RU2474822C1 - Method for prediction of clinical outcome of extracorporeal fertilisation and embryo transfer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention may be used for prediction of the clinical outcomes of the extracorporeal fertilisation (EF) program and embryo transfer (ET) in females suffering sterility of various geneses. Substance of the invention consists in the fact that before the ovarian stimulation, peripheral venous blood leukocytes of a sterile female are examined for class II HLA gene polymorphism; if detecting the carrier state of DQA1*0101-DQB1*0501 or DQA1*0102-DQB1*0502 haplotypes, an unfavourable outcome of the EF and ET programs (pregnancy failure) is predicted, while no DQA1*0101-DQB1*0501 and DQA1*0102-DQB1*0502 haplotypes ensure predicting the favourable outcome of the EF and ET programs (pregnancy).

EFFECT: simplified and enhanced prediction of the clinical outcomes of the EF and ET programs in females suffering sterility of various geneses that enables considerably higher effectiveness of the assisted reproductive treatment ensured by more accurate selection of the patients at the stage of planning for pregnancy.

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Description

The invention relates to medicine, namely to obstetrics, gynecology, medical genetics and can be used to predict the outcome of in vitro fertilization (IVF) and embryo transfer (PE) in women with infertility of various origins.

Relevance. According to the results of epidemiological studies, the frequency of infertility in marriage varies from 8 to 29% (Kulakov V.I. Reproductive health of the population of Russia // Obstetrics and Gynecology. - 2002. - No. 2. - P.4-7). In this regard, in recent years, assisted reproductive technologies (ART), in particular the IVF and PE method, have become more widespread. However, despite the widespread use and experience in carrying out this technique, the frequency of pregnancy after IVF and PE remains low and, according to various authors, ranges from 20% to 40%. Moreover, even one cycle of IVF is associated with significant economic costs and a certain risk to a woman’s health associated with ovarian stimulation (Grishenko V.I., Petrushko M.P., Pinyaev V.I. Effectiveness of the IVF program depending on quantity and quality tolerated embryos // Problems of reproduction. - 2000. - No. 1. - P.44-47). The development of prognostic criteria for the outcome of IVF will significantly increase its effectiveness and avoid unnecessary material costs due to a more careful selection of patients at the stage of pregnancy planning.

There is a method for predicting the outcome of an IVF and PE program (RF Patent No. 2193777. A method for predicting the outcome of an IVF and PE program / K.Yu. Sagamonova, V.I. Orlov, T.N. Pogorelova, A.V. Kuzmin, S.V. Lomteva // Inventions. Utility models. - 2002. - No. 33) by qualitative and quantitative assessment of the contents of the follicles, characterized in that the activity of peptide hydrolases is studied in a follicular fluid aspirated separately from the right and left ovaries and at its level not less than 75 mg / ml / h in one of the ovaries predict a favorable outcome.

This method has several disadvantages:

1. When implementing the method, there is a high risk of complications associated with performing transvaginal puncture of the follicles, which is carried out under general anesthesia, and may be complicated by bleeding.

2. The method is carried out at the stage of follicle puncture, when it is impossible to adjust the patient management tactics.

3. The need to study two samples of follicular fluid (from the right and left ovaries).

There is a method for predicting the results of infertility treatment by in vitro fertilization method (RF Patent No. 2357673. A method for predicting the results of infertility treatment by in vitro fertilization method / Potekhina Yu.P., Scherbatyuk TG, Khruleva NV // Inventions. Utility models. - 2009 - No. 16), including transvaginal puncture of follicles under ultrasound control followed by examination of the obtained follicular fluid, characterized in that the follicular fluid is subjected to chemiluminescent analysis according to according to the standard method, the free radical activity of the samples is determined and they give a prognosis of pregnancy on the basis of the total antioxidant activity: when the indicators of total antioxidant activity are 0.05 and higher, they give an unfavorable prognosis, when the indicators of total antioxidant activity are less than 0.05 they give a favorable forecast - the probability of pregnancy is more than 50 %

The disadvantages of the method are:

1. There is a high risk of complications associated with performing transvaginal puncture of the follicles, which is carried out under general anesthesia, and may be complicated by bleeding.

