Regulation of the functional activity of peripheral B lymphocytes in pregnant women with a history of recurrent miscarriage

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BACKGROUND

Miscarriage remains an urgent problem until the present time, with an incidence that varies from 10% to 25% [1]. Concurrently, approximately 2%–5% of married couples repeatedly lose pregnancy, which is called recurrent miscarriage [2]. In recent years, the attention of scientists was attracted by the immunological aspects of recurrent miscarriage, which are detected in various parts of the immune system and rank first in the etiology of unexplained reproductive losses [3]. B-lymphocytes are significant in uncomplicated pregnancy through humoral immune responses and antibody production. In addition, B cells can cause various obstetric complications, such as spontaneous miscarriage, preeclampsia, intrauterine growth retardation, stillbirth, and preterm birth due to the production of autoantibodies [4].

Member 13 of the tumor necrosis factor superfamily (TNFSF) includes two soluble ligands that are involved in the development of B-lymphocytes, the B-cell-activating factor ([BAFF] or TNFSF13B) and the proliferation-inducing ligand ([APRIL] or TNFSF13). These two ligands signal through three receptors, namely the specific BAFF receptor (BAFF-R) and receptors that recognize both BAFF and APRIL, transmembrane activator-1 and calcium modulator and cyclophilin ligand interacting with CD267 (TACI), and B-cell maturation antigen ([BCMA], CD269) [5].

All three BAFF-R are predominantly expressed on B-lymphocytes, whereas TACI is present on both B cells and activated T lymphocytes. BAFF-R can be found on regulatory and activated T lymphocytes. By binding to these receptors, BAFF mediates the activation of the canonical nuclear factor-kappa, which is an amplifier of the light chain of activated B cells (NF-êB), non-canonical NF-êB, N-terminal kinase c-Jun, extracellular signal of the regulated kinase (ERK), and phosphoinositide 3-kinase (PI3K), namely signaling protein kinase B (Akt) [5].

After binding to BAFF-R, BAFF activates Akt and ERK depending on IêB kinase (IKK)-1 in the primary B cells. Thereby promoting B-cell survival [6] and affecting the physiological activation of T cells by activating the PI3K–Akt signaling pathway, which reflects one of the pathological mechanisms of T lymphocyte-mediated autoimmune diseases [5].

BAFF maintains the homeostasis of the B-cell link of immunity, acting as a survival and adaptation factor for B-lymphocytes, starting from the transitional stage of development. Whereas, APRIL acts at a later stage, modulating the function and survival of B cells after an antigen contact and participates in the creation and survival of long-lived plasma cells in the bone marrow [7]. Both cytokines contribute to the formation of humoral effector mechanisms.

Increased levels of BAFF and APRIL in the blood serum are associated with autoimmune diseases and correlate with their severity and levels of pathogenic autoantibodies [7]. Thus, an increased level of BAFF and APRIL was noted in patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus [8], systemic scleroderma, etc. [9].

Currently, only a few data are available regarding the BAFF content in recurrent pregnancy loss, such as in patients with repeated spontaneous miscarriage. Its level in the decidual membrane and trophoblast was significantly reduced compared to healthy pregnant women [10]. In a pregnancy complicated by fetal growth retardation, the serum BAFF level was also reduced [11]. Currently, no literature data is available regarding the APRIL content during pregnancy.

This work aimed to identify the aspects of functional activity regulation of the peripheral B-lymphocytes in women at 5–12 weeks of gestation with a threatened spontaneous miscarriage and a history of recurrent miscarriage.

MATERIALS AND METHODS

In the V.N. Gorodkov Ivanovo Research Institute of Motherhood and Childhood, 88 women aged 18–40 years, with a gestational age of 5–12 weeks, were examined. The main group consisted of 36 patients with threatened spontaneous miscarriage during the examination and a history of recurrent miscarriage, the control group included 28 female patients with an uncomplicated pregnancy, and the comparison group consisted of 24 primigravidae with threatened spontaneous miscarriage at the time of examination. Exclusion criteria include chromosomal rearrangements of spouses’ karyotypes established at the pregravid stage, autoimmune diseases, severe allergic reactions, chronic disease exacerbations, and acute inflammatory diseases, and severe extragenital pathology. The peripheral venous blood was collected upon hospital admission, before the start of conservation therapy. BAFF and APRIL levels in the blood serum were determined by enzyme immunoassay, and CD19⁺BAFFR⁺ B-lymphocytes levels in the lymphocyte gate were determined by flow cytofluorometry using monoclonal antibodies.

