RU2761539C1 - Method for diagnosing external genital endometriosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to gynecology, and is intended for the diagnosis of external genital endometriosis. In the blood plasma, the number of microvesicles of leukocyte origin with the CD54+CD14+ phenotype is determined. With their content of 5.22% and above, external genital endometriosis is diagnosed.

EFFECT: invention provides an expansion of the arsenal of tools, an increase in the specificity and accuracy of early non-invasive diagnosis of external genital endometriosis.

1 cl, 4 ex

Description

The invention relates to medicine, namely to gynecology, and improves the accuracy and efficiency of non-invasive diagnosis of external genital endometriosis.

Endometriosis is a disease characterized by the presence of tissue similar to the endometrium outside the uterine cavity, is most often diagnosed at reproductive age and is a process with a chronic, recurrent and progressive course. Clinically, the disease manifests itself as chronic pelvic pain, dysmenorrhea and infertility, but in 20-25% of cases it may have an asymptomatic course [Bulun SE, Yilmaz BD, Sison C., Miyazaki K., Bernardi L., Liu S., Kohlmeier A., Yin P., Milad M., Wei J. Endometriosis. Endocr Rev. 2019, 40 (4): 1048-1079].

Given the varied symptoms of this disease, which can mimic various chronic inflammatory diseases of the pelvic organs, intestines, and bladder, the gold standard for definitive diagnosis consists of a surgical step followed by histopathological examination of the material obtained. This can explain the significant diagnostic delay of 8-12 years, leading to the delayed appointment of adequate treatment [Culley L., Law C., Hudson N., Denny E., Mitchell H., Baumgarten M., Raine-Fenning N. The social and psychological impact of endometriosis on women's lives: a critical narrative review. Hum Reprod Update. 2013, 19 (6): 625-639].

Until now, no reliable laboratory biomarkers of this gynecological pathology have been found. Therefore, new non-invasive biomarkers are constantly being developed for early diagnosis, adequate treatment and selection of patients for surgery [Rogers R.A., Adamson GD, Al-Jefout M., Becker S.M., d'Hooghe T.M., Dunselman GA , et al. Research priorities for endometriosis: recommendations from a global consortium of investigators in endometriosis. Reprod Sci. 2016; 24: 202-26].

The central role in endometriosis belongs to the local inflammatory process, which contributes to the onset and further development of the disease, causing pain and infertility [Riccio L., Santulli P., Marcellin L., Abrao M.S., Batteux F., Chapron C. Immunology of endometriosis. Best Pract Res Clin Obstet Gynaecol. 2018, 50: 39-49]. In support of this statement, based on a large number of studies, it has been shown that the peritoneal fluid of patients with endometriosis contains an increased number of macrophages and other immune cells that secrete growth factors, cytokines and angiogenic factors [Gazvani R., Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction. 2002, 123 (2): 217-226]. Therefore, proinflammatory cytokines are considered as potential non-invasive biomarkers in the blood or peritoneal fluid of patients with endometriosis.

It has been demonstrated that a group of markers, rather than a single indicator, have a higher sensitivity and specificity for the diagnosis of endometriosis. It was shown that the disease can be diagnosed using a combination of indicators (interleukin (IL) -6, IL-8, tumor necrosis factor (TNF) a, tumor antigen (CA) -125, CA-19-9 and highly sensitive C-reactive protein) with specificity 60-71% and a sensitivity of 87-92% [Mihalyi A., Gevaert O., Kyama CM, Simsa P., Pochet N., de Smet F. et al. Noninvasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers. Hum Reprod. 2010; 25: 654-64]. Using a multivariate statistical approach, we investigated the specificity and sensitivity of 28 biomarkers in plasma samples from women with and without endometriosis. A panel of biomarkers (vascular endothelial growth factor (VEGF), annexation V, CA-125, glycodelin and soluble intracellular adhesion molecule 1 (ICAM-1)) was determined, which showed a specificity of 63-81% and a sensitivity of 81-90% for the ability to diagnose endometriosis in women [Vodolazkaia A., El-Aalamat Y., Popovic D., Mihalyi A., Bossuyt X., Kyama C. et al. Evaluation of a panel of 28 biomarkers for the noninvasive diagnosis of endometriosis. Hum Reprod. 2012; 27: 2698-711]. However, the authors found increased levels of other proinflammatory markers, including TNFa, IL-6 and IL-1β, in the control group of patients, rather than in the group of women with endometriosis, noting that the composition of peripheral blood cytokines can be influenced by various inflammatory processes in the small pelvis.

Thus, proinflammatory cytokines cannot be considered as highly specific and highly sensitive markers for non-invasive diagnosis of endometriosis, since their level can be increased in acute and chronic inflammatory processes in the small pelvis, accompanied by pain or asymptomatic.

