Antiphospholipid syndrome and miscarriage of pregnancy

Antiphospholipid syndrome is the most common cause of thrombophilic complications and the associated habitual loss of pregnancy. There is a distinction between primary antiphospholipid syndrome and secondary - in the presence of an autoimmune disease (most often this is systemic lupus erythematosus). There is no big difference in all parameters between primary antiphospholipid syndrome and secondary, only the symptoms of an autoimmune disease are added to the secondary. There is also a "catastrophic antiphospholipid syndrome".

The cause of antiphospholipid syndrome remains unclear, but viral infections are thought to play a role. The pathogenesis of antiphospholipid syndrome is associated with autoantibodies with heterogeneous specificity directed against negatively charged phospholipids or phospholipid-binding proteins.

Based on numerous studies, a working group of experts in this field adopted the following criteria for antiphospholipid syndrome at its last symposium in September 2000 in France to enable comparison of studies conducted in different countries.

Criteria for classification and definition of AFS

Clinical criteria

Vascular thrombosis - one or more clinical episodes of arterial, venous in any tissue or organ. Thrombosis should be confirmed by Doppler or histological examination, with the exception of thrombosis of small superficial veins. For histological confirmation, thrombosis should not be accompanied by inflammatory processes in the vascular wall.

During pregnancy:

• One or more unclear deaths of a morphologically normal fetus older than 10 weeks of gestation, with normal morphology as determined by ultrasound or direct examination of the fetus.
• One or more premature births of morphologically normal neonates before 34 weeks of gestation due to preeclampsia or eclampsia, or severe placental insufficiency.
• Three or more unclear causes of spontaneous miscarriages before 10 weeks of pregnancy in the mother after excluding anatomical, hormonal and genetic causes of termination.

Laboratory criteria:

• Anticardiolipin antibodies of IgG and/or IgM isotypes in the blood, in medium or high titer 2 or more times in a row when tested at an interval of 6 weeks, tested by the standard enzyme immunoassay for beta2-glycoprotein-1-dependent anticardiolipin antibodies.
• Lupus anticoagulant present in plasma on 2 or more consecutive occasions, when tested 6 weeks apart, as determined according to the International Society for Thrombosis and Hemostasis guidelines as follows:
  • Prolongation of phospholipid-dependent coagulation in coagulation tests: activated partial thromboplastin time (APTT); goat clotting time; snake venom test; prolongation of prothrombin time, Textarin time.
  • Failure to correct clotting time in a screening test when mixed with normal platelet-poor plasma.
  • Shortening or correction of prolonged coagulation time by adding excess phospholipids to the screening test.
  • Exclusion of other coagulopathies, i.e. factor VIII inhibitor, heparin, etc.

The following tests are excluded from the laboratory criteria: low levels of anticardiolipin antibodies, IgA anticardiolipin antibodies, anti-beta2-glycoprotein-1, antibodies to prothrombin, annexin or neutral phospholipids, false-positive Wasserman reaction.

The working group believes that these methods require further study. As for anti-beta2-glycoprotein-1, which most researchers believe plays a key role in the development of thrombophilia, this test requires internal laboratory standardization and technical improvement. Perhaps in the future, this test will be the main criterion in the diagnosis of antiphospholipid syndrome.

Currently, studies have appeared on the role of anti-beta2-glycoprotein-1 IgA and IgG in the development of antiphospholipid syndrome. In groups of women with a clinical picture of antiphospholipid syndrome in the absence of cardiolipin antibodies and LA, a high level of these antibodies was detected.

According to literary data, the incidence of antiphospholipid syndrome among patients with recurrent pregnancy loss is 27-42%.

The population frequency of this condition has not been studied in our country, but in the USA it is 5%.

There are two classes of antiphospholipid antibodies formed under the influence of endogenous stimuli:

1. Antiphospholipid antibodies that prolong in vitro phospholipid-dependent coagulation reactions by affecting the Ca ²⁺ -dependent binding of prothrombin and factors Xa, Va during the assembly of the prothrombin-activator complex (prothrombinase) - lupus anticoagulant (LA);
2. Antiphospholipid antibodies that are determined by immunological tests based on cardiolipin are anticardiolipin antibodies (ACA).

