Immunologic studies in miscarriage of pregnancy

Indications for immunological examination: habitual miscarriage of unknown genesis; history of anembryony; previous pregnancy with intrauterine growth retardation; intrauterine fetal death at any stage of pregnancy; autoimmune diseases and conditions; history of arterial and venous thrombosis; thrombocytopenia; failure of in vitro fertilization (IVF).

Treatment with antibiotics, some physiotherapeutic procedures lead to a decrease in cellular and humoral immunity. The period of restoration of immunity indicators to the initial level varies from 3 to 6 months, therefore immunological studies must be carried out before the start of therapy.

If all immunity parameters are reduced, then treatment with antibacterial agents must be combined with immunomodulatory agents. Pregnancy can be recommended only after the cellular and humoral immunity parameters are restored, since an adequate immune response of the body is necessary for the normal course of pregnancy.

Immunological examination tests for patients with recurrent miscarriage.

• Immunophenotyping

Immunophenotyping of subpopulations of peripheral blood lymphocytes allows us to identify deviations from the norm and, in particular, to assess the level of activated cells that are responsible for the production of proinflammatory cytokines and autoantibodies.

• Determination of antibodies.

There are 5 classes of antibodies:

1. IgM - are the first to appear in response to antigen stimulation and are effective in binding and agglutinating microorganisms (antigens). They have a larger molecule than other immunoglobulins and do not penetrate the placenta to the fetus.
2. IgG antibodies appear after IgM during an immune response, penetrate into extravascular spaces and pass through the placenta to the fetus.
3. IgA - the main antibodies contained in secretions in the intestines, lungs, urine. Their main function is to prevent the penetration of antigens from the surface into tissues.
4. IgE - normally makes up less than 1/10,000 of all serum immunoglobulins, but in case of allergy its content increases many times, more than 30 times, and the content of specific IgE more than 100 times.
5. IgD - act on the surface of B cells, performing a regulatory function.

Determination of immunoglobulins of three main classes (A, M, G) is necessary when assessing the immune status. An increase in the IgM level is observed during a primary infection or during an exacerbation of a persistent viral infection. A low IgA level is the basis for refusing to use immunoglobulin during treatment, since anaphylactic complications are possible. The greatest importance in obstetric practice is the determination of specific antibodies to viral and parasitic infections.

The presence of IgG immunoglobulins specific to the herpes simplex virus, cytomegalovirus, and toxoplasma means that the patient has encountered these antigens in the past and has immunity, and when the herpes simplex virus and/or cytomegalovirus infection are activated, the fetus will not suffer severely, and if there are IgG antibodies to toxoplasma, the fetus will not suffer from this disease at all.

The presence of specific IgM in the absence of IgG means that there is a primary infection. In the presence of both specific IgM and IgG antibodies, there is most often an exacerbation of a chronic viral infection. It is possible that there is no exacerbation, but there is a long-term persistence of IgM antibodies.

Particular attention should be paid to patients who do not have antibodies to infections that can cause severe damage to the fetus during pregnancy - HSV, CMV, toxoplasmosis, rubella. These patients are called seronegative. When in contact with an infectious agent, infection occurs for the first time and, accordingly, antibodies are produced. First, IgM antibodies appear, the so-called conversion occurs and the patient from seronegative becomes seropositive for a specific infection. In this case, if the infection causes fetal malformations, most often it is necessary to terminate the pregnancy rather than maintain it, especially if the conversion was observed in the first trimester.

Therefore, when determining the carriage of the virus, the presence and class of specific antibodies should be determined simultaneously.

Evaluation of interferon status appears to be an extremely important aspect of the examination.

Interferon-y is a group of proteins produced in response to a viral infection, as well as under the influence of lipopolysaccharin, etc., produced by macrophages IFN-a, fibroblasts IFN-R and T cells (Th-1 helpers) IFN-y. Interferons stimulate cells to secrete proteins that block transcription of viral messenger RNA. Interferons are more species-specific than other cytokines.

