RU2748191C1 - Method for determining the severity of chronic endometritis in women with uterine factor of infertility after unsuccessful extracorporeal fertilization in the anamnesis (options) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to pathomorphology, gynecology and reproductology, it can be used to assess the severity of chronic endometritis (hereinafter – CE) in women with uterine factor of infertility after an unsuccessful extracorporeal fertilization (hereinafter – ECF) in anamnesis. In the first embodiment of the invention, a papel biopsy of the endometrium is performed in the middle stage of the proliferation phase. In the obtained material, the levels of CD138 and CD56 expression are determined. If the number of CD138+ cells is zero, then “0 points” is assigned; if the number of CD138+ cells is 1, then “1 point” is assigned; if the number is 2-3, then “2 points” is assigned; if the number is 4 or more, then “3 points” is assigned. If the expression of CD56+ is up to 10 cells, then “0 points” is assigned; if the expression of CD56+ exceeds the normal number of cells by 2 times, then “1 point” is assigned; if it exceeds the normal number by 3 times, then “2 points” is assigned; if it exceeds the normal number by 4 times or more, then “3 points” is assigned. The sum of the obtained scores of the CD138 and CD56 expression levels is determined. With the sum of “5-6 points”, the presence of an expressed degree of chronic endometritis is determined; with “3-4 points”, the presence of a moderately expressed degree is determined; with “2 points”, the presence of a weakly expressed degree is determined. In the second embodiment of the invention, a papel biopsy of the endometrium is performed in the middle stage of the proliferation phase. In the obtained material, the level of CD56 expression is determined and the degree of endometrial fibrosis is determined, for which a histochemical study is performed by means of Mallory staining. If the expression of CD56+ is up to 10 cells, then “0 points” is assigned; if the expression of CD56+ exceeds the normal number of cells by 2 times, then “1 point” is assigned; if it exceeds the normal number by 3 times, then “2 points” is assigned; if it exceeds the normal number by 4 times or more, then “3 points” is assigned. If the severity of endometrial fibrosis is 0%, then “0 points” is assigned; if it is 1-15%, then “1 point” is assigned; if it is 16-49%, then “2 points” is assigned; if it is 50% or more, then “3 points” is assigned. The sum of the obtained scores of the CD 56 expression level and the severity of endometrial fibrosis is determined. With the sum of “5-6 points”, the presence of an expressed degree of chronic endometritis is determined; with “3-4 points”, the presence of a moderately expressed degree is determined; with “2 points”, the presence of a weakly expressed degree is determined.

EFFECT: group of inventions makes it possible to determine accurately the severity of CE in women with uterine factor of infertility and unsuccessful ECF attempts in the anamnesis.

2 cl, 4 tbl, 2 ex

Description

The technical field to which the invention relates

The invention relates to medicine, namely to pathomorphology, gynecology and reproductive medicine and can be used to assess the severity of chronic endometritis (CE) in women with uterine factor infertility after failures of in vitro fertilization (IVF) in history based on the assessment of markers of chronic persistent inflammation and plasma cells for the purpose of specific diagnosis of the endometrial infertility factor in the uterine form of infertility associated with ChE.

The prevalence of ChE in the female population reaches 24.4% [1-4], among those who are in the reproductive period, the indicator varies from 8% to 72% of cases. The incidence of the disease in infertile women reaches 55.7% [2, 5]. It should be recognized that ChE is the most common cause of implantation failures in IVF programs. This is evidenced by the high prevalence among patients with repeated loss of pregnancies and repeated implantation failures (PNI), 13 and 30%, respectively [6]. Studies have shown that patients with a combination of PNI and verified ChE in IVF programs demonstrated a lower frequency of satisfactory implantations in comparison with the group where women had a history of PNI without ChE (11.5 and 32.7%, respectively) [5]. In fairness, it should be noted that persistent inflammation of the endometrium with uterine factor of infertility remains unknown, and the frequency of occurrence varies in different publications [4].

The world literature presents many pathogenetic pathways for the development of ChE and associated negative reproductive outcomes: changes in populations of immunocompetent cells in the endometrium, the production of pro-inflammatory cytokines, a negative effect on endometrial decidualization [2]. Of particular importance is a change in the reception of sex steroid hormones, a violation of the pattern of paracrine factors and a violation of adhesion. Thus, ChE disrupts decidualization, initiates resistance to progesterone, while at the same time disrupting the balance of adhesion factors [7]. The listed anomalies of the endometrial environment cause an increased proliferative potential with a reduced ability to differentiate, asynchronous peristalsis of the uterus, which is evidence that the endometrium is not ready for implantation [8]. To date, methods for diagnosing ChE and endometrial receptivity are far from ideal, since they do not have specific markers. All of the above determines the relevance of the chosen topic, indicates the need to develop new methods for diagnosing and predicting strategies for overcoming the uterine factor of infertility in CE in patients after IVF failures in history.

