RU2746644C1 - Method for assessing the implantation viability of endometrium in repeated implantation failures associated with chronic endometritis - Google Patents

Abstract

FIELD: medicine; pathomorphology; gynecology; reproductology.

SUBSTANCE: invention relates to the field of medicine, namely to pathomorphology, gynecology and reproductology, it can be used to detect violations of the implantation capabilities of the endometrium in chronic endometritis (CE) among patients with repeated implantation failures (RIF). A papel biopsy of the endometrium is performed during the “implantation window”. An immunohistochemical study of the obtained biomaterial is performed by staining the sections using polyclonal antibodies to the following markers: MUC1 in the endometrial glands, NOHA-10 in the stroma and in the glands. The percentage of colored cells is calculated. If the expression of MUC1 is less than 6%, then “1 point” is assigned, if it is 6% or more, then “0 points” is assigned. If the expression of NOHA-10 in the glandular compartment of the endometrium is less than 25%, then “1 point” is assigned, if it is 25% or more, then “0 points” is assigned. If the expression of NOHA-10 in the stromal compartment of the endometrium is 19% or more, then “1 point” is assigned, if it is less than 19%, then “0 points” is assigned. The received points are summarized. If the sum is “3 points”, a low implantation potential of the endometrium is determined, indicating a negative prognosis for blastocyst implantation. If the sum is “2 points”, a moderate implantation potential of the endometrium is determined, indicating a doubtful prognosis of blastocyst implantation. If the sum is “0-1 points”, a high implantation potential of the endometrium is determined, indicating a positive prognosis of blastocyst implantation.

EFFECT: method allows assessing accurately the implantation potential of the endometrium during the “implantation window” among patients with repeated implantation failures associated with chronic endometritis, based on the interpretation of the expression levels of specific immunohistochemical markers of endometrial receptivity.

1 cl, 3 tbl, 3 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 detect violations of the implantation capabilities of the endometrium in chronic endometritis (CE) in patients with repeated implantation failures (PNI) based on the assessment of markers of cell adhesion for the purpose of specific diagnostics endometrial infertility factor associated with ChE.

The prevalence of ChE ranges from 8% to 72% in women of reproductive age [1]. The relationship between ChE and infertility is undeniable. In fact, 2.8-56.8% of women with infertility [1, 2], 14-67.5% of women with repeated implantation failures [3-5] and 9.3-67.6% of women with habitual miscarriage of pregnancy histologically confirmed the diagnosis of ChE [6, 7]. It was convincingly shown that when using ART programs in patients with PNI, the frequency of implantation in in vitro fertilization (IVF) cycles was significantly lower with histologically confirmed ChE than in those women where there were no chronic inflammatory disorders of the endometrium with PNI (11.5% versus 32.7%) [4].

Summarizing the data of researchers on altered reception to sex steroid hormones, impaired pattern of paracrine factors and impaired adhesion, it becomes clear that ChE impairs decidualization and induces resistance to progesterone, while simultaneously affecting adhesion factors [8-12]. As a result, the potential for proliferation in cells increases with deterioration of differentiation, and in general, the synchronous peristaltic movement of the entire endometrial tissue and even myometrium in the transition zone is disturbed, which indicates its unpreparedness for implantation [14-16]. The existing methods for diagnosing endometrial receptivity are not ideal, since they do not have a set of specific markers of adhesion and proliferation, which dictates the need to search for the latter in order to determine the presence and predict the overcoming of the endometrial factor of infertility in chronic endometritis in women with repeated implantation failures.

State of the art

A known method for assessing the readiness of women with chronic endometritis for in vitro fertilization based on the assessment of the concentration of alpha-2-macroglobulin (a2-MG) by the method of enzyme-linked immunosorbent (RF patent 2617061, 2015.12.16). It was found that the level of α2-macroglobulin 4.5 mg / l and more was recorded in 80% of patients with ChE with a negative outcome of the program. In patients with ChE with a level of α2-macroglobulin less than 4.5 mg / l in 17-20%, a positive result of the IVF program was achieved, as well as in women from the comparison group with other forms of infertility (without the presence of an inflammatory component). The invention makes it possible to predict the onset of pregnancy in in vitro fertilization programs in women with chronic endometritis.

The disadvantage of this prediction method is the focus only on the indicator of the inflammatory response - a marker of exudation α2-macroglobulin, without taking into account the markers of receptivity, making it possible to predict the outcome only in patients with active ongoing inflammation. In addition, the method does not employ receptor markers specific for infertility associated with repeated implantation failures.