A known method for the diagnosis of external genital endometriosis in women with infertility [patent RU No. 2291439, G01N 33/577, priority date 07/04/2005, publication date 01/10/2007] by examining the peripheral venous blood and determining the relative amount of CD16 + monocytes in the monocyte gate and at values this indicator, equal to or less than 45%, is diagnosed with external genital endometriosis.

This method is nonspecific, since the number of CD16 + monocytes varies significantly in venous blood for various diseases, pathological and extreme conditions (viral infections, autoimmune diseases, gastroenterological diseases, inflammatory processes, stress), and is also not sensitive enough, since the measurement of the diagnostic indicator is carried out in relative quantities.

Known diagnostic test, which takes into account the absolute number of macrophages in the peritoneal fluid - 13⋅10 ⁶ , allowing to diagnose the presence of endometriosis [Oosterlynk DJ, Lacquaet FA, Waer M. et al. Lymphokine - activated killer activity in women with endometriosis. Gyn. Obstet. Invest. 1994; 37 (3): 185-190].

The disadvantage of this method is its invasiveness, since for the diagnosis of endometriosis by this method, it is necessary to obtain peritoneal fluid during the laparoscopic operation.

A known method for the diagnosis of external genital endometriosis (EGE) [patent RU No. 2211454, G01N 33/53, priority date 29.10.2001, publication date 27.08.2003] in women, in which to establish the diagnosis of endometriosis in the serum of patients determine the concentration of interferon-γ and when its content is equal to 1000 pkg / ml or less, the disease is diagnosed.

The disadvantages of this method are: non-specificity, since the content of interferon-γ in the blood varies depending on many endo- and exogenous factors: stress, the presence of bronchial asthma, allergies, autoimmune diseases, viral infections, immunodeficiency conditions [Ershov F.I. The interferon system is normal and pathological. - M .: Medicine, 1996. - 240 s]. The above-mentioned concomitant diseases can be observed in many women, combined with endometriosis, which leads to an insufficiently high predictive accuracy (77%) of this marker in the diagnosis of the disease.

The above methods for diagnosing IGE are taken as a prototype of the present invention.

The technical result of the invention is to expand the arsenal of tools, increase the specificity and accuracy of early non-invasive diagnosis of EGE.

The specified technical result is achieved in a method for the diagnosis of endometriosis, including the study of blood plasma, to determine the number of microvesicles of leukocyte origin with the phenotype CD54 + CD14 + and when their content is 5.22% and higher, EGE is diagnosed.

Microvesicles (MB) - extracellular structures that are produced by almost all types of cells during their life, intercellular interactions, as well as during apoptosis [Burger D., Schock S., Thompson C.S., Montezano A.C., Hakim A.M., Touyz R.M. Microparticles: biomarkers and beyond. Clinical science 2013,124 (7): 423-441]. It was shown that cell activation leads to an increase in the rate and intensity of MB production. At the moment they are considered as the most important regulators of intercellular interactions and participants in physiological and pathophysiological processes [Antonyak MA, Cerione R.A. Microvesicles as mediators of intercellular communication in cancer. Methods in molecular biology. 2014, 1165: 147-173]. It has been determined that MB can act as diagnostic markers of various diseases. Interacting with recipient cells, MBs are able to transmit "biological information" to them, in the form of proteins, nucleic acids, signaling molecules, membrane-associated receptors, bioactive lipids, and thereby change their phenotypic and functional characteristics [Mause SF, Weber C . Microparticles: protagonists of a novel communication network for intercellular information exchange. Circulation research. 2010, 107 (9): 1047-1057].

Currently, there are a limited number of studies to study the composition of microvesicles in the peripheral blood (PC) of patients with endometriosis. In one of the studies, the quantitative composition of MB in PC was studied in patients with deep infiltrative endometriosis (HIE) and in patients with endometriomas, comparing them with patients without this disease. Higher levels of circulating MB were found in patients with DIE in comparison with patients with and without endometriomas, which can be explained by a more intense degree of inflammation and angiogenesis in this aggressive form of endometriosis. It remains unknown whether these MB levels are cause or effect in patients with DIE. However, these results indicated that circulating MB levels may play a role in the pathophysiological mechanisms of DIE [Munros J., Martinez-Zamora MA, Tassies D., Coloma JL, Torrente MA, Reverter JC, Carmona F., Balasch J. Total circulating microparticle levels are increased in patients with deep infiltrating endometriosis. Hum Reprod. 2017, 32 (2): 325-331].

