RU2236013C1 - Method for diagnosing the cases of chronic endometritis and inflammation nature - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining local immunity indices in applying immunohistocytochemical approach that is, number of lymphocytes expressing natural killer cells markers CD56+, CD16+ and HLA-DR(II)+ activation markers.

EFFECT: improved diagnostic tests; observed immune system involvement into inflammation process.

Description

The invention relates to medicine, to the section of gynecology, pathological anatomy and immunology.

The problem of studying inflammatory diseases of the endometrium in women of reproductive age is relevant, as it causes not only a violation of menstrual function, but also generative. The frequency of occurrence of chronic endometritis in infertility ranges from 20 to 60% (Smetnik V.P., Tumilovich L.G. Non-operative gynecology. - 2nd edition. - St. Petersburg., 1995.- Book 2 - P. 46-52) , with habitual miscarriage from 60.5 to 86.7% (Demidova EM. Habitual miscarriage (Pathogenesis, obstetric tactics) abstract for the degree of Doctor of Medical Sciences. - St. Petersburg, 1993). The clinical manifestations of chronic endometritis are nonspecific, the disease is often diagnosed by chance during a screening examination of women.

For the diagnosis of chronic endometritis, a standard histological method is used that takes into account the totality of the general morphological signs of chronic inflammation: the presence of focal lymphocytic leukocyte infiltration; focal fibrosis of the stroma; the presence of plasma cells and sclerotic changes in the walls of the spiral arteries (Khmelnitsky OK Pathomorphological diagnosis of gynecological diseases. - St. Petersburg: Sotis., 1994. P.136-144; Kuznetsova A.V. Chronic endometritis. Archive of Pathology, 2000, No. 3, P. 48 -52). The main disadvantage of the existing method is the low diagnostic accuracy. The presence of only one or two of the above criteria for inflammation always raises doubts among morphologists in the diagnosis of chronic endometritis. Other methods for verifying the diagnosis do not exist today (Savelyeva G.M. et al. Hysteroscopy / Savelyeva G.M., Brusenko V.G., Kaplusheva L.M. - M .: GZOTAR Medicine, 1999).

Literature data on changes in the parameters of local immunity in chronic inflammatory processes of the endometrium (endometritis) are scarce (Kuznetsova A.V. et al. Morphological features of chronic endometritis. Pathology Archive, 2001, No. 5, P.8-13; Ellinidi V.N. ., Kalinina NM [Endometrial immunomorphology. Treatment of infertility: unsolved problems. Saratov, 2001, S. 158-162).

The purpose of the invention is the development of criteria for the diagnosis of chronic endometritis.

The solution of this goal is achieved by identifying inflammation in the endometrial tissue based on a comprehensive clinical and morphological examination of women, determining the indices of local immunity characteristic of inflammation by the immunohistocytochemical method and clarifying the nature of the inflammatory process when comparing indices of local and systemic immunity.

According to the proposed method, the markers of immunocompetent endometrial cells are determined by the immunohistochemical method, which can reliably identify the presence of chronic inflammation in the endometrium and its severity; for this purpose, lymphocytes expressing markers of natural killer cells CD56 + and CD16 + and lymphocytes expressing an activation marker of class HLA-DR + II involved in antigen recognition are examined in the endometrium.

The rationale for the proposed diagnostic method is the fact that inflammation often develops only locally, causing organ dysfunction, and is always accompanied by changes in the parameters of systemic and local immunity.

The proposed method for determining the expression on the membranes of immunocompetent cells of specific antigens, that is, the surface phenotype, was chosen due to the fact that it is a method for identifying cells. The advantage of the proposed method is the determination of cells involved in inflammation. The immunohisto-cytochemical method is a modern, highly sensitive method for identifying and determining the localization in the cell and tissue of various structures with antigenic properties, based on the antigen-antibody reaction. The method is based on highly avid and highly specific monoclonal antibodies directed to the same epitope of antigen. In this regard, the method allows the identification of various immunocompetent cells (T, B lymphocytes, natural killer cells) involved in inflammation. In order to visualize the antigen-antibody reaction, an avidin-biotin-peroxidase complex is used, and the manifestation of horseradish peroxidase is carried out by diaminobenzidine. As a result, the reaction product is detected in the form of a brown persistent staining of the membrane. The immunohistochemical method is described in detail in the guidelines (Ellinidi V.N., Anikeeva N.V., Maksimova N.A .: Practical immunohistochemistry (guidelines), St. Petersburg, 2002, S.1-36).

According to the proposed method, along with the use of the immunohistocytochemical method, immunocompetent cells expressing activation markers and natural killer markers are quantified, which makes it possible to assess the severity of the inflammatory process, and to evaluate the effectiveness of the therapy by the dynamics of changes in the studied parameters.