High serum interferon level disrupts normal development of placentation, limiting trophoblast invasion and exerting direct toxic effect on the embryo. Serum interferon, spontaneous IFN reaction of leukocytes, leukocyte production of IFN-a during induction by Newcastle disease virus (NDV), production of alpha and beta IFN in response to immunomodulators are assessed to select the most effective inducers for a specific patient (neovir, polyoxidonium, cycloferon, ridostin, lorifan, imunofan, derinat, temurit); lymphocyte production of IFN-y during induction by phytohemagglutinin (PHA), concvalin (ConA), staphylococcal enterotoxin (SEA).

An imbalance of the interferon system is present in almost all women with habitual miscarriage, especially in chronic viral infections and autoimmune disorders. This imbalance is expressed by a sharp increase in serum interferon or a sharp decrease in the production of all types of interferon by blood cells in response to various inducers.

• Determination of levels of proinflammatory and regulatory cytokines is carried out by enzyme immunoassay (Elisa) in blood serum, mucus and cells of the cervical canal, supernatants of in vitro activated lymphocytes.

Currently, more than 30 cytokines are known. Traditionally, based on biological effects, it is customary to distinguish:

• interleukins - regulatory factors of leukocytes (17 of them have been studied);
• interferons - cytokines with predominantly antiviral activity;
• tumor necrosis factors that have immunoregulatory and direct cytotoxic effects;
• colony-stimulating factors - hematopoietic cytokines;
• chemokines;
• growth factors.

Cytokines differ in structure, biological activity, and origin, but have a number of similar features characteristic of this class of bioregulatory molecules.

The normal functioning of the cytokine system is characterized by: individual nature of formation and reception of cytokines; cascade mechanism of action; locality of functioning; redundancy; interrelation and interaction of components. Normally, cytokines formed during the primary immune response practically do not enter the bloodstream, do not have systemic effects, i.e. their action is local.

The detection of high levels of cytokines in the peripheral blood always indicates a violation of the principle of local functioning of the cytokine network, which is observed in intense, long-term inflammatory, autoimmune diseases accompanied by generalized activation of immune system cells.

The redundancy of the cytokine system is manifested in the fact that each type of immune system cell is capable of producing several cytokines, and each type of cytokine can be secreted by different cells. In addition, all cytokines are characterized by polyfunctionality with strong overlapping effects. Thus, the manifestation of general and local signs of inflammation is caused by a number of cytokines: il-1, il-6, il-8, TNFa, colony-stimulating factors.

IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, TNFa participate in the proliferation of T-lymphocytes. Such duplication ensures the reliability of the cytokine cascade. Under the influence of specific antigens, T-helpers differentiate into two subpopulations: Th1 and Th2, which differ in the antigens of the main histocompatibility complex and the cytokines produced. Th1 secrete mainly proinflammatory cytokines, and Th2 - regulatory, causing mainly humoral reactions of hematopoiesis, angiogenesis.

The generalized nature of cytokine release is manifested by a number of systemic effects. It is known that mortality in septic shock is determined not so much by the effect of endotoxin as by the increased level of proinflammatory cytokines that arise in response to its introduction.

The most important antagonists of proinflammatory cytokines are regulatory cytokines - il-4, il-10.

Thus, the cytokine system, despite all its diversity, represents a single and integral network, disturbances in which can lead to a breakdown in self-regulation, a change in the direction of the immune response, which acquires particular significance in the early stages of embryonic development.

Therefore, it is extremely important that all cytokine parameters are within normal limits on the eve of pregnancy. The normal course of pregnancy is largely determined by the ratio of immunomodulatory and immunosuppressive effects in the endometrium, trophoblast, and subsequently in the placenta, in the regulation of which the components of the cytokine system take direct part.

• Study of autoantibodies.

Autoimmunity is a mirror image of tolerance, indicating the loss of tolerance by the body, immunity to its own antigens. Normally, the immune system restrains the autoreactivity of lymphocytes using regular mechanisms. Their disruption can lead to autoimmune pathology. The literature describes many variants of the development of autoimmunity. It is assumed that intracellular viral infection changes the antigenic nature of "its" cell, as a result of which antibodies against "its" cell may appear. It is possible that microorganisms have common antigens with the human body, in which case there is insufficient elimination of all autoreactive B-lymphocytes and the appearance of autoantibodies. The presence of genetic influences at the level of B-lymphocytes, subpopulations of T-cells, macrophages, target tissues and hormones is assumed.