A concentrated population of CD19⁺ B-lymphocytes was obtained by direct magnetic separation using DETACHABEAD CD19 magnetic particles (Invitrogen). Total RNA was isolated from CD19⁺ B-lymphocytes by the standard phenol method, and then RNA was converted into complementary DNA using a commercial kit manufactured by Fractal Bio (St. Petersburg, Russia). The obtained samples determined the expression level of Akt mRNA using the reverse transcriptional quantitative real-time polymerase chain reaction. All expression results of the studied genes were presented as the number of copies of pairs of a specific gene in the sample of ×10⁴/μl, normalized to the level of mRNA expression of the housekeeping gene (â₂-microglobulin).

 

Table. Characteristics of indicators of the functional activity of B-lymphocytes in women with threatened spontaneous miscarriage and history of recurrent miscarriage

Indicator	Control group (n=28)	Main group (n=36)	Comparison group (n=24)
BAFF-R, %	95.3 ± 0.5	97.89 ± 0.30 p1 = 0.0001	95.41 ± 0.94 р2 = 0.02
BAFF, ng/ml	1.5 ± 0.1	1.3 ± 0.05 p1 = 0.03	1.9 ± 0.21 р2 = 0.01
APRIL	1.2 ± 0.8	0.34 ± 0.06	0.31 ± 0.43
мРНК Akt	0.10 ± 0.06	1.39 ± 0.92 р1 < 0.05	0.24 ± 0.06 р2 < 0.05

Note: p₁ — level of statistical significance of differences between the main and control groups; р₂ — level of statistical significance of differences between the comparison and the control groups.

 

Informed voluntary consent for study participation was obtained from all participants. The study was approved at a meeting of the local ethical committee of the V.N. Gorodkov Ivanovo Research Institute of Motherhood and Childhood on November 21, 2018.

Statistical processing of the obtained data was performed using licensed applied programs Microsoft Office 2010, Statistica for Windows 13.0. Data on normal distribution were tested using the Kolmogorov and Shapiro–Wilk tests. Quantitative description of the values was made as mean and the standard error of the mean (M ± m). Statistically significant differences were assessed using the Student’s and the Mann–Whitney tests; differences were considered statistically significant at a p-value of <0.05.

STUDY RESULTS

The women who were examined were comparable in age, extra genital pathology, and menstrual function formation. Women with a threatened spontaneous miscarriage and recurrent miscarriage in the anamnesis in the range of extra genital pathology more often had thyroid diseases and anemia and were also married to an older spouse (p < 0.05) compared to the rest of the patients. No significant differences were revealed when analyzing the childhood ­diseases of surgical (extra genital and genital) interventions and allergic reactions in the history of women in the ­studied groups. Female patients of the main group had a history of chronic endometritis, sexually transmitted infections, ­uterine cavity curettage, non-developing pregnancies, and spontaneous miscarriages (p < 0.05) in all cases compared with the rest of the patients. Women with a history of threatened sporadic miscarriage had primary and ­secondary infertility more often than the rest of the patients. The clinical manifestations of a threatened spontaneous miscarriage were more pronounced in primigravidae, thus the frequency of spotting was 8.5% among the participants of the main group and 70.8% in the comparison group (p < 0.05). In addition, signs of ovum detachment, according to ultrasound examination of the pelvic organs were noted in 18.2% of cases in the main group and 50% in the comparison group. Expansion of the internal os of the cervical canal was recorded only among women in the comparison group (13.04% of cases).

The immunological examination data analysis showed that women with a threatened spontaneous miscarriage at the time of examination and a history of recurrent miscarriage had an increased level of BAFF-R on peripheral CD19⁺ B-lymphocytes compared to patients in the control group and primigravidae with the threat of pregnancy termination (Table). Contrarily, the serum BAFF level was lower in the main group compared to that of healthy pregnant women and primigravidae with threatened sporadic miscarriages. The difference in serum BAFF concentration between the comparison and the control groups was statistically significant. No significant differences were found in the APRIL content in the peripheral blood of the study groups but a pronounced tendency toward a decreased APRIL level in the main and comparison groups relative to the indicator of healthy pregnant women in all cases.

The expression analysis of Akt mRNA in the peripheral CD19⁺ B-lymphocytes found that women with a history of recurrent miscarriage and threatened spontaneous miscarriage at the time of examination are characterized by its increased level compared to healthy pregnant women and primigravidae with threatened pregnancy termination.

