RU2763707C1 - Method for predicting premature rupture of membranes in the period from 22 to 28 weeks of gestation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to obstetrics. In pregnant women at a gestational age of 22-28 weeks, the relative abundance of bacteria of the genus Lactobacillus and Staphylococcus, expressed as a percentage (%), is determined in the vaginal microbiome, and the probability of premature rupture of the membranes is calculated within 28 weeks. pregnancy p according to the formula. If the p value is 57.42% or more, the development of premature rupture of the membranes is predicted in the period from 22 to 28 weeks of pregnancy.

EFFECT: method allows increasing the accuracy and specificity of prognosis of PRM at the preclinical stage, when the membranes have not yet ruptured, and due to this, the possibility of timely optimization of pregnancy management tactics and improvement of perinatal outcomes.

1 cl, 3 ex, 1 dwg

Description

The invention relates to medicine, namely to obstetrics and gynecology, and can be used to predict premature rupture of the membranes (PROM) in the period of 22-28 weeks. pregnancy.

Premature rupture of the membranes (PROM) is a violation of the integrity of the membranes of the fetus and the outflow of amniotic fluid before the onset of labor, regardless of the gestational age.

The share of PROM in preterm pregnancy accounts for 20-40%, which is combined with 30-40% of preterm births. Birth before 28 weeks of gestation (or extremely early preterm birth) is a global health problem [March of Dimes, PMNCH, Save the Children, WHO. Born Too Soon: The Global Action Report on Preterm Birth. Geneva: World Health Organization; 2012.: http.//www.who.int/pmnch/media/news/2012/201204_borntoosoon-report.pdf.] and complicate approximately 0.4-0.7% of all pregnancies. This condition is associated with a very high neonatal mortality rate, as well as an increased risk of both short-term and long-term neonatal morbidity. Among children born at 22-24 weeks of gestation, mortality within a few weeks is 97-98%, with only 1% surviving without neurodevelopmental disorders. Among those born at 24-25 weeks, 55% of newborns survive, but only 32% do not have neurological deficits at the age of 18-22 months of life [Noelle Younge et al., 2017]

According to the literature, the question of the etiology of PROM does not have a clear answer. To date, it is the infectious-inflammatory factor that is the most studied and widespread component of the pathogenesis of PROM. Indirect evidence of the significant role of the infectious-inflammatory factor is the high incidence of chorioamnionitis in pregnant women with PROM. Histological chorioamnionitis is observed in almost half of all cases of PROM occurring before 34 weeks of gestation, and the incidence of clinical chorioamninitis, even with a short latent period (less than 7 days in 75.7%), is 17.8% [Yu N. et al., 2015] .

If we talk about the species structure of the pathogen, then the issue of diagnosis is still relevant, and this is due not only to the very fact of verification of the pathogen, but to the formation of strains resistant to antibacterial drugs. Recent studies have shown that culture studies are less likely to detect microorganisms (34%) compared to the polymerase chain reaction method (45%) [DiGiulio D.B. et al., 2010], and in cases with microorganisms of the Mycoplasma class, the use of only bacteriological examination can lead to false negative results in the study of amniotic fluid in 91% of cases [Oh K.J. et al., 2010; Kasper D.C. et al., 2011]. This fact is especially important, since according to Romero R. et al. are among the most frequently detected microorganisms in women with PROM [Romero R. et al., 2015].

M. et al., 2008]. Кроме того, стоит отметить следующую закономерность: чем меньше гестационный возраст при ПРПО, тем выше вероятность микробного воспаления [Romero R. et al., 2015].Sneathia amnii [Romero R. et al., 2015], coagulase-negative Staphylococcus, Streptococcus viridans [

M. et al., 2008]. In addition, it is worth noting the following pattern: the lower the gestational age in PROM, the higher the likelihood of microbial inflammation [Romero R. et al., 2015].