State of the art

There is a method for diagnosing ChE, described in RF patent 2236013 dated 09/10/2004 (prototype), the essence of which is that, using immunohistochemical (IHC) staining of endometrial tissue sections, local immunity parameters are determined: the number of lymphocytes expressing markers of natural killer cells, CD56 +, CD16 + and markers of activation HLA-DR (II) +, and thereby diagnose various variants of the course of ChE.

The disadvantage of this method is the absence of a complex of IHC and histochemical assessment of ChE, which underlies the implantation failure of women with uterine factor infertility after IVF failures in history.

A known method for diagnosing and predicting the development of ChE in female patients (RF patent 2587325 dated 20.06.2016). In women of reproductive age, by means of IHC studies of endometrial slices, according to the percentage of positively stained endometrial cells in points, the indices of local innate immunity TLR2, TLR4, TLR9 are determined. The obtained IHC pattern of TLR2, TLR4, TLR9 expression together with morphological data makes it possible to diagnose either the absence of ChE, or the presence of risk factors and predisposition to its development, or the presence and progression of the disease, or the stage of remission of this condition, or the presence of long-term persistent inflammation of the endometrium, during which characterized by the development of sclerotic vascular changes and fibrosis of the endometrial stroma of varying severity.

The disadvantage of this method is the absence of a comprehensive histochemical and IHC assessment of ChE associated with the uterine factor of infertility and a history of in vitro fertilization failures in these women.

There is a method for determining the stage of the clinical course of ChE, the degree of inflammation activity and the severity of fibrosis with the establishment of the prognosis of implantation and impairment of endometrial receptivity, described in RF patent 2674155 dated September 27, 2017. The essence of the method consists in assessing the presence of inflammatory lymphocytic infiltration and determining the severity of fibrosis, by quantitative assessment, its prevalence and presence in the main topographic zones of the endometrium in points, the sum of which determines the indices of ChE activity and the severity of fibrosis, according to the value of which three stages of activity are distinguished and clinical course of ChE.

The disadvantage of this method is the lack of a systemic determination of pathognomonic markers present in the chronic inflammatory process in the endometrium.

The technical problem solved in the present invention is the elimination of the indicated disadvantages: increasing the accuracy of verification of the severity of ChE in women with uterine factor of infertility after IVF failures in history.

Disclosure of the essence of the invention

Due to the absence of highly specific and sensitive methods for assessing the severity of ChE with associated infertility in patients with repeated IVF failures in history, the proposed method based on the analysis of a complex of pathognomonic markers of the severity of inflammatory and post-inflammatory (adhesive) disorders in the endometrial pattern of this cohort ...

We conducted our own study, which demonstrated the high specificity of our proposed method in assessing the severity of ChE in infertile patients with a history of IVF failures. The groups of markers CD138 + and CD56 +, as well as CD56 + (with CD138 equal to zero, and the presence of fibrosis of varying severity in the stroma of the endometrium), in combination with Mallory's histochemical stain, reliably reflect the severity of ChE in patients with this disease and IVF failures in history ...

The developed variants of the method are based on a complex pathomorphological study of the endometrium in the middle stage of the proliferation phase, including, in the first version, histological staining with hematoxylin and eosin, IHC study with antibodies to CD138, CD56, and in the second embodiment of the method, histological staining with hematoxylin and eosin, IHC study with antibodies to CD138, CD56 and Mallory histochemical staining.

The complex of IHC studies includes analysis of the expression levels of key proteins: CD138 + - a marker of a plasma cell, CD56 + - a marker of natural killer cells.

The need to perform the second variant of the method arises in the presence of long-term chronic inflammation of the endometrium, accompanied by the proliferation of fibrous tissue. In this case, CD138 + cells are absent and there are only CD56 + cells. In this case, it is required to take into account the severity of endometrial fibrosis, for which it is necessary to perform a histochemical study by means of Mallory staining.

