Immunological tests: what is informative?

Immunological mechanisms play a crucial role in establishing and maintaining pregnancy: the embryo is partially genetically "foreign" to the mother, so normal pregnancy requires the development of local and systemic tolerance of the maternal immune system to fetal antigens. Disruptions to this delicate balance can lead to implantation failure, early spontaneous abortions, and complications in late pregnancy. [1]

However, the term "immunologic miscarriage" encompasses both documented autoimmune syndromes clearly associated with pregnancy loss and a wide range of laboratory and morphologic findings whose clinical significance remains controversial. Governing bodies emphasize the need for caution: not all immunologic tests provide reliable and interpretable information for clinical practice. [2]

In practice, three groups of situations are often confused: 1) well-proven immune causes for which treatment alters pregnancy outcome; 2) conditions with an association, but without clear evidence of treatment effectiveness; 3) laboratory parameters that researchers suggest using, but which professional societies do not recommend for widespread clinical use. Proper distribution of tests across these groups helps avoid unnecessary examinations and harmful interventions. [3]

The purpose of this text is to clearly describe modern clinical practice with an emphasis on immunological testing: which tests are necessary, which are optional in certain clinical situations, and which are not recommended. It is based on international guidelines and extensive literature reviews. [4]

Definitions and prevalence

The term "recurrent miscarriage" most often refers to two or more consecutive spontaneous miscarriages during a clinically confirmed pregnancy. For epidemiological studies, a threshold of three or more losses is traditionally used; however, in clinical practice, many guidelines recommend beginning testing after two losses, taking into account age and desired future reproductive plans. [5]

The prevalence of recurrence is low: approximately 1-3% among women of reproductive age. Immunological factors account for only a fraction of the identified causes—for example, antiphospholipid syndrome occurs in 10-15% of patients with recurrent miscarriage across different populations. This emphasizes the importance of focused diagnostics—first ruling out genetic, anatomical, endocrine, infectious, and thrombophilic causes. [6]

It is important to remember that a proportion of positive laboratory findings does not necessarily indicate a causal relationship. Many immunological markers demonstrate associations in control studies, but intervention studies often fail to confirm that correcting these markers improves pregnancy outcomes. Therefore, the evidence base determines which tests to include in routine testing. [7]

The key practical point: not all "immunological" tests are equally useful. Guidelines identify mandatory and recommended tests with proven clinical value, as well as a list of tests that should only be used in research protocols or for specific indications. [8]

Key immune factors and mechanisms that influence pregnancy

1. Antiphospholipid syndrome is an immune disorder in which persistent antibodies to phospholipids increase placental vascular thrombogenicity and impair placental function, leading to early and late pregnancy losses. APS is one of the few immunological causes of miscarriage for which targeted therapy (aspirin and low-molecular-weight heparin during pregnancy in positive patients) has been shown to be beneficial. [9]
2. Thyroid autoimmunity—the presence of antibodies to thyroid enzymes, particularly thyroid peroxidase—has been associated with an increased risk of pregnancy loss and preterm birth in observational studies. However, randomized data on the benefit of levothyroxine treatment in euthyroid women with positive antibodies are inconclusive, so treatment recommendations depend on hormone levels and the clinical situation. [10]
3. Chronic endometritis—microscopic inflammation of the endometrium associated with the presence of plasma cells—is frequently found in women with recurrent implantation losses or failures. Evidence is accumulating in favor of diagnosis using CD138 immunohistochemistry and treatment, including targeted antibiotic therapy, although standardized protocols are still being developed. [11]
4. Natural killer cells and cytokine imbalances – studies show that uterine natural killer cell levels and activity may differ in women with recurrent pregnancy losses. However, methodological differences in measurements and the lack of compelling intervention data make NK cell-targeted testing and therapy controversial in routine clinical practice. [12]

Practical examination: which immunological tests are recommended and why

Below is a summary list of tests most commonly recommended for evaluation of patients with recurrent miscarriage. The basic approach is based on international guidelines and major reviews. [13]