Despite the frequent identification of an infectious factor, in most cases the cause of PROM and associated perinatal complications remains unknown. The main problem that researchers faced when trying to analyze the genetic material in a biological sample (for example, in vaginal fluid) was to distinguish bacterial DNA from all others - primarily from the DNA of the body's own cells. This problem was solved only thanks to the discovery of the American scientist Carl Richard Woese, made at the end of the last century (1985): ribosomal RNA (rRNA) is unique for each type of nuclear and non-nuclear cells, including bacteria, and besides is outside the influence of natural selection, and therefore its genes are the most stable [Goldenfeld N., Race NR Carl R. Woese, 2013]. The 16S component of the bacterial rRNA subunit turned out to be the most rational for analysis, because neither eukaryotes nor archaea have it; it is characteristic only of bacteria. Thanks to the method of metagenomic sequencing of 16S rRNA, the possibility of early prediction of premature rupture of the membranes has become possible.

The further identification of early diagnostic markers of PPROM seems to be relevant in order to carry out timely therapeutic measures. Early, preclinical diagnosis and prediction of the development of PROM will optimize the management of pregnant women, which will help to identify this complication in a timely manner and improve perinatal outcomes.

From the existing state of the art, a method is known for predicting premature rupture of the membranes by examining the contents of the vagina, characterized in that, in order to improve the accuracy of the forecast, the activity of neurominidase is determined and, at a value of 2.5 mmol/l and higher, premature rupture of the membranes is predicted (patent RF No. 2014601, 1994.06.15).

The disadvantages of this method: the method of sampling the vaginal contents is not standardized; the gestational age for sampling the contents of the vagina and the gestational age at which premature rupture of amniotic fluid is predicted - from 30 weeks.

A known method for predicting premature rupture of amniotic fluid in pregnant women by studying biological material, characterized in that in women at a gestational age of 32-36 weeks, the level of IL-8 is determined in the blood serum and at its value equal to or less than 15.4 ng / ml, predict premature rupture of amniotic fluid (RF patent No. 2405453, publ. 10.12.2010).

The disadvantage of this technical solution is that the prediction of PROM by examining peripheral venous blood in pregnant women is carried out at a gestational age of 32-36 weeks, and thus does not allow predicting very early and early preterm birth. Which significantly reduces the importance of this method for the choice of obstetric tactics in this particular period of pregnancy.

A known method for predicting preterm birth by the joint determination of extracellular DNA and interleukin-8 in peripheral blood plasma, characterized in that this method uses the simultaneous measurement of the total amount of extracellular DNA by real-time polymerase chain reaction and IL-8 by enzyme immunoassay in blood plasma in pregnant women at 22-36 weeks of gestation to determine the variable P by the formula:

P \u003d 1 / (1 + exp (- (-4.1 + 4 × X1 + 0.0001 × X2))), where

X1 is the concentration of IL-8 in peripheral blood plasma, expressed in pg/ml;

X2 is the concentration of ovDNA in peripheral blood plasma, expressed in GE / ml,

at P>0.5, preterm labor will occur; at P≤0.5, preterm birth will not occur (RF patent No. 2682713, published on 03/21/2019).

The disadvantage of this technical solution is that the prediction of PROM is carried out at 32-36 weeks of gestation, and thus does not allow predicting very early and early preterm birth. Which significantly reduces the importance of this method for the choice of obstetric tactics in this particular period of pregnancy.

A known method for predicting preterm birth in women with a threat of miscarriage, including a blood test, characterized in that in the blood serum of a pregnant woman at 18-22 weeks the level of IL-33 is determined by the enzyme immunoassay method and, if its value is above 0.98 pg / ml, the outcome of pregnancy is predicted in the form of premature birth (patent No. 2710244, published on 12/25/2019).

The disadvantage of this technical solution is that the prediction of preterm birth is performed without specifying the onset date, which is important in further obstetric management tactics.

A known method for predicting the duration of the latent period in ultra-early and early preterm labor, complicated by rupture of the membranes, characterized in that as predictors are used indicators of highly sensitive C-reactive protein - XhsCRP and anti-inflammatory cytokine IL-6 - XIL6, which are determined in venous blood serum and according to the formula: Ydlp = 450.1 - 14.84 * XhsCRP - 14.54 * XIL6, the duration of the latent period in hours is calculated (patent No. 2716973, published on 03/17/2020).

