RU2627472C1 - Method for prediction of miscarriage of infectious genesis at early stages of gestation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: level of IL-28B interferon-like cytokine is determined in the serum of pregnant women with a 6-11 week gestation period, by means of enzyme immunoassay. At IL-28B concentration above 261.4 pg/ml compared to the concentration established for a physiologically occurring pregnancy, a threat of miscarriage in the first trimester of gestation is projected. The invention provides improved accuracy of miscarriage threat prediction using an increase in the IL-28B cytokine concentration as a quantitative measure. The method is minimally invasive, convenient and simple to use, sufficiently informative and provides high reliability of the forecast.

EFFECT: use of this method will facilitate a more rapid and safe diagnosis of the threat of pregnancy termination of infectious genesis in the first trimester.

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Description

The present invention relates to obstetrics and relates to predicting the threat of miscarriage of infectious genesis in the early stages of gestation.

Pregnancy is a complex unique process of many immunoregulatory mechanisms to protect the semi-allogenic fetus from the mother’s immune system, while protecting the mother’s body from various pathogens. It is known that during physiological pregnancy suppression of adaptive immunity occurs, accompanied by activation of innate immunity. (Makarov O.V., Kovalchuk L.V., Gankovskaya L.V., Bakhareva I.V., Gankovskaya O.A. Miscarriage, infection, innate immunity, Moscow, “GEOTAR-Media”, 2007, p. 176).

The formation of a balance of pro- and anti-inflammatory cytokines is necessary for the normal course of pregnancy. Currently, the main causes of miscarriage are considered to be sexually transmitted infections and immune disorders in the body of a woman (Sidelnikova V.M. Habitual loss of pregnancy. Moscow. Triada-X, 2000, p. 50). In the presence of infection in the body of a pregnant woman, the cytokine balance shifts toward proinflammatory cytokines, their production increases, which leads to a high concentration of cytokine-mediated prostaglandins, which cause uterine mimetry hypertonicity.

A known method for predicting miscarriage in the early stages of infectious genesis (RU 2341799, C1, 12/20/2008) in which the level of interleukin 12 (IL-12) in the mucus of the cervical canal is determined in pregnant women with identified urogenital infections in the 1st and 2nd trimesters. If the level of IL-12 is higher than 102.0 pg / ml, a high risk of miscarriage in 1 and 2 trimesters is predicted. The disadvantage of this method is the taking of material for research from the cervical canal.

A known method for predicting miscarriage in the early stages of infectious genesis (RU 2281504, C1, 08/10/2006), in which pregnant women 1 trimester with verified urogenital infection, conduct simultaneous determination of serum peripheral blood and cervical mucus neopterin, which is an early marker of cell activation , is an indicator of the inflammatory process of infectious genesis. With a neopterin value in the blood above 3.2 ± 0.7 ng / ml, in cervical mucus above 10.1 ± 6.4 ng / ml, a high risk of miscarriage in 1 trimester is predicted. The disadvantage of this method is the taking of material for research from the cervical canal.

At the moment, a known method for predicting miscarriage in the early stages (RU 2501017, C1, 12/10/2013). In pregnant women with a revealed urogenital infection in the first trimester, the level of chemokine of macrophages MIP-1α is determined in the mucus of the cervical canal. If the level of MIP-1α in cervical mucus is higher than 36.9 pg / ml, a high risk of miscarriage in 1 trimester is predicted. However, in this work, there are no reference values of MIP-1α, in addition, at the same values, the authors predict both a threat of miscarriage and a normal course. The disadvantage of this method is also the taking of material for research from the cervical canal.

This method is accepted by us as a prototype.

From the foregoing, it is obvious that today the main drawbacks of methods for predicting miscarriage are taking material for research from the cervical canal, which can provoke abortion, as well as the absence of reference values for cytokines and chemokines, which makes it difficult to predict the threat of pregnancy termination.

These disadvantages are eliminated in the claimed method.

. IL-28 and IL-29: Newcomers to the interferon family // Biochimie V. 89, Issues 6-7, 2007, P. 729-734).The first line of defense against viral infection is mediated by IFNs, which are produced as soon as possible in response to infection. Recently, a group of IFN type III (IFN-λ) has been actively studied, including IFN-λ1, IFN-λ2, and IFN-λ3 (IL-29, IL-28A, or IL-28B, respectively) (Gilles

. IL-28 and IL-29: Newcomers to the interferon family // Biochimie V. 89, Issues 6-7, 2007, P. 729-734).

IFN-λ, in addition to antiviral activity, has immunomodulatory properties. IFN-λ acts as an autocrine signal to enhance the antiviral response by dendritic cells by increasing the production of IFN-α and IFN-λ, which ensures the survival and functionality of plasmacytoid DCs. In addition, IFN-λ, DC is produced more prolonged for the implementation of long-term antiviral protection (Yin Z, Dai J, Deng J, Sheikh F. Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells // 2012. J. Immmunol. V l89 (6) p. 2735-45).

