RU2731203C1 - Method for evaluating receptivity of endometrium in secretory phase in women with primary infertility caused by endometrial hypoplasia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to pathomorphology, obstetrics and gynecology. Disclosed is a method of assessing endometrial receptivity in a secretory phase of a cycle in females suffering a uterine infertility caused by endometrial hypoplasia, in which by immunohistochemical method, p53 and PTEN receptor expression is determined in glands and stroma, followed by calculating prognostic index (PI) by formula PI=X1×-0.69+0.59×X2+0.99×X3+0.37×X4-69.3. If PI is more than 0, a conclusion is made on the endometrium's readiness for successful implantation of the embryo and possibility of directing the woman into the high-tech reproductive technologies (HRT) program. If PI is less than 0, low endometrial receptiveness and high risk of reproductive failure in HRT programs in women with primary infertility caused by endometrial hypoplasia are predicted.

EFFECT: invention provides more effective prediction of reproductive failures in females with endometrial hypoplasia and uterine infertility.

1 cl, 1 tbl, 3 ex

Description

The invention relates to medicine, namely to pathomorphology, obstetrics and gynecology, and is intended to assess the receptivity of the endometrium during the "implantation window" in women with primary infertility due to endometrial hypoplasia. EFFECT: possibility of correction of ART protocols in women with endometrial hypoplasia and infertility.

Disruption of full-fledged implantation during the transfer of good quality embryos due to non-receptive endometrium is the most significant cause of reproductive failures of ART, occupying up to 70% in their structure (Boyarsky K.Yu., Gaidukov S.N., Palchenko N.A. Modern view of the problem receptivity and thin endometrium in ART programs. Literature review. Problems of reproduction. 2013; 4: 3-7. Link active on 28.08.2017.). Therefore, in recent years, studies devoted to the study of endometrial receptivity in the pathology of implantation associated with infertility, caused, among other things, by endometrial hypoplasia, are of particular interest.

The analogs of this invention are described in the scientific, medical and patent literature methods for assessing the receptivity of the endometrium by studying the morphological picture and expression of hormone receptors.

A method for predicting endometrial receptivity in IVF cycles (Patent RU 2562559, publ. 05/27/2014) based on the determination of the optical density of expression of the inhibitory factor leukemia (LIF) in the surface and glandular epithelium of the endometrium during the implantation window in the cycle preceding in vitro fertilization, and the content of vascular endothelial growth factor (VEGF) in cervical mucus on the day of transvaginal follicle puncture, systolic-diastolic ratio (S / D) and resistance index (IR) of the spiral arteries on the day of ovulation trigger administration.

The method allows predicting the onset of pregnancy only in ART programs, taking into account the day of transvaginal puncture, the day of the ovulation trigger introduction, however, there is no data on predicting spontaneous pregnancy. The disadvantage of the method is that it is carried out directly during the ART protocol, which does not allow predicting the outcome of the protocol in advance; only LIF is analyzed, which does not reflect the mechanisms of endometrial receptivity impairment.

From patent RU 2580629, published on May 15, 2015, a method is known for predicting endometrial receptivity based on an assessment of the expression level and intracellular localization of ezrin in the endometrium of women with infertility and repeated failures of the IVF program. It was shown that during the "window of implantation" the receptor endometrium is characterized by the middle stage of the secretion phase, mature pinopodia by more than 20% of the surface epithelium and a high level of ezrin expression both in the epithelium and pinopodia. The invention makes it possible to predict the onset of pregnancy in in vitro fertilization (IVF) programs and natural cycles in women with infertility.

The disadvantage of the forecasting method is the focus on only one of the indicators from the whole spectrum of molecules of endometrial receptivity - the expression of the adhesion factor - ezrin. In addition, the prediction method is based only on the study of patients with tubal-peritoneal factor infertility without taking into account other factors of impaired endometrial receptivity.

From patent RU 2617061, publ. 12/16/2015, a known method for assessing the readiness of women with chronic endometritis for in vitro fertilization. In women with chronic endometritis, on days 19-21 of the menstrual cycle preceding the IVF program, a lavage is obtained from the uterine cavity, in which the content of alpha-2-macroglobulin (a2-MG) is determined by the method of enzyme-linked immunosorbent assay and the content of a2-MG is equal to or more than 4.5 mg / l assess women's readiness for IVF as low, recommend additional treatment and postponement of IVF, and with a2-MG content less than 4.5 mg / l, assess women's readiness for IVF as high and recommend initiation of an IVF program. The implementation of the invention provides a non-invasive method for assessing the readiness for IVF, which makes it possible to increase the efficiency of IVF by choosing the optimal start period of the program.