Autoantibodies to phospholipids can arise under the influence of exogenous and endogenous stimuli. Exogenous stimuli are associated mainly with infectious antigens, they lead to the formation of transient antibodies that do not cause thromboembolic disorders. An example of such exogenous antiphospholipid antibodies are antibodies detected in the Wasserman reaction.

Antibodies formed under the influence of endogenous stimuli are associated with the disruption of endothelial hemostasis. These antiphospholipid antibodies cause thromboembolic disorders, often associated with strokes, heart attacks in young people, other thromboses and thromboembolisms, and the development of Snedon's syndrome. An explanation for this phenomenon was obtained in recent years, when it was established that for the binding of antibodies present in the sera of patients with autoimmune, but not infectious diseases, to cardiolipin, the presence of a plasma component (cofactor) is necessary, which was identified as beta-glycoprotein-1 beta1-GP-1). In a more detailed study of this phenomenon, scientists showed that antibodies to cardiolipin isolated from the sera of patients with autoimmune diseases reacted with cardiolipin only in the presence of uGP-1, while the binding of antibodies to cardiolipin (AKA) synthesized in patients with various infectious diseases (malaria, infectious mononucleosis, tuberculosis, hepatitis A and syphilis) did not require a cofactor in the system. Moreover, the addition of beta2-GP-1 in some cases inhibited the interaction of sera of patients with infectious diseases with cardiolipin. In a clinical analysis of the obtained results, it turned out that the development of thrombotic complications was associated with the synthesis of cofactor-dependent antibodies to cardiolipin. However, according to other data, even in patients with antiphospholipid syndrome, despite the presence of beta2-GP-1, the ability of antibodies to phospholipids (APA) to interact with cardiolipin is determined by a number of other factors. Thus, the binding of low-avidity antiphospholipid antibodies to cardiolipin depends to a greater extent on the presence of a cofactor in the system than is required in the case of the presence of high-avidity antibodies in the sera of patients. On the contrary, A.E. Gharavi (1992) emphasizes that cofactor dependence is characteristic of high-avidity antibodies. Earlier, when studying the sera of patients with antiphospholipid syndrome, it was shown that in their blood serum, in addition to antiphospholipid antibodies, there is a large number of various phospholipid-binding proteins that react with anionic phospholipids (apolipoproteins, lipocortins, placental anticoagulant protein, coagulation inhibitors, C-reactive protein, etc.).

The above data suggest the existence of at least two populations of cardiolipin-binding antibodies. Some of them (the "infectious" antibodies) have the ability to directly recognize negatively charged epitopes of phospholipids, while others (the "autoimmune" antibodies) react with a complex epitope consisting of a phospholipid and beta2-GP-1, and possibly other phospholipid-binding proteins.

The development of thrombotic complications is associated with the synthesis of “autoimmune” (cofactor-dependent) antibodies.

In obstetric practice, lupus anticoagulant is of great importance. It is believed that the 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.

An extremely interesting approach to interpreting the immunological aspects of miscarriage is presented in the works of A. Beer and J. Kwak (1999, 2000). The authors identify 5 categories of immune disorders that are the cause of habitual miscarriage, IVF failures and some forms of infertility.

1. Category I - compatibility of spouses according to the HLA system and the connection of currently known HLA antigens with reproductive dysfunction. HLA compatibility, according to the authors, leads to ineffective "camouflage" of the placenta and makes it accessible to the mother's immune attack.
2. Category II - antiphospholipid syndrome associated with the circulation of antiphospholipid antibodies. The incidence of antiphospholipid syndrome among patients with habitual miscarriage is 27-42%. The pathogenetic basis for unsuccessful completion of pregnancy in APS is thrombotic complications occurring at the level of the uteroplacental pool. In addition, phosphatidylserine and phosphatidylethanalamine play an important role in the implantation process, as a "molecular glue". In the presence of antibodies to these phospholipids, differentiation of cytotrophoblast into syncytiotrophoblast may be disrupted, which leads to the death of pregnancy in the early stages.
3. Category III of immunological disorders includes antinuclear, antihistone antibodies, which account for 22% of miscarriages of immune genesis. In the presence of these antibodies, there may be no manifestations of autoimmune diseases, but inflammatory changes are found in the placenta.
4. Category IV - the presence of antisperm antibodies. This category of immunological disorders occurs in 10% of patients with habitual miscarriage and infertility. Antisperm antibodies are detected in women with antiphospholipid antibodies to serine or ethanolamine.
5. Category V is the most severe, it includes 45% of women with IVF failures due to implantation disorders. This category is divided into several sections.

