptide is present in non-myelinated C- type and small myelinated A-delta type and are synthesized by the cells of the dorsal horns of the ganglia, and then transported along the axons to the nerve endings, where they accumulate in dense vesicles (Gomazkov O.A., 1995). Substance P (SP) is the main mediator of pain neurotransmission. Female genital organs are innervated by autonomous (adrenergic, cholinergic) and sensory nerves, as well as peptidergic nerve fibers containing substance P, a vasoactive intestinal peptide, calcitonin-gene binding peptide, neuropeptide Y (Klukovits et al., 2002, Zupko et al., 2005, Bae et al., 2001). Information on the amount of substance of P-dependent nerve fibers in the uterus during pregnancy is quite contradictory. It is known that uterine denervation occurs during gestation, the physiological significance of which is the preservation, growth and development of the fetus (C. Schmidt, E. Lobos, K. Spanel-Borowski, 2003). However, the innervation of the cervix is more pronounced, and there is no loss of nerve endings, i.e. their function is preserved (Kulakov V.I., Murashko L.E., 2002). SP receptors were found in the cervix at the endothelium of postcapillary venules, and the effect of neuropeptides on microcirculation is suggested (Collins et al., 2002, Pokabia et al., 2002). So, the introduction of SP in non-pregnant rats in the experiment caused vasodilation, plasma extravasation and migration of leukocytes in the cervix, i.e. changes characteristic of a “maturing” cervix (Collins et al., 2002). As a pro-inflammatory agent, SP stimulates T-cell proliferation (Payany et al., 1985), an increase in the synthesis of immunoglobulins causes chemotaxis of monocytes, neutrophils, fibroblasts, induces mast cell degranulation and histamine release, stimulates macrophage phagocytosis, IL-1 secretion (IL -6, tumor necrosis factor-α), monocytes (Goode et al., 1998). This neuropeptide serves as a local “controller” of the microenvironment due to its effect on endothelial cells, macrophages, lymphocytes, synoviocytes, mast cells (Geppetti P.A., Holzer P., 1996; McDonald D.M. et al., 1996). The spectrum of action of SP is from regulatory effects on the immune system to the control of vascular tone, which explains how the neuropeptide generates and supports the inflammatory process called “neurogenic” (Meggs W.J., 1995). From the point of view of physiological pregnancy, the division of cytokines into "harmful" and "useful" is unjustified. It turned out that the increase in the production of certain cytokines (including the so-called harmful ones) by the placental tissue accompanies not only pathological processes, such as miscarriage and premature birth, but is also a characteristic sign of normal timely delivery.

In experiments on rodents, the abortogenic effect of substance P was convincingly proved by increasing the production of tumor necrosis factor-α, interleukin-1 by macrophages and reducing the content of transforming growth factor β2, which has immunosuppressive properties (Arck PC, 1997; Markert UR, 1997), t. about. substance P disrupts the "cytokine balance" in the uterus that exists during pregnancy, an increase in pro-inflammatory cytokines occurs, thereby rejecting the fetus. Violation of this balance as a result of exogenous or endogenous factors can lead to pregnancy complications, the extreme manifestation of which is its spontaneous premature termination (Pavlov O.V., Selkov S.A., Lalayan D.V., Arzhanova O.N., 2003 )

In the course of our own research, we obtained very interesting results.

By analyzing studies of the transforming growth factor β2 (TGF-β2), we can state the predictability of laboratory results. TGF - β2, being an immunosuppressive factor, promotes the formation of a suppressor dominant, blocks cellular immunity reactions and contributes to the prevalence of the effects of Th2-regulatory cytokines. In the course of our study, it was revealed that during normal pregnancy, high levels of TGF-β2 in the blood serum were recorded, amounting to 2756.75 (2461.75-3628.88) pg / ml and 2 times lower with the threat of miscarriage - 1375, respectively 5 (756-2707.5) pg / ml (p <0.001), and after a course of complex therapy it increased slightly to 1980.5 (936.5-3092) pg / ml.

The study of substance P during physiological gestation showed a low level of its content, equal to 0.19 (1.14-0.21) ng / ml. In the blood serum of pregnant women with the threat of miscarriage, this indicator increased almost 2 times and amounted to 0.35 (0.2-0.93) ng / ml (p <0.001), and maintained a tendency to increase, despite the therapy. Upon repeated examination, the concentration of substance P increased to 0.43 (0.19-1.03) ng / ml and significantly differed from the control (p <0.001). Thus, we can state that the threat of miscarriage in the second trimester develops against the background of a stably low level of pro-inflammatory cytokines, but, at the same time, an increased content of substance P in the blood serum. The low level of pro-inflammatory cytokines at the systemic level can be explained by the fact that the condition of our patients was not critical.