The disadvantages of this method are the lack of histological examination of the endometrium to verify the exact diagnosis, the orientation in predicting the only parameter - alpha 2 microglobulin, as well as the use of the prediction method only for ART cycles, but not for natural cycles. In addition, the proposed prognosis method determines only the contingent of women with chronic endometritis, without taking into account other factors of impaired endometrial receptivity. The test has a rather low sensitivity - about 80%.

From patent RU 2617515, publ. 04/22/2016, a known method for predicting endometrial receptivity based on the assessment of the expression of gonadotropin-releasing hormone (GnRG) and its receptor (GnRGR) into the "implantation window" in the endometrium of women with infertility and unsuccessful attempts at IVF programs. 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 lack of accounting for the expression of a number of molecules of endometrial receptivity that characterize the main physiological stages of implantation, the lack of data on the method of counting cells with pinopodia. The prediction model was determined only for patients with tuboperitoneal factor infertility; other factors of infertility leading to impaired endometrial receptivity were not taken into account.

The closest analogue (prototype) of the claimed invention is described by O.A. Melkozerova et al. (2018) a method for assessing endometrial receptivity during the "implantation window" RU 2651762 C1, publ. 04/23/2018, which consists in the fact that on the 20-23th day of the menstrual cycle, a pipel biopsy of the endometrium is performed and examined by immunohistochemistry using a two-stage streptavidin-biotin-peroxidase method, the following markers: ERb 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, after which the prognosis of endometrial receptivity is calculated.

The objective of the invention is to develop a method for assessing the receptivity of the endometrium in the secretory phase in women with primary infertility due to endometrial hypoplasia on the basis of immunohistochemical study of the level of expression in the endometrium (T-lymphocytes - CD3 +, p53 - a marker of apoptosis induction and PTEN - phosphatase with dual substrate specificity), with the subsequent calculation of the predictive index (PI).

The technical result when using the invention is to improve the efficiency of predicting reproductive failures in women with endometrial hypoplasia and uterine infertility.

Said technical result is achieved by the claimed method of estimating the receptivity of endometrium to a secretory phase of the cycle in women with uterine form of infertility due hypoplastic endometrium, characterized by determining the expression of p53 and PTEN receptors - in glands and stroma, with subsequent calculation prognostic index (PI ) according to the formula:

PI = X1 × -0.69 + 0.59 × X2 + 0.99 × X3 + 0.37 × X4-69.3,

Where:

X1 is the H-score of PTEN expression in the glands;

X2 - H-score of PTEN expression in the stroma

X3 - p53 in the endometrial stroma (% of stained nuclei in the field of view),

X4 - p53 in the endometrial glands (% of stained nuclei in the field of view),

-69.3– Const.

If PI is more than 0, a conclusion is made about the readiness of the endometrium for successful embryo implantation and the possibility of sending a woman to the ART program.

If PI is less than 0, low endometrial receptivity and a high risk of reproductive failures in ART programs are predicted in women with primary infertility due to endometrial hypoplasia.

The method is carried out as follows: Immunohistochemical study of endometrial biopsies taken by the pipel biopsy method on the 20-22 day of the 28-day menstrual cycle (during the supposed "implantation window"), was carried out using a two-stage streptavidin-biotin-peroxidase method with antigen unmasking and application standard kits of monoclonal and polyclonal antibodies from Bond RTU Primary and DAKO, USA. The following markers were detected on paraffin sections using a standard technique with preliminary antigen unmasking using the corresponding standard antibodies: PTEN and p53.

The manifestation of the reaction was carried out by the "Dako Cytomation" visualization system. For visualization of primary antibodies, a Super Sensitive Polymer-HPR Detection System (Biogenex) was used.

Serial paraffin sections were used for immunohistochemical studies. The results of the reaction of receptors to PTEN were identified by nuclear or membrane staining of cells for the corresponding markers with an estimate of the percentage of stained cells and the intensity of cell staining. To analyze the results of IHC reactions, the histological H-score method was used according to the formula: HS = 1a + 2b + 3c, where a is the% of weakly stained cells, b is the% of moderately stained cells, c is the% of strongly stained cells, 1, 2, 3 - the intensity of staining, expressed in points.

To assess the level of expression of p53 antigens in the glands and stroma, the indices of proliferation and apoptosis were calculated - the ratio of the number of stained cell nuclei to the total number of nuclei in percentage when counting at least 400 nuclei.