Section 1 is associated with an increase in the content of natural killers CD 56 in the blood over 12%. According to the authors, with an increase in CD 56+ above 18%, the embryo always dies. This type of cells is determined both in the blood and in the endometrium. In addition to the cytotoxic function, they synthesize proinflammatory cytokines, including TNFa. As a result of excess proinflammatory cytokines, implantation processes are disrupted, trophoblast cells are damaged, with subsequent development of trophoblast insufficiency, placenta and death of the embryo/fetus (similar data were obtained by other authors).

The 2nd section of the V category is associated with the activation of CD19+5+ cells. The level above 10% is considered pathological. The main significance of these cells is associated with the production of antibodies to hormones that are essential for the normal development of pregnancy: estradiol, progesterone, chorionic gonadotropin. In addition, antibodies to thyroid hormones and growth hormones may appear. With pathological activation of CD 19+5+, luteal phase insufficiency, inadequate response to ovulation stimulation, "resistant ovary" syndrome, premature "aging" of the ovaries, and premature menopause develop. In addition to the direct effect on the listed hormones, with excessive activity of these cells, there is a deficiency of preparatory reactions for implantation in the endometrium and myometrium, and later in the decidual tissue. This is expressed in inflammatory and necrotic processes in the decidua, in disruption of fibrinoid formation, and in excessive deposition of fibrin.

Section 3 is associated with a high content of CD 19+5+ cells, which produce antibodies to neurotransmitters, including serotonin, endorphins, and enkephalins. These antibodies contribute to ovarian resistance to stimulation, affect the development of the myometrium, and contribute to a decrease in blood circulation in the uterus during implantation. In the presence of these antibodies, patients may have depression, fibromyalgia, sleep disorders, and panic states.

Such a differentiated approach allows an individual approach to solving the issue of the role of different immune aspects in the genesis of habitual pregnancy loss. Unfortunately, such a clear division does not work in clinical practice. Most often, patients with antiphospholipid syndrome may have antibodies to hCG and antithyroid antibodies, etc.

In recent years, the problem of alloimmune relations regarding compatibility by HLA antigens has been widely discussed. Many researchers question the existence of this problem, taking into account that HLA antigens are not expressed on the trophoblast. Research on this problem was raised back in the 1970s. A number of researchers believed that leukocyte sensitization, like erythrocyte sensitization, is accompanied by spontaneous abortion. In Rh- and ABO-conflict pregnancy, the most frequent complication of pregnancy is the threat of its termination. But even without sensitization, the threat of termination is its most frequent complication. Even in case of severe damage to the fetus and its death from hemolytic disease, termination of pregnancy often does not occur spontaneously. The work we have conducted over a number of years has shown that habitual miscarriage, as a rule, does not have a direct etiological connection with Rh- and ABO-sensitization. Frequent interruptions, especially after 7-8 weeks (the time when the Rh factor appears in the fetus), can lead to sensitization, which complicates the course of pregnancy. When managing such a pregnancy, complex problems arise. Is it worth examining and treating habitual miscarriage if the patient has Rh sensitization, since by maintaining the pregnancy in the early stages, you can get a fetus with an edematous form of hemolytic disease in its later stages.

Particular attention in the literature is paid to the role of histocompatibility antigens in miscarriage. The probability of allosensitization of the maternal organism to fetal leukocyte antigens is quite high, given their early formation and ability to penetrate the placenta. The question of the etiological role of leukocyte sensitization is considered extremely controversially. Many researchers etiologically associate leukosensitization with miscarriage and recommend immunosuppressive therapy.