Since there are still no standard normative indicators of proinflammatory cytokines in the available literature, and their role in embryogenesis varies sharply depending on the concentration, our forecasting method allows only 2 indicators to be determined in the second trimester of pregnancy, namely, the substances P and TGF-β2, which is sufficient to calculate the prognostic criterion. Substance P is an important modulator of inflammation, because induces the synthesis of "pro-inflammatory" mediators (interleukin 1, 6, tumor necrosis factor, etc.) by macrophages and activates phagocytosis, while at the same time reduces the content of immunosuppressive factors. It is known that only with a high content of TGF-β2 prolongation of pregnancy is possible.

The method is as follows.

Blood is taken from a pregnant woman in the morning on an empty stomach from the cubital vein (preferably by gravity into a plastic or silicone tube), then blood serum is obtained by the standard method. Determine TGF-β2 and substance R. If TGF-β2 is less than 1806.8 pg / ml, then the threat of termination of pregnancy is predicted, if TGF-p2 is more than 1806.8 pg / ml and SP is more than 0.274 ng / ml, then predict the threat of abortion in the second trimester and prescribe adequate therapy.

The level of TGF-β2 in blood serum is determined by enzyme-linked immunosorbent assay kits from BIOSOURCE (USA). Wash buffer, standard solutions, streptavidin-peroxidase conjugate are prepared according to the instructions for the set. The samples are preliminarily diluted with a working buffer 50 times, then the samples are oxidized with 1N hydrochloric acid, incubated for 15 minutes and the samples are neutralized with a solution of 1 N sodium hydroxide.

100 μl of standards, controls and test samples were added to the corresponding wells of the plate coated with polyclonal antibodies to human TGF-β2. Cover the tablet with a film and incubate for 3 hours at room temperature. At the end of the incubation, wash the wells of the plate with wash buffer 3 times on an ELISA WASHER HUMAN microplate washer (USA).

Then, 100 μl of a solution of biotinylated antibodies to human TGF-β2 are added to all wells. Incubate the plate at room temperature for 2 hours. Wash the plate 3 times with wash buffer as described above. 100 μl of a solution of streptavidin - peroxidase conjugate are added to all wells and the plate is incubated for 20 minutes at room temperature. Then, washing is carried out as described above. 100 μl of substrate solution was added to all wells and incubated at room temperature for 30 minutes. Stop the color reaction by adding 100 μl stop reagent to each well.

Measurement of optical density at 450 nm and calculation of the results are carried out on a MULTILABEL COUNTER 1420 photometer (Denmark).

Numerical values of the concentrations of TGF-β2 in standard solutions are introduced into the computer software of the device and the concentration of TGF-β2 in the experimental samples is obtained after measurement. The results are multiplied by a dilution factor (50) and the level of TGF-β2 in the patient's blood serum is expressed in pg / ml.

The level of substance P (SP) in the blood serum of patients is determined by enzyme-linked immunosorbent assay kits from VASNEM GROUP (USA).

Preparation of standard solutions, conjugates, washing and working buffer is carried out according to the instructions for the set. 25 μl of primary antiserum are added to all wells of the plate and the plate is incubated for 1 hour at room temperature. Then, 50 μl of standard solutions or test samples are added to the corresponding wells of the plate, incubated for 2 hours at room temperature. 25 μl of a solution of biotinylated conjugate are added to all wells of the plate, the plate is covered with a film and left overnight at a temperature of 2-8C.

At the end of the incubation, the wells of the plate are washed 5 times with washing buffer on an ELISA WASHER HUMAN microplate washer (USA). 100 μl of streptavidin-peroxidase conjugate solution are added to all wells of the plate and the plate is incubated for 1 hour at room temperature. Wash the plate as described above. 100 μl of TMB solution are added to all wells of the plate, incubated in the dark for 15 minutes, the color reaction is stopped by adding 100 μl of 2N HCl solution to each well.

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

Numerical values of the concentrations of the substance P in standard solutions are introduced into the computer software of the device and, after measuring, the concentration of the substance P in the experimental samples in ng / ml is obtained.