The disadvantage of this technical solution is that only the possible duration of pregnancy prolongation is predicted after ascertaining the onset of premature rupture of the membranes (PROM), and the fact of the onset of PROM is not predicted with intact membranes.

A known method for assessing the risk of premature rupture of the membranes in pregnant women, including performing a scraping of the epithelium of the cervical canal, assessing the local immune status based on the determination of the level of cytokine gene mRNA expression using real-time polymerase chain reaction, characterized in that in pregnant women with a gestational age of 37 weeks or more determine the levels of expression of mRNA genes IL-18 and TLR-2, normalized to the reference gene B2M; based on the results obtained, the risk of premature rupture of the membranes R is calculated by the formula: units; X2 - the value of the expression level of the mRNA of the TLR-2 gene in relative units; with a P value greater than or equal to 0.5, the risk is assessed as high; with a value of P less than 0.5 - as low (RF patent No. 2740950, pub. 01/21/2021).

The disadvantage of this method is that it is applicable only for gestational age of 37 weeks or more. It cannot solve the problem of predicting PRPO at very early gestation.

Closest to the claimed technical solution is a method for predicting premature rupture of the membranes according to the patent of the Russian Federation No. 2014601, publication 15. 06.1994, adopted by us as a prototype.

These disadvantages of the prototype method are eliminated in the claimed technical solution.

The objective of the claimed invention is to develop a method for predicting premature rupture of the membranes in the early stages to identify pregnant women at risk for the formation of this pathology.

This problem is solved due to the fact that in pregnant women at gestational age from 22 to 28 weeks, a discharge from the posterior vaginal fornix is obtained, microbes are analyzed and the relative representation of bacteria of the genus Lactobacillus and Staphylococcus is determined, the probability of premature rupture of the membranes is calculated up to 28 weeks. pregnancy p according to the formula:

p \u003d 1 / (1 + e ^-z ) * 100%, where

e - Euler constant ≈2.718281,

z \u003d 2.99 - 0.06 * X _{Lactobacillus} + 6.71 * X _{Staphylococcus} , where

X _{Lactobacillus} - relative representation of bacteria of the genus Lactobacillus, numerical value;

X _{Staphylococcus} - relative representation of bacteria of the genus Staphylococcus, numerical value;

if the p value is 57.42% or more, the risk of premature rupture of the membranes at 22-28 weeks of gestation is predicted.

The technical result provided by the above set of features is to increase the accuracy and specificity of predicting PRPO at the preclinical stage, when the membranes have not yet ruptured. Due to this, it is possible to carry out timely optimization of pregnancy management tactics and improve perinatal outcomes.

One of the most studied components of the pathogenesis of PROM is the infectious-inflammatory factor. Indirect evidence of the significant role of the infectious-inflammatory factor is the high incidence of chorioamnionitis in pregnant women with PROM. Histological chorioamnionitis is observed in almost half of all cases of PROM occurring before 34 weeks of gestation, and the incidence of clinical chorioamninitis, even with a short latent period (less than 7 days in 75.7%), is 17.8% [Haiyan Yu et al., 2015].

There are data in the literature on the results of a study of the vaginal microbiome in women with PROM at 24-33.6 weeks in a hospital in Vancouver, Canada. Microbe - a set of genes of microorganisms that inhabit a certain habitat (in this case, the vagina). [Teenus Paramel Jayaprakash et al., 2016]. Pregnant women had a mixed vaginal microbe, but the specific type of profile in PROM did not correlate with the length of the anhydrous period.

A study in Korea [Young-Ah You et al., 2019] found that women at 22.0-36.6 weeks of gestation with PROM had more and more bacterial diversity at admission than women with preterm birth. Women who delivered prematurely had a bacterial community dominated by Bacteroides and Lactobacillus crispatus. It is noted that women with an abundance of Weissella in a bacterial community dominated by bacteroids gave birth at term. In addition, in bacterial communities with Lactobacillus, the prevalence of Weissella and Rickettsiales was associated with term birth, and the prevalence of Bacteroides and Escherichia-Shigella was associated with preterm birth. Based on the data available in the literature, microbiome analysis was carried out according to general rules using the SILVA v132 global database to identify known bacteria.

