RU2709246C1 - Method for periconceptional prediction of recurrent early reproductive losses and primary placental insufficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine. At the periconceptional stage, polymorphisms of the folate cycle and PAI-1 genes are determined in the patients of the risk group, which is separated by anamnestic data. Anamnestic data are analyzed. Each parameter is evaluated as 1 point. If total is more than 2 points, folate cycle and PAI-1 gene polymorphisms are diagnosed. If a homozygous form of PAI-1 gene polymorphism is combined with a homozygous form of one of the folate cycle genes, recurrent early pregnancy loss is predicted. If folate and PAI-1 gene polymorphisms are combined, and at least one of them is represented by a homozygous form, primary placental insufficiency is predicted. If observing only heterozygous forms of folate cycle and PAI-1 genome polymorphisms, uncomplicated course of early pregnancy is predicted.

EFFECT: method provides higher objectivity and diagnostic value of prediction of recurrent early reproductive losses and primary placental insufficiency by evaluating complex of the most significant indicators.

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Description

The invention relates to medicine, namely to obstetrics and perinatology, and can be used in patients at risk at the pregravid stage to predict early pregnancy loss and other placenta-dependent complications due to primary placental insufficiency.

Currently, it is considered an axiom that the further growth and development of the placenta and fetus depends on the nature of the processes in the first weeks of gestation (implantation, early placentation). If these processes are disturbed, primary placental insufficiency develops, which determines the formation of placenta-dependent complications (preeclampsia, fetal growth retardation, detachment of a normally located placenta, premature birth, placenta previa, and other complications) and causes adverse perinatal outcomes.

Attachment and invasion of the fetal egg begins on the 7th day of pregnancy. Before invasion, changes in the endometrium occur, aimed at preventing the formation of hemorrhages with active trophoblast invasion. From the embryo, proteases are synthesized that destroy the extracellular matrix upon implantation. The uncomplicated course of these processes is possible with synchronization of the coagulation and fibrinolytic activity of the blood, as well as with the full proliferative activity of trophoblast cells.

The hemotrophic phase of implantation begins on day 12 of gestation. The trophoblast is introduced into the spiral arteries of the endometrium, from which the blood pours into lacunae, which then increase, close and transform into the intervillous space. The placenta begins to form, the fetal growth is actively progressing.

In pathological conditions, excessive activation of intravascular coagulation occurs, which determines the desynchronization of fibrin formation and fibrinolysis during implantation. In such a situation, the activity of the proteases synthesized by the blastocyst becomes insufficient to destroy the extracellular matrix in the endometrium and the fetal egg cannot penetrate to a sufficient depth. In this regard, implantation processes are disrupted. This situation is characteristic of preembryonic and early embryonic losses, the development of primary placental insufficiency.

Of all the possible variants of thrombophilic disorders during pregnancy, genetically determined hypofibrinolysis makes up more than 50% of all genetic polymorphisms, which determines its leading role in the failure of pregnancy and its course in the early stages of gestation. The genes predisposing the development of hypofibrinolysis are primarily PAI-1.

In addition to genetically determined hypofibrinolysis, a significant role in the pathology of early pregnancy is associated with folate cycle disorders. On the one hand, impaired metabolism of folic acid is associated with an increase in homocysteine levels and, as a consequence, a tendency to thrombosis. Homocysteine is involved in damage to the vascular wall, upsets the balance of procoagulant and anticoagulant mechanisms, the formation of the prothrombotic potential of the endothelium, a decrease in the fibrinolytic activity of the blood and due to a decrease in the fibrinolysis inhibitors PAI-1, the synthesis of which during pregnancy is mainly carried out by trophoblast.

On the other hand, the non-thrombogenic properties of folate cycle substances (methionine, homocysteine, cysteine), which are realized in the activity of rapidly dividing cells of the trophoblast, embryo, and affect the formation of endothelial dysfunction, are important. Methionine, an essential amino acid, plays a key role in intracellular metabolism, which is closely associated with redox metabolism and cell growth. It is not surprising that disturbances in the metabolism of methionine are associated with a number of serious pathologies, such as malformations of the fetus, trophoblast and the entire fetoplacental complex, oncological, cardiovascular and neurodegenerative diseases. Folic acid affects the synthesis of the endothelial relaxing factor - nitric oxide and the development of endothelial dysfunction.