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

Example 1

The first pregnant X-oh, 22 years old, at the next reception in the antenatal clinic does not complain. Clinical and laboratory examination without pathology. Ultrasound protocol: gestational 22 weeks. Fetometry is consistent. Organs of the fetus without features. The placenta along the back wall, ambilateral, 0 degree of maturity, the thickness of the placenta is 20 mm. Normative Dopplerometry: hemodynamic impairment not detected. When a vaginal examination: the cervix is rejected posteriorly, the vaginal part of the cervix is 2.5 cm long, dense, the external pharynx is closed. The uterus is enlarged according to the gestational age, out of tone. Slated to be the head of the fetus, movable above the first plane of the pelvis. Discharge mucous, scanty. In order to solve the question of the appropriateness of preventive therapy, an examination was conducted to determine the level of TGF-β2 in the blood serum (equal to 2672 pg / ml). Additionally investigated the level of substance P (equal to 0.154 ng / ml). Because the level of TGF-β2 is greater than 1806.8 pg / ml, and the level of substance P is less than 0.274 ng / ml, then this patient can be assigned to a group of healthy pregnant women who do not require drug-preserving therapy. The course of this pregnancy was favorable.

Example 2

Re-pregnant P-th, 28 years old, with a history of 1 urgent delivery, 2 artificial abortions (without complications), is observed in the antenatal clinic from 8 weeks of pregnancy. The course of gestation without pathology. Data from a general clinical examination within standard values. Ultrasound: gestational 13 weeks. CRL-75 mm - 13 weeks. 4 days Chorion on the front wall, 2 cm above the internal pharynx. Dopplerometry: hemodynamic impairment not detected. When a vaginal examination: the cervix is rejected posteriorly, the vaginal part of the cervix is 2.5 cm long, dense, the external pharynx is closed. The uterus is enlarged according to the gestational age, out of tone. Discharge mucous, scanty. In order to solve the issue of tactics of conducting a real pregnancy, an examination was conducted to determine the level of TGF-β2 in the blood serum (equal to 695 pg / ml), i.e. TGF-β2 is less than 1806.8 pg / ml, which, according to our method, allows this pregnant woman to be at risk of miscarriage. A pregnant woman was recommended a course of preservation therapy. However, the patient did not comply with the recommendations, referring to subjectively good health. After 10 days, the patient complained of pain in the lower abdomen, uterine tone. Ultrasound: gestational age 14 weeks. 3 days Fetometry is consistent. Placenta along the posterior wall, ambilateral, 0 degree of maturity, normal thickness. Normative Dopplerometry: violation of utero-placental hemodynamics 1A Art. When a vaginal examination: the cervix is rejected posteriorly, the vaginal part of the cervix is 2.5 cm long, dense, softened on the periphery, the external pharynx is 0.1 cm. The uterus is enlarged according to the gestational age, in tone. Discharge mucous, scanty. A patient in a hospital conducted a course of conservation therapy. Pregnancy is prolonged.

Example 3

The first pregnant Ok, 29 years old, at the next appointment with the district obstetrician-gynecologist, has no complaints. Registered in pregnancy is from 7-8 weeks. The course of gestation without pathology. In a period of 16 weeks. On her own initiative, she performed an ultrasound: on gestation 16 weeks. 3 days Fetometry is consistent. The placenta along the posterior wall is ambilateral, 0 degree of maturity, normal thickness. Normative Dopplerometry: hemodynamic impairment not detected. When a vaginal examination: the cervix is rejected posteriorly, the vaginal part of the cervix is 2.5 cm long, dense, the external pharynx is closed. The uterus is enlarged according to the gestational age, out of tone. Discharge mucous, scanty. In order to solve the issue of tactics of conducting a real pregnancy, an examination was performed to determine in serum the level of TGF-β2 (equal to 4312.5 pc / ml). Because the level of TGF-β2 is greater than 1806.8 pg / ml; the level of substance P (0.47 ng / ml) was additionally investigated in blood serum. Because the level of substance P is more than 0.274 ng / ml, according to our methodology, this pregnant woman should be attributed to the risk group for miscarriage. A course of preventive therapy. Pregnancy proceeded without signs of a threatening late abortion.

The reliability of the method is confirmed by clinical and laboratory studies.

The aim of our work was to develop diagnostic criteria based on the study of the components of "neurogenic" inflammation. As part of the solution of the tasks, we conducted a comprehensive examination of 79 pregnant women in a period of 12 to 28 weeks. gestation. The main group included 49 pregnant women with clinical and ultrasound signs of a threat of abortion who were hospitalized in the Department of Pregnancy Pathology of the Federal State Institution "Russian Medical Research Institute of Medical Technologies" (Rostov-on-Don). The control group consisted of 30 women with a physiological pregnancy during the same gestational periods according to a retrospective analysis of their observation in the framework of the Obstetric Monitoring program, which carries out the outpatient stage of monitoring and management of pregnant women. All women received informed consent, in addition, approval for research was obtained from the local ethics committee of the Rostov Research Institute of Obstetrics and Pediatrics. All patients underwent clinical and laboratory examination, including dynamic clinical, hormonal, biochemical, genetic, ultrasound and Doppler studies, as well as the content of substance P, interleukin-1β, -6, tumor necrosis factor-α and transforming growth factor in the blood serum β2. In the main clinical group, a double immunological examination was carried out, respectively, before and after a course of standard therapy aimed at prolonging pregnancy.