CD138 (also known as syndecan-1) is a transmembrane heparan sulfate proteoglycan implicated in the modulation of inflammatory diseases, cancers, and infections. It also participates in the regulation of cyclical changes in the endometrium and is used to predict reproductive outcomes as a specific and sensitive antigen of plasma cells. In addition, it was suggested that the counting of CD138 + plasma cells can improve the accuracy of the diagnosis of ChE and reproductive outcomes in infertile patients with a high number of CD138 + cells. Corresponding studies have shown that IHC staining with this marker is the most sensitive method for diagnosing ChE [9, 10].

CD56 - large granular lymphocytes - a marker of natural killer cells. In the proliferation phase, the proportion of CD56 + cells is 8% of all endometrial cells, and in the secretory phase - 60-70%. Normally, natural killers under the influence of progesterone migrate into the endometrium and are constitutively present in it. The increase in the number of CD56 + cells in the endometrium of women with impaired reproductive function characterizes the intensity of the persistent process of inflammation in the tissue and is an unfavorable factor that prevents normal adhesion and implantation of the blastocyst [11].

Mallory staining is a histochemical technique of polychromatic staining of preparations, which makes it possible to identify the structures of connective tissue: collagen, reticular fibers, cartilage, bone, amyloid. This method involves sequential treatment with fuchsin, phosphomolybdic acid and a mixture of aniline blue, orange and oxalic acid.

Thus, the proposed method for assessing the severity of ChE in women with uterine factor of infertility and unsuccessful IVF attempts in history, based on the determination of pathognomonic markers that determine the pathogenetic features of endometrial disease, should be considered as an additional option for assessing the severity of ChE in this group of patients.

The technical result, achieved with the help of a group of inventions, is an accurate determination of the severity of ChE in women with uterine factor of infertility and unsuccessful IVF attempts in history.

The specified technical result is achieved due to the following set of essential features:

Option for performing method 1

to determine the severity of chronic endometritis (CE) in women with uterine factor infertility after a history of unsuccessful in vitro fertilization (IVF), a pipel biopsy of the endometrium is performed in the middle stage of the proliferation phase,

in the obtained material, the levels of expression of CD138 and CD56 are determined,

if the number of CD138 + cells is zero, then "0 points" are assigned; if the number of CD138 + cells is 1, then "1 point"; if 2-3 - "2 points"; if 4 or more - "3 points";

if the expression of CD56 + is up to 10 cells, then assign "0 points"; if the expression of CD56 + exceeds the normal number of cells by 2 times - "1 point"; if it exceeds 3 times - "2 points"; if it exceeds 4 times or more - "3 points";

determine the sum of the scores of the levels of expression of CD138 and CD56,

with the sum of "5-6 points", the presence of a pronounced degree of chronic endometritis is determined; with "3-4 points" - moderately expressed; with "2 points" - poorly expressed.

Option for performing method 2

to determine the severity of chronic endometritis (CE) in women with uterine factor infertility after a history of unsuccessful in vitro fertilization (IVF), a pipel biopsy of the endometrium is performed in the middle stage of the proliferation phase, with immunohistochemical determination of the CD138 level equal to zero, but in the presence of a different degree the severity of fibrosis in the stroma,

in the obtained material, the level of expression of CD56 is determined and the severity of endometrial fibrosis is determined, for which a histochemical study is carried out by means of Mallory staining,

if the number of CD56 + is up to 10 cells, then "0 points" are assigned; if the expression of CD56 + exceeds the normal number of cells by 2 times - "1 point"; if it exceeds 3 times - "2 points"; if it exceeds 4 times or more - "3 points";

if the severity of endometrial fibrosis is 0%, then "0 points" are assigned; 1-15% - "1 point"; 16-49% - "2 points"; 50% or more - "3 points";

determine the sum of the received points of the level of expression of CD138 equal to zero, CD56 + (0-3 points) and the severity of endometrial fibrosis,

with the sum of "5-6 points", the presence of a pronounced degree of chronic endometritis is determined; with "3-4 points" - moderately expressed; with "2 points" - poorly expressed.

The developed methods for assessing the severity of ChE are that in the middle stage of the proliferation phase (on days 7-11 of the menstrual cycle), a pipe biopsy of the endometrium is performed. The obtained histological sections are stained with hematoxylin and eosin, after which the material is examined by IHC using sets of monoclonal antibodies to the following markers: positively stained plasma cells CD138; natural killer cells CD56 in the endometrium.