Autoimmune diseases are more common and more severe in women. In recent years, the attention of scientists around the world has been focused on autoimmune processes in the human body, particularly in obstetric practice. A great deal of research has been done to understand the significance of these disorders, including in obstetric pathology.

The most significant autoimmune disorder for obstetric practice is antiphospholipid syndrome. The incidence of antiphospholipid syndrome among patients with habitual miscarriage is 27-42%.

Lupus anticoagulant is determined by hemostasiological methods. Lupus anticoagulant is of great importance in obstetric practice. It is believed that detection of lupus anticoagulant in the blood is a qualitative manifestation of the effect of certain levels of autoantibodies to phospholipids (cardiolipin, phosphatidylethanol, phosphatidylcholine, phosphatidylserine, phosphatidylinasitol, phosphatidylic acid) on the state of hemostasis.

The risk group for the presence of autoantibodies to phospholipids is the following category of patients whose medical history includes: habitual miscarriage of unknown genesis, intrauterine fetal death in the second and third trimesters of pregnancy, arterial and venous thrombosis, cerebrovascular diseases, thrombocytopenia of unknown genesis, false-positive reactions to syphilis, early toxicosis of the second half of pregnancy, intrauterine growth retardation, autoimmune diseases.

Anticardiolipin antibodies, antibodies to other phospholipids, phosphoethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylic acid are determined by the Elisa enzyme immunoassay.

As researchers believe, the same pool of immune system cells produces not only antibodies to phospholipids, but also other antibodies: anti-DNA antibodies, antinuclear antibodies, antithyroid, antisperm. It is believed that these antibodies account for up to 22% of habitual miscarriages of immune genesis and about 50% of infertility of unclear genesis and IVF failures.

These antibodies can be directed against both double and single DNA molecules, as well as against polynucleotides and histones. They are most often detected in autoimmune diseases, but there may be antibodies without manifestation of an autoimmune disease. Other researchers do not share this point of view. According to their research, these autoantibodies are non-specific, often transient, there is no scientific data explaining the mechanism of their action in habitual miscarriage. According to research, these antibodies should be kept in mind, since they can be markers of autoimmune trouble, and although there is no scientific explanation for the mechanism of their action yet, pregnancy always proceeds with complications in the form of placental insufficiency, intrauterine growth retardation.

In recent years, there have been studies on the importance of antibodies to hormones. A pool of cells producing CD 19+5+ antibodies. Activation of these cells leads to the appearance of autoantibodies to hormones that are essential for the normal course of pregnancy: estradiol, progesterone, human chorionic gonadotropin, thyroid hormones, and growth hormone.

With excess CD19+5+ due to the presence of autoantibodies to hormones, a number of clinical manifestations of this syndrome are observed: luteal phase deficiency, inadequate response to ovulation stimulation, "resistant ovary" syndrome, premature "aging" of the ovaries and premature menopause. When autoantibodies appear, the action of activated CD19+5+ leads to early implantation disorders, necrosis and inflammation in the decidua, disruption of fibrinoid formation, and excessive fibrin deposition. During IVF, these patients experience slow division and fragmentation of embryos, slow increase in the level of human chorionic gonadotropin during pregnancy, damage to the yolk sac, and subchorionic hematomas.

In our clinic we can only determine antibodies to human chorionic gonadotropin and we attach great importance to this aspect in habitual miscarriage.

The same pool of cells produces autoantibodies to neurotransmitters, including serotonin, endorphins and enkephalins. In the presence of these antibodies, resistance of the ovaries to stimulation, decreased blood circulation in the uterus, thinning of the endometrium, frequent depression, fibromyalgia, sleep disorders, including night sweats, panic attacks, etc. are observed.

Unfortunately, many methods for detecting autoantibodies are not standardized and require clarification of the mechanism of action. Practitioners need to know about the existence of this direction of research in miscarriages of unclear genesis, refer to specialized laboratories and departments, and not solve this problem by prescribing no-shpa and progesterone.

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