RU2636527C1 - Method of determining personal "implantation window" in women based on analysis of transcryption profile of genes - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: method of determining personal "implantation window" in women involves measurement of expression levels of mRNA of functional human genes REIHER, DPP4, HLA-DOB, MSX1 in endometrial tissue samples obtained by pipelle biopsy in 7-8 day after the peak of luteinizing hormone. Further, the obtained values of mRNA expression levels are used to calculate the endometrial receptivity index. If the value of endometrial receptivity index is less than or equal to the threshold value, a conclusion is made about the incompatibility of endometrium of "implantation window" stage; if the value of endometrial receptivity index is greater than the threshold value, a conclusion is made about the correspondence of endometrium of "implantation window" stage, the threshold value is determined from the experimental data by ROC analysis.EFFECT: invention provides the cumulative assessment of violations of fertility and gives the ability to determine the personal implantation window through study only one biological sample, namely endometrial tissue, using only one research methods - real-time polymerase chain reaction.3 dwg, 1 ex

Description

1. Field of technology.

The invention relates to the field of medicine (in particular, reproductive medicine and assisted reproductive technologies (ART), as well as to the field of science (in particular, molecular biology and embryology) and can be used to determine the personal “implantation window” in women in in vitro fertilization programs and transfer of embryos into the uterine cavity.

2. The prior art.

The functional layer of the endometrium under the influence of steroid hormones cyclically undergoes the processes of regeneration, secretory transformation and rejection during the menstrual cycle. The purpose of these physiological changes is to synchronize the maturation processes of the blastocyst and endometrial tissues, as well as to ensure the receptive status of the endometrium, susceptible to implantation of the embryo. Blastocyst implantation is carried out in a strictly defined period of the “implantation window”, corresponding to 7-8 days after the peak of luteinizing hormone (LH + 7-8), in women with a 28-day menstrual cycle this corresponds to 19-21 days of MC.

Morphological changes in the endometrium during the menstrual cycle are described by Noyes R.W. more than 60 years ago and are widely used to date. Criteria Noyes R.W. et al. based on morphological parameters such as the presence of vacuoles, stromal edema, secretion of glands, etc. [1, 2]. However, the accuracy of these criteria has been called into question in randomized trials [3, 4]. Therefore, at present, histological examination of the functional layer of the endometrium is insufficient and should be supplemented by new highly informative methods for assessing the receptivity of the endometrium.

In recent years, studies have been conducted of global transcriptional gene profiles in various diseases that affect the condition of the endometrium. In addition, a large amount of information has been accumulated on endometrial receptivity markers and the transcriptional profile of genes at different stages of the menstrual cycle.

Most researchers studied transcriptional profiles in total endometrial tissue without dividing it into separate components. Ponnampalam A.R. et al. The first to use microarray analysis to study transcriptional signatures (fingerprints) of genes at all stages of the menstrual cycle [5]. They isolated 425 genes, the expression of which is increased in at least one phase of the menstrual cycle by more than 2 times. Based on the analysis of these data, they identified 7 groups of genes with maximum expression levels at a certain stage of the menstrual cycle: menstruation, early proliferation, medium proliferation, late proliferation, early secretion, middle secretion, late secretion.

Research Team Talbi S. et al. 4 clusters of genes with a high level of expression were distinguished, which made it possible to discriminate 4 stages: proliferation, early, middle, and late secretion [6]. In addition, they described the relationship of the level of expression of these genes with their functions and biological processes characteristic of each of the selected stages.

A significant part of the publications is devoted to comparing global transcriptional gene signatures of the “implantation window” stage with other stages of the menstrual cycle: with the proliferation stage [7, 8, 9], early secretion [10, 11, 12, 13, 14, 15, 16], late secretion [16, 17].

In addition to the accumulation of information about the features of the expression profiles of the “implantation window” stage, some diagnostic platforms have already been proposed for practical use for determining the personal “implantation window” in women implementing reproductive function in IVF and PE programs. An example is the Endometrial Receptivity Array (ERA) test, which is a microchip containing 238 genes. The use of this test is recommended in the natural cycle on LH + the 7th day of the menstrual cycle (MDC) or in the cycle with the use of hormone replacement therapy on the 5th day of the appointment of progesterone (18). The use of this chip made it possible to increase the frequency of implantation of embryos, according to various estimates, by 10–20% [19].

Using microarray analysis, it was found that every fourth patient with unsuccessful attempts at in vitro fertilization has a shift of the implantation window by 2 days, which has fatal consequences for the implementation of the assisted reproductive technology program [20]. The concept of universal correspondence of the stage of the “implantation window” to 19-21 days of the menstrual cycle proved to be untenable. In this regard, the definition of a personal “implantation window” and the correction of embryo transfer time can be one of the ways to increase the effectiveness of ART programs.

