RU2845997C1 - Method for determining probability of antenatal foetal death for unspecified cause on term of full-term pregnancy - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to medicine, namely to pathological anatomy, obstetrics and gynaecology, and can be used for determining a probability of antenatal foetal death of an unspecified cause in the full-term pregnancy. In placental tissue samples, determining expression level of microRNA-126, microRNA-451, microRNA-125b with subsequent calculation of index d. If d > 0, a probability of antenatal foetal death of an unspecified cause at the full-term pregnancy is determined.

EFFECT: method enables determining a probability of the antenatal foetal death caused by an unspecified cause at the full-term pregnancy due to the assessment of placental expression of microRNA-126, microRNA-451, microRNA-125b.

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Description

The invention relates to medicine, namely to the field of pathological anatomy, obstetrics and gynecology. A method is presented for determining the role of epigenetic programming in the occurrence of antenatal fetal death due to an unspecified cause at full-term pregnancy based on an assessment of the expression level of microRNA-126, microRNA-451, microRNA-125b in the placenta to assess the preventability of this adverse outcome.

According to WHO, about 2.6 million cases of stillbirths are registered annually in the world, this figure remains stable and shows no tendency to decrease. About 80% are due to antenatal losses, characterized by polyetiology, a variety of risk factors, and incompletely studied thanatogenesis. At the same time, the share of antenatal fetal death due to an unspecified cause (sudden fetal death) ranges from 30% to 60% [Bulletin. WHO recommendations “Every child matters. Audit and analysis of stillbirth and neonatal death cases”. 2016; O.V. Remneva, E.G. Ershova, A.E. Chernova. Antenatal death of a full-term fetus: risk factors, possibilities of telemedicine in its prediction. Modern problems of science and education. 2018; 5: 33-36]. It is important that the incidence of sudden fetal death increases with gestational age from 26% to 50% [E.A. Sonchenko, A.F. Mikhelson, E.Yu. Lebedenko. “Mysterious” antenatal fetal death (literature review). Health and education in the 21st century. 2017; 10: 154-156].

Antenatal death of a full-term fetus is the death of a fetus that occurs in utero at a gestational age of 37 weeks 0 days - 41 weeks 6 days before the development of regular labor [G.V. Matrokhina. Antenatal death of a full-term fetus: risk factors, the possibilities of telemedicine in its prediction. Bulletin of Medical Science. 2019; 4 (16)].

There is currently insufficient literature data on the term “sudden fetal death syndrome,” which is the death of a fetus without diagnosed clinical manifestations of intrauterine hypoxia, the causes of which are not determined even after a pathological examination. In Russia, this term was introduced in 2011 by M.A. Kurtser et al. [M.A. Kurtser, Yu.Yu. Sudden fetal death syndrome. Obstetrics and Gynecology. 2011; 7(1)]. More modern studies of sudden fetal death syndrome describe the impact of certain factors that influence the increased incidence of this syndrome. In the study by N. Bednarczuk et al. (2020) Maternal smoking is considered as one of the causes of sudden fetal death due to an abnormal response to hypoxia and hypercarbia and a reduced response to arousal mediated by the effects of carbon monoxide and nicotine on the development of the nuclei of the brainstem, cerebral cortex and autonomic nervous system of the fetus [Bednarczuk N., Milner A., Greenough A. The Role of Maternal Smoking in Sudden Fetal and Infant Death Pathogenesis. Front Neurol. 2020; 23(11)]. In 2022, A.M. Gatti et al. conducted an in-depth examination of the brain of a stillborn fetus, which revealed various anomalies in the development of the brain centers that control vegetative and cardiorespiratory functions [Gatti A.M., Ristic M., Stanzani S., Lavezzi A.M. Novel chemical-physical autopsy investigation in sudden infant death and sudden intrauterine unexplained death syndromes. Nanomedicine (Lond). 2022; 17(5):275-288]. The authors of the study discovered nanosized environmental pollutants (NEPs) that can penetrate the blood-brain barrier and disrupt the normal development of vital centers of the fetal brain and, therefore, participate in thanatogenesis of intrauterine fetal death. Interesting data were presented by a team of Russian authors who showed a relationship between increased thickness of the nuchal translucency in the fetus in the absence of chromosomal abnormalities with an increased risk of antenatal fetal death even during normal pregnancy [E.V. Kudryavtseva, V.V. Kovalev, I.I. Baranov, K.S. Vshivtsev, E.V. Arebyov, N.N. Bayazitova. Relationship between first trimester prenatal screening indicators and the risk of pregnancy complications. Obstetrics and gynecology: news, opinions, training. 2020; 8(1):38-46].