In 48 patients, the threat of termination of pregnancy was stopped, and in only one woman, despite the therapy, the pregnancy was terminated. The successful course of the gestational process, in our opinion, is due to the tendency to increase the content of TGF-β2, which has immunosuppressive properties, contributing to the switching of the immune response to the Th2-regulatory mechanism. Summing up our work, we can state with a high degree of certainty (p <0.001) that the most sensitive and statistically significant laboratory indicators of the threat of termination of pregnancy are serum levels of TGF-β2 and SP, the change of which plays a leading and paramount role in the development of this pathology. And one more confirmation of our conclusion can be considered the presence of pain in all pregnant women of the main group, but SP is the main mediator of nociception.

The procedure for statistical processing of the obtained empirical data provided for the sequential execution of several stages. The distribution of the source variables was checked for normality. Further, a procedure of descriptive statistics was carried out, with the help of which medians, modes and confidence intervals were found, and sample means for various groups were checked. The reliability of the results was calculated with a confidence level of 0.95. The results are presented in tables 1-3.

* - significance of differences p <0.05 compared with the control group,

** - significance of differences p <0.01 compared with the control group,

*** - significance of differences p <0.001 compared with the control group.

The content of components of neurogenic inflammation in the blood serum of pregnant women in the control group.

Table 1 indicator FNOα (pg / ml) IL-1β (pg / ml) IL-6 (pg / ml) TGF-2β (pg / ml) SP (ng / ml) median 29.31 3.37 * 9.25 * 2756.75 *** 0.19 *** 1 quartile 20.02 1.10 6.49 2461.75 0.14 2 quartile 68.00 6.72 27.68 3628.88 0.21

The content of components of neurogenic inflammation in the blood serum of pregnant women of the main group before therapy.

table 2 indicator FNOα (pg / ml) IL-1β (pg / ml) IL-6 (pg / ml) TGF-2β (pg / ml) SP (ng / ml) median 26.2 1.78 * 7.9 * 1375.5 *** 0.35 *** 1 quartile 22.7 1.31 6.6 756.5 0.2 2 quartile 31.1 2.82 9.7 2707.5 0.93

The content of components of neurogenic inflammation in the blood serum of pregnant women of the main group after therapy.

Table 3 indicator FNOα (pg / ml) IL-1β (pg / ml) IL-6 (pg / ml) TGF-2β (pg / ml) SP (ng / ml) median 29.8 1.75 * 7.12 ** 1980.5 *** 0.43 *** 1 quartile 24 1.28 6.19 936.5 0.19 2 quartile 36.7 2.27 9.6 3092 1,03

Processing of the initial series of features was carried out using the PolyAnalyst 3.5 PRO and EXCEL 2003 packages.

To compare the intergroup differences, the Wilcoxon method for independent groups was used using the AtteStat 6.4 package. To analyze multidimensional nonlinear dependencies, we used the PolyAnalist 3.5 Pro package, one of the functions of which includes the technology of “evolutionary programming”.

A special module continuously converts the “best” program at the moment from an internal representation into an external PolyAnalyst language - the language of symbolic rules (Symbolic Rule Language), understandable to humans: mathematical formulas, conditional constructions, and so on. This allows the user to understand the essence of the obtained dependence, to control the search process, and also to receive a graphical visualization of the results.

The advantage of the proposed method is that in pregnant women in the second trimester, on the basis of highly sensitive modern laboratory markers, prediction of miscarriage is made with the aim of early appointment of adequate therapy. In clinical practice, this will reduce the risk of perinatal losses due to the timely identification of pregnant risk groups for the development of this pathology, and conduct early preventive treatment that will significantly reduce the rate of miscarriage and will positively affect the rates of perinatal morbidity and mortality in the face of an exacerbated demographic situation. The ability to simultaneously prepare for the study of a large number of blood samples and a small investment of research time, which allows the use of the method in screening studies.

This method is non-traumatic, has a speed of execution using standard test systems, which allows it to be widely used in practical health care.

Claims (1)

A method for predicting miscarriage in the second trimester of pregnancy by examining blood serum, characterized in that the content of transforming growth factor-β2 (TGF-β2) in the blood serum is determined, and if TGF-β2 is less than 1806.8 pg / ml, a threat of termination is predicted pregnancy, if TGF-β2 is more than 1806.8 pg / ml, then the substance level P (SP) is additionally determined and if it is more than 0.274 ng / ml, the threat of termination of pregnancy in the second trimester is also predicted.