The same group of authors published the results of a pilot study in 2019 in patients with endometriomas. It has been shown that the number of MB PC increases after removal of endometriomas by excision, but not when using laser ablation. The increase in circulating MB levels is temporary and returns to basal levels 3 months after the removal procedure. These results show that compared with laser evaporation, the excision method induces a more pronounced short-term inflammatory response [Munros J., Martinez-Zamora MA, Tassies D., Reverter JC, Rius M., Gracia M., Ros C. , Carmona F. Total Circulating Microparticle Levels After Laparoscopic Surgical Treatment for Endometrioma: A Pilot, Prospective, Randomized Study Comparing Stripping with CO2 Laser Vaporization. J Minim Invasive Gynecol. 2019, 26 (3): 450-455].

The results of these studies do not allow us to draw conclusions about changes in the MB content in the PC of patients with EGE.

To verify the proposed method, a study was carried out to determine the number of microvesicles of leukocyte origin with the CD54 + CD14 + phenotype among:

- 97 patients with external genital endometriosis of I / II degree of prevalence. The prevalence of the process was determined in points according to the revised classification of the American Fertility Society in accordance with the results of a laparoscopic examination. The diagnosis in all patients was established on the basis of laparoscopic surgery and histologically confirmed.

- 20 patients of the control group, examined in connection with male factor infertility before IVF, in whom, based on the intraoperative examination, no gynecological diseases were found.

Patients with grade I and II EGE are characterized by an increase in the relative content of MB with CD54 + CD14 + receptors in PC by 1.55 times compared with these indicators in patients from the control group: 5.7 (2.0; 9.2) and 3 , 71 (1.48; 5.1), respectively (p <0.01).

Monocytes are the source of CD54 + CD14 + microvesicles. They are large leukocytes of the mononuclear macrophage system. Activated macrophages are involved in the pathogenesis of endometriosis by synthesizing soluble mediators such as cytokines, prostaglandins, complement components, and enzymes. Through the secretion of these immune mediators, macrophages can cause inflammation, tissue repair, neovascularization, and also promote the recruitment of fibroblasts and endothelial cells [Riccio L., Santulli P., Marcellin L., Abrao M.S., Batteux F., Chapron C. Immunology of endometriosis. Best Pract Res Clin Obstet Gynaecol. 2018, 50: 39-49]. It has been shown that macrophages of the peritoneal fluid of women with endometriosis have an increased ability to secrete monocytic chemotactic protein-1 (MCP-1), which plays a role in attracting peripheral blood monocytes to sites of injury and inflammation.

To assess the quality (diagnostic efficiency) of the predictive model, the method of constructing a curve of the mutual dependence of the probabilities of false-positive and true-positive results (ROC-curve) was used. The area under the ROC-curve of the corresponding relationship between the prognosis of IGE and the content of microvesicles with the CD54 + CD14 + phenotype in blood plasma was 0.7 ± 0.056 with 95% CI: 0.59-0.81. The resulting model corresponds to a good predictive quality and was statistically significant (p <0.01). The threshold value of the content of microvesicles with the CD54 + CD14 + phenotype at the classification threshold point was 5.22%. The relative content of microvesicles with the CD54 + CD14 + phenotype in blood plasma equal to 5.22% or exceeding this value corresponded to the prediction of the presence of IGE. The sensitivity and specificity of the method were 80.5% and 71%, respectively.

Thus, the data of our study show that it is possible to diagnose degree I / II EGE using the determination of the content of microvesicles in the PC with the CD54 + CD14 + phenotype in patients with pain syndrome, as well as infertility with high sensitivity (80.5%) and acceptable specificity ( 71%).

The method is clinically effective and can be recommended for use in gynecological practice. Early non-invasive diagnosis of minimal or mild endometriosis (r-ASRM) will facilitate timely referral of patients for surgery, which in turn will prevent the disease from progressing to moderate to severe and improve fertility rates.

The method is carried out as follows.

To determine the number of microvesicles, blood samples are collected from the cubital vein on an empty stomach into a polypropylene tube containing 3.2% sodium citrate. To isolate microvesicles using the method described by the authors Gelderman M. Semak J (Gelderman M. P, Simak J. Flow cytometric analysis of cell membrane microparticles. Methods Mol Biol. 2008, 484: 79-93). All solutions for working with microvesicles are pre-filtered through an ultrafilter with a pore diameter of 0.2 μm (Corning, USA), and if their content is 5.22% and higher, IGE is diagnosed.

