RU2743880C1 - Method for prediction of preterm delivery in multiple pregnancies - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to obstetrics and gynecology, and aims at prediction of preterm delivery in multiple pregnancies. M-RNA expression levels of the innate immunity genes are determined IL1B, IL10, IL18, TNFa, TLR4, GATA3, CD68, B2M by molecular-biological analysis of PCR-RT in epithelial cells of separated vagina. Values of discriminant functions F and F1 are calculated. If F>F1, preterm labor is predicted, and F<F1 shows the term birth.EFFECT: invention provides prediction of preterm delivery in multiple pregnancies with high accuracy, reduced number of hospitalizations and unreasonable therapeutic interventions at low risk of preterm delivery, timely preventive measures in case of premature birth threat and pathogenetic justified choice of the therapy method of threatening preterm delivery with high risk of spontaneous preterm delivery.1 cl, 3 ex, 1 tbl

Description

The invention relates to the field of medicine, namely to obstetrics and gynecology, and can be used to predict premature birth in multiple pregnancies, is intended for outpatient obstetricians-gynecologists, hospitals, perinatal centers.

Currently, the problem of premature birth (PR) in multiple pregnancies has acquired the status of one of the most important areas in modern obstetrics. The priority of the issue was determined by the global trend of a steady increase in the frequency of multiple pregnancies associated with the introduction of assisted reproductive technologies (ART). In turn, the predominance of PR in the category of complications and the significance of their consequences approved them as a leading factor in perinatal losses in multiple pregnancies [Romero R., Conde-Agudelo A., El-Refaie W., El-Refaie W., Rode L., Brizot M, Cetingoz E., Serra V., Da Fonseca E., Abdelhafez M., Tabor A., Perales A., Hassan S., Nicolaides K. Vaginal progesterone decreases preterm birth and neonatal morbidity and mortality in women with a twin gestation and a short cervix: an updated meta-analysis of individual patient data. Ultrasound Obstet Gynecol. 2017].

Finding ways to reduce the number of premature births with multiple births is an important but also extremely difficult task. The polyethiological nature of pathogenetic mechanisms makes it difficult to determine the most effective strategies for predicting, identifying and diagnosing preterm labor [Krasnopolsky, V.I., Novikova, S.V., Tsivtsivadze, EB, Zharova, A.A. (2015). Management of pregnancy and childbirth in multiple pregnancies. Almanac of Clinical Medicine, (37), 32-40].

Expansion of ideas about pathological mechanisms determined infectious and inflammatory processes a dominant role in the initiation of premature labor [Pustotina OA, Ostromensky V.V. Infectious factor in the genesis of miscarriage // Effective pharmacotherapy. 2019.Vol. 15.No.13. S. 26-33].

Inflammatory processes play an important role in the development of physiological labor: remodeling of the cervix during its maturation, rupture of the membranes and the occurrence of regular uterine contractions. There is a large amount of evidence that by the beginning of labor in the amnion, chorion, decidua, the number of pro-inflammatory cytokines and prostaglandins increases. In the cervix, subpopulations of leukocytes, macrophages, neutrophils are attracted. Leukocytosis within the myometrium and the synthesis of cytokines play an important role in the regulation of labor and its progression [Ailamazyan E., Kuzminykh T. 2013. Discussion issues of preterm birth. Journal of Obstetrics and Women's Diseases, 62 (4), 97-105].

During pregnancy, the response to the introduction of infectious agents can reproduce a cascade of reactions leading to the early onset of labor. The resulting inflammatory response becomes a trigger of uterine contractile activity, an increase in the level of enzymes leads to a shortening of the cervix, rupture of membranes and, as a result, premature birth [Beznoshchenko GB Syndrome of systemic inflammatory response in obstetric clinic: resolved issues and unresolved problems. Russian Bulletin of Obstetrician-Gynecologist. 2018.Vol. 18.No.4. S. 6-10].