We have established prognostic parameters for the development of this obstetric pathology of pregnancy by calculating a diagnostic index based on determining the relative representation of bacteria of the genus Lactobacillus and Staphylococcus in the vaginal microbiome. Of the analyzed all genera of bacteria in the vaginal microbiome, a decrease in the relative representation of bacteria of the genus Lactobacillus and an increase in the relative representation of bacteria of the genus Staphylococcus in the vaginal microbiome of pregnant women with premature rupture of the membranes were revealed compared with the control group.

Thus, our results made it possible to judge the change in the relative representation of bacteria of the genus Lactobacillus and Staphylococcus in the vaginal microbiome in pregnant women with premature rupture of the membranes compared to women whose pregnancy proceeds without complications and to develop a method for predicting this pathology in pregnant women from 22 to 28 weeks of gestation.

To classify patients according to the risk of developing PPROM, a prognostic model was built using the binary logistic regression method based on the results of determining the relative representation of the most informative combination of bacterial genera in the vaginal microbiome {Lactobacillus and Staphylococcus).

The totality of all microorganisms (bacteria, archaea, protozoa, fungi, etc.) of various areas of the human body is commonly called the microbiota, and together with the genome, the microbiome [Eamonn M M Quigley, 2017; Ron Sender et al., 2016], thus, a microbial is a set of genes of microorganisms inhabiting a certain habitat (https://bn-cl.ru/images/2020/01/20/microbiom.pdf) (in our case - vagina).

The relative representation of bacteria of the genus is the relative content (proportion) of the microorganism genus, expressed as a percentage (%) of the total composition of the vaginal microbiome, taken as 100%.

The probability of developing PPROM is described by the following regression equation:

in which z \u003d 2.99 - 0.06 * X _{Lactobacillus} + 6.71 * X _{Staphylococcus}

where p is a prognostic index characterizing the probability of having PPROM, e is the Euler number, a mathematical constant (≈2.718), z is the coefficient of the regression function, X _{Lactobacillus} is the relative abundance of the genus Lactobacillus, a numerical value; X _{Staphylococcus} - relative representation of the genus Staphylococcus, numerical value; if the p value is 57.42% or more, the development of premature rupture of the membranes in the period from 22 to 28 weeks of pregnancy is predicted.

The resulting regression model is statistically significant (p<0.001). Based on the value of the Nigelkirk determination coefficient, the model takes into account 65.9% of the factors that determine the likelihood of developing a disease (PRPO). In order to improve the diagnostic efficiency of the model, we adjusted the classification threshold based on the results of the analysis of the ROC curve. The threshold value was -57.42%. If the p value is 57.42% or more, the risk of premature rupture of the membranes is predicted.

Using ROC-analysis, threshold values for the level of relative representation of bacteria of the genus Lactobacillus and Staphylococcus were determined. The AUC value was 92.9%. Model sensitivity - 83.3%, specificity - 86.7% (Fig. 1).

Thus, the belonging of patients to the group of high or low risk of developing PPROM was determined based on the calculated values of the diagnostic regression function: with a prognostic index value of 57.42% or more, the pregnant woman was at high risk of developing PPROM due to an imbalance in the bacterial balance in the vaginal microbiome, with function value less than 57.42% - to the low-risk group.

Using the claimed method, we examined 60 pregnant women in the period of 22-28 weeks, in which a study of the bacterial balance in the vaginal microbiome was carried out by the claimed method. In 40 pregnant women, the value of the prognostic index was equal to or more than 57.42%, i.e. pregnant women were at high risk of developing premature rupture of the membranes. Thus, the proposed method allows timely identification of pregnant women at risk for the formation of this pathology. In 20 pregnant women, the value of the prognostic index was less than 57.42%, i.e. they belonged to the low-risk group for developing PPROM. The obtained data were processed using the IBM SPSS Statistics 23 program.