The proposed diagnostic method allows, by comparing local and systemic immunity, to clarify the nature of inflammation, to distinguish patients with an autoimmune nature of inflammatory processes.

The proposed method for the diagnosis of chronic endometritis and the nature of inflammatory diseases is as follows.

Lymphocytes expressing the markers CD56 +, CD16 + and HLA-DR (II) + are determined in the endometrium. The study is performed using Novocastra antigen-specific monoclonal antibodies to these markers on paraffin sections of endometrial tissue using the immunohistocytochemical method, which includes the following steps.

Deparaffinization of the sections is carried out, followed by unmasking of the tissue antigen, by boiling in citrate buffer (pH 6.0) under pressure in a pressure cooker for 2 minutes. Sections were incubated with normal serum for 30 minutes. Sections were incubated with the first antigen-specific monoclonal antibodies CD56 +, CD16 + and HLA-DR (II) + from Novocastra for one hour. Sections were incubated with second biotinylated antibodies for 30 minutes. An incubation with avidin-biotin-peroxidase complex is carried out for 30 minutes. The achievement of the staining of the reaction substrate (horseradish peroxidase) for visualization is carried out using diaminobenzidine 3 minutes. The reaction product appears as a persistent membrane staining in brown. The results of the study of the parameters of local immunity (CD56 +, CD16 + and HLA-DR (II) +) are measured quantitatively by scanning the endometrial tissues (biopsy, scrapings, aspirates) stained with an immunohistochemical method with a light microscope with an increase in lens × 40, eyepiece × 10 in 3 fields view.

Count the number of positively stained cells (expressing the studied markers) in each field of view. In the case of a slight scatter of quantitative indicators in different fields of view, the arithmetic mean is determined.

The number of lymphocytes expressing markers CD56 +, CD16 + and HLA-DR (II) + is determined in the following way.

The number of lymphocytes expressing CD56 + markers is equal to the sum of the number of lymphocytes expressing CD56 + markers in the first, second and third fields of view, divided by 3.

The number of lymphocytes expressing CD16 + markers is equal to the sum of the number of lymphocytes expressing CD16 + markers in the first, second and third fields of view, divided by 3.

The number of lymphocytes expressing HLA-DR (II) + markers is equal to the sum of the number of lymphocytes expressing HLA-DR (II) + markers in the first, second and third fields of view, divided by 3.

Example. The number of CD56 + lymphocytes in the first field of view is 21, in the second field of view is 23, in the third field of view is 25.

The calculation is carried out as follows: (21 + 23 + 25): 3 = 23.

By increasing the quantitative indicators of the studied markers, the severity of the inflammatory process is determined.

In order to analyze the results, the revealed changes in the parameters of local immunity of natural killer cells (CD56 +, CD16 +) and lymphocytes expressing an activation marker (HLA-DR (II) + in the endometrial tissue are compared with systemic immunity in the examined women, for which the subpopulation composition is examined peripheral blood lymphocytes (flow cytometry method), cytokine production and serum immunoglobulins of classes A, M, G, E, secretory immunoglobulin A (sIgA) (enzyme-linked immunosorbent assay).

According to the proposed method, when the number of cells expressing CD56 +, CD16 +, HLA-DR (II) + from 0 to 10 in the field of view, the absence of endometritis is diagnosed. Autoimmune chronic endometritis is diagnosed with the number of cells expressing CD56 + above 10, and with the number of cells expressing CD16 + and HLA-DR (II) + from 0 to 10 in the field of view. With the number of cells expressing CD56 +, CD16 +, HLA-DR (II) + above 10 in the field of view, an exacerbation of autoimmune chronic endometritis is diagnosed. When the number of cells expressing CD16 + and HLA-DR (II) + is above 10, and when the number of cells expressing CD56 + from 0 to 10 in the field of view, chronic endometritis with exacerbation or acute endometritis is diagnosed. With the number of cells expressing CD16 +, CD56 + above 10, and with the number of cells expressing HLA-DR (II) + from 0 to 10 in the field of view, chronic inflammation with an autoimmune component, but without activation of the process, is diagnosed. With the number of cells expressing CD16 + above 10, and with the number of cells expressing CD56 + and HLA-DR (II) + from 0 to 10, chronic endometritis is diagnosed in the field of view.

The method is illustrated by the following examples.