In the case of a negative IHC reaction with the CD138 marker, but with a detected violation of local immunity based on an abnormal number of CD56 cells, it is necessary to carry out histochemical staining according to Mallory in order to identify the severity of endometrial fibrosis, which may indicate the duration of the chronic inflammatory process in it. The interpretation of the obtained data is based on comparison with reference values.

This study included 74 patients aged 24 to 40 years (mean age was 32 ± 4.1 years).

The main group (n = 53) consisted of patients with uterine infertility (ICD10 code N 97.2), with de facto one or more IVF failures in history and suspected ChE. The average duration of infertility was 2.9 ± 0.9 years.

The comparison group (n = 21) consisted of fertile women before planned laparoscopic myomectomy, with a history of 1 or more deliveries to full-term healthy children, who gave voluntary informed consent to participate in the study.

Histological sections obtained by pipel biopsy in the middle stage of the proliferation phase of the menstrual cycle (on days 7-11) were examined under a DLMB light binocular microscope with a digital camera and a Leica image analyzer (Germany). Morphometric analysis was performed using an ocular measuring test grid by G.G. Avtandilov [12, 13] and using the Leica Quin digital image analyzer and the Sima Scan morphometric program.

The results obtained during the morphometric study were processed by standard statistical methods and using the Statistica 10 for Windows software (Stat Soft, USA). Statistical processing of the results was carried out with an assessment of the type of distribution, determination of the mean M, standard error ± se (on the diagrams ± confidence interval). The significance of differences between independent samples with normal distribution was determined by Student's t-test. The identification of correlations was carried out according to the Spearman coefficient (Rs). The critical level of significance was taken equal to 0.05.

IHC study of endometrial biopsies, taken by the pipel biopsy method in the middle stage of the proliferation phase of the menstrual cycle (on days 7-11), was carried out using a two-stage streptavidin-biotin-peroxidase method with antigen unmasking and using standard sets of polyclonal antibodies from Ventana, Cell Marque and Diagnostic BioSystems USA. IHC staining was carried out in a Ventana BenchMark ULTRA IHC / ISH immunostainer (USA) on paraffin sections according to a standard technique with preliminary unmasking of antigens in a microwave oven using appropriate standard antibodies, markers were detected (Table 1).

Table 1.Scroll monoclonal antibodies used for IHC studies

Antibodies Characteristic CD138 monoclonal Plasma cell marker CD56 monoclonal Natural killer marker

We analyzed the expression of markers CD138, CD56 and the severity of endometrial fibrosis, the degree of prevalence of fibrosis in the endometrium in women with a uterine infertility factor and IVF failures in history, and in patients with preserved reproductive function before planned laparoscopic myomectomy, the latter made up the comparison group (Table 2) ...

Table 2. The level of expression of CD138, CD58, as well as the prevalence of fibrosis in the endometrium in patients with uterine factor infertility and IVF failures in history (main group), and fertile women before planned laparoscopic myomectomy (comparison group)

Marker Main group, n = 53 Comparison group, n = 21 CD138 1.8 ± 1.26 cells / p.z. 0.1 ± 0.3 cells / p.z. CD56 26 ± 9 cells / p.z. 0 cl / p.z. The severity of endometrial fibrosis 26.5 ± 15.4% 0%

When IHC study of endometrial biopsies of women from the main group (infertility and IVF failures), obtained in the middle stage of the proliferation phase of the menstrual cycle (on days 7-11), CD138 + cells were found in 44 (83%) of 53 patients, CD56 + - in 51 (96.2%) of 53. At the same time, each of the 9 patients of the main group with the absence of CD138 + cells showed an increase in CD56 +, on average, 2.2 times, and also during the subsequent histochemical study (Mallory stain), stromal fibrosis was noted ... Among the biopsies of women from the comparison group, single CD138 + cells were observed only in 2 (9.5%) patients out of 21, while the expression of the natural killer cell marker CD56 did not exceed the reference values in any of them (Table 3).

Table 3. Results of IHC and histochemical studies in the middle stage of the endometrial proliferation phase in women with uterine factor infertility and IVF failures in history, and in fertile women before elective laparoscopic myomectomy

Markers Main group, n = 53 Comparison group, n = 21 Received values Number of patients Number of patients CD138, the number of cells per field of view 0 9 (17%) 19 (90.5%) one 15 (28.3%) 2 (9.5%) 2-3 23 (43.4%) 0 (0%) four 6 (11.3%) 0 (0%) CD56, the number of cells per field of view Up to 10 cells 2 (3.8%) 21 (100%) Excess by 2 times 26 (49%) 0 (0%) Excess by 3 times 17 (32.1%) 0 (0%) Excess by 4-5 times 8 (15.1%) 0 (0%) The severity of endometrial fibrosis, % 0 0 (0%) 21 (100%) 1-15 3 (33.3%) 0 (0%) 16-49 5 (55.6%) 0 (0%) ≥50 1 (11.1%) 0 (0%)

Thus, according to the results of morphometric studies in the middle stage of the proliferation phase of the menstrual cycle, CE was verified in patients of the main group by the combination of the presence of plasma cells, natural killer cells, and endometrial fibrosis.