DISCUSSION

Isolated data recently appeared that B-lymphocytes can play a direct role in the pathogenesis of recurrent ­miscarriages [12]. BAFF is actively involved in the homeostasis of the entire pool of B-lymphocytes under physiological conditions by promoting the survival and maturation of B cells. In addition, data presented that increased B-cell activation leads to excessive formation of plasma cells and potentiation of humoral reactions, which may contribute to the development of autoimmune diseases [13]. Moreover, the BAFF/BAFF-R interaction activates the PI3K-Akt signaling pathway, which leads to increased viability and activation of T lymphocytes, showing a direct involvement in the mechanisms of recurrent miscarriages. Thus, BAFF is not only involved in maintaining the homeostasis of the B-cell link of immunity but is also an important factor in the pathogenesis of recurrent miscarriages.

An increased level of BAFF-R in women with threatened spontaneous miscarriages and recurrent miscarriages may indicate increased readiness for B-lymphocyte differentiation, and a simultaneously decreased serum BAFF concentration may indicate either accelerated binding of the ligand to the receptor or impaired homeostasis of both the B- and T-cell link of immunity. Decreased serum APRIL level in women with threatened pregnancy loss (both in primiparous women and in the group of recurrent miscarriage) probably indicates a reduced survival rate of mature high-efficiency forms of B-lymphocytes.

The PI3K/AKT pathway has recently attracted increasing attention from researchers, as it plays a critical role in the regulation of various cellular functions, including metabolism, growth, proliferation, survival, transcription, and protein synthesis [14]. A successful pregnancy is known to be associated with a sufficient level of relaxin produced by the corpus luteum, uterus, decidual tissue, and placenta. Insufficiency of this hormone is associated with the threat of termination of both the first and repeated pregnancies due to necrosis and apoptosis of the cytotrophoblast [15]. The registered increased expression of Akt mRNA in the peripheral CD19⁺ B-lymphocytes in women with recurrent miscarriages may indicate increased functional activity of B cells associated with a response to fetal antigens entering the mother’s bloodstream. Similarly, however, less pronounced changes occurred during a threatened miscarriage in primigravidae.

In addition, hormone-mediated B-cell lymphopenia was described in normal pregnancy, which was accompanied by a lower amount of BAFF-R and, accordingly, an increased level of circulating BAFF, which is necessary to differentiate immature transitional B cells into naive mature ones [16]. According to our data, B-cell lymphocytosis was detected in women with threatened sporadic and recurrent miscarriage, along with a pronounced decreased proportion of mature naive B-lymphocytes, indicating an increased expression of BAFF-R and decreased serum BAFF concentration. Mature naive B cells give rise to highly efficient subpopulations, memory cells, and plasma cells. A possible deficiency of plasma cells as the main producers of antibodies may be due, among other things, to a deficiency of APRIL and, probably, leads to insufficient provision of the immune tolerance of the maternal organism in response to a semi-allogeneic fetus.

Therefore, a recurrent threatened miscarriage in women with a history of recurrent miscarriage is associated with an impaired regulatory influence of BAFF and APRIL, which is expressed in a change in B-cell homeostasis and a weakened regulation of humoral effector mechanisms.

About the authors

Anna I. Malyshkina

Ivanovo Research Institute of Maternity and Childhood named after V.N. Gorodkov

Email: anna_im@mail.ru
ORCID iD: 0000-0002-1145-0563

MD, Dr. Sci. (Med.), Professor

Russian Federation, 20 st. Victory, Ivanovo

Natalia Y. Sotnikova

Ivanovo Research Institute of Maternity and Childhood named after V.N. Gorodkov

Email: niimid.immune@mail.ru
ORCID iD: 0000-0002-0608-0692

MD, Dr. Sci. (Med.), Professor

Russian Federation, 20 st. Victory, Ivanovo

Dmitriy N. Voronin

Ivanovo Research Institute of Maternity and Childhood named after V.N. Gorodkov

Email: niimid.immune@mail.ru
ORCID iD: 0000-0003-2836-8694

Cand. Sci. (Biol.)

Russian Federation, 20 st. Victory, Ivanovo

Alena V. Kust

Ivanovo Research Institute of Maternity and Childhood named after V.N. Gorodkov

Author for correspondence.
Email: avkMD@yandex.ru
Russian Federation, 20 st. Victory, Ivanovo

References

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