RU2763837C1 - Method for immunopathological placental insufficiency simulation - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine in the field of obstetrics-gynecology, reproductive medicine, immunology, histology. A method for modeling immunopathological placental insufficiency is that IgG antibodies isolated from anti-TPO-positive blood sera with a titer of more than 1000 units are injected into outbred ICR mice, obtained from patients with diagnosed autoimmune thyroiditis who have fetal loss and a history of miscarriage. The introduction of IgG is carried out intravenously into the tail vein on the 4th day of pregnancy at a dose of 100 mcg/mouse, while an adequate model of immunopathological placental insufficiency with morphological signs of placental damage associated with antibodies is obtained on the 16th day of pregnancy.

EFFECT: invention provides a direct damaging effect of the injected antibodies on the morphogenesis of the placenta, the absence of a direct damaging effect of the injected antibodies on the target organ - the thyroid gland, minimizes the systemic immune response, has a high reliability of extrapolation to the human population, is affordable and reproducible.

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Description

The invention relates to experimental medicine, namely to modeling methods, and can be used to model immunopathological placental insufficiency. It is intended for obstetricians-gynecologists, reproductologists, immunologists, histologists to study the features of placental morphogenesis and pregnancy outcome for the fetus in the presence of autoantibodies, to study the mechanisms underlying repeated reproductive losses in patients with autoimmune diseases.

Currently, the percentage of reproductive losses due to unrecognized factors does not tend to decrease and exceeds 30%. According to most researchers, the basis of the pathogenesis of these reproductive losses is damage to the placental tissue caused by immunological disorders. It is the immunological conflict that is considered as a key factor in damage to placental tissue, including in women with autoimmune diseases. Thus, of particular interest is the study of the features of placental morphogenesis and the outcome of pregnancy for the fetus in the presence of autoantibodies.

The relationship of autoimmune diseases (AIT - autoimmune thyroiditis, SLE - systemic lupus erythematosus, APS-antiphospholipid syndrome) with reproductive failures has been confirmed by numerous studies, however, the question of the possible direct effect of autoantibodies continues to be the subject of discussion. Supporters of the established principled position consider autoantibodies as markers and criteria for immune disorders at the level of organs and tissues, and reproductive losses are regarded as the result of the influence of systemic disorders in the body associated with the underlying autoimmune disease. However, recently there is more and more evidence in favor of the possible direct effect of autoantibodies on the organs of the reproductive system. Thus, meta-analyses have been published showing that the presence of antibodies to thyroid peroxidase (TPO), even with unchanged thyroid function, is an independent risk factor for an increase in the percentage of miscarriage and preterm birth [Dhillon-Smith RK, Coomarasamy A. TPO antibody positivity and adversepregnancy out comes . Best Pract Res ClinEndocrinolMetab. 2020 Jul; 34(4):101433. doi: 10.1016/j.beem.2020.101433. Epub 2020 Jun 18. PMID: 32883611.; Chen L, Hu R. Thyroid autoimmunity and miscarriage: a meta-analysis. ClinEndocrinol (Oxf). 2011 Apr; 74(4):513-9. doi: 10.1111/j.l365-2265.2010.03974.x. PMID: 21198746].

Many experimental animal models have been developed to study preventive and therapeutic measures, due to the impossibility of conducting such studies in humans for ethical reasons. One of the most common types of experimental animals in this area are mice. This is due to the similarity in the organization of mouse and human placentas, as well as the high fecundity and short gestation period of mice. Thus, the human placenta belongs to hemochorial villous, where an increase in the contact area of the trophoblast with the mother's blood is achieved due to the multiple branching of the chorionic villi, and the mouse placenta belongs to the hemochorial labyrinth, where an increase in the contact of the trophoblast with the mother's blood is achieved due to the formation of a complex labyrinth of channels.

Existing models of placental insufficiency include a wide range of different damaging factors: from uterine vessel ligation to the use of vector systems for gene delivery to the trophectoderm.

The conditions closest to those in the body of a patient with an autoimmune disease are created in models using genetic or immunological damaging factors.

A known method for modeling immunopathological placental insufficiency (placental injury) using the BPH/5 mouse line, which is an inbred subline obtained as a result of numerous closely related crossings of the known BPH/2 mouse line, characterized by high blood pressure (patent US 2004133929 A1). This line of experimental animals demonstrates moderately elevated blood pressure before pregnancy and the spontaneous development of gestational hypertension, proteinuria, renal glomerulosclerosis, and endothelial dysfunction.

Obviously, any inbred line has immunological disorders, however, the presence of a genetically determined predisposition to hypertension in these animals makes it difficult to extrapolate data to patients.

A known method for modeling immunopathological placental insufficiency by crossing females of the CBA/J line and males of the DBA/2 line (patent CN 110100789 A). This combination of mouse strains is characterized by a high rate of spontaneous abortions due to immunological causes.