Thus, the published data on the leading etiopathogenetic role of polymorphisms of the PAI-1 genes and the folate cycle in disrupting the processes of trophoblast invasion and early placentation are more theoretically justified. The practical use of this information is extremely difficult due to insufficient information. There is no data indicating the role of isolated polymorphisms of the folate cycle genes, as well as their combination with PAI-1, or a combination with defects of various components of the hemostatic system on the nature and time of occurrence of pregnancy complications. There is no connection between the severity of gestational complications depending on the hetero- or homozygous forms of polymorphisms of the PAI-1 genes and the folate cycle.

In addition, it is incorrect to diagnose hemostatic disorders only when thrombogenic mutations are detected. A genetic predisposition to venous thromboembolic complications does not always guarantee their implementation. True thrombophilia suggests the presence of at least 1-2x clinical or laboratory signs in combination with completed thrombosis or reproductive loss. In addition, folate cycle gene polymorphism is quite widespread in the population and for this reason it makes no sense to screen all pregnant women (or in the population).

Our studies have confirmed the high importance of polymorphisms of the PAI-1 genes and folate cycle in case of complications in the early stages of gestation and proved the feasibility of diagnosing polymorphisms of these genes in patients at risk.

There are various methods for predicting the complicated course of early pregnancy, but most of them suggest predicting an already developed pathological process in the early stages of gestation. Methods for assessing the course of pregnancy in the early stages of gestation are of high diagnostic value, but they do not reveal the root cause, but reflect the consequences of the disturbed course of implantation and placentation, and prevent preventive measures, since nothing can be changed during pregnancy.

The known "Method for early prediction of the implementation of gestational complications", patent No. 2530624, which consists in determining the threat of termination of a real pregnancy in women carriers of polymorphisms of the folate cycle genes, by the level of hCG hormone, lactoferrin and the relative content of lymphocytes in the mother’s peripheral blood in the dynamics of 1 trimester.

The proposed method has several disadvantages.

The study is conducted during pregnancy and, for this reason, there is no way to rectify the situation and prevent the development of primary placental insufficiency;

Difficulties in calculating the prognostic index using a formula with 5 coefficients, since they require the use of computer technology;

It is proposed to use a cumbersome examination algorithm, including clinical anamnestic data, determination of the carrier of polymorphisms of the folate cycle genes, determination of the level of lymphocytes in peripheral blood, the level of lactoferrin and the hCG hormone.

The well-known "Method for predicting intrauterine growth retardation", patent No. 2526178, which propose to calculate the prognostic index in the blood of a pregnant woman by the level of immunocompetent factors (CD 3+, CD 16+, CD 56+, level of the CZ component of complement and tumor necrosis factor) and then give a forecast about the prospect of fetal growth.

The disadvantages of this forecasting method can be considered that the forecast is based on the results of a survey of pregnant women. In addition, the prognosis results do not have a clear etiopathogenetic orientation. Only indicators of the overall immune status of a pregnant woman are analyzed, without taking into account local immune and inflammatory changes in the mother.

A known method for predicting the complicated course of pregnancy in patients with habitual miscarriage at the pregravid stage (Patent No. 2652894 “Method for screening for thrombophilia in patients with habitual miscarriage”).

This method cannot be considered objective, because it is based on the analysis of subjective information, namely on clinical and medical history data. In addition, it has low specificity and can predict thrombophilia in a woman if her close relatives had thrombophilic disorders, but does not take into account the peculiarities of obstetric and gynecological complications in the woman herself. The low efficiency of the method (according to the authors, up to 40%) is primarily determined by the fact that the prognosis is based only on subjective historical data without taking into account the objective results of the examination for the woman herself.