Detailed description of the method and examples of its clinical implementation

The method is carried out as follows. In pregnant women in the period of 22-28 weeks of gestation, the discharge from the posterior fornix of the vagina is taken using a sterile disposable universal urogenital probe. Samples are placed in sterile test tubes with transport medium "transport medium with mucolytic" (state institution Central Research Institute of Epidemiology, Russia) and stored at +4°C until DNA is isolated (vaginal microbiome). Total DNA was isolated from the samples using the RIBO-prep kit (Central Research Institute of Epidemiology, Russia) according to the manufacturer's recommendations. 16S DNA libraries were prepared according to the Illumina protocol "16S metagenomic sequencing library preparation" (Part #15044223 Rev.B). Bioinformatic analysis of sequencing data was carried out using programming languages: R v.3.6 and Python 3.6.9. In the first step, the primer sequences were truncated from the beginning of the pairwise end reads, with the pairs of reads not containing the primer sequence being discarded. The final 25 base pairs from the end of each read were then trimmed as low-quality bases and processed using the DADA2 workflow to accurately identify sequence variants. After exact sequence variants were deduced, the forward and backward reads were concatenated and the resulting sequences were used for Bayesian taxonomic comparison using the SILVA v132 database as a reference.

The determined values of the relative representation are the relative content (proportion) of the microorganism genus, expressed as a percentage (%) of the total composition of the vaginal microbiome, taken as 100%. At the same time, X _{Lactobacillus} - the relative representation of the genus Lactobacillus and X _{Staphylococcus} - the relative representation of the genus Staphylococcus by absolute numbers (numerical values) are substituted into the formula for calculating the z - coefficient of the regression function:

z \u003d 2.99 - 0.06 * X _{Lactobacillus} + 6.71 * X _{Staphylococcus} , after which they calculate

p - prognostic index characterizing the probability of developing PPROM, which is calculated by the formula:

If the p value is 57.42% or more, a high probability of developing PROM is established in the very early stages of pregnancy (up to 28 weeks).

This allows you to timely identify pregnant women at risk for the formation of this pathology and carry out the necessary set of therapeutic and preventive measures, which will help reduce the risk of perinatal mortality and morbidity, and reduce the economic costs of treating and nursing premature newborns.

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

Example 1

Pregnant M-ova, aged 26, examined according to the claimed method at a scheduled outpatient appointment in the outpatient department of the GBU RO "PC" in the period of 22 weeks of gestation. In a laboratory study of the vagina separated from the posterior fornix, the following indicators of the relative representation of labor in the vaginal microbiome were obtained:

X _{Lactobacillus} = 45.38%;

X _{Staphylococcus} = 0.09%;

z \u003d 2.99 - 0.06 * 45.38 + 6.71 * 0.09 \u003d 0.87.

The value of the prognostic index was: p=1/(1+e ^-0.87 )*100%=70.5%. This value exceeds 57.42%, which, according to the claimed method, indicates the need to include this patient in a high risk group for developing PROM.

In the period of 25 weeks, 4 days, she was hospitalized at the Perinatal Center in the Department of Pregnancy Pathology with complaints of light amniotic fluid leakage. Diagnosis at admission: Pregnancy 25 weeks 4 days. PRPO. The threat of premature birth. Mild anemia.

The woman was admitted to the hospital, observed and received therapy in accordance with the standard of medical care, in the period of 26 weeks 1 day regular labor activity developed.

The pregnancy ended with premature operative delivery at 26 weeks gestation 1 day and the birth of a live premature baby weighing 850 g, 37 cm long, with an Apgar score of 4/5 points.

Example 2

Pregnant G-va, 25 years old, at a scheduled outpatient appointment at 24 weeks of gestation was examined by the claimed method. In a laboratory study of the vagina separated from the posterior fornix, the following indicators of the relative representation of labor in the vaginal microbiome were obtained:

X _{Lactobacillus} 45.9%; X _{Staphylococcus} 0.02%;

z \u003d 2.99 - 0.06 * 47.09 + 6.71 * 0.02 \u003d 0.2988. The value of the prognostic index was: p=1/(1+e ^-0.37 )*100%=57.42%.