2. The method is carried out at the stage of follicle puncture, when it is impossible to adjust the patient management tactics.

Closest to the claimed method is a method for predicting the results of in vitro fertilization in patients with autoimmune diseases (RF Patent No. 2345715. A method for predicting the results of in vitro fertilization in patients with autoimmune diseases / Yarygina T.V., Bashmakova N.V., Mazurov A.D. // Inventions. Utility models. - 2009. - No. 4), characterized in that the woman before the start of ovarian stimulation establish the presence of adnexitis in the history, determine the presence of B18, Cw3, Cw4 and K4 HLA antigens in the phenotype lichestvo platelets in coagulogram and Prognostic Index is calculated according to the formula F

F = 1.4 × X1-1.3 × X2-2.4 × X3-2.1 × X4 + 0.019 × X5-2.5,

where X1 is the presence (or absence) of the female phenotype of the HLA B18 antigen (1 or 0);

X2 - the presence (or absence) of the CW3 HLA antigen (1 or 0) in the female phenotype;

X3 - the presence (or absence) of the CW4 HLA antigen (1 or 0) in the female phenotype;

X4 - the presence (or absence) in the history of adnexitis (1 or 0);

X5 - platelet count in the coagulogram (10 ⁹ / l);

and if F is greater than 0, a successful outcome of IVF and PE is predicted, and for values of F less than 0, the inexpediency of IVF at this stage is judged.

The disadvantages of the method are:

1. The method can be used only in patients with autoimmune diseases.

2. For the analysis of HLA antigens, the method of serological histotyping is used, which is based on the reaction of cell lysis with subsequent microscopy, the accuracy of which depends on the subjective assessment of the researcher.

3. The method involves the determination of a large number of variable indicators: HLA antigens B18, Cw3, Cw4, platelet count in a coagulogram.

4. The complexity of the mathematical calculation of the prognostic index.

The technical result is aimed at simplifying (a single study, the result of which will be valid for possible repeated IVF) and expanding the ability to predict the outcome of the IVF and PE program in women with infertility of various genesis, which will significantly increase the effectiveness of the ART program due to more accurate patient selection for pregnancy planning stage.

The technical result is achieved by the fact that in women with infertility, prior to the start of ovarian stimulation, peripheral blood leukocytes determine the polymorphism of class II HLA genes and, when carriage of DQA1 * 0101-DQB1 * 0501 or DQA1 * 0102-DQB1 * 0502 haplotypes is detected, they predict an unfavorable outcome of the IVF program and PE (non-pregnancy), and in the absence of carriage of the haplotypes DQA1 * 0101-DQB1 * 0501 and DQA1 * 0102-DQB1 * 0502 predict a favorable outcome of the IVF and PE program (pregnancy) with an accuracy of 81.1%.

The novelty of the proposed method lies in the fact that for the first time it is proposed to predict the outcome of the IVF and PE program in women with infertility of various genesis by determining the polymorphism of class II HLA genes in peripheral blood leukocytes.

Previously, the study of antigens of the main histocompatibility complex was used to predict the risk of developing a number of diseases in the pathogenesis of which the autoimmune component is of great importance. There is evidence of the importance of certain alleles, genotypes and haplotypes of the HLA system in the formation of a predisposition or resistance to diseases such as insulin-dependent diabetes mellitus (Akhmedova Sh.U., Rakhimova DA. Polymorphism of class II HLA genes in patients with insulin-dependent diabetes mellitus in Uzbek populations // Immunology. - 2002. - No. 5. - P.298-300), ankylosing spondylitis, rheumatoid arthritis, Reiter's disease, multiple sclerosis, oncopathology (Konenkov V.I. Medical and environmental immunogenetics. - Novosibirsk, 1999. - 250 s.). The problem of alloimmune relations during pregnancy is widely debated, taking into account the compatibility of spouses and, accordingly, the mother and fetus on the HLA system antigens (Immunological riddle of pregnancy / edited by N.Yu.Sotnikova. - Ivanova: MIC, 2005. - 276 p.).