At the same time, in the endometrial samples of women from the comparison group, the opposite picture was observed: single CD138 + cells were found in 2 patients, in the absence of IHC reaction to the CD56 marker and endometrial fibrosis; in the remaining women in this group, morphometric studies did not reveal any deviations.

In the endometrium in the middle stage of the proliferation phase in patients with uterine factor of infertility and IVF failures in history (main group), reliable IHC and histochemical determinants were established in comparison with biopsies of fertile women before elective laparoscopic myomectomy (comparison group).

During IHC study of biopsy specimens of eutopic endometrium in women with a history of IVF failures, pathognomonic limits of sensitivity were established for the marker of plasma cells (CD138), natural killer cells (CD56) and detection of endometrial fibrosis (Mallory stain) (Tables 4, 5).

Table 4 . Pathognomonic limits of sensitivity for markers CD138, CD56 and Mallory stain in the diagnosis of ChE

Marker Pronounced ChE Moderate ChE Weakly pronounced ChE Sensitivity limit CD138 + ≥4 - 3 points 2-3 cells -2 points Single cells - 1 point 0 cells CD56 + The number of cells is exceeded 4-5 times - 3 The number of cells is exceeded 3 times - 2 The number of cells is exceeded 2 times - 1 point ≤10 cells Mallory stain ≥50% - 3 points 16-49% - 2 points 1-15% - 1 point Lack of fibrosis

Implementation of the invention

Endometrial biopsy is performed in the middle stage of the proliferation phase (on days 7-11 of the menstrual cycle) in a spontaneous cycle, or in the cycle preceding IVF, using an aspiration curette. The material obtained after histological examination in an automatic histoprocessor Leica ASP 30 is embedded in paraffin at the Leica EG 1150 station (Germany). IHC staining is performed in a Ventana BenchMark ULTRA IHC / ISH immunostainer (USA) on dewaxed and dehydrated sections 4-6 μm thick using the avidin-biotin immunoperoxidase method. First, the levels of expression of CD138 and CD56 in the endometrium are assessed in accordance with the methods described above using the appropriate monoclonal antibodies. The percentage of positively stained cells is calculated. The interpretation of the IHC reaction for the described markers is carried out by comparing the obtained data with the following parameters:

• if the number of CD138 + cells is zero, then the probability of the presence of ChE is minimal - 0 points, if there are single positive-stained cells, then ChE is weakly expressed - 1 point, if 2-3 - moderately expressed - 2 points, if ≥4 - pronounced - 3 points;

• if up to 10 CD56 + cells are detected, then the probability of the presence of ChE is minimal - 0 points, if the number of cells is 2 times higher than the norm, then ChE is weakly expressed - 1 point, if 3 times - moderately expressed - 2 points, if in 4-5 times or more - expressed - 3 points.

In the event that positively stained CD138 cells are not detected, but there is a violation of local immunity based on an abnormal number of CD56 cells, a histochemical study (Mallory stain) is performed, interpreted as follows: connective tissue stains blue, fibrin from dark red to blue ("young and mature" fibrin in red, "old" - in blue).

The prevalence of endometrial fibrosis: no (0%) - the probability of the presence of ChE is minimal - 0 points, 1-15% - ChE is weakly expressed - 1 point, 16-49% - moderately pronounced - 2 points, ≥50% - pronounced - 3 points.

Thus, to determine the severity of ChE, the scores obtained are CD138 and CD56 in the endometrium, or CD56 + (with CD138 equal to zero, and the presence of fibrosis of varying severity in the endometrial stroma), Mallory staining and the following conclusions are made:

• 5-6 points - pronounced ChE;

• 3-4 points - moderately pronounced ChE;

• 2 points - weak ChE;

• 0-1 point - ChE is unlikely.