There is a known method for predicting endometrial receptivity based on the assessment of the expression of GnRG and GnRGR in the "implantation window" in the endometrium of women with infertility and unsuccessful attempts at the IVF program (RF patent 2617515, 22.04.2016). Lagging secretory transformation of the endometrium, a decrease in the number of cells containing mature pinopodia in the surface epithelium, less than 20%, as well as low expression (<2 points) of GnRG and GnRGR are considered as prognostically unfavorable indicators of endometrial receptivity.

The disadvantages of this method are the absence of systemic pathognomonic determination of specific molecules characterizing the receptivity of the endometrium, which underlie the regulation of implantation, as well as the absence of markers specific for infertility in chronic endometritis in patients with PNI. The established model for the purpose of predicting implantation disorders was based only on the analysis of the endometrial pattern in women with tubo-peritoneal factor infertility.

There is a known method for assessing the receptivity of the endometrium during the "implantation window" (RF patent 2651762, 23.04.2018). To achieve this, the researchers analyzed the endometrial pipe biopsy method on days 20-23 of the menstrual cycle based on immunohistochemistry using a two-stage streptavidin-biotin-peroxidase method with antigen unmasking and using key biomolecules responsible for implantation: a standard set of monoclonal antibodies and polyclonal ERα in the stroma, PR in the stroma, p53 in the glands, bcl 2 in the stroma, CD95 in the stroma, Ki67 in the stroma, LIFR in the glands, LIF in the endometrial glands. Based on the results of the study, the authors calculated the prognosis of endometrial receptivity according to the formula: d = -1.389x1 + 0.022x2 - 0.044x3 - 0.019x4 + 0.041x5 + 0.006x6 + 0.015x7 - 3.248, where x1 is the ER / PR index in the endometrial stroma ( H-score points for determining the nuclear expression of ER and PR), х2 - p53 in the endometrial glands (% of stained nuclei in the field of view), х3 - bcl 2 in the stroma of the endometrium (% of stained nuclei in the field of vision), х4 - CD95 + in the stroma of the endometrium (% of stained cells), х5 - Ki67 in the stroma of the endometrium (% of stained nuclei in the field of view), х6 - LIF-R in the endometrial glands (H-score points), х7 - LIF in the endometrial glands (H-score points). For d> 0, the endometrium was characterized as receptive, with d ≤ 0, the endometrium was characterized as non-receptive.

The disadvantages of this method were the lack of assessment of markers pathognomonic for infertility associated with chronic endometritis, reflecting adhesion disorders due to impaired decidualization and induction of progesterone resistance in the endometrium in patients with repeated implantation failures, leading to false results in the study of endometrial receptivity in this cohort of patients.

The technical problem to be solved was the need to improve the accuracy of assessing the implantation viability of the endometrium in inflammatory and adhesive disorders in the endometrium of patients with repeated implantation failures in CE associated with endometrial infertility during the "implantation window".

Disclosure of the essence of the invention

Due to the lack of highly specific and sensitive methods for assessing the implantation viability of the endometrium in infertility associated with chronic endometritis in patients with repeated implantation failures, the proposed method, based on the analysis of a complex of pathognomonic markers of the endometrial factor of failure, expands the possibilities of assessing receptivity in this cohort of patients, and also allows predicting the susceptibility of the endometrium to blastocyst implantation during the "implantation window".

According to our own study, a high specificity for infertility associated with chronic endometritis of the MUC1 glycoprotein, as well as a marker of receptive solvency HOXA10, which comprehensively reflects impaired adhesion and realization of progesterone resistance, determined by proliferative-inflammatory disorders in chronic endometrium and unsuccessful endometrium, was demonstrated. implantation.

The proposed method is based on an immunohistochemical study in the endometrium during the "window of implantation" of the expression levels of key molecules responsible for adhesion - MUC1 and receptivity - HOXA-10.

The glycoprotein MUC1 is a molecule that regulates adhesion in the endometrium. In healthy women, MUC1 expression is reduced to relatively low levels during the "implantation window" to allow for contact between the embryo and the endometrium. On the other hand, our study, as well as numerous studies of foreign colleagues, show that extremely low MUC1 expression during the "implantation window" is associated with repeated implantation failures compared to fertile women. A low MUC1 level may explain why patients experience repeated implantation failures despite the absence of factors predisposing to infertility. In addition, the dramatically reduced MUC1 expression disrupts the regulation of the local immune response during implantation, thereby adversely affecting the implantation process. MUC1 is expressed in T cells and acts as an immunomodulator, protecting blastocysts during implantation. Thus, an extreme decrease in MUC1 expression can reduce the protective effect in patients with chronic endometritis, leaving blastocysts unprotected from infections. Thus, MUC1 expression plays an important role in determining endometrial receptivity in patients with chronic endometritis and repeated implantation failures [18].