In the studies carried out to date, data have been obtained on the presence of an association between the imbalance of pro-inflammatory and anti-inflammatory cytokines, changes in the level of mRNA expression of their genes and the risk of spontaneous preterm birth [Kalinka J, Sobala W, Wasiela M, Brzezin 'skaBlaszczyk E. in cervicovaginal fluid, as measured in midgestation, are associated with preterm delivery. AJRI 2005; 54: 70-76, Katkova N.Yu., Bodrikova O.I., Sergeeva A.B, Andosova L.D., Shakhova K.A., Bezrukova I.M., Pokusaeva K.B., Ryzhova N.K. The state of the local immune status, the content of neopterin and cortisol in various types of preterm labor. Journal of Obstetrics and Women's Diseases. - 2017. - T. 66. - No. 3. - S. 60-70].

At the same time, these studies did not include a comprehensive assessment of indicators, and the study cohort was represented by women with singleton pregnancies. Thus, the problem of determining the prognostic value of the integral assessment of inflammatory biomarkers in relation to the implementation of preterm birth in multiple pregnancies seems to be an unsolved problem.

The technical result of the invention is to predict premature births with multiple pregnancies with a high degree of accuracy, reduce the number of hospitalizations and unreasonable therapeutic interventions with a low risk of premature birth, timely implementation of preventive measures with the threat of premature birth and pathogenetically justified choice of a method of therapy for threatening premature births with a high risk of premature spontaneous birth childbirth.

The specified technical result is achieved in a method for predicting premature birth in multiple pregnancies, in which the expression levels of mRNA of the innate immunity genes IL1B, IL10, IL18, TNFa, TLR4, GATA3, CD68, B2M are determined by the method of molecular biological analysis of RT-PCR in epithelial cells of the discharge vagina, the values of the discriminant functions F and F1 are calculated using the following formulas:

F = -151.299 + 45.199 x X -11.166 x Y -45.49 x Z-64.028 x M + 10.267 x K-71.962 x L + 129.403 x S + 5.985 x R;

F1 = -153.151 + 45.155 x X -11.123 x Y -45.375 x Z-63.212 x M + 11.983 x K-72.564 x L + 129.102 x S + 5.209 x R, where:

F - the likelihood of premature birth;

F1 - the likelihood of urgent delivery;

X is the level of expression of the IL1B gene; Y is the level of gene expression, IL10;

Z - TNFA express gene level; M is the expression level of the TLR4 gene;

K is the expression level of the GATA3 gene; L - gene expression level - CD68;

S is the expression level of the B2M gene; R - expression level of the IL18 gene

and with F> F1, preterm labor is predicted, with F <F1 - urgent labor.

The method is based on the integral assessment of markers of the local inflammatory process - indicators of the most significant mechanism for the implementation of preterm birth, which ensures the effectiveness of predicting spontaneous preterm birth in multiple pregnancies.

In order to assess the presence and severity of local inflammation, 30 pregnant dichorionic twins were examined. The minimum gestation period was 22 weeks, the maximum - 36 weeks. The average gestational age was (M ± m) 29.4 ± 2.1 weeks, the median was 30 weeks. All pregnant women underwent a test based on determining the mRNA expression profile of innate immunity genes in epithelial cells of the lower reproductive tract (IL1B, IL10, TNFA, TLR4, GATA3, CD68, B2M, IL18).

Based on the data obtained, a prognostic model of the likelihood of preterm birth in multiple pregnancies was built based on the level of mRNA of innate immunity genes in epithelial cells of the lower parts of the reproductive tract. Linear discriminant analysis was used to identify the predictive value of the studied parameters, to derive the formula for calculating the probability of preterm birth and to check the operation of the algorithm for predicting it.

As a result of discriminant analysis, a mathematical model of the likelihood of the realization of premature birth was obtained, represented by the following formulas.

The likelihood of premature birth:

F = -151.299 + 45.199 x X -11.166 x Y -45.49 x Z-64.028 x M + 10.267 x K-71.962 x L + 129.403 x S + 5.985 x R.