Due to the high cost of research based on microarray analysis, including the cost of kits, equipment and software support, this method is not widespread in our country. As an alternative to the expensive and laborious method of microarray analysis, a reverse transcription and quantitative polymerase chain reaction (RT-PCR) method is proposed - a method that is not inferior in sensitivity and specificity to microarray analysis, but more affordable for routine use. To determine the personal “implantation window” in women with unsuccessful attempts in the IVF and PE program, in contrast to the Endometrial Receptivity Array (ERA) counterpart, a study is proposed of not one hundred, but one or two dozen genes using real-time RT-PCR method , as well as an integral criterion for assessing the transcriptional profile by the ratio of the levels of transcript representation.

At the time of filing an application from free sources, the following closest analogues were known:

- A method for diagnosing endometrial receptivity disorders in patients with uterine myoma and infertility (RF patent No. 2553340). Popov Yu.V., Burykina P.N., Kogan E.A., Demura T.A., Chuprynin V.D., Askolskaya S.I. The invention relates to medicine, namely to gynecology, is intended for the diagnosis of reproductive dysfunction in patients with uterine myoma. The method consists in determining the number of pinopodia in the surface epithelium, the level of expression of progesterone and estrogen-alpha receptors in the endometrial stroma, their ratio, as well as the level of expression of the implantation factor LIF on days 21-24 of the menstrual cycle by ELISA. Fertility impairment associated with uterine myoma is diagnosed with pinopodia less than 35%, the expression ratio of progesterone and estrogen receptors in the endometrial stroma is less than 3.5, and the level of LIF expression is less than 2 points. This method is provided only for patients planning a pregnancy with uterine myoma not deforming the uterine cavity and the size of myomatous nodes up to 5.0 cm in diameter.

The described method is based on methods of morphological and immunohistochemical studies of endometrial tissues obtained by pipeliopsy. The disadvantage of this diagnostic method is the lack of an integral criterion for assessing impaired fertility. The interpretation of the results remains unclear when not all indicators are changed.

- A method for predicting endometrial receptivity in in vitro fertilization cycles (RF patent No. 2562559). Ailamazyan E.K., Dzhemlikhanova L.Kh., Gzzzyan A.M., Niauri D.A., Kogan I.Yu., Sharfi Yu.N. The invention relates to medicine, namely to a method for predicting endometrial receptivity in in vitro fertilization cycles (IVF). The advantage of this method is the integrated assessment of several parameters: 1) determination of the optical density of expression of leukemia inhibitory factor (LIF) in the surface and glandular epithelium of the endometrium during the “implantation window” in the cycle prior to in vitro fertilization, 2) determination of the content of vascular endothelial growth factor (VEGF) in cervical mucus on the day of transvaginal puncture of the follicles, 3) determination of systolodiastolic ratio (S / D) and spiral resistance index (IR) GOVERNMENTAL arteries on the day of ovulation trigger. Based on the obtained indicators, the value of the regression function (Z) is calculated by the formula. With a value of Z> 0, it is predicted that the endometrium is receptive, and with Z <0, the endometrium is not receptive.

The described method is based on the methods of Dopplerometric study of blood flow in the uterine vessels, immunohistochemical determination of LIF in endometrial tissues obtained by pipeliopsy, as well as the study of mucus of the cervical canal for VEGF content by multiplex ELISA analysis. The proposed method differs from this analogue in that the study can be carried out in one sample of biological material (endometrial tissues obtained by pipe-biopsy) using one research method - RT-PCR in real time.

The method described in the analogue allows to predict the receptivity of the endometrium and the onset of pregnancy as a result of a stimulated IVF cycle. The proposed method makes it possible to determine a personal “implantation window” and its subsequent use in natural cycles and hormone replacement therapy cycles during transfers of cryopreserved / thawed embryos, as well as correction of the transfer time of the embryo.

3. Description of the invention.

A method is proposed for evaluating the endometrial receptivity in patients undergoing infertility treatment in the IVF and PE program according to the level of transcripts of individual functional genes in endometrial tissues obtained by pipe-biopsy on days 7-8 after the peak of luteinizing hormone (LH + 7-8) s the goal of optimizing the time of transfer of embryos into the uterine cavity, primarily cryopreserved / thawed embryos.

In the course of the study of 71 endometrial samples corresponding to the 19-21 day of the menstrual cycle, the most informative markers of endometrial receptivity were selected and a method was proposed for determining the personal “implantation window” in women implementing reproductive function in the assisted reproductive technology program by measuring the mRNA level of progestogen genes associated endometrial protein RAEP, dipeptidyl peptidase DPP4, major histocompatibility complex class II DO-beta chain HLA-DOB and homeobox 7 HOX7 (MSX1) compared correlation with the level of representation of reference genes, for example, B2M, GUSB, TBP.