The prototype of our proposed method for predicting is the "Method for predicting the outcome of pregnancy in women with thrombophilia" (patent No. 2577759). It is proposed to use the prototype in order to predict the pathology of the embryo and extraembryonic structures in the first trimester in pregnant women with thrombophilia caused by a mutation of the MTHFR gene.

Among the disadvantages of the prototype, the following can be distinguished: 1 - forecasting is carried out in the first trimester of pregnancy; 2 - a limited group of women is analyzed, because only one gene is distinguished from three known genes in the folate cycle; 3 - there is no information about the homozygous or heterozygous form of the gene; 4 - does not take into account the pathogenetically substantiated combined effect of the folate cycle and PAI-1 genes. For these reasons, the proposed method has low reliability and prognostic value.

The technical result of the invention is to increase the objectivity and diagnostic value of predicting the recurrence of early reproductive loss and primary placental insufficiency.

The technical result is achieved by the fact that at the pregravid stage, patients at risk of developing reproductive losses and gestational complications, which is identified according to anamnestic data, determine the polymorphisms of the folate cycle and PAI-1 genes.

The method is as follows: at 1 (preliminary) stage, screening is conducted according to anamnestic data. At the same time, 7 parameters of the general, family, and obstetric-gynecological history of the patient are evaluated, which indirectly characterize genetically determined etiopathogenetic factors that affect the process of implantation, early placentation, and the development of structures of the ovum in early pregnancy. These include:

1) one or more spontaneous miscarriages in terms of 10 weeks or more;

2) two or more spontaneous miscarriages at the preembryonic or early embryonic stage (up to 10 weeks) in patients with a burdened personal or family history of thrombotic complications.

3) a combination of early spontaneous miscarriage and stillbirth;

4) antenatal fetal death as a complication of primary placental insufficiency in a patient with a history of clinically significant thrombosis;

5) severe early preeclampsia or detachment of a normally located placenta against a background of sub- or decompensated placental insufficiency;

6) history of stroke, thrombosis in close relatives;

7) endothelium-dependent extragenital pathology of the patient

Each parameter is rated one point. With a total of 2 points or more, the patient is allocated to the risk group and she is determined (stage 2) by PCR polymorphisms of the folate cycle and PAI-1 genes.

Peripheral venous blood is sampled from the cubital vein on an empty stomach, in the morning, in a clean, dry test tube.

The presence of polymorphisms: methylenetetrahydrofolate reductase MTHFR (677 C> T (A222V); 1298 A> C (E429A)), methionine synthase reductase MTRR (66 A> G (I22M)); MTR methionine synthases (2756 A> G (D919G)); plasminogen activator inhibitor (SERPINE 1 (PAI-1: -675 5G> 4G)) is determined using a real-time polymerase chain reaction using reagents of a test system from InterLabService LLC (RF) using a CFX 96 TouchDeepWell analyzer from Bio- RadLaboratories (USA).

If a combination of the homozygous form of the PAI-1 gene polymorphism with the polymophisms of two or three folate cycle genes, at least one of which is presented in the homozygous form, is diagnosed, a relapse of early pregnancy is predicted.

If a combination of polymorphisms of two or three folate cycle genes with PAI-1 gene polymorphism is diagnosed and at least one of them is represented by a homozygous form, then the development of primary placental insufficiency is predicted.

If only heterozygous forms of polymorphism of the PAI-1 gene and one of the three genes of the folate cycle are diagnosed, then an uncomplicated course of early pregnancy is predicted.