Probability of urgent delivery F1 = -153.151 + 45.155 × X -11.123 × Y -45.375 × Z-63.212 × M + 11.983 × K-72.564 × L + 129.102 × S + 5.209 × R.

The table shows the parameters of the functions F and F1 in the model of the probability of the realization of preterm birth.

With F> F1 - a conclusion about a high risk of preterm birth, with F <F1 - a conclusion about a low risk of preterm birth.

When checking the work of this formula, the program assigned the patients to the group threatened by preterm birth in 100% of cases. When calculating the probability of urgent delivery, the program determined correctly in 86.7% of cases. In 13.3%, a false-positive result was obtained for premature birth. Thus, when calculating according to this formula, if the probability of control (urgent labor) is less than the probability of preterm birth, then with a probability of 100% it can be assumed that this patient will have spontaneous preterm labor. If the probability of control is greater than the probability of premature birth, then with a probability of 86.7% it can be assumed that this patient will have a term birth.

The method is carried out, for example, as follows.

To assess the local immune status, vaginal discharge is taken from the subjects with a sterile disposable probe. The biomaterial is placed in 1.5 ml plastic tubes, which contain 500 μl of RNA stabilization medium. Carry out the formulation of the test "ImmunoKvantex" (LLC "NPO DNA-Tekhnologiya", Moscow, Russia). In this test system, after isolation of the total pool of DNA and RNA, a reverse transcription reaction is carried out to obtain complementary DNA from the mRNA template, which is further amplified by PCR. After the amplification stage, according to the indicator cycle, the program calculates the level of mRNA expression of the innate immunity genes IL1B, IL10, IL18, TNFa, TLR4, GATA3, CD68, B2M.

The obtained values of the expression levels are substituted into the formulas for calculating the values of the discriminant functions F and F1, and with F> F1, preterm labor is predicted, and with F <F1, urgent labor.

The essence of the method is confirmed by the following clinical examples.

Example 1. Pregnant dichorionic twins K., 33 years old, was admitted to the hospital with clinical manifestations of threatening premature birth at 32 weeks of gestation (the patient noted pulling pains in the lower abdomen, according to ultrasound cervicometry, a "short" cervix 10 mm long was stated). A standard clinical and laboratory examination was carried out, as well as a molecular biological analysis of RT-PCR of the mRNA expression profile of innate immunity genes in epithelial cells of the lower parts of the reproductive tract. The following values of gene expression were determined: IL1B -4.6, IL10-5.1, IL 18-5.8, TNFA-3.1, TLR-0, GATA3-3.8, CD68-3.6, B2M-3.9 lg GE / ml.

When calculating by the formulas:

F = -246.922

(-151.299 + 45.199 x 4.6 -11.166 x 5.1 -45.49 x 5.8 -64.028 x 3.1 + 10.267 x 0 -71.962 x 3.8 + 129.403 x 3.6 + 5.985 x 3, 9), F1 = -251.958

(-153.151 + 45.155 x 4.6 -11.123 x 5.1 -45.375 x 5.8 -63.212 x 3.1 + 11.983 x 0 -72.564 x 3.8 + 129.102 x 3.6 + 5.209 x 3.9) ...

Considering that F> F1, it was concluded that there is a high risk of preterm birth.

The patient, at 33 weeks, had a spontaneous premature birth, which began with a premature rupture of amniotic fluid. Live premature babies were born with weight-height indicators of 2070/45 and 2210/45, with Apgar scores of 6/6 and 6/6 points.

Example 2. Pregnant dichorionic twins L. 30 years old, was admitted to the hospital with a period of 28 weeks with clinical manifestations of threatening premature birth (the patient noted pulling pains in the lower abdomen. According to ultrasound cervicometry, the length of the cervix was 37 mm. A standard clinical and laboratory examination was carried out, and a molecular biological analysis of RT-PCR of the mRNA expression profile of innate immunity genes in epithelial cells of the lower parts of the reproductive tract The following values of gene expression were determined: IL1B -4.2, IL10-1, IL18-5.1, TNFA-2.6, TLR- 1.3, GATA3-3.9, CD68-4.7, B2M-4.5 lg GE / ml.