The start of the expression of antigens of the main histocompatibility complex occurs approximately 96 hours after fertilization: cells of the developing embryo begin to express antigens of paternal origin. Moreover, the maternal immune system not only does not reject, but also protects the fetus (Billington WD The nature and possible functions of MHC antigens on the surface of human trophoblast // Reproductive immunology. - New Delhi: Narosa Publishing House, 1999. - P.71 -77). HLA antigens of the embryo expressed on the surface of trophoblast cells are recognized by the mother's T-lymphocytes, and she forms a clone of immune cells that produces special “protective” anti-paternal (blocking) alloantibodies (ARCA - antipaternal cytotoxic antibodies, Ab2 - anti-anti HLA antibodies, MLR -Bf - mixed lymphocyte reaction blocking factor), which cover the surface of the trophoblast. (Characterization of mixed lymphocyte reaction blocking antibodies (MLR-Bf) in human pregnancy / M.K. Pandey, V. Saxena, S. Agrawal et all. // BMC Pregnancy Childbirth. - 2003. - Vol.19. - P.2.)

To date, it has been proven that a large number of matching HLA antigens in spouses leads to insufficient immunostimulation of the mother's body, resulting in reduced production of blocking antibodies and triggering an immune response directed against the embryo antigens. This may cause a halt in the development of pregnancy in its early stages (Beer A.E., Kwak J.Y. H. Reproductive Medicine Program. Finch University of Health Science. - Chicago. 1999. - 132 p.).

The presence of certain alleles, genotypes, and haplotypes for the genes of the main histocompatibility complex is associated with a change in the activity of humoral immunity. For example, carriers of the haplotype DRB1 * 1601-DQB1 * 0502-DQA1 * 0102 more often have a weak immune response to HBs vaccination with the antigen (The contribution of HLA-Class II antigens in humoral non-response and delayed response to HBsAG vaccination. / B. Vidan-Jeras, V. Brinovec, B. Jurca et all. // Pflugers Arch. - 2000. - Vol.440, Suppl.5. - P.188-189. 11005666 Cit: 2, Blood Transfusion Center of Slovenia, Ljubljana )

Thus, it can be assumed that in women carriers of the DQA1 * 0101-DQB1 * 0501 / DQA1 * 0102-DQB1 * 0502 haplotypes, due to a disturbed immune response (decreased production of “blocking” alloantibodies - ARCA - antipatemal cytotoxic antibodies), the blastocyst implantation process is impaired, which determines the adverse outcomes of the IVF and PE procedure.

The inventive method is carried out according to the standard method as follows.

I. Blood sampling for research.

Blood from patients is obtained by venipuncture of the ulnar vein (volume 3 ml) in disposable plastic tubes with a preservative. As a preservative, a 0.5M EDTA solution is used in a preservative: blood ratio of 1:10.

II. Isolation and purification of DNA.

DNA is isolated from peripheral blood leukocytes using the reagent kit “Set of reagents for DNA extraction from blood samples” (“DNA-technology”, Russia) according to the manufacturer’s protocol.

III. Polymerase chain reaction and detection of amplification products.

Amplification of all studied DNA fragments is carried out by the PNR method on a programmable thermal cycler MS2 of the DNA Technology company (Russia). Preparation of the reaction mixture, programming of the thermal cycler and detection of amplification products is carried out according to the manufacturer's protocol (DNA Technology, Russia). If carriers of the DQA1 * 0101-DQB1 * 0501 or DQA1 * 0102-DQB1 * 0502 haplotypes are identified, an unfavorable outcome of the IVF and PE program (non-occurrence of pregnancy) is predicted, and in the absence of the carrier haplotypes DQA1 * 0101-DQB1 * 0501 and DQA1 * 0102-DQB1 0502 predict a favorable outcome of the IVF and PE program (pregnancy).