Over the past 50 years, various studies have been conducted to detect genetic aberrations in fetal cells: from cytogenetic methods (karyotyping, fluorescent hybridization) to molecular genetics (quantitative fluorescence polymerase chain reaction, chromosome microarray analysis, next-generation sequencing technologies) [Silver R.M., Varner M.W., Reddy U. Work-up of stillbirth: a review of the evidence. Am J Obstet Gynecol. 2007; 196(5): 433-444; Bauld R., Sutherland G.R., Bain A.D. Chromosome studies in investigation of stillbirths and neonatal deaths. Arch Dis Child. 1974; 49(10): 782-788; Sahlin E., Gustavsson P., Liedén A. Molecular and Cytogenetic Analysis in Stillbirth: Results from 481 Consecutive Cases. Fetal diagnosis and therapy. 2014; 36(4): 326-332]. However, each method has its limitations, for example, karyotype analysis is only possible in the presence of viable cells, and chromosome abnormalities smaller than 5-10 Mb are technically impossible to detect at this stage.

Next-generation sequencing (NGS) technologies will make it possible to detect potential “independent” causes in various pathological processes, including stillbirth [Sahlin E., Gréen A., Gustavsson P. Identification of putative pathogenic single nucleotide variants (SNVs) in genes associated with heart disease in 290 cases of stillbirth. PLoS One. 2019; 14(1)]. Moreover, whole-exome analysis will identify genes that are not currently associated with a human disease, but can potentially influence the potentiation of antenatal fetal death. Unfortunately, at this stage in the development of medical science, an insufficient number of studies have been conducted in cohorts of patients with a history of stillbirth using these technologies [Wilkins-Haug L. Genetic innovations and our understanding of stillbirth. Human Genetics. 2020; 139(9): 1161-1172; Hays T., Wapner J.R. Genetic testing for unexplained perinatal disorders. Curr Opin Pediatr. 2021; 33(2): 195-202].

It is important to note that more than 60% of human protein-coding genes contain at least one miRNA binding site, therefore, the expression of these genes depends on the level of miRNA expression [Gonzalez T.L., Eisman L.E., Joshi N.V., Flowers A.E., Wu D., Wang Y., Santiskulvong C., Tang J., Buttle R.A., Sauro E., Clark E.L., DiPentino R., Jefferies C.A., Chan J.L., Lin Y., Zhu Ya., Afshar Ya., Tseng H-R., Taylor K., Williams III J., Pisarska M.D. High-throughput miRNA sequencing of the human placenta: expression throughout gestation. Epigenomics. 2021; 13: 995-1012].

It is known that placenta formation is controlled by various types of microRNAs, changes in the expression levels of which lead to placental dysfunction, which is directly related to pregnancy complications [Gillet V., Ouellet A., Stepanov Y., Rodosthenous R.S., Croft E.K., Brennan K., Abdelouahab N., Baccarelli A., Takser L. MiRNA profiles in extracellular vesicles from serum early in pregnancies. Complicated by gestational diabetes mellitus. The Journal of Clinical Endocrinology & Metabolism. 2019; 104(11): 5157-5169; Vaiman D. Genes, epigenetics and miRNA regulation in the placenta. Placenta 2017; 52:127-133; Ardekani A.M., Naeini M.M. The role of microRNAs in human diseases. Avicenna J. Med. Biotechnol. 2010; 2(4):161-179].

MicroRNAs are non-coding RNAs of 18 to 25 nucleotides in length that perform post-transcriptional regulation of gene expression, most often through negative feedback [Ali A., Hadlich F., Abbas M.W. MicroRNA-mRNA networks in pregnancy complications: a comprehensive downstream analysis of potential biomarkers. Int. J. Mol. Sci. 2021; 22(5): 2313; Ezhilarasan D. MicroRNA interplay between hepatic stellate cell quiescence and activation. Eur. J. Pharmacol. 2020; 885:173-507; Hayder H., O'Brien J., Nadeem U., Peng C. MicroRNAs: Crucial regulators of placental development. Reproduction. 2018; 155:259-271]. Currently, 762 placenta-specific microRNAs expressed by placental trophoblasts have been identified [Morales-Prieto D.M., Ospina-Prieto S., Schmidt A., Chaiwangyen W., Markert U.R. Elsevier trophoblast research award lecture: origin, evolution and future of placenta miRNAs. Placenta. 2014; 35:39-45; Morales-Prieto D.M., Chaiwangyen W., Ospina-Prieto S., Schneider U., Herrmann J., Gruhn B., Markert U.R. MicroRNA expression profiles of trophoblastic cells. Placenta. 2012; 33: 725-734; Chen P.S., Chiu W.T., Hsu P.L., Lin S.C., Peng I.C., Wang C.Y., Tsai S.J. Pathophysiological implications of hypoxia in human diseases. Journal of Biomedical Science. 2020; 27(1)].