Defective expression of HOXA-10 in the endometrium during the midsecretory phase is associated with many inflammatory diseases leading to impaired receptivity and repeated implantation failures [17].

Thus, the proposed method for assessing the implantation viability of the endometrium in inflammatory disorders in chronic endometritis associated with repeated implantation failures, based on the determination of pathognomonic markers that determine the pathogenetic features of dysfunction of the endometrial receptor apparatus, should be considered as an addition to the standard diagnosis of endometrial receptivity in this group of patients.

The achieved technical result is an accurate determination of the susceptibility of the endometrium to blastocyst implantation during the "implantation window" based on the assessment of its proliferative-inflammatory pattern in patients with repeated implantation failures due to chronic endometritis.

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

- perform pipel biopsy of the endometrium during the "window of implantation",

- carry out an immunohistochemical study of the obtained biomaterial by staining the sections using polyclonal antibodies to the following markers: MUC1 in the endometrial glands, HOXA-10 in the stroma and in the glands,

- calculate the percentage of stained cells (i.e. the level of expression),

- if the expression of MUC1 is less than 6%, then "1 point" is assigned, if 6% or more, then "0 points",

- if the expression of HOXA-10 in the glandular compartment of the endometrium is less than 25%, assign "1 point", if 25% or more, assign "0 points",

- if the expression of HOXA-10 in the stromal compartment of the endometrium is 19% or more, assign "1 point", if less than 19%, assign "0 points",

- summarize the received points,

- with the sum: "3 points", the low implantation potential of the endometrium is determined, indicating a negative prognosis of blastocyst implantation; "2 points", determine the moderate implantation potential of the endometrium, indicating a dubious prognosis of blastocyst implantation; "0-1 point", characterizes the high implantation potential of the endometrium, indicating a positive prognosis of blastocyst implantation.

A method has been developed for assessing the receptivity of the endometrium during the "implantation window", that is, on the 7th day after the peak of luteinizing hormone determined in blood serum during a dynamic study in the perio-ovulatory period (as a rule, this period corresponds to 20-22 days of the menstrual cycle), a pipe biopsy is performed endometrium. The resulting material is examined by immunohistochemistry (IHC) using sets of polyclonal antibodies to the following markers: MUC1 in the endometrial glands, HOXA-10 in the stroma and glands.

The percentage of stained cells is calculated, after which the response is assessed: no response - ≤1%, low expression - 2-10%, moderate expression - 11-50%, pronounced expression - ≥51%. The interpretation of the IHC reaction is carried out by comparing the obtained data with the norms of values available from healthy fertile women without chronic endometritis.

The present study included 67 women aged from 29 to 40 years (average age was 35 ± 3.8 years) with a morphologically verified diagnosis of chronic endometritis (ICD10 code - N71.1), as well as infertility (ICD10 code N 97.7 , established in the heading other forms of female infertility) and repeated implantation failures.

The main group (n = 33) consisted of patients with chronic endometritis and infertility caused by the endometrial factor (mean duration of infertility 4.5 ± 0.8), with a history of 3 or more (2 or more in patients over 35) unsuccessful attempts to transfer 1 embryo of good quality into the uterine cavity during IVF programs (CE PNI group - patients with repeated implantation failures due to chronic endometritis).

The comparison group (n = 34) consisted of patients with chronic endometritis, who realized their reproductive function and had no history of infertility, who applied for the treatment of menstrual irregularities associated with chronic endometritis (HEF group - fertile patients with chronic endometritis).

The morphological control group (n = 15) consisted of fertile women without chronic endometritis, with a history of 2 or more deliveries of full-term healthy children, who gave voluntary informed consent to participate in the study.

Immunohistochemical study of endometrial biopsies taken by the pipel biopsy method during the period of the supposed "implantation window" - on the 7th day after the peak of luteinizing hormone determined in blood serum during a dynamic study in the periovulatory period (as a rule, this period corresponds to 20-22 days of the menstrual cycle) - 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. Immunohistochemical staining was performed in a Ventana BenchMark ULTRA IHC / ISH immunostainer (USA), markers were detected on paraffin sections according to the standard using the appropriate standard antibodies (Table 1).