When calculating by the formulas:

F = -3.2791

(-151.299 + 45.199 × 4.2 -11.166 × 1 -45.49 × 5.1.8-64.028 × 2.6 + 10.267 × 1.3-71.962 × 3.9.8 + 129.403 × 4.7 + 5.985 × 4.5), F1 = -7.5885

(-153.151 + 45.155 x 4.2 -11.123 x 1 -45.375 x 5.1-63.212 x 2.6 + 11.983 x 1.3-72.564 x 3.9 + 129.102 x 4.7 + 5.209 x 4.5) ...

Considering that F> F1, it was concluded that there is a high risk of preterm birth.

The patient had spontaneous preterm labor at 31 weeks. Live premature babies were born with weight-height indicators of 1630/42 and 1530/38, with Apgar scores of 6/6 and 6/6 points.

Example 3. Pregnant dichorial twins S. 32 years old, was admitted to the hospital at a period of 27 weeks. The patient did not report any complaints. According to ultrasound cervicometry, the length of the cervix was 12.5 mm. A standard clinical and laboratory examination was carried out, as well as a molecular biological analysis of RT-PCR expression profile of innate immunity genes in epithelial cells of the lower parts of the reproductive tract. The following values of gene expression were determined: IL1B -6.1, IL10-3.2, IL18-4.1, TNFA-3.7, TLR-4.4, GATA3-3.4, CD68-4.3, B2M-5.9 lg GE / ml.

When calculating by the formulas:

F = 57.5195

(-151.299 + 45.199 × 6.1-11.166 × 3.2-45.49 × 4.1-64.028 × 3.7 + 10.267 × 4.4-71.962 × 3.4 + 129.403 × 4.3 + 5.985 × 5.9),

F1 = -58.6583

(-153.151 + 45.155 × 6.1-11.123 × 3.2-45.375 × 4.1-63.212 × 3.7 + 11.983 × 4.4-72.564 × 3.4 + 129.102 × 4.3 + 5.209 × 5, 9).

Considering that F <F1, it was concluded that there is a low risk of preterm birth.

The patient had spontaneous urgent labor at 37 weeks gestation. Live full-term babies were born with weight-height indicators of 2410/47 and 2480/48, with Apgar scores of 7/8 and 7/8 points.

EFFECT: method ensures efficiency of preterm birth prediction, reduces the number of hospitalizations and unreasonable therapeutic interventions at low risk of preterm birth and ensures timely preventive measures, pathogenetically justified choice of a method of therapy for threatening preterm birth with a high risk of spontaneous preterm birth.

Claims (14)

A method for predicting premature birth in multiple pregnancies, which consists in determining the levels of mRNA expression of the innate immunity genes IL1B, IL10, IL18, TNFa, TLR4, GATA3, CD68, B2M by the method of molecular biological analysis of PCR-RT in the epithelial cells of the vaginal discharge, calculate the values of the discriminant functions F and F1 according to the following formulas: F = -151.299 + 45.199 x X -11.166 x Y -45.49 x Z -64.028 x M + 10.267 x K -71.962 x L + 129.403 x S + 5.985 x R; F1 = -153.151 + 45.155 x X -11.123 x Y -45.375 x Z -63.212 x M + 11.983 x K -72.564 x L + 129.102 x S + 5.209 x R, where: F - the likelihood of premature birth; F1 - the likelihood of urgent delivery; X — expression level of the IL1B gene, lg GE / ml; Y - expression level of the IL10 gene, lg GE / ml; Z — expression level of the TNFa gene, lg GE / ml; M is the expression level of the TLR4 gene, lg GE / ml; K - expression level of the GATA3 gene, lg GE / ml; L - gene expression level - CD68, lg GE / ml; S is the expression level of the B2M gene, lg GE / ml; R - expression level of the IL18 gene, lg GE / ml and with F> F1, preterm labor is predicted, with F <F1 - urgent labor.