Table 1. Basic set of immunological studies in case of repeated losses

Test	Indication	What gives the result?
Laboratory markers of antiphospholipid syndrome: lupus anticoagulant, anti-cardiolipin IgG/IgM, anti-β2-glycoprotein I IgG/IgM	With ≥2 consecutive losses or late loss, as well as with thrombotic history	Diagnosis of APS determines specific therapy during pregnancy. [14]
TSH and thyroid peroxidase antibodies (TPO-Ab)	Thyroid function screening before or when planning pregnancy	Detection of subclinical disorders and autoimmune status; treatment decision depends on TSH. [15]
Chronic endometritis testing: CD138 histology or endometrial biopsy if suspected	In case of repeated losses or implantation failures	Detection of plasma cells guides antibacterial therapy. [16]
Common autoantibodies: antinuclear antibodies (ANA) in clinical suspicion	If a systemic autoimmune disease is suspected	Helps identify systemic autoimmune diseases that may require targeted evaluation. [17]

(table: ESHRE and ASRM reference guidelines). [18]

Below is a detailed description of the key tests and their correct interpretation. [19]

Table 2. Antiphospholipid antibody testing: algorithm and interpretation

Test	What to look for	Interpretation
Lupus anticoagulant (coagulation tests)	The presence of an inhibitory effect on phospholipids in a special panel	Positive with repeated positive tests after ≥12 weeks confirms the laboratory criterion for APS.[20]
Anti-cardiolipin antibodies IgG/IgM	High titers of IgG or IgM	High titers are associated with a risk of obstetric complications and require confirmation by repeat testing.[21]
Anti-β2-glycoprotein I IgG/IgM	High credits	As above, included in the APS laboratory criteria. [22]

A key clinical principle: a positive result from one test does not establish a diagnosis of APS; a combination of clinical manifestations and persistent positive laboratory results at least 12 weeks apart are required. A definitive diagnosis of APS changes the management of pregnancy and the effectiveness of therapy. [23]

Tests of limited utility or not recommended for routine practice

There are numerous laboratory and immunological markers that are popular in research and private practice, but which professional societies do not recommend for inclusion in the standard panel for all patients. These include: peripheral NK cell counts, blood cytokine measurements, many immunoglobulin panels without clinical context, partner HLA matching tests, and experimental serological markers. [24]

Studies of uterine natural killer cells have shown differences in activity in some patients, but methodological issues and inconsistency in results between laboratories make the test's use controversial. Guidelines note that therapies aimed at reducing NK activity lack reliable evidence of effectiveness and may be harmful, so such interventions should only be considered within the context of clinical trials. [25]

Serum cytokine assays and Th1/Th2/Th17 profiles exhibit high variability and are method-dependent; they correlate poorly with local processes in the uterus. Therefore, these tests are not indicated for routine diagnosis and therapeutic decision-making. [26]

Finally, it is important to avoid the widespread use of unproven immunotherapies: intravenous immunoglobulin without strict indications, high-dose steroids, and other immunomodulators can have serious side effects and do not provide proven benefit for most patients. The decision to use such methods should be made in a multidisciplinary manner and based on scientific justification. [27]

Table 3. Tests and interventions that are not routinely recommended

Test or intervention	Why it is not recommended
Peripheral NK cells and their percentage	Methodological heterogeneity, lack of proven predictive value. [28]
Measurement of a wide range of cytokines in the blood	High interlaboratory variability and poor clinical correlation.[29]
Intravenous immunoglobulin without strict indications	Lack of convincing evidence of benefit; possible risks and high cost. [30]