This value coincides with the boundary value of 57.42%, which, according to the claimed method, indicates the need for this patient to be attributed to the risk group for developing PROM. The pregnant woman underwent an adjustment of the treatment plan, a two-stage therapy was carried out (antibacterial drugs at the first stage, then the use of drugs with probiotic strains of lactobacilli), which made it possible to prevent the development of PROM in a timely manner.

Upon further observation, according to objective, vaginal, ultrasound examinations, as well as according to microbiological and microscopic examination of discharge from the vagina and cervical canal during pregnancy, no abnormalities were detected, the fetus's condition was assessed as normal, the pregnancy ended with urgent delivery through the natural birth canal in a period of 40 weeks and the birth of a live full-term boy weighing 3400 g, height 52 cm, Apgar score 9/10 points. Child's diagnosis at birth: healthy. Example 3

Pregnant G-eva, 28 years old, at a scheduled outpatient appointment at 26 weeks of gestation was examined according to the claimed method. In a laboratory study of the vaginal discharge from the posterior fornix, the following indicators of the relative representation of the vaginal microbiome delivery were obtained:

X _{Lactobacillus} = 71.82%; X _{Staphylococcus} = 0.03%;

z \u003d 2.99 - 0.06 * 71.82 + 6.71 * 0.03 \u003d -1.12.

The value of the prognostic index was: p=1/(1+e ^1.12 )*100%=24.64%. This value is less than 57.42%, which, according to the claimed method, indicates that this pregnant woman has no risk of developing PROM.

Upon further observation, according to objective, vaginal, ultrasound examinations, as well as according to microbiological and microscopic examination of discharge from the vagina and cervical canal during pregnancy, no abnormalities were detected, the fetus's condition was assessed as normal, the pregnancy ended with urgent delivery through the natural birth canal in term of 39 weeks and the birth of a live full-term boy weighing 3800 g, height 50 cm, Apgar score 10/10 points. Child's diagnosis at birth: healthy.

The claimed method allows to obtain reliable results and is more efficient than the known ones, having a number of advantages:

1. The method is specific - it allows predicting the risk of PROM formation using the relative abundance of only two genera of bacteria (Lactobacillus and Staphylococcus) in the vaginal microbiome, which are predictors of PROM, which makes it a highly specific method.

2. The method is highly sensitive, makes it possible to predict PROM at the preclinical stage, timely identify pregnant women at risk for the formation of this pathology, which makes it possible to carry out preventive measures aimed at preventing this pathological condition.

3. The method is minimally invasive, since the diagnosis is carried out according to the results of a study of the vaginal discharge from the posterior fornix.

The claimed method for predicting premature rupture of the membranes allows, based on highly sensitive modern laboratory markers, to timely implement the necessary set of therapeutic and preventive measures, which will help reduce the risk of perinatal mortality and morbidity, and reduce the economic costs of treating and nursing premature newborns.

Approbation 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 application in wide clinical practice.

Claims (7)

A method for predicting premature rupture of membranes in the period from 22 to 28 weeks of pregnancy by studying biological material, characterized in that in pregnant women at gestational age of 22-28 weeks, the relative representation of bacteria of the genus Lactobacillus and Staphylococcus is determined in the vaginal microbiome and the probability of premature rupture of membranes is calculated up to 28 weeks. pregnancy p according to the formula: p=1/(1+e ^-z )*100%, where e - Euler constant ≈2.718281, z=2.99-0.06*X _{Lactobacillus} +6.71*X _{Staphylococcia} , where X _{Lactobacillus} - relative representation of the genus Lactobacillus, numerical value; X _{staphylococcus} - relative representation of the genus Staphylococcus, numerical value; if the p value is 57.42% or more, the development of premature rupture of the membranes is predicted in the period from 22 to 28 weeks of pregnancy.

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