For long-term storage of microvesicles obtained from peripheral blood, the method of plasma cryopreservation is used. The samples are stored in a Dewar flask at a temperature of -196 ° C. Plasma samples stored in liquid nitrogen are thawed immediately. The procedure is carried out in a water bath at 37 ° C. Next, the resulting plasma is centrifuged for 20 minutes at 19800g + 10 ° C in order to sediment microvesicles. Next, the obtained MB are washed with cold Hanks solution without CaCl ₂ (Sigma, USA) containing sodium heparin at a concentration of 30 U / 1 ml of solution by centrifugation for 20 minutes at 19800g + 10 ° C. The resulting MB precipitate is resuspended in Hanks solution (Biolot, Russia) containing 0.35% serum albumin (Sigma, USA) and sodium heparin at a concentration of 30 U / 1 ml of solution, and treated with antibodies to CD14 and CD54 in accordance with the manufacturer's instructions ... The expression of these markers is assessed using a BD FACS Canto II flow cytometer (BD, USA). Gate MB is isolated by light scattering parameters using polystyrene beads calibrated in sizes 1.0 µm and 0.2 µm (Invitrogen, USA).

If the content of microvesicles with the CD54 + CD14 + phenotype is 5.22% and higher, IGE is diagnosed.

The essence of the method is illustrated by the following clinical examples:

Example 1. Patient M., 32 years old, complained of painful menstruation from the menarche period, periodic pulling pains in the lower abdomen not associated with the cycle for 3 years, as well as the absence of pregnancy during 2 years of regular sex life without contraception. Patient 1 has a history of pregnancy, which ended in artificial termination with curettage of the uterine cavity. Preliminary diagnosis: External genital endometriosis? The content of microvesicles with the CD54 + CD14 + phenotype in the patient's peripheral blood before the surgical stage was 7.55%. Completed: Operating laparoscopy. Electrocoagulation of foci of endometriosis, chromohydrotubation, hysteroscopy, endometrial biopsy. Postoperative diagnosis: external genital endometriosis of the II degree, which confirms the diagnosis established according to the declared method. The diagnosis was verified based on the results of the histological examination.

Example 2. Patient 3., 28 years old, applied for an appointment with complaints about the absence of pregnancy during 1 year of regular sex life without contraception, pain during sexual activity and painful menstruation that bother the patient for 2 years. It is known from the anamnesis that the patient underwent hysteroresectoscopy, polypectomy. According to the results of histology: glandular - fibrous polyp of the endometrium. The patient was recommended to undergo laparoscopy in connection with the presumptive diagnosis of external genital endometriosis. Before the surgery, the content of microvesicles with the CD54 + CD14 + phenotype was determined in the patient's peripheral blood, which was 11.2%. During the surgical stage, ablation of endometriosis foci, right ovariolysis, chromohydrotubation, hysteroscopy, endometrial biopsy were performed. The diagnosis of external genital endometriosis of the II degree was established, which was confirmed by the results of histological examination, and corresponded to the diagnosis established by the declared method.

Example 3. Patient V., 29 years old, complained about the absence of pregnancy within 3 years of regular sex life without contraception. From the anamnesis, menstruation is moderately painful, sometimes requiring the use of non-steroidal anti-inflammatory drugs, regular, no dyspareunia, no pain in the small pelvis. Spermogram of the spouse without pathology. The patient twice underwent artificial insemination with ovarian stimulation without effect. Presumptive diagnosis: external genital endometriosis? In order to clarify the cause of infertility, the patient was offered an operation. Before the surgery, the patient underwent a peripheral blood test to determine the number of microvesicles with the CD54 + CD14 + phenotype, which was 5.5%. Performed: surgical laparoscopy, ablation of foci of endometriosis, chromohydrotubation, hysteroscopy, endometrial biopsy. Postoperative diagnosis: grade I external genital endometriosis. The diagnosis was verified on the basis of a histological examination. Thus, the diagnosis according to the stated method was confirmed.

Example 4. Patient T., 27 years old, complained of pulling pains in the lower abdomen not associated with the menstrual cycle, which bother her for 3 years, periodically painful menstruation. Not interested in pregnancy at the moment. The patient used non-steroidal anti-inflammatory drugs, as well as low-dose hormonal contraceptives for a year, but the pain syndrome persisted during therapy. Presumptive diagnosis: external genital endometriosis? In order to identify the cause of the pain syndrome, as well as to eliminate it, it was proposed to conduct a laparoscopic operation. Before the surgery, the content of microvesicles with the CD54 + CD14 + phenotype was determined in the woman's peripheral blood, which was 2.6%. Performed: operating laparoscopy, bilateral salpingoovariolysis, chromohydrotubation, left salpingectomy. Postoperative diagnosis: Chronic adnexitis, hydrosalpinx on the left, adhesions of the pelvic organs of the 2nd degree. Thus, the diagnosis of external genital endometriosis was not confirmed, which corresponds to the declared method.

Claims (1)

A method for diagnosing external genital endometriosis, including the study of blood plasma, characterized in that the number of microvesicles of leukocyte origin with the CD54 + CD14 + phenotype is determined in the blood plasma, with their content of 5.22% and higher, external genital endometriosis is diagnosed.