Practical interpretation and clinical algorithmization

1. Initial examination. If the patient has had two or more losses, a basic examination is performed: karyotypes of the partners if indicated, ultrasound examination of the uterus, screening for infectious causes if clinically indicated, endocrine screening including thyroid-stimulating hormone and TPO antibody levels, and testing for antiphospholipid antibodies. It is the combination of these approaches that yields the greatest clinical benefit. [31]
2. If the laboratory shows at least one positive marker for antiphospholipid syndrome, confirmation is required after ≥12 weeks and an assessment of the clinical history for thrombosis or obstetric complications. Confirmed APS is an indication for specific management during pregnancy, with the use of low-dose aspirin and low-molecular-weight heparin in most cases. [32]
3. If positive thyroid peroxidase antibodies are detected, the decision to prescribe levothyroxine depends on the thyroid-stimulating hormone level and is discussed with an endocrinologist. Current data do not provide clear grounds for prescribing levothyroxine to all euthyroid women with TPO antibodies; an individual approach is essential. [33]
4. If chronic endometritis is suspected, an endometrial biopsy with CD138 immunohistochemistry is performed. A positive result leads to a course of antibiotic therapy according to the protocol. After confirmation of eradication during a repeat assessment, further pregnancy management may be attempted. Systematic reviews show benefits of treatment in subgroups, but standards are still being developed. [34]

Table 4. Approximate clinical algorithm for recurrent miscarriage

Step	Action	Comment
1	Confirmation of losses and collection of anamnesis	Find out the number and timing of losses, thrombotic episodes, chronic diseases. [35]
2	Basic examination: ultrasound, endocrinology, aPL, karyotypes if indicated	Standard tests that provide the greatest clinical benefit. [36]
3	Additional tests for clinical clues: CD138, ANA, infectious tests	Directed research with appropriate data. [37]
4	Interpretation and treatment based on results	APS - specific therapy; endometritis - antibiotics; thyroid dysfunction - treatment according to indications. [38]

Current research and areas requiring attention

In recent years, there has been a growing number of studies on the role of the uterine immune microbiome, the local immune response, and the endometrial cellular profile. These studies promise more accurate targeted diagnostics; however, most results remain at the stage of scientific validation and are not ready for widespread clinical application. Guidelines are updated as new data emerges. [39]

The debate surrounding NK cells and immunotherapies continues: multicenter studies and standardized assessment methods may change recommendations in the future, but at this stage, widespread use of such tests and methods is not supported by evidence. Patient participation in an informed discussion of the risks and uncertainties is essential before prescribing any experimental immunomodulatory interventions. [40]

Research priorities for the coming years include standardizing methods for assessing the local immune status of the uterus, randomized trials of targeted interventions using confirmed biomarkers, and integrating microbiome and immune data into a unified diagnostic strategy. These areas promise to reduce the proportion of "unexplained" losses. [41]

Table 5. Promising research directions and clinical goals

Direction	Practical purpose
Local immune profile of the endometrium	Development of tests with predictive value for implantation. [42]
The uterine microbiome	Assessing the role of dysbiosis in chronic endometritis and pregnancy loss. [43]
Randomized trials of immunomodulation	Evaluation of the effectiveness of targeted interventions with strictly verified markers. [44]

Practical recommendations for doctors and patients

1. The initial approach should focus on basic, evidence-based testing: antiphospholipid antibodies, thyroid function with TPO antibodies, assessment of uterine anatomy, and, if indicated, endometrial biopsy with CD138. Other immunological tests should be prescribed on a case-by-case basis and only after discussion with the patient. [45]
2. Avoid prescribing extensive immunopanels and expensive immunomodulation treatments outside of clinical trials. Intravenous immunoglobulin, immunosuppressants, and other interventions require evidence-based support before widespread use. [46]
3. In the presence of confirmed antiphospholipid syndrome, ensure coordination of the pregnant woman's care between the obstetrician-gynecologist and a thrombosis specialist or immunologist, and apply confirmed treatment and monitoring protocols. [47]
4. Discuss expectations and the likelihood of success with the patient. Most patients with two or three losses, with adequate evaluation and treatment, can expect a favorable outcome in a subsequent pregnancy, especially if specific underlying causes are identified and corrected. [48]

Conclusion

Immunological testing for recurrent miscarriage is important, but should be used selectively, based on evidence-based medicine and international guidelines. Currently, the priorities are testing for antiphospholipid syndrome, thyroid function screening based on TPO antibodies, and diagnosis of chronic endometritis when indicated. Other immunological markers remain primarily research-based. [49]