Thus, microRNA plays the role of an epigenetic factor, being a mediator of the influence of the environment on the development and functioning of the placenta and one of the main regulators of the course of pregnancy [Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T) method. Methods. 2001; 25(4): 402]. In this regard, it is logical to assume that antenatal death of the fetus at full-term pregnancy can also be directly or indirectly associated with dysregulation of the expression of a number of placental microRNAs.

Recent studies have identified microRNAs that affect proliferation, apoptosis, migration, and invasion of placental trophoblast. In particular, aberrant expression of microRNA-125b in trophoblast cells leads to inhibition of their proliferation and inadequate invasion, which disrupts the processes of spiral artery reconstruction and angiogenesis [Yu.K. Antenatal fetal death: clinical and biochemical parallels and features of delivery. Chief Physician of the South of Russia. 2020; 5(75): 18-23; 35. Wong S.T.K., Tse W.T., Lau S.L., Sahota D.S., Leung T.Y. Stillbirth rate in singleton pregnancies: a 20-year retrospective study from a public obstetric unit in Hong Kong. Hong Kong Med J. 2022; 28(4): 285-293; Wilkins-Haug L. Genetic innovations and our understanding of stillbirth. Hum Genet. 2020; 139(9): 1161-1172]. In turn, active expression of microRNA-126, on the contrary, has a positive effect on the proliferation, migration, invasion and differentiation of placental trophoblast and enhances the anti-apoptotic effects [Yan T., Liu Y., Cui K., Hu B., Wang F., Zou L. MicroRNA-126 regulates EPCs function: implications for a role of miR-126 in preeclampsia. J. Cell Biochem. 2013; 114: 2148–2159], and microRNA-451 indirectly regulates glucose metabolism [Guo H., Nan Y., Zhen Y. Zhang Y., Guo L., Yu K., Huang Q., Zhong Yu. MiRNA-451 inhibits glioma cell proliferation and invasion by downregulating glucose transporter 1. Tumour Biol. 2016; 37(10):13751–13761].

An analogue of the proposed invention is the "Method for predicting antenatal fetal death", patent RU22009140816A, presented by Sagamonova K.Yu. et al. (2011). The method is represented by determining the level of D-dimer in the blood serum of a pregnant woman, starting from 22-23 weeks of gestation: at a value of 400-740 ng/ml, antenatal fetal death is predicted.

This method considers hemostasis disorder during pregnancy as the cause of antenatal death of the fetus, without taking into account the time of occurrence of this unfavorable outcome. In addition, it has now been proven that changes in D-dimer levels during pregnancy have no diagnostic or prognostic significance [To M.S., Hunt B.J. Obstetric case reports a negative D-dimer does not exclude venous thromboembolism (VTE) in pregnancy Early fundoscopy, magnetic resonance imaging and venometry in the diagnosis of venous sinus thrombosis. 2008; 28; Damodaram M. D-dimers as a screening test for venous thromboembolism in pregnancy: Is it of any use? 2009; 29: 101-103].

Another analogue is the method for early prediction of losses in the first trimester of pregnancy, patent RU2014102019A, proposed by Korotova S.V. et al. (2015), which is based on the definition of a number of signs that are interconnected with the formation of primary fetoplacental insufficiency, the consequence of which is antenatal death of the fetus, namely: chronic endometritis, carriage of TORCH infection, uterine scar, blood group, PID, chlamydia, spontaneous miscarriage, Rh factor, cardiovascular diseases. When calculating the prognostic coefficients, the patient belongs either to the group of high risk of reproductive losses, or to the group with a physiological course of pregnancy.

The proposed method is based on a number of clinical and anamnestic signs that are not specific and indirectly indicate the probability of an unfavorable pregnancy outcome. In addition, the risk is assessed only for the first trimester of pregnancy and does not assess the risks of antenatal fetal death at full-term pregnancy.

The prototype of this invention is the invention "Method for predicting fetal growth retardation" by Kuznetsova N.B. et al. (patent RU2738674C1, 12/15/2020), based on the study of venous blood in pregnant women at 18-22 weeks of gestation using quantitative PCR to determine the expression of three placenta-specific microRNAs: microRNA -103a-3p, microRNA -26a-5p, microRNA -125b-5p. The claimed method improves the accuracy and specificity of predicting fetal growth retardation at the preclinical stage.