The data analysis showed that in healthy multiparous women, antileukocyte sensitization is observed much more often than in pregnant women with habitual miscarriage (33.6% and 14.9%, respectively). At the same time, a number of features are revealed: in women who had multiple pregnancies that ended in normal births, leukosensitization was 4 times more often than in those whose pregnancies were terminated by artificial abortion (33.6% versus 7.2%, respectively). Frequent detection of these antibodies in the blood of healthy multiparous women indicated their harmlessness for reproductive processes. On the other hand, an increase in the frequency of occurrence of lymphocytotoxic and leukoagglutinating antibodies in the blood of healthy women as the number of normal pregnancies ending in birth increases indicates a physiological rather than pathological significance of this type of isosensitization. The production of anti-leukocyte antibodies is a natural process, since the fetus necessarily contains transplantation antigens that are incompatible with the mother, and they apparently protect the fetus from the damaging effects of the mother's immune lymphocytes.

According to the research data, when studying the indices of cellular immunity in pregnant women with miscarriage, it was not possible to find any noticeable differences between them and women with physiological pregnancy. The value of the blast transformation reaction with phytohemagglutinin, the intensity of the blast transformation reaction in a mixed lymphocyte culture, and the content of serum immunoglobulins did not differ statistically. At the same time, in case of miscarriage, women's serum significantly more often stimulated cellular immunity, and the serum blocking factor was detected in uncomplicated pregnancy. In physiological pregnancy, 83.3% of women had sensitization of lymphocytes to fetal antigens. In pregnant women with habitual miscarriage, cell sensitization was weaker and less common, and the blocking effect of serum was usually absent.

The differences revealed indicate a weakening of the blocking properties of the serum of pregnant women in the case of threatened spontaneous abortion. Apparently, the immunoregulatory properties of the blood serum play a decisive role in the development of pregnancy. With a decrease in the blocking properties of the serum, mechanisms leading to termination of pregnancy are activated. Similar data have been obtained by many researchers.

This theory about the role of the serum's blocking properties in maintaining pregnancy is not accepted by many researchers. Their main motivation is that there are women with normal pregnancies who do not have blocking antibodies.

Moreover, the methods for determining blocking antibodies are not standardized and have low sensitivity to accurately and in different laboratories to obtain similar results. The determination of blocking antibodies by the reaction of mixed lymphocyte culture also has a number of defects:

1. variability of responses among different patients and even among the same patients but performed at different times;
2. difficulties in assessing the degree of suppression relative to blocking activity;
3. the sensitivity of the method is unknown;
4. there is no standardization of the method and standards for evaluating the result;
5. There is no single method for interpreting data.

Despite this, many research groups consider this problem among immunological factors of miscarriage. It is believed that blocking antibodies can act in several ways. They can be directed against antigen-specific receptors on maternal lymphocytes, which prevents their reaction to fetoplacental tissue antigens; or they can react with fetoplacental tissue antigens and block their recognition by maternal lymphocytes. It is also believed that blocking antibodies are anti-idiotypic antibodies, directed against antigen-specific sides (idiotypes) of other antibodies, i.e. antigen receptors on the surface of T-lymphocytes can be bound and therefore their action against the fetus is prevented. There is evidence that they can be bound to anti-HLA-DR antigens and to anti-Fc receptors of antibodies.

In addition to blocking antibodies, there is data on the role of lymphocytotoxic antibodies against the husband's lymphocytes. Most researchers believe that they, like blocking antibodies, are a consequence of a normal pregnancy. In 20%, they are detected after the first normal pregnancy, and they are found in 64% of women who give birth successfully and many times. In women with habitual miscarriage, they are much less common (from 9 to 23%).

In addition, there are studies indicating that the presence of neutrophil-specific antibodies against paternal antigens in the mother may be accompanied by severe neutropenia in the fetus. Neutrophil-specific antigens NA1, NA2, NB1 and NC1 were first characterized by Lalezari et al. (1960). Other neutrophil antigens NB2, ND1, NE1 were discovered by Lalezari et al. (1971), Verheugt F. et al. (1978), ClaasF. et al. (1979), respectively.

N antigens are independent of other antigens present on the surface of neutrophils, such as HLA f. The most important antigens that induce antibody production are NA 1 and NB1 antigens. The frequency of detection of neutrophil-specific antibodies varies in different studies from 0.2% to 20%. This difference is due to the fact that methods for detecting these antibodies have only recently become available and because severe neutropenia in neonates is rare. Most often, these children develop an infection early and very quickly progress to sepsis. Therefore, the authors recommend that all neonates with unclear neutropenia, especially premature infants, should have their mother tested for antibodies to neutrophils. The presence of antibodies to neutrophils in the mother does not cause neutropenia, like Rh antibodies, provided that they are not autoimmune.