Example 1

Patient A., 34 years old. I went to the doctor obstetrician-gynecologist for a consultation at the pregravid stage. There are no complaints. A woman has arterial hypertension 1 tbsp., 1 tbsp., Risk 2; varicose veins of the lower extremities. The patient’s father has deep leg vein thrombosis. Menarche at the age of 13; menstrual cycle - 28 days, menstruation duration - 5 days: regular, moderate, painless. A history of 3 non-developing pregnancies. The first pregnancy ended at 4-5 weeks. (Features of the course of pregnancy: threatening early miscarriage, myometrial hypertonicity), subsequent 2nd and 3rd at 6-8 weeks, were characterized by the presence of retrochorial hematoma, antenatal death of the embryo. Curettage of the uterine cavity was made without visible complications. According to histological examination: chorionic tissue with immature, involutive-dystrophic changes in the chorionic villi. It is suggested that the patient is at high risk of developing fetal loss syndrome - 4 points (a woman has arterial hypertension 1 tbsp., 1 tbsp., Risk 2 (1 point); varicose veins of the lower extremities (1 point); the patient’s father has thrombosis deep veins of the leg (1 point); a history of 3 non-developing pregnancies (1 point)). A woman was examined for the presence of polymorphisms of the folate cycle and PAI-1 genes. A combination of 4 polymorphisms was revealed: 3 folate cycle genes - 2 associated with a heterozygote (MTR, MTRR) and 1 associated with a homozygote (MTHFR) and PAI-1 associated with a homozygous. A woman predicts an adverse pregnancy outcome and early embryo loss. The patient was offered a set of preventive measures, which was not used by the patient. Subsequent pregnancy in a woman occurs spontaneously. At the period of 7-8 weeks of gestation, the patient was diagnosed with chorionic detachment, an dissociated type of development of the elements of the fetal egg, and the absence of a fetal heartbeat during ultrasound examination. After 1 day there was a complete spontaneous miscarriage. The forecast was confirmed.

Example 2

Patient N., 29 years old, at a scheduled appointment with an obstetrician-gynecologist. There are no complaints. The patient has varicose veins of the lower extremities. In the history of 1 pregnancy, it occurred spontaneously, proceeded against the background of the threat of termination of pregnancy, dissociated development of elements of the ovum, asymmetric type of blood flow in the uterine arteries. At a period of 12-13 weeks of gestation, a late spontaneous complete miscarriage occurred. Given the high risk of developing reproductive losses and pregnancy complications - 2 points (varicose veins of the lower extremities (1 point, spontaneous complete miscarriage at 12-13 weeks - 1 point), the patient was examined for the presence of genetic polymorphisms, as a result of which a combination of 2 polymorphisms: MTHFR associated with homozygote and PAI-1 associated with heterozygous, a high risk of developing primary placental insufficiency and associated gestational complications is predicted. The woman was recommended, but did not perform, a fetal loss syndrome and placenta-related complications.The second pregnancy of the patient occurred spontaneously, proceeded against the background of a recurring threat of abortion: myometrial hypertonicity, signs of chorionic detachment, dissociated type of development of elements of the ovum, signs of primary placental insufficiency (decrease in levels HCG and PAPP-A). From 20 weeks of gestation, fetal growth retardation syndrome 1-2 tbsp. Pregnancy ended in antenatal fetal death for a period of 29 weeks and premature birth. During vaginal birth, a dead premature boy weighing 850 g, 32 cm long was born. According to the pathological study, fetal death occurred in connection with acute intrauterine hypoxia against the background of decompensated fetoplacental insufficiency. In the study of the placenta: immature villi, involutive-dystrophic changes in the chorionic villi, the functional structure of the villi. The forecast was confirmed.

Example 3

Patient O., 32 years old. I turned to the obstetrician-gynecologist for a antenatal consultation in order to prepare for a second pregnancy. There are no complaints. The patient has a violation of fat metabolism. Menstrual function without features. The patient had a history of 1 missed pregnancy for a period of 11-12 weeks, which occurred against the background of a recurring threat of interruption: chorion detachment for a period of 8-9 weeks, myometrial hypertonicity. Curettage of the uterine cavity was made without visible complications. According to histological examination: chorionic tissue with immature, involutive-dystrophic changes in the chorionic villi. The patient is at a high risk of developing reproductive loss and pregnancy complications 2 points: the patient has a violation of fat metabolism (1 point); 1 frozen pregnancy for a period of 11-12 weeks (1 point). A study of genetic polymorphisms revealed a combination of 3: 2 folate cycle genes - MTR, MTHFR associated with heterozygote, and PAI-1 associated with homozygous. A woman was recommended a course of preventive measures, which the patient was ignored. The 2nd pregnancy, which also occurred spontaneously, in the same marriage, occurred against the background of moderate early toxicosis, the threat of termination at different gestational periods, and signs of primary placental insufficiency (decreased levels of hCG and PAPP-A). Pregnancy ended with an operative delivery by cesarean section on an emergency basis for a period of 33-34 weeks due to more severe preeclampsia, fetal growth retardation syndrome. A live premature baby was born weighing 1650 g, 40 cm long, with an Apgar score of 7-8 points. According to the histological examination of the placenta: immature villi, involutive-dystrophic changes in the chorionic villi, the afunctional structure of the villi. The forecast was confirmed.