An analysis of the endometrial receptor apparatus was carried out in women with repeated implantation failures associated with chronic endometritis, fertile women with chronic endometritis and fertile women without chronic endometritis, the latter constituted the morphological control group (Table 2).

In the endometrium during the "window of implantation" in patients with repeated implantation failures associated with chronic endometritis (ChE PNI group) in comparison with biopsies of women with chronic endometritis and preserved fertile function (HEF group), reliable immunohistochemical proliferative determinants were established. Against the background of the desynchronous transformation of the epithelial-mesenchymal compartment, a statistically significant decrease in the expression of MUC1 in the glands was determined in the ChE PNI group compared with the HEF group (3.4 times; 5.5% versus 18.7%, respectively). In addition, against the background of resistance to progesterone in the endometrium of women in the study groups, a violation of the receptive consistency of this pattern is determined, due to a statistically significant decrease in the expression of HOXA10 in the stromal and glandular compartments (3.4 times; 64.7% versus 19% in the stroma, and in 2.0 times 24.8% and 44.2%, respectively, p <0.05).

Thus, on the basis of the statistically significant results obtained, signs of adhesion disorders and implantation failure of the eutopic endometrium in women with repeated implantation failures associated with chronic endometritis were established.

An immunohistochemical study of biopsy specimens of eutopic endometrium in women with repeated implantation failures associated with chronic endometritis revealed pathognomonic limits of sensitivity for the adhesion marker (MUC1) and endometrial receptivity marker in inflammatory diseases (HOXA-10) (Table 3).

In order to predict the onset of implantation, the sum of the points obtained is calculated:

• 3 points - high risk of implantation failure (negative prognosis);

• 2 points - moderate risk of implantation failure (doubtful prognosis);

• 0-1 point - low risk of implantation failure (positive prognosis).

Thus, in patients with repeated implantation failures and chronic endometritis, there is a violation of endometrial receptivity due to local inflammatory processes, as well as adhesion disorders that disrupt the synchronization of the development of endometrial glands and stroma and decidual transformation, which is realized in implantation failure.

Implementation of the invention

Endometrial biopsy is performed during the period of the alleged "implantation window" - on the 7th day after the peak of luteinizing hormone determined in blood serum during a dynamic study in the periovulatory period (as a rule, this period corresponds to 20-22 days of the menstrual cycle) in a spontaneous cycle, or in a cycle before 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. The expression level of HOXA-10 in the stroma and glands, MUC1 only in the endometrial glands is determined in accordance with the methods described above using the corresponding polyclonal antibodies. The percentage of stained cells is calculated, after which the response is assessed: no response - ≤1%, low expression - 2-10%, moderate expression - 11-50%, pronounced expression - ≥51%. The interpretation of the IHC reaction for an adhesion marker and a marker of receptive insolvency is carried out by comparing the obtained data with the following parameters:

• if MUC1 expression is <6%, then the risk of implantation failure is high - 1 point, if ≥ 6%, low - 0 points;

• if the expression of HOXA-10 in the glands is <25%, then the risk of implantation failure is high - 1 point, if ≥ 25%, low - 0 points;

• if the expression of HOXA-10 in the stroma is <19%, then the risk of implantation failure is high - 1 point, if ≥ 19%, low - 0 points.

Further, in order to predict the onset of pregnancy, the sum of the received MUC1 + HOXA-10 points in the glands + HOXA-10 in the stroma is calculated and the following conclusions are made:

• 3 points - high risk of implantation failure (negative prognosis);

• 2 points - moderate risk of implantation failure (doubtful prognosis);

• 0-1 point - low risk of implantation failure (positive prognosis).

Clinical example 1

Patient A., 32 years old, suffers from secondary infertility for 3 years, a history of 3 IVF attempts without effect. According to the immunohistochemical study, moderate ChE was diagnosed. Diagnosis: Secondary infertility. Chronic endometritis. Complicated gynecological history (1 induced abortion with curettage of the uterine cavity, 3 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 3 unsuccessful high quality embryo transfer attempts. With hysteroscopy after 3 unsuccessful attempts and targeted endometrial biopsy, there are signs of chronic endometritis. Before the next embryo transfer attempt, a control pipel biopsy of the endometrium was performed on day 21 of the menstrual cycle.