1. No. of medical history 4620.01 of the outpatient department of the Institute of Obstetrics and Gynecology. D.O. Otta RAMS, G.L.N., 28 years old. Clinical diagnosis: Primary infertility. The data of history, objective examination, gynecological examination did not reveal signs of chronic endometritis. There is no data for the transmitted urogenital infection. The patient was examined on the 19th day of the menstrual cycle. Histological examination N2277.01: endometrium of the middle stage of the proliferation phase, focal lymphoid infiltration of the stroma. Immunohistochemical study: the number of cells expressing CD56 + - 5 in the field of view, CD16 + - 10 in the field of view, HLA-DR (II) + - 0 in the field of view, which indicates the absence of inflammation. After specific hormonal therapy, pregnancy occurred, ending 05.11.2002 with the birth of a full-term baby.

2. No. of case history 3170.01 outpatient department of the Institute of Obstetrics and Gynecology. D.O. Otta RAMS, V.Yu.V., 28 years old. Clinical diagnosis: Primary infertility. The data of anamnesis, objective and gynecological examination did not reveal signs of chronic endometritis. There is no data for the transmitted urogenital infection. Examined on the 24th day of the menstrual cycle. Histological examination N1887.01: endometrium of the middle stage of the secretion phase - late proliferation, lag in the development of the stroma, iron-in-iron structures, single mononuclear cells in the stroma, lymphoid stromal infiltration. Immunohistochemical study: the number of cells expressing CD56 + - 5 in the field of view, CD16 + 0 -10 in the field of view, HLA-DR (II) + 0 in the field of view, which indicates the absence of inflammation. After carrying out specific hormone therapy, pregnancy occurred; on January 15, 2003, it progressed for 25 weeks.

3. No. of case history 3440.02 - outpatient department of the Institute of Obstetrics and Gynecology. D.O. Otta RAMS, I.Sh.N., 27 years old. Clinical diagnosis: Habitual miscarriage. Before the examination, she received complex therapy for urogenital infection: mycoplasmas and ureaplasmas were isolated from the cervical canal, a diagnostic titer of immunoglobulin G to chlamydia 1/64 was determined in the blood. Examined on the 12th day of the menstrual cycle: Histological examination N: 1515.02: endometrium of the middle stage of the proliferation phase, stroma with a tendency to fibrosis, pronounced diffuse-focal mononuclear infiltration. Immunohistochemical study: the number of cells expressing CD56 + - 50 in the field of view, CD16 + - 60 in the field of view, HLA-DR (II) + - 5 in the field of view, which indicated a chronic inflammatory process in the endometrium without signs of activation, but with an autoimmune component. After the complex therapy, pregnancy occurred, on January 15, 2003, it progresses - 29 weeks.

4. No. of case history 4408.02 - outpatient department of the Institute of Obstetrics and Gynecology. D.O. Otta RAMS, P.M., 32 years old, a history of primary infertility of 7 years. Clinical diagnosis: Primary infertility. During bacteriological examination, data for infection were not received. Histological examination N2033.02: day 7 of the cycle: endometrium of the middle stage of the proliferation phase with moderate diffuse focal mononuclear infiltration and stromal fibrosis. Immunohistochemical study: the number of cells expressing CD56 + - 3 in the field of view, CD16 + - 17 in the field of view, HLA-DR (II) + - 30 in the field of view, which indicated chronic endometritis without an autoimmune component, but with signs of exacerbation (active inflammation) .

After the complex therapy, pregnancy has begun, at the moment it is progressing - 26 weeks.

5. No. of case history 58.01 - out-patient department of the Institute of Obstetrics and Gynecology. D.O. Otta RAMS, T.E.V., 24 years old. Clinical diagnosis: Secondary infertility. A history of spontaneous early miscarriage. During bacteriological examination, data for the presence of genital infection was not obtained. Histological examination N1316.01: day 22 of the cycle: endometrium of the early stage of the secretion phase, tangles of spiral arteries are well defined, weak diffuse mononuclear infiltration of the stroma, tendency to fibrosis under the surface epithelium. Immunohistochemical study: the number of cells expressing CD56 + - 45 in the field of view, CD16 + 0 - 45 in the field of view, HLA-DR (II) + - 45 in the field of view, which indicated autoimmune chronic endometritis in the acute phase. After complex therapy, pregnancy occurred, which ended in the birth of a full-term baby on 05.02.2002.

After conducting complex therapy at the moment, 19 women have different periods of progressive pregnancy, the pregnancy ended in urgent delivery in 16 women.

Based on the proposed method for the diagnosis of chronic endometritis, a survey of 156 women of reproductive age was carried out in clinics of the Research Institute of Obstetrics and Gynecology named after D.O. Otta RAMS (of which 5 healthy women are a control group).