Statistical processing of the obtained data was carried out on a personal computer using the standard Microsoft Excel package and the Statisticafor Windows application package version 6.0, StatSoftInc. (USA) and SPSS-19 using parametric and nonparametric statistics methods.

To accomplish this task, a cohort comparative study of 26 women with primary infertility and endometrial hypoplasia, including those with unsuccessful attempts at ART, was carried out. All patients had a refractory endometrium, the thickness of which did not exceed 7 mm during the "implantation window" (peak secretion of luteinizing gonadotropin - LH + 7 - LH + 9 day of the cycle). The control group consisted of 24 patients with hypoplasia and infertility who became pregnant as a result of ART, who voluntarily took part in the study and signed an informed consent.

The most significant immunohistochemical parameters of the functional state of the endometrium, which determine the receptivity of the endometrium, were determined by the method of stepwise discriminant analysis with the exclusion of variables (Table 1).

Table 1

Results of stepwise discriminant analysis

Indicators Wilkes lambda Private
lambda F exceptions p-level
significance Tolerance 1-tolerance
(R-square) H-score of PTEN expression in glands 0.688611 0.112124 158.3736 0.000000 0.042849 0.957151 H-score of PTEN expression in the stroma 0.336036 0.229767 67.0446 0.000000 0.065319 0.934681 p53 in the stroma of the endometrium (% of stained nuclei in the field of view), 0.128400 0.601322 13,2601 0.001624 0.289231 0.710769 p53 in the endometrial glands (% of stained nuclei in the field of view), 0.133011 0.580479 14.4543 0.001118 0.407920 0.592080

Number of steps-4

Number of variables in the model: 4

F (1.20) = 5.462790 p <.0299

Lambda Wilks:, 0772099 approx. F (4.20) = 59.75849 p <.0000

Using the identified indicators developed a mathematical model for estimating receptivity of endometrium to a secretory phase in women with uterine form of infertility due hypoplastic endometrium, wherein the immunohistochemistry determine expression of p53 and PTEN receptors - in glands and stroma, with subsequent calculation Prognostic Index ( PI) by the formula:

PI = X1 × -0.69 + 0.59 × X2 + 0.99 × X3 + 0.37 × X4-69.3, where

X1 is the H-score of PTEN expression in the glands;

X2 - H-score of PTEN expression in the stroma

X3 - p53 in the endometrial stroma (% of stained nuclei in the field of view),

X4 - p53 in the endometrial glands (% of stained nuclei in the field of view),

-69.3 - Const

If PI is more than 0, a conclusion is made about the readiness of the endometrium for successful embryo implantation and the possibility of sending a woman to the ART program.

If PI is less than 0, low endometrial receptivity and a high risk of reproductive failures in ART programs are predicted in women with primary infertility due to endometrial hypoplasia.

The sensitivity, specificity and diagnostic efficiency of the method is 100%.

Clinical examples

Example 1 Patient T., 41 years old, diagnosed with primary infertility of 1 degree within 8 years, two unsuccessful IVF attempts in history, diagnosis of endometrial hypoplasia, the height of the endometrium according to M-echo less than 8 mm. The patient had no complaints, she was not somatically burdened. The material was obtained by the Pipel biopsy method on the 20th day of the menstrual cycle. With extended histological and IHC examination, the HSCORE PTEN score in the glands is 120; HSCORE PTEN score in stroma 190; p53 in the stroma of the endometrium (% of stained nuclei in the field of view) -31; p53 in the endometrial glands (% of stained nuclei in the field of view) -10,

Using the formula, the value of the prognostic index was determined:

PI = 120 × -0.69 + 0.59 × 190 + 0.99 × 31 + 0.37 × 10 -69.3 = -5.61

PI <0, which indicates a high risk of reproductive failures in ART programs in women with primary infertility due to endometrial hypoplasia.

In the next two menstrual cycles, the patient underwent ART in the cryoprotocol program, and two good quality thawed embryos were transferred. No embryo implantation took place.