In women with recurrent miscarriage, autoantibodies against their own lymphocytes may be detected - lymphocytotoxic autoantibodies, which are detected in 20.5% of cases in women with recurrent miscarriage, whereas they are not detected in physiologically normal pregnancies.

The decrease in the blocking properties of the serum is associated with the compatibility of the spouses by the antigens of the HLA system (Human leukocyte antigens). The HLA system or the old name "major histocompatibility complex" is a group of genes whose proteins serve as identity markers on the surface of various cells with which T-lymphocytes interact through their own receptors in the immune reaction. They were first identified in the transplant rejection reaction. HLA consists of a group of genes of classes I, II and III located on the 6th chromosome. This system has a huge polymorphism and only within one chromosome, the number of possible combinations of its genes is 3x10 ⁶.

HLA class I includes the HLA-AB and -C loci - these genes represent a family of peptides that react with T-cytotoxic (CD8+) cells.

Class II includes the HUDP, -DQ and DR loci - they mainly interact with T-helpers (CD4+). The region of class III genes takes a major part in inflammation processes, contains alleles of complement components C2, C4 and Bf (properdin factor), as well as TNF (tumor necrosis factor) and a number of isoenzymes. In addition, it was recently discovered that class I molecules also interact with NK cells, preventing cell lysis.

A large group of immunoglobulins similar to NK cell receptors are found on chromosome 19 - these are the so-called non-classical loci HLA-E, -F and G. They also participate in immune responses, and the HLA-G locus of the fetus is expressed on the trophoblast.

Allelic variants of genes have different frequencies of occurrence. The allele frequency indicator is used as a genetic marker for a number of pathological conditions.

In recent years, the connections of the HLA system with various diseases have been studied very intensively. It has been established that autoimmune diseases such as arthritis and Reiter's disease are observed in 95% of patients who have the HLA B27 allele, i.e. almost 20 times more often than this antigen is found in the population.

In 86.4% of patients with antiphospholipid syndrome, HLA DQ4 is determined. If the husband has HLA DQ 201, anembryony will occur in 50% of cases.

If the spouses have HLA B14, they should be tested for the adrenogenital syndrome gene; with HLA B18, there is a high probability of having a child with developmental abnormalities.

In habitual miscarriage, an increase in the frequency of occurrence of some alleles and HLA phenotypes was noted: A19, B8, B13, B15, B35, DR5, DR7, their frequency is 19%, 9.5%, 19%, 17.5%, 22.2%, 69.6% and 39.1% versus 6.3%, 3.8%, 10.3%, 16.7%, 29.9% and 22.7%, respectively, in women with uncomplicated pregnancy.

In addition to the HLA phenotype, many researchers believe that the compatibility of spouses by HLA antigens plays a very important role. The main idea is that with compatibility by the HLA system, antibodies that play the role of a blocking factor do not develop. With compatibility of spouses by more than 2 HLA antigens, the risk of miscarriage is almost 100%.

Compatibility of spouses according to the HLA system and its importance in reproduction have long been in the field of attention of immunologists and obstetricians. There is a whole direction of research on the role of lymphocyte therapy in the treatment of habitual miscarriage using lymphocytes of the father or donor or both. There are many supporters of this therapy.

At the same time, there are many opponents of this therapy, who believe that compatibility hardly plays a role and lymphocyte therapy does not give the same effect as that obtained by the proponents of this therapy.

Different results have been obtained from methodologically different approaches to solving this problem: different groups of patients, different amounts of lymphocytes administered, different gestational periods at which therapy is carried out, etc.

There is another original point of view in the literature regarding the HLA system. According to Chiristiansen OB et al. (1996), the effect of compatibility of parental antigens may be of non-immunological origin. In experiments on mouse embryos, the authors demonstrated the existence of a lethal recessive gene closely associated with HLA. Mouse embryos homozygous for certain HLA alleles die at different stages of embryogenesis. Humans may also have a similar HLA complex. If so, parental compatibility for HLA may be secondary, reflecting homozygosity for the embryo for the lethal gene associated with HLA.

Further research in this area will allow us to more accurately determine the place of HLA in the reproductive system.

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