Distinctive features of the method. In the peripheral blood leukocytes of women, polymorphism of class II HLA genes is determined and, if carriage of the DQA1 * 0101-DQB1 * 0501 or DQA1 * 0102-DQB1 * 0502 haplotypes is detected, an unfavorable outcome of the IVF and PE program is predicted, and in the absence of the DQA1 * 0101-DQB1 haplotype carriers 0501 and DQA1 * 0102-DQB1 * 0502 predict a favorable outcome of the IVF and PE program.

The essence of the proposed method is illustrated by the following examples.

Example 1. Patient S., 30 years old, underwent examination and treatment at the Federal State Institution "Ivanovo Research Institute of Motherhood and Childhood named after V.N. When analyzing HLA genes of the II class of carriage, the haplotypes DQA1 * 0101-DQB1 * 0501 and DQA1 * 0102-DQB1 * 0502 were not found, a favorable prognosis was given. Superovulation was stimulated using a short protocol. As a result of transvaginal puncture, 7 mature oocytes were obtained. As a result of the standard in vitro fertilization procedure, 7 zygotes with two pronuclei were obtained. On the 3rd day, 2 embryos were transferred into the uterine cavity, maintenance therapy was prescribed. On the 14th day after embryo transfer, a blood test for the content of chorionic gonadotropin showed the onset of pregnancy (341 mm U / ml, positive).

Example 2. Patient B., 36 years old, underwent examination and treatment at the Federal State Institution "Ivanovo Research Institute of Maternity and Childhood named after VN Gorodkova "about infertility of endocrine genesis. When analyzing class II HLA genes, the carriage of the DQA1 * 0101-DQB1 * 0501 haplotype was revealed, and an unfavorable prognosis was given. Superovulation was stimulated using a short protocol. As a result of transvaginal puncture, 8 mature oocytes were obtained. As a result of the standard in vitro fertilization procedure, 7 zygotes with two pronuclei were obtained. On the 3rd day, 2 embryos were transferred into the uterine cavity, maintenance therapy was prescribed. On the 14th day after embryo transfer, a blood test for the content of chorionic gonadotropin showed that pregnancy did not occur (1.2 mM units / ml, negative result).

Example 3. Patient F., 32 years old, underwent examination and treatment at the Federal State Institution "Ivanovo Research Institute of Motherhood and Childhood named after VN Gorodkova "about idiopathic infertility. When analyzing class II HLA genes, the carriage of the DQA1 * 0102-DQB1 * 0502 haplotype was revealed, and an unfavorable prognosis was given. Superovulation was stimulated using a short protocol. As a result of transvaginal puncture, 6 mature oocytes were obtained. As a result of the standard in vitro fertilization procedure, 5 zygotes with two pronuclei were obtained. On the 3rd day, 2 embryos were transferred into the uterine cavity, maintenance therapy was prescribed. On the 14th day after embryo transfer, a blood test for the content of chorionic gonadotropin showed that pregnancy did not occur (1.7 mM u / ml, negative result).

The results of clinical testing of the method are presented in table No. 1.

Table 1 Indicator amount Total surveyed 53 True positive result thirty False positive one True negative result 13 False negative result 9

Method sensitivity: 76.9%

Method specificity: 92.9%

Method accuracy: 81.1%

The advantages of the proposed method:

1. The method can be used to predict the outcome of an IVF and PE program in women with infertility of various origins.

2. Since the method is based on the assessment of gene polymorphism, a single study is sufficient, the result of which will be valid when conducting possible repeated IVFs.

3. High accuracy - 81.1%, specificity - 92.9% and sensitivity - 76.9% of the method.

Claims (1)

A method for predicting the outcome of an in vitro fertilization and embryo transfer program in women with infertility by examining the histocompatibility complex antigens in peripheral venous blood prior to stimulation of superovulation, characterized in that polymorphism of class II HLA genes is determined in leukocytes and upon detection of carriage of DQA1 * 0101-DQB haplotypes 0501 or DQA1 * 0102-DQB1 * 0502 predict an unfavorable outcome, and in the absence of carriage of the haplotypes DQA1 * 0101-DQB1 * 0501 and DQA1 * 0102-DQB1 * 0502 - a favorable outcome of the IVF and PE program.