Clinical example 1

Patient A., 33 years old, has been suffering from secondary infertility for 4 years, a history of 2 IVF attempts without effect. Diagnosis: Secondary infertility. Complicated gynecological history (1 induced abortion with curettage of the uterine cavity, 2 attempts at IVF, 1 operating laparo / hysteroscopy, diagnostic hysteroscopy). In order to overcome infertility, IVF and cryopreservation of the resulting embryos were carried out. History of 2 unsuccessful attempts to transfer high quality embryos. According to hysteroscopy, after 2 unsuccessful attempts and targeted endometrial biopsy, there are no signs of ChE. Before the next attempt at embryo transfer, a control pipel biopsy of the endometrium was performed on the 8th day of the menstrual cycle.

On the basis of a comprehensive pathomorphological study, which included histological staining with hematoxylin and eosin, IHC and Mallory stain, the following data were obtained: CD138 + = 3 cells (2 points); CD56 + = 4 times exceeded (3 points).

The total of points was "5", which indicates a pronounced ChE.

Clinical example 2

Patient M., 39 years old, turned to a reproductive specialist in April 2019 with complaints about the absence of pregnancy for 4 years, burdened by an obstetric and gynecological history: in 2008 - spontaneous miscarriage at 3-4 weeks, in 2014 - spontaneous miscarriage in 8 weeks of pregnancy. Gynecological diseases: cervical ectopia, RV coagulation 2009 Chlamydia - treated in 2006 Operations - laparoscopic left-sided adnexectomy for tubovarial abscess, in 2006. History of 3 unsuccessful IVF attempts with high quality embryo transfer (4AB). The first transfer was performed in a stimulated cycle, the second - in a cryocycle. Diagnostic hysteroscopy on the 9th day of the menstrual cycle after 2 unsuccessful attempts at embryo transfer - without signs of ChE. In order to assess the implantation capabilities of the endometrium, before the next attempt at embryo transfer, a control pipe-biopsy of the endometrium was performed on the 7th day of the menstrual cycle.

Based on a comprehensive pathomorphological study, which included histological staining with hematoxylin and eosin, IHC and Mallory stain, the following data were obtained: CD138 + = 0 cells (0 points); CD56 + = exceeded 3 times (2 points). The material was additionally stained according to Mallory's technique in order to detect endometrial fibrosis. The prevalence of endometrial fibrosis was -36% (2 points).

The total of points was "4", which indicates a moderately pronounced ChE.

Bibliography

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Claims (2)

1. A method for determining the severity of chronic endometritis (CE) in women with uterine factor infertility after a history of unsuccessful in vitro fertilization (IVF), including performing pipel biopsy of the endometrium in the middle stage of the proliferation phase, characterized in that the levels of expression of CD138 are determined in the obtained material and CD56, if the number of CD138 + cells is zero, then "0 points" is assigned; if the number of CD138 + cells is 1, then "1 point"; if 2-3 - "2 points"; if 4 or more - "3 points"; if the expression of CD56 + is up to 10 cells, then assign "0 points"; if the expression of CD56 + exceeds the normal number of cells by 2 times - "1 point"; if it exceeds 3 times - "2 points"; if it exceeds 4 times or more - "3 points"; determine the sum of the received points of the levels of expression of CD138 and CD56, with the sum of "5-6 points" determine the presence of a pronounced degree of chronic endometritis; with "3-4 points" - moderately expressed; with "2 points" - poorly expressed. 2. A method for determining the severity of chronic endometritis (CE) in women with uterine factor infertility after a history of unsuccessful in vitro fertilization (IVF), including performing a pipel biopsy of the endometrium in the middle stage of the proliferation phase, characterized in that in the material obtained with a CD138 level equal to zero and the presence of fibrosis in the endometrial stroma, it is determined the level of expression of CD56 and determine the severity of endometrial fibrosis, for which a histochemical study is carried out by means of Mallory staining, if the number of CD56 + is up to 10 cells, then "0 points" is assigned; if the expression of CD56 + exceeds the normal number of cells by 2 times - "1 point"; if it exceeds 3 times - "2 points"; if it exceeds 4 times or more - "3 points"; if the severity of endometrial fibrosis is 0%, then "0 points" are assigned; 1-15% - "1 point"; 16-49% - "2 points"; 50% or more - "3 points"; determine the sum of the received points of the level of expression of CD56 and the severity of endometrial fibrosis, with the sum of "5-6 points" determine the presence of a pronounced degree of chronic endometritis; with "3-4 points" - moderately expressed; with "2 points" - poorly expressed.