Disadvantage of the method: this invention also uses microRNA-125b, but only as a tool for predicting fetal growth retardation, as one of the predictors of antenatal fetal death at full-term pregnancy, but not a tool for directly predicting antenatal fetal death at full-term pregnancy. In addition, this method does not specify the gestational age at which the onset of antenatal fetal death is predicted. The use of blood as a substrate for determining the level of microRNA expression in relation to predicting any pathology is currently controversial: it is difficult to determine the specific direction of microRNAs circulating in the blood and to prove that aberrant expression levels of the detected microRNAs indicate the development of the process under study, and not a parallel phenomenon.

Disclosure of the essence of the invention

The objective of the present invention is to develop a method for determining the probability of antenatal death of a fetus due to an unspecified cause at full-term pregnancy.

Technical result: makes it possible to establish the role of epigenetic programming in the occurrence of antenatal death of a full-term fetus for an unspecified reason.

A method is declared for determining the probability of antenatal death of a fetus due to an unspecified cause at full-term pregnancy, including determining the level of expression of microRNA-126, microRNA-451, microRNA-125b in placental tissue samples, followed by calculation of the d index using the formula:

Where:

d - the probability of death of a full-term fetus due to an unspecified cause;

x ₁ - expression level of microRNA-126, ng/µl;

x ₂ - expression level of microRNA-451, ng/μl;

x ₃ - expression level of microRNA-125b, ng/μl;

11,318 is a constant value,

at d > 0, the probability of antenatal death of the fetus due to an unspecified cause at full-term pregnancy is determined.

Fig. 1 shows the ROC curve for determining the sensitivity and specificity of the method for determining the epigenetic programming of antenatal fetal death at full-term pregnancy.

The predictive accuracy of the obtained predictive rules was assessed using the sliding examination method.

Based on the ROC analysis carried out using the IBM SPSS 22 software package, the sensitivity of the rule was determined to be 96.7%, and the specificity, respectively, 96.2% (Fig. 1).

Implementation of the invention

The method is carried out as follows. The expression level of microRNA-126, microRNA-451, microRNA-125b in placental tissue samples is determined, with deparaffinization of the placental tissue fragment, RNA extraction, reverse transcription reaction, and polymerase chain reaction (PCR) in real time being sequentially carried out.

Deparaffinization of placental tissue is carried out using mineral oil: 1 ml of mineral oil is added to a 1.5 ml test tube with two paraffin sections 5 μm thick and mixed thoroughly for 10 seconds. Then the tubes are incubated for 2 minutes in a thermoshaker at a temperature of 65 ° C and a rotation speed of 1300 rpm and centrifuged at 13,000-15,000 rpm for 4 minutes. After removing the supernatant, 1 ml of 96% ethanol is added to the sediment and mixed again for 10 seconds, then centrifuged under the same conditions as the previous time. After which the supernatant is removed again, 1 ml of 70% ethanol is added to the sediment and centrifuged at 13,000-15,000 rpm for 2 minutes. Nucleic acids are then isolated from the obtained deparaffinized placental tissue using the RealBest Extraction 100 reagent kit (Vector-Best JSC, Russia). 700 μl of the lysis solution are added to the samples and they are thoroughly mixed in a TS-20 thermoshaker (Biosan, Latvijas Republika) at 90°C for 60 minutes at 1300 rpm. The tubes are then placed in a centrifuge (angle rotor F-45-12-11 MiniSpin Eppendorf, Germany) for 2 minutes at 15,000 rpm. 600 μl of the supernatant are transferred to new tubes, another 600 μl of isopropanol and 10 μl of the magnetic particle suspension are added. Then they are mixed and left at room temperature for 5 minutes. The tubes are then centrifuged at 13,000 rpm for 10 min, the supernatant is poured off, and the sediment is washed first with 500 μl of 70% ethanol and then with 300 μl of acetone. The isolated sediment is dried and dissolved in 300 μl of the eluting solution, then the tubes are placed in a thermoshaker and incubated for 5 minutes at 65°C with a rotation speed of 1300 rpm. Then the tubes are centrifuged at 13,000-15,000 rpm for 1 minute. The RNA concentration is determined on a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA). The RNA concentration of the isolated preparations was in the range of 75.6-123.6 ng/μl.