We set the task of finding a statistically significant marker for predicting the threat of miscarriage of infectious origin in the early stages of gestation associated with IFNλ3 (IL-28B) type.

The technical result of the invention is to increase the accuracy of predicting the threat of miscarriage due to the use of an increase in the concentration of IL-28B cytokine as a quantitative indicator.

At the first stage, a group of women with a physiologically proceeding pregnancy in the first trimester was examined and the normal concentration of IL-28B in serum was established (Table 1).

In a study of a group of women with a threat of termination of pregnancy of infectious genesis of early gestation, we first revealed that an increase in the serum IL-28B cytokine content above 261.4 pg / ml is associated with miscarriage in the first trimester (Table 1). An increased concentration of IL-28B leads to the overproduction of type I IFN, pro-inflammatory cytokines and prostaglandins. This in turn causes uterine hypertonicity and miscarriage.

The prognostic signs revealed by us turned out to be suitable for clinical purposes - predicting miscarriage of infectious genesis in the early stages of gestation and, accordingly, timely correction of management of pregnant women.

The invention consists in the following.

To predict miscarriage of infectious genesis in the early stages of gestation in the blood serum of pregnant women with a period of 6-11 weeks of gestation, the level of interferon-like cytokine IL-28B is determined by enzyme-linked immunosorbent assay. When the concentration of IL-28B is higher than 261.4 pg / ml compared with the concentration established for the physiologically occurring pregnancy, the risk of miscarriage in the first trimester of gestation is predicted.

The method is as follows.

To study the level of IL-28B in blood serum, the following technique was used, which included several successive stages: venous blood sampling by puncture of the ulnar vein, obtaining blood serum, and enzyme-linked immunosorbent assay.

Taking venous blood and obtaining blood serum is carried out in a conventional manner.

To determine the concentration of IL-28B in serum, we used the ELISA Kit for ELISA 28-ELISA ELISA Kit for Interleukin 28B (Cloud-Clone Corp., USA) "Sandwich" method strictly according to the protocol of the manufacturer.

For this, a series of dilutions of IL-28B standards was prepared by diluting the standard included in the kit. The dilution of the standards corresponded to the following concentrations - from 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL and 7.813 pg / mL to construct a calibration curve. Then, 100 μL of standards and samples were introduced into the corresponding wells, the plate was closed with an adhesive film, incubated for 2 hours at 37 ° C. Then the film was removed, the tablet was emptied and dried by tapping on filter paper. A working concentration of detection reagents was prepared, for which the Assay Diluent A and Assay Diluent B were diluted 1: 100 in the Detection Reagent A and Detection Reagent B kit. Then, 100 μL Detection Reagent A was added to the wells, covered with a film, incubated for 1 hour at 37 ° C. After incubation, the film was removed, 350 μL wash buffer was added to each well with a multichannel pipette. Washing was repeated 3 times. Then, at 100 μL, Detection Reagent B was added at the working concentration, covered with a film and incubated for 30 minutes at 37 ° C. Then the film was removed, the plate was emptied, and washing was performed with wash buffer 5 times. After washing, 90 μL streptavidin peroxidase conjugate (TMB Substrate) was added to each well. Covered with a film, incubated for 25 min at 37 ° C in the dark. Then, to stop the reaction, 50 μL Stop reagent was added to each well. The measurement of optical density at 450 nm, the construction of a calibration curve and the calculation of the concentration are carried out using an Anthos 2020 microplate reader (BioChemMack, RF).

Table 1 shows the level of the serum IL-28B cytokine in the physiological course of pregnancy and in the case of miscarriage in the early stages of gestation.

To prove the possibility of realizing the claimed purpose and achieving the specified technical result, we provide the following data.

Example 1

Pregnant R., 34 years old. It is observed in the antenatal clinic with a diagnosis of physiological pregnancy, 12 weeks. This pregnancy is the first. Menarche at the age of 14, established immediately, for 5 days, b / w 29 days, regular, moderate, painless. Gynecological diseases - ectopia of the cervix. From the anamnesis: in childhood - chicken pox, rubella. Clinical and laboratory examination results are within normal limits. HELL - within normal limits. Somatic diseases denied. Outpatiently examined for urogenital infection by PCR diagnostics - negative.

In this pregnant woman, the level of cytokine IL-28B in the blood serum was 162.19 [pg / ml]. Thus, in this pregnant woman with a physiological course of gestation, the prognostic indicator is within normal limits. Miscarriage is not predicted. Pregnancy complications were not revealed in the future.