According to the immunohistochemical study, the following results were obtained: MUC1 in the glands = 4% (1b); HOHA-10 in the glands = 19% (1b); NOHA-10 in the stroma = 13% (1b).

The sum of points is 3 points, which indicates a high risk of implantation failure of the endometrium and a negative prognosis of the likelihood of implantation.

After the transfer of a good quality embryo into the uterine cavity, pregnancy did not occur in the next menstrual cycle.

Clinical example 2

Patient M., 31 years old, turned to a reproductive specialist in March 2020 with complaints about the absence of pregnancy for 3 years, burdened by an obstetric and gynecological history: in 2012 - a spontaneous miscarriage at a period of 2-3 weeks, in 2015 - an aborted miscarriage in 8 weeks of pregnancy. Gynecological diseases - ectopia of the cervix, RV coagulation 2013 Chlamydia - treated in 2009 Operations - laparoscopic right-sided adnexectomy for tubovarial abscess, in 2010 History of 3 unsuccessful implantation attempts during high-quality embryo transfer (4AB): 1 transfer in a stimulated cycle, 2 - in a cryocycle. The results of diagnostic hysteroscopy and histological examination on the 9th day of the menstrual cycle after 3 unsuccessful attempts at embryo transfer indicate the presence of moderate chronic endometritis with a minimum degree of activity. 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 22nd day of the menstrual cycle.

According to the immunohistochemical study, the following results were obtained: MUC1 in the glands = 12% (0b); HOHA-10 in the glands = 39% (0b); NOHA-10 in the stroma = 27% (0b).

The sum of points is 0 points, which indicates a low risk of implantation failure of the endometrium and a positive prognosis of the likelihood of implantation.

In the next menstrual cycle, a good quality embryo transfer was performed, and a clinical progressive uterine pregnancy occurred (confirmed by imaging of the embryo's heartbeat and the corresponding period of the level of hCG in the blood). Thus, fertility has been achieved.

Clinical case 3

Patient A., 28 years old. She complained about the absence of pregnancy for 4 years of regular sex life without contraception. History: at the age of 20 years, a medical abortion at a gestational age of 8-9 weeks, without complications. History of 4 unsuccessful implantation attempts during high quality embryo transfer (4AB): 2 transfers in a stimulated cycle, 2 in a cryocycle. Examined. There is no pathogenic and opportunistic flora. The fallopian tubes are passable. Examination of the partner did not reveal any pathology. According to the results of histological examination of the endometrium, the diagnosis was made: Infertility 2. Chronic endometritis. Burdened obstetric and gynecological history. According to the data of ultrasound and hysteroscopic studies, signs of chronic endometritis were found. In order to assess the implantation capabilities of the endometrium, before the next attempt at embryo transfer, a control pipe endometrial biopsy was performed on the 22nd day of the menstrual cycle.

According to the immunohistochemical study, the following results were obtained: MUC1 in the glands = 3% (1b); HOHA-10 in the glands = 15% (1b); NOHA-10 in the stroma = 12% (1b).

The sum of points is 3 points, which indicates a high risk of implantation failure of the endometrium and a negative prognosis of the likelihood of implantation.

After the transfer of a good quality embryo into the uterine cavity, pregnancy did not occur in the next menstrual cycle.

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Claims (1)

A method for assessing the implantation potential of the endometrium in patients with repeated implantation failures associated with chronic endometritis, including performing a pipel biopsy of the endometrium during the "implantation window", characterized in that an immunohistochemical study of the obtained biomaterial is carried out by staining sections using polyclonal antibodies to the following markers: MUC1 in the endometrial glands, HOXA-10 in the stroma and in the glands, the percentage of stained cells is calculated, if the expression of MUC1 is less than 6%, then “1 point” is assigned, if 6% or more, then “0 points”, if the expression of HOXA-10 in the glandular compartment of the endometrium less than 25%, assign "1 point", if 25% or more, assign "0 points", if the expression of HOXA-10 in the stromal compartment of the endometrium is 19% or more, assign "1 point" if less than 19% , assign "0 points", summarize the received points and with the sum: "3 points", determine the low implantation potential of the endometrium, indicating a negative prognosis blastocyst implantation; "2 points", determine the moderate implantation potential of the endometrium, indicating a dubious prognosis of blastocyst implantation; "0-1 point", characterizes the high implantation potential of the endometrium, indicating a positive prognosis of blastocyst implantation.