However, given the linearity of experimental animals, one must be aware of the limitations in extrapolating data to the human population.

A known method for modeling immunopathological placental insufficiency, which consists in removing the zona pellucida of the mouse blastocyst and introducing sFLTl-encoding nucleic acid into the trophectoderm (patent US 20130198874).

However, this modeling method is expensive and difficult to reproduce, in contrast to the claimed one. In addition, the claimed model is favorably distinguished by the fact that the used line of mice is heterozygous for an indefinite number of genes, which makes it possible to more reliably extrapolate the data obtained in the experiment to a heterogeneous human population.

A known method of modeling immunopathological placental insufficiency by introducing female linear and non-linear mice glycoside muramyldipeptide - N-acetyl-β-1-O-heptylmuramyl-L-alanyl-D-isoglutamine (patent RU 2511107 C1). The introduction of a molecule similar in structure to the lipopolysaccharide (LPS) of the bacterial wall provokes hyperactivation of the systemic immune response, activation of innate immunity and an adjuvant effect. Unlike the known method, the claimed method minimizes the systemic immune response due to passive immunization of mice due to the absence of non-specific adjuvant action and activation of adaptive immunity factors. This approach reduces the likelihood of a negative effect of the mother's immune system on placental tissue.

A known method for modeling immunopathological placental insufficiency, based on the introduction of galectin-9 to pregnant mice intraperitoneally (patent CN 111514277 A).

The introduction of galectin-9 indirectly modulates immunopathological placental insufficiency, with subsequent disruption of trophoblast invasion and remodeling of spiral arteries, however, this occurs mainly due to the activation of CD14 ⁺ macrophages, which is more characteristic of an anti-infective immune response. In the claimed modeling method, the activation of this link of the immune system is excluded, which brings the proposed model closer to the clinical picture of an autoimmune condition.

A known method for modeling immunopathological placental insufficiency, based on the introduction of nephrotoxic antibodies (patent SU 1267467 A1).

The disadvantage of this method is that the introduction of nephrotoxic antibodies observed violations of the renal blood flow, resulting in secondary changes in blood circulation in the placenta.

The technical result of the invention is a confirmed direct damaging effect of injected antibodies on placental morphogenesis, the absence of a direct damaging effect of injected antibodies on the target organ - the thyroid gland, minimization of the systemic immune response due to passive immunization of mice, high reliability of extrapolation to the human population, availability and reproducibility.

This technical result is achieved in a method for modeling immunopathological placental insufficiency, including the administration of antibodies to pregnant mice, in which outbred ICR mice are injected with IgG antibodies isolated from anti-TPO-positive blood sera with a titer of more than 1000 units obtained from patients diagnosed with autoimmune thyroiditis, with a history of fetal loss and miscarriage, IgG is administered intravenously into the tail vein on the 4th day of pregnancy at a dose of 100 μg / mouse, while an adequate model of immunopathological placental insufficiency with morphological signs of placental injury associated with antibodies is obtained at 16- day of pregnancy.

The advantage of administering IgG antibodies isolated from anti-TPO-positive blood sera with a titer of more than 1000 units obtained from patients diagnosed with autoimmune thyroiditis who have fetal loss and a history of miscarriage lies in the absence of a direct damaging effect of the administered antibodies on the target organ - the thyroid gland.

The presence of antibodies to thyroglobulin and phospholipids in patients was excluded. The comparison group used Commercial IgG (Jackson, ImmunoResearchLaboratories) and PBS (BiologicalIndustries, IsraelBeit-Haemek Ltd).

The experiment used female mice aged 10-12 weeks. Animals were reared under standard conditions at 23°C (±1°C) in 12-hour light cycles (7:00 AM-7:00 PM) with unlimited access to food and water. Females and males were planted in a ratio of 3-4:1. After mating, the presence of a vaginal plug was assessed (the day of its detection was considered the 0th day of pregnancy).

IgG antibodies were injected intravenously into the tail vein on the 4th day of pregnancy (the initial stage of implantation) at 100 µg/mouse. The chosen term for the introduction of antibodies is due to the desire to simulate a state close to clinical in relation to pregnancy that occurred against the background of circulating antibodies, meaning the ability to assess the effect of injected antibodies at all stages of placentogenesis. At the same time, the introduction of antibodies at the time corresponding to the gestational period makes it possible to minimize the systemic immune response caused by immunization of the maternal organism, which inevitably develops within 10-14 days after induced passive immunization.

The study included the following groups of mice (n=15):

1) Mice injected with IgG isolated from anti-TPO positive sera obtained from female patients.

2) Mice injected with commercial IgG mixture.

3) Mice injected with PBS (Phosphate-bufferedsaline).

4) Intact control.