To obtain complementary DNA, a reverse transcription reaction is carried out in 30 μl test tubes using ready-made reaction mixtures "RealBest Master Mix OT" (Vector-Best JSC, Russia). Mix 3 μl of previously isolated RNA, 25.5 μl of the reaction mixture for reverse transcription, 1.5 μl of a 10 μM solution of the corresponding primer for reverse transcription of a specific RNA. The reaction mixture in a volume of 3 μl is used as a matrix for Real-time PCR on a CFX 96 amplifier (Bio-Rad, USA) in order to measure the levels of microRNA expression.

Into 30 μl test tubes are successively added 3 μl of the obtained complementary DNA, 14 μl of H2O, 3 μl of 10x PCR buffer, 3 μl of 4 mM deoxynucleoside triphosphate solution, 3 μl of 10% BSA solution, 1 μl of Taq polymerase in complex with monoclonal antibodies to its active center (Clontech, USA), 3 μl of a solution of forward and reverse primers (5 μM) and a probe (2.5 μM). The primer and probe systems were developed by Vector-Best JSC (Russia), the reaction efficiency is 90-100%. The obtained data of threshold real-time PCR cycles are analyzed by the 2(-ΔCt) method. The reference gene is selected using the geNorm algorithm, which allows identifying the most stable genes in the sample under study. In this case, the geometric mean of at least three most stable genes is used as the reference gene.

The obtained results are analyzed by calculating the index d using the formula:

Where:

d - index of epigenetic programming of antenatal fetal death at full-term pregnancy; microRNA-126 expression level, ng/µl; microRNA-451 expression level, ng/µl; expression level of microRNA-125b, ng/µl;

11,318 is a constant value.

The decision is made as follows: if d > 0, the probability of antenatal death of the fetus due to an unspecified cause at full-term pregnancy is determined.

Clinical example 1.

Patient P., 31 years old, pregnant for the first time. The somatic history is not burdened.

The course of the current pregnancy was complicated by the development of moderate preeclampsia at 38 weeks of pregnancy, an increase in blood pressure of 140 and 90 mm Hg, daily proteinuria of 0.3 g/l were recorded. Antihypertensive therapy was prescribed, preparation of the cervix with antiprogestins was started. The condition of the fetus according to CTG and Doppler data was compensated. Antenatal death of a full-term fetus was recorded within 24 hours.

During the pathological examination of the placenta, changes characteristic of a full-term pregnancy were revealed, while the immediate cause of fetal death was not identified. In this regard, a molecular biological study of the placenta was conducted to determine the expression levels of microRNA-126, - 451 and 125b.

The index d is determined by the formula:

d = 2.02

Considering that d > 0, there is a probability of antenatal death of the fetus due to an unspecified cause at full-term pregnancy.

Clinical example 2.

Patient S., 34 years old, multiparous. Obstetric history: the second birth of the second pregnancy is forthcoming. The first birth ended in spontaneous timely delivery, without complications. The somatic history is burdened by first-degree obesity, chronic pyelonephritis in remission.

The course of the current pregnancy was complicated by placental insufficiency (II degree uterofetal blood flow disorder), registered during the II ultrasound screening at 20 weeks of pregnancy. Doppler ultrasound showed IA-IB degree uterofetal blood flow disorder in dynamics. At 38 weeks of pregnancy, the patient was taken to the hospital by an ambulance team due to the absence of fetal movement for 6 hours. Antenatal death of the fetus was recorded upon admission.

A pathological examination of the placenta did not reveal any characteristic histological changes, and therefore an additional molecular biological study of the placenta was carried out to determine the expression levels of microRNA-126, -451 and 125b.

The index d is determined by the formula:

d= -5.3×3.18-0.5×6.91-5.387×2.691+11.318

d = -23.4

Considering that d > 0, there is no probability of antenatal death of the fetus due to an unspecified cause at full-term pregnancy.

Claims (9)

A method for determining the probability of antenatal death of a fetus due to an unspecified cause at full-term pregnancy, including determining the expression level of microRNA-126, microRNA-451, microRNA-125b in placental tissue samples, followed by calculating the d index using the formula: d = -5.3 × x ₁ – 0.5 × x ₂ - 5.387 × x ₃ + 11.318, Where: d - the probability of death of a full-term fetus due to an unspecified cause; x ₁ – expression level of microRNA-126, ng/µl; x ₂ – expression level of microRNA-451, ng/µl; x ₃ – expression level of microRNA-125b, ng/µl; 11,318 is a constant value, at d > 0, the probability of antenatal death of the fetus due to an unspecified cause at full-term pregnancy is determined.