Example 2

Pregnant, I. 27 years old was admitted to the gynecological department with a diagnosis of Pregnancy 10-11 weeks. Beginning of a spontaneous miscarriage. Complained of drawing pains in the lower abdomen, blood discharge from the genital tract. From the anamnesis: this pregnancy is the first. Menarche (the first menstruation) at 14 years of age, established after 1 year, 4 days, w / o 30 days, regular, moderate, painful. Examined at the UGI: detected HPV 16/18/32, Herpes simplex virus 2. From the anamnesis: in childhood - rubella, chickenpox; gastritis. Gynecological history: cervical erosion, condylomas. Ultrasound - pregnancy 10-11 weeks. Hypertonic myometrium.

Despite the therapy aimed at prolonging pregnancy, after three days the patient noted an increase in cramping pains in the lower abdomen and blood secretions, with clots. Diagnosed abortion, removal of the remains of the fetal egg.

The level of IL-28B was 343.7 [pg / ml]. 2.2-fold increase in the concentration of IL-28B cytokine.

Conclusion: in comparison with the group of healthy pregnant women, patient I. revealed a significant increase in the level of antiviral cytokine in the blood serum in the presence of urogenital infection of viral origin. It was predicted the development of a miscarriage, which took place at 11 weeks of gestation. The forecast has been confirmed.

Example 3

Pregnant, Z. 35 years old, was admitted to the gynecological department of 1GB with complaints of dizziness, profuse blood discharge from the genital tract, cramping pains in the lower abdomen. Diagnosed with: Abortion during pregnancy during 9-10 weeks. Habitual miscarriage. The remnants of the ovum were removed. From the anamnesis: the third pregnancy, proceeded with a threat of interruption, was carried out therapy aimed at prolonging the pregnancy (duphaston, magneB6, valerian). In the period of 5-6 weeks she underwent acute respiratory viral infections with fever up to 37.2, was treated with folk remedies. The first pregnancy in 2006 ended in spontaneous miscarriage for a period of 8-9 weeks. The second in 2007 - non-developing pregnancy for a period of 6-7 weeks. Menarche at 13 years old, established immediately, for 5 days, b / w 28 days, regular, moderate, painless. Examined at a UGI: Ureaplasma urealiticum was detected, no treatment was performed. From the anamnesis: in childhood - rubella, chicken pox; chronic tonsillitis.

Despite the therapy aimed at prolonging pregnancy, the patient subsequently noted increased pain in the lower abdomen and blood discharge. Diagnosed abortion, removal of the remains of the fetal egg.

The level of IL-28B was 471.54 [pg / ml]. 3 times increase in IL-28B cytokine concentration.

Conclusion: in comparison with the group of healthy pregnant women in patient 3. a significant increase in the content of the cytokine IL-28B in the presence of urogenital infection was revealed. It was predicted the development of a miscarriage, which took place at 12 weeks of gestation.

Example 4

Pregnant, B. 28 years old was admitted to the Department of Gynecology 1RD with a diagnosis of Pregnancy 8-9 weeks. Toxicosis of the middle art. gravity. Complaints of weakness, nausea, vomiting up to 8 times a day, weight loss of 4 kg. From the anamnesis: second pregnancy. The first in 2013 - ended in spontaneous miscarriage for a period of 7/8 weeks. Menarche at age 12, established immediately, for 6-7 days, b / w 28-30 days, moderate, painless. Ectopia of the cervix uteri since 2010. Examined at the IGO: detected HPV 16/18/32, Herpes simplex virus 2. Treatment was not carried out. From the anamnesis: in childhood - chicken pox; chronic gastritis. Ultrasound - Pregnancy 8-9 weeks, myometrial hypertonicity. Antiemetic, infusion, sedative therapy was carried out.

The level of IL-28B was 324.43 [pg / ml]. 2.1-fold increase in the concentration of IL-28B cytokine.

Conclusion: in comparison with the group of healthy pregnant women, patient B. revealed a significant increase in the content of IL-28B cytokine in the presence of urogenital infection of viral origin. Miscarriage was predicted. Despite the therapy, the miscarriage occurred at 10 weeks of gestation. The forecast has been confirmed.

As a result of studies on the assessment of indicators of innate immunity in a group of 18 pregnant women with a threat of abortion, we predict the development of a miscarriage with a probability of 66.6%. In addition, among the women studied, 80% of all miscarriages occurred at 6–7 and 10–11 weeks of gestation.

Thus, this method is minimally invasive, convenient and easy to use, quite informative and has a high reliability of the prognosis of miscarriage of infectious genesis of early gestation. The claimed invention allows to solve an important practical problem of early prediction of the threat of miscarriage in the first trimester, which is important in practical terms, and is based on specific indicators. Using this method will contribute to a faster and safer diagnosis of the threat of termination of pregnancy of infectious origin in the first trimester.

Claims (1)

A method for predicting miscarriage of pregnancy of infectious origin at an early stage of gestation, characterized in that in the blood serum of pregnant women with a period of 6-11 weeks of gestation an enzyme-linked immunosorbent assay determines the level of interferon-like cytokine IL-28B and at a concentration above 261 pg / ml they predict the risk of miscarriage in first trimester of gestation.