Example 4

Patient J., 30 years old. There are no complaints. Somatic, family history is not burdened. Menarche at the age of 12; the menstrual cycle is 28 days, the duration of menstruation is 4-5 days: regular, moderate, painless. Chronic inflammatory diseases of the pelvic organs, STI denies. In the study of genetic polymorphisms, 1 MTRR polymorphism associated with heterozygote and PAI-1 associated with heterozygote were revealed. The risk of fetal loss syndrome is low. Subsequently, pregnancy occurred spontaneously, proceeded against mild anemia in the third trimester, and ended in childbirth through the natural birth canal for a period of 39–40 weeks, which was uneventful. A live full-term girl was born weighing 3,500 g, 51 cm long, with an Apgar score of 9-10 points. The postpartum period was uneventful. During histological examination, the placenta corresponds to a gestational period. The forecast was confirmed.

Thus, the proposed method is minimally invasive, allowing to select a risk group at the pregravid stage, and then predict pregnancy outcomes by objective genetic criteria. Prediction of SPP or the development of placenta-dependent complications even at the pregravid stage allows timely correction of the revealed disorders before pregnancy, which makes it possible to avoid the pathological course of early pregnancy and the development of undesirable gestational complications.

Claims (1)

A method for pre-gestational prediction of recurrence of early reproductive losses and primary placental insufficiency, including examining women with polymorphisms of the folate cycle genes, characterized in that at the stage of preparation for pregnancy, a risk group is identified taking into account 7 parameters of the patient’s general, family and obstetric-gynecological history, which indirectly indicate for the presence of polymorphisms of folate cycle and PAI-1 genes adversely affecting the implantation process, early placentation: one or more spontaneous n miscarriages in terms of 10 weeks or more; two or more spontaneous miscarriages at the preembryonic or early embryonic stage up to 10 weeks of pregnancy in patients with a burdened personal or family history of thrombotic complications; a combination of early spontaneous miscarriage and stillbirth; antenatal fetal death as a complication of primary placental insufficiency in a patient with a history of clinically significant thrombosis; severe early preeclampsia or detachment of a normally located placenta against a background of sub- or decompensated placental insufficiency; history of stroke, thrombosis in close relatives; endothelium-dependent extragenital pathology of the patient; each parameter is evaluated at 1 point, and with a total of 2 points or more, the patient is allocated to the risk group and she is tested for folate cycle polymorphisms: methylenetetrahydrofolate reductase MTHFR (677 C> T (A222V); 1298 A> C (E429A)), methionine synthase reductase MTRR (66 A> G (I22M)); methionine synthase MTR (2756 A> G (D919G)) and plasminogen activator inhibitor (SERPINE 1 (PAI-1: -675 5G> 4G)) associated with a homo- or heterozygote, and if a combination of the homozygous form of the PAI-1 gene polymorphism is diagnosed with polymorphisms in two or three folate cycle genes, at least one of which is presented in a homozygous form, relapse of pregnancy is predicted in the early stages, and if a combination of polymorphism in two or three folate cycle genes with PAI-1 gene polymorphism is diagnosed, and , at least one of them is represented by homozygous forms oh, they predict the development of primary placental insufficiency, and if only heterozygous forms of polymorphism of the PAI-1 genes and one of the three folate cycle genes are diagnosed, then an uncomplicated course of early pregnancy is predicted.