Before performing invasive examinations (laparoscopy, hysteroscopy, hysterosalpingography, endometrial biopsy), all women were clinically examined. Examination was carried out for the exclusion of syphilis, HIV infection, hepatitis B and C. Genital infection was excluded during bacteriological examination of the vaginal and cervical canal secretions, including mycoplasma, chlamydia, culture diagnostics, herpes I and II types were detected by immunofluorescence, serological examination was performed blood for the presence of anti-Chlamydia antibodies of classes G and A, antibodies of class G to the causative agents of herpetic infection, and if available, the avidity index was determined. A total of 44.5% of women were infected, and 2 or more pathogens were detected in 66% of those infected. Prior to the immunological examination, all infected women were treated for detected genital tract infections.

The parameters of local immunity - the number of natural killer cells CD56 + and CD 16+, lymphocytes expressing an activation marker (HLA-DR (II) +) were studied in the endometrium of all 156 women.

Using the usual method of examination (histological method), the diagnosis of chronic endometritis was established only in 18 out of 151 women (11.9%). The use of immunohistochemical studies made it possible to establish a diagnosis of chronic endometritis in 81 out of 151 women (53.6%), which increased the accuracy of clinical diagnosis and contributed to the development of the correct treatment tactics for women.

After complex therapy, at present, 28 women have different periods of progressive pregnancy; pregnancy ended in urgent delivery in 27 women.

Thus, the proposed method allowed to develop criteria for assessing indicators of local immunity of the endometrium by the immunohistochemical method on paraffin sections. The proposed method for diagnosing chronic endometritis and the nature of inflammatory changes in the endometrium in women by the immunohistocytochemical method is different in that it allows you to accurately establish the involvement of the immune system in the inflammatory process in the studied tissue (endometrium). A comparison of indices of local immunity (lymphocytes expressing differentiating antigens to CD56 +, CD16 +, HLA-DR (II) +) and systemic immunity allowed confirming the nature of the autoimmune inflammatory process in the endometrium.

List of references

1. Demidova E.M. Habitual miscarriage (Pathogenesis, obstetric tactics): Abstract for the degree of Doctor of Medical Sciences. SPb - 1993. - 42 pp.

2. Kuznetsova A.V. Chronic endometritis Archive of Pathology, 2000, N3, pp. 48-52.

3. Kuznetsova A.V., Spiders S.V., Voloshchuk I.N., Demidova E.M., Kazaryan S.M. Morphological features of chronic endometritis. Archive of Pathology, 2001, N5, pp. 8-13.

4. Savelyeva G.M. et al. Hysteroscopy / Savelyeva G.M., Breusenko V.G., Kaplusheva L.M.-M .: GZOTAR Medicine, 1999. - 176 p.

5. Smetnik V.P., Tumilovich L.G. Non-operative gynecology. - 2nd edition. - SPb., 1995. - Book 2. - S. 46-52.

6. Freidlin I.S., Totolyan A.A. Cells of the immune system. - St. Petersburg: Nauka, 2001 .-- 390 p. - (T.3; T.4; T.5).

7. Khmelnitsky O.K. Pathomorphological diagnosis of gynecological diseases. - St. Petersburg: Sotis., 1994. - S.136-144.

8. Ellinidi V.N., Anikeeva N.V., Maksimova N.A. Practical immunohistochemistry (guidelines), St. Petersburg, 2002, pp. 1-36.

9. Ellinidi V.N., Kalinina N.M. Immunomorphology of the endometrium. Infertility treatment: unsolved problems. Saratov, 2001 .-- pp. 158-162.

Claims (1)

A method for diagnosing chronic endometritis, including clinical and morphological examination, characterized in that the local immunity parameters are determined by the immunohistocytochemical method in the endometrial tissue: the number of lymphocytes expressing CD56 +, CD16 + natural killer cell markers and HLA-DR (II) + activation markers, while counting carried out in a light microscope with an increase in the lens x40, eyepiece x10 in three fields of view, and with the number of cells expressing CD56 +, CD16 +, HLA-DR (II) +, from 0 to 10 in the field of view diagnosed with no vie endometritis; with the number of cells expressing CD56 + above 10 and with the number of cells expressing CD16 + and HLA-DR (II) + from 0 to 10, autoimmune chronic endometritis is diagnosed in the field of view; with the number of cells expressing CD56 +, CD16 +, HLA-DR (II) +, above 10 in the field of view, an exacerbation of autoimmune chronic endometritis is diagnosed; with the number of cells expressing CD16 + and HLA-DR (II) + above 10 and with the number of cells expressing CD56 + from 0 to 10, chronic endometritis with exacerbation or acute endometritis is diagnosed in the field of view; with the number of cells expressing CD16 +, CD56 + above 10 and with the number of cells expressing HLA-DR (II) + from 0 to 10 in the field of view, chronic inflammation with an autoimmune component, but without activation of the process, is diagnosed; with the number of cells expressing CD16 + above 10 and with the number of cells expressing CD56 + and HLA-DR (II) + from 0 to 10, chronic endometritis is diagnosed in the field of view.