In the receptive endometrium (endometrium of the stage of middle secretion) compared with the prereceptive (endometrium of the stage of early secretion), the expression of PAEP and DPP4 mRNA is increased by 144 and 46 times, and the expression of mRNA of the HLA-DOB and MSX1 genes is reduced by 17.5 and 2.8 times, respectively (p <0.001).

The level of mRNA representation of the proposed genes can serve as an additional as well as an independent studied parameter when assessing endometrial receptivity in samples obtained by pipeliopsy on days 7-8 after the peak of luteinizing hormone (LH + 7-8), diagnosed by ultrasound and / or Clear Blue urine test (Unipaht Ltd, UK) in natural or hormone replacement therapy cycles prior to transferring the cryopreserved / thawed embryos.

To assess the level of mRNA representation of the studied genes in the endometrium, the real-time polymerase chain reaction method with a preliminary stage of reverse transcription can be used. The obtained values of transcript representation levels and their ratios are used to group samples by cluster and multivariate analysis methods.

As an example, to distinguish clusters of samples, the method of binary logistic regression can be used. In this case, the endometrial receptivity index can be calculated by the formula:

(формула 1), где р - индекс рецептивности эндометрия;

(formula 1), where p is the endometrial receptivity index;

z is the value of the regression function, is determined by the formula:

(формула 2),

(formula 2),

– соотношение уровней экспрессии мРНК соответствующих генов.Where

- the ratio of the levels of mRNA expression of the corresponding genes.

If the value of the function p is less than or equal to the threshold value (p≤cut off), conclude that the endometrium does not match the stage of the “implantation window”; if the value p> cut off, a conclusion is made about the good receptivity of the endometrium and its compliance with the stage of the implantation window. The threshold value is determined based on experimental data using ROC analysis.

4. The implementation of the invention

Taking material by endometrial pipe-biopsy on the 7-8th day after the peak of the luteinizing hormone (LH + 7-8 d.m.ts.). Tissue fragments are placed in a 1.5 ml plastic tube into which 700 μl of transport medium is preliminarily added to stabilize RNA in biological samples (for example, IntactRNA, Eurogen, Russia, cat. No. NL001), process and store according to the manufacturer's instructions. RNA isolation is carried out by any standard method, for example, according to the Instruction for the RNA and DNA Isolation Kit “PROBA-NK / PROBA-NK-PLUS” (NPF DNA-Technology CJSC, Russia, registration certificate No. ФСР 2010/08867 dated 21.09 .2010 g /, TU 9398-035-46482062-2009). It is advisable to immediately use the obtained RNA preparations for the formulation of the reverse transcription reaction.

Reverse transcription is carried out using any standard method, for example, according to the Instructions for the reagent kit using AMV reverse transcriptase (Fermentas, Lithuania, cat. No. EP0641) in combination with 5 x reverse transcription buffer (composition: 250 mM Tris-HCl (pH 8.5 at 25 ° C), 40 mM MgCl ₂ , 150 mM KCl, 5 mM DTT). The composition of the RT mixture includes dNTP in a final concentration of 0.2 mM each and a mixture of random hexamers in a final concentration of 0.5 rM.

To determine the level of mRNA representation of the studied genes, the standard “real-time” PCR method is used under standard conditions with primers and fluorescently-labeled samples (probes) specific to the sequences of the determined RNAs. To avoid processing the samples with DNase, it is better to use oligonucleotides that do not interact with genomic DNA. For example, for a final reaction volume of 35 μl, the composition of the reaction mixture may be as follows: 66 mM Tris-HCl, pH 8.8, 16.6 mM ammonium sulfate, 0.01% Tween-20, 0.001% gelatin, 3 mM magnesium chloride , 250 μM each of dNTPs 12.5 pmol of each primer and probe, 0.8 units of Taq polymerase.

To increase the sensitivity and specificity of the reaction, it is desirable to use a “hot” start, which is ensured either by using Taq polymerase activated at high temperature or by the method of preparation of the reaction mixture consisting of two layers separated by a paraffin layer. The lower layer contains dNTP, primers and a probe, the upper layer contains a matrix and Taq polymerase. The layers are mixed and converted into a reaction mixture only when paraffin is melted, which eliminates non-specific annealing of the primers on the target DNA during initial heating of the tube.

The manifestation of the accumulation of reaction products is carried out using fluorescently-labeled oligonucleotides (probes) or intercalating dyes. The level of transcript representation and their ratio can be calculated using the ΔCq method.

The obtained values of transcript representation levels and their ratios are used to group samples by cluster and multivariate analysis methods.