Example 2. Patient S., 35 years old (diagnosis of primary infertility of 1 degree within 7 years, IVF attempts were not made, endometrial hypoplasia). Complaints about scanty, short menses. Somatically vegetative-vascular dystonia type 1. The material was obtained by the Pipel biopsy method on the 20th day of the menstrual cycle. With manual histological examination: fragments of the endometrium correspond to the initial stage of the secretion phase. With extended histological and IHC examination, the HSCORE PTEN score in the glands is 125; HSCORE PTEN score in stroma 170; p53 in the stroma of the endometrium (% of stained nuclei in the field of view) -25; p53 in the endometrial glands (% of stained nuclei in the field of view) -10,

Using the formula, the value of the prognostic index was determined:

PI = 125 × H-0.69 + 0.59 × 170 + 0.99 × 25 + 0.37 × 10 -69.3 = -26.8

PI <0, which indicates a high risk of reproductive failures in ART programs in women with primary infertility due to endometrial hypoplasia.

There was no treatment, the woman was referred for ART. After hormonal stimulation, embryo transfer without effect, pregnancy did not occur.

Example 3. Patient A. 29 years old. Diagnosis: endometrial hypoplasia, infertility 1 within 5 years, IVF attempts were not made. Somatically genetic thrombophilia: genes MTR AG, MTHFR AC. The material was obtained by the Pipel biopsy method on the 20th day of the menstrual cycle. With extended histological and IHC examination, the HSCORE PTEN score in the glands is 10; HSCORE PTEN score in stroma 130; p53 in the stroma of the endometrium (% of stained nuclei in the field of view) -15; p53 in the endometrial glands (% of stained nuclei in the field of view) -16,

Using the formula, the value of the prognostic index was determined:

PI = 10 × -0.69 + 0.591 × 30 + 0.99 × 15 + 0.37 × 16-69.3 = 21.27

PI> 0, which indicates that the endometrium is ready for successful embryo implantation in women with primary infertility due to endometrial hypoplasia.

In the process of observation, successful transfer of 2 frozen embryos, at the moment pregnancy with one fetus.

Thus, predicting the readiness of the endometrium for successful embryo implantation in women with uterine infertility using computer morphometry, the proposed method will reduce the number of ineffective treatment protocols and ART cycles.

The inventive method is simple to implement and does not require expensive equipment.

Unlike the prototype:

- to calculate the prognostic index, 2 immunohistochemical indicators are used;

- does not require lengthy and laborious preparation of samples before research and the presence of bulky equipment, additional media;

- is used in everyday clinical practice without special training as a pathologist.

Sources of information

1. Boyarsky K.Yu., Gaidukov S.N., Palchenko N.A. Modern view on the problem of receptivity and thin endometrium in ART programs. Literature review. Reproduction problems. - 2013.- Volume 4.-S. 3-7.

2. RF patent No. 2562559, 05/27/2014, A method for predicting endometrial receptivity in in vitro fertilization cycles. Ailamazyan E. K.,

Niauri D.A., Kogan I. Yu. Et al.

3. Patent RU 2580629, published on May 15, 2015. A method for predicting endometrial receptivity based on assessing the expression and intracellular localization of ezrin in the endometrium of women with infertility and repeated failures of the IVF program. Perminova S.G., Kogan E.A., Demura T.A., Seidova L.A.

4. RF patent No. 2617061, 16.12.2015, Method for assessing the readiness of women with chronic endometritis for in vitro fertilization. Tretyakova Ya.N., Likhacheva V.V., Zorina V.N. and others.

5. RF patent No. 2617515, 22.04.2016, 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 IVF program attempts. Demura T.A., Savelyeva E.M., Perminova S.G.

6. RF patent No. 2651762, 23.04.2018 Method for assessing endometrial receptivity during the implantation window. Melkozerova O.A., Bashmakova N.V., Esareva A.V., Chistyakova G.N., Barlit O.G., Giniyatova A.A.

Claims (10)

A method for assessing the receptivity of the endometrium in the secretory phase of the cycle in women with uterine infertility caused by endometrial hypoplasia, characterized in that the expression of p53 and PTEN receptors in the glands and stroma is determined by the immunohistochemical method, followed by the calculation of the prognostic index (PI) according to the formula: PI = X1 × -0.69 + 0.59 × X2 + 0.99 × X3 + 0.37 × X4-69.3, Where: X1 is the H-score of PTEN expression in the glands, X2 is the H-score of PTEN expression in the stroma, X3 - p53 in the endometrial stroma (% of stained nuclei in the field of view), X4 - p53 in the endometrial glands (% of stained nuclei in the field of view), -69.3 is a constant if PI is more than 0, a conclusion is made about the readiness of the endometrium for successful embryo implantation and the possibility of sending a woman to the program of high-tech reproductive technologies (ART), with PI less than 0, low endometrial receptivity and a high risk of reproductive failures in ART programs are predicted in women with primary infertility due to endometrial hypoplasia.