Animals were withdrawn from the experiment on the 16th day of pregnancy, corresponding to the stage of maximum development of the labyrinthine zone of the placenta in mice, which determines the exchange between maternal and fetal blood flow and, therefore, is responsible for the target effect of the model.

The uterine horns with ovaries were isolated and the percentage of viable and resorbed fetuses was estimated according to the formula:

Nres./(Nres.+Nl.) × 100%, where:

Npez. - number of resorbed fruits;

Nlife - number of viable fetuses.

Fetal resorption was defined as growth arrest or regression to a necrotic mass. Then viable fetuses and placentas were weighed. For histological evaluation, 2 placentas and 2 fetuses from each mouse from each group were selected, the biomaterial was immediately fixed in 4% formaldehyde. After that, the samples were subjected to dehydration in 96% ethanol followed by treatment with xylene. The samples were then immersed in paraffin. Paraffin blocks with samples were cut into slides ranging in size from 3 to 5 μm and stained with H&E.

Micropreparations were examined under a microscope with Keller illumination setting at 100×, 200× and 400× magnification in order to obtain a general idea of the results of histological examination. The results were quantified on micrographs obtained using a microscopic image fixation system consisting of an Olympus BX46 microscope and CellSens 47 Entry software. Fields of view containing tissue defects, staining defects, and artifacts were excluded from photography. Photographs were taken at a magnification of 400× (eyepiece 10×, lens 40×), with full opening of the aperture diaphragm, in the Photo mode, exposure time 1/38 s, camera sensitivity - maximum, image size 2080×1544 pixels, image format JPEG (normal ). The proportion of the area occupied by spongiotrophoblast cells, trophoblast cells of the labyrinthine part of the placenta, and giant trophoblastic cells was calculated using the VideoTest-Morphology 5.0 program (Russia).

From each area of the placenta under study, 5 microscopic images were taken, and the relative area of cells (%) was estimated as the ratio of the area of cells to the total area of the preparation according to the formula:

Erelative = S cells / S preparation × 100%.

Data were evaluated using ranks, means, and standard deviation. A p<0.05 value was taken as an indication of the significance of the difference.

In the course of the study, the following results were obtained.

In the group of mice that received antibodies from patients with AIT, a total of 96 fetuses were obtained, and in the control group - 201 fetuses (p<0.05). Among all fetuses, 27% (n=26) in the study group underwent resorption and only 3% (n=6) of fetuses in the control group (p<0.05). Thus, the number of viable fetuses in the comparison group (n=195) exceeded the number of viable fetuses in the study group (n=70) by almost 3 times. The average weight of fetuses and placentas in the study group was 351 mg and 112 mg, respectively, while in the control group 503 mg and 125 mg, respectively (p<0.05). Histological assessment of the placentas showed that, in contrast to the comparison group, the identified violations of placentogenesis in the study group affected all placental zones. These changes were characterized by an uneven thickness of the zones, a violation of the ratio of the placental zones, as well as an inhomogeneous distribution of spongiotrophoblast cells and glycogen cells, indicating a violation of cell migration processes. The most significant violations were noted in the labyrinth zone. These disorders were mainly represented by uneven thickness of intervascular membranes and uniform blood filling of lacunae. In other cases, hypervascularization of varying severity was noted.

The method is illustrated in Fig. 1-10 where:

figure 1 - the accumulation of spongiotrophoblast cells in the labyrinth zone of the placenta, the anti-TPO group. H&E, x200;

fig. 2 - labyrinthine zone of the placenta without accumulations of trophoblastic cells, group IgG, H&E, x400;

fig. 3 - gaps in the labyrinthine zone of the placenta, the arrow indicates the accumulation of glycogen cells in the labyrinthine zone of the placenta, the anti-TPO group. H&E, x200;

fig. 4 - uniform blood filling of vascular lacunae without signs of hypervascularization and thickening of intervascular membranes in the labyrinthine zone of the placenta, rpynnalgG. H&E, x400;

fig. 5 - accumulation of glycogen cells in the labyrinth zone of the placenta, anti-TPO group. H&E, x200;

fig. 6 - incomplete migration of spongiotrophoblast cells from the labyrinthine zone of the placenta, anti-TPO group, H&E, x200;

fig. 7 - violation of the ratio of placental zones in the placenta, anti-TPO group. the labyrinth zone/connection zone ratio is close to 1.5:1. (norm-3:1), a-labyrinth zone, b-connective zone/spongiotrophoblast H&E, x40;

Fig. 8 - expression of thyroperoxidase by syncytiotrophoblast cells of the labyrinth zone of mouse placentas x400;

Fig. 9 - expression of thyroperoxidase by spongiotrophoblast cells of the connective zone of mouse placentas x400;

Fig. 10 - expression of thyroperoxidase by trophoblast giant cells of xbOO mouse placentas.

In the presence of anti-TPO antibodies, the area of the nuclei of trophoblast giant cells and spongiotrophoblast cells, as well as the area of the cells themselves, was significantly lower (p<0.01) than in all compared groups. At the same time, in the labyrinthine part of the placenta, the average area of the nucleus was significantly higher (p<0.01), but the area of the trophoblastic cell itself did not have significant differences between the groups, there was only a tendency to increase in the anti-TPO group. The data obtained (Tables 1, 2) indicate damage to the placental tissue at the cellular level, which has a negative impact on the state of blood flow.

For the first time, immunohistochemical evaluation of mouse placentas using antibodies to mouse thyroperoxidase revealed the presence of thyroperoxidase in syncytiotrophoblast, spongiotrophoblast, and trophoblast giant cells. In this case, these data allow us to confirm the assumption that all layers of mouse placentas contain a potential target for the direct damaging effect of antithyroid antibodies. The results obtained are consistent with a study showing that thyroperoxidase is present in the human placenta [Rahnama R, Mahmoudi AR, Kazemnejad S, Salehi M, Ghahiri A, Soltanghoraee H, Vafaei S, Rezaei A, Zarnani AH. Thyroid peroxidase in human endometrium and placenta: a potential target for anti-TPO antibodies. ClinExp Med. Feb 2021; 21(l):79-88. doi: 10.1007/sl0238-020-00663-y. Epub 2020 Sep 26. PMID: 32980989]. The authors of this study, using immunohistochemical analysis of human placentas, found thyroperoxidase in syncytiotrophoblast cells and on the cell surface of invasive trophoblast. In the present study, thyroperoxidase was found in similar structures in mouse placentas.

Thus, the results of assessing the validity of the model are based on indicators of reproductive losses associated with a detailed description of the target effect - morphological indicators of placental damage, supplemented by morphometry and IHC study. Previously, in known models for studying the mechanisms of miscarriage, the assessment of the influence of the damaging factor was considered, as a rule, only in comparison with the level and nature of fetal losses. The analysis of the state of the placental tissue was carried out, as a rule, in single works, fragmentarily, in a descriptive accompanying format.

An increased percentage of fetal resorption and a lag in the mass-growth indicators of the fetus are a traditional sign of placental damage. However, the structural and functional changes in the placental tissue identified in this work serve as objective evidence of impaired placental morphogenesis and allow us to determine the pathogenetic mechanisms of development and possible modeling of insufficiency of the uteroplacental system in experimental animals after the administration of IgG from patients with AIT. The presence of thyroperoxidase detected for the first time in the structures of placental tissue damaged in experimental animals during immunization with antibodies to thyroperoxidase can serve as a sufficient basis for assuming a direct damaging effect of these antibodies on placental morphogenesis.

The results obtained allow us to conclude that the claimed model is an adequate experimental model that reproduces antibody-dependent damage to the placenta. The model format can technically be replicated with other antibodies obtained from patients with a specific autoimmune disease and a history of miscarriage. This method can be used to assess the role of autoantibodies as a factor in placental damage with subsequent fetal loss in various autoimmune diseases, including in clinical settings to verify idiopathic reproductive losses.

The proposed method has a number of advantages:

- allows you to more reliably extrapolate the data obtained in the experiment to the human population, in contrast to experiments with linear laboratory animals due to the fact that these mice are heterozygous for an indefinite number of genes;

- allows you to determine antibodies as an independent immunological factor in damage to placental tissue;

- allows to detect disorders of placentogenesis associated with the circulation of autoantibodies;

- eliminates the influence of possible factors damaging the placental tissue (genetic, toxic, drug, chemical, infectious-inflammatory, disrupting blood circulation, etc.);

- allows you to study the independent mechanisms of the influence of reproductively significant antibodies on placentogenesis;

- allows you to identify new approaches to the predicted risk of complicated pregnancy, childbirth, the perinatal period in women with autoimmune diseases and to ensure the prevention of early recurrent pregnancy losses.

The claimed method provides a direct damaging effect of the injected antibodies on the morphogenesis of the placenta, the absence of a direct damaging effect of the injected antibodies on the target organ - the thyroid gland, minimizes the systemic immune response, has a high reliability of extrapolation to the human population, is affordable and reproducible.

Claims (1)

A method for modeling immunopathological placental insufficiency, including the administration of antibodies to pregnant mice, characterized in that outbred ICR mice are injected with IgG antibodies isolated from anti-TPO-positive blood sera with a titer of more than 1000 units obtained from patients diagnosed with autoimmune thyroiditis and having fetal loss and a history of miscarriage, IgG is injected intravenously into the tail vein on the 4th day of pregnancy at a dose of 100 μg / mouse, while an adequate model of immunopathological placental insufficiency with morphological signs of placental damage associated with antibodies is obtained on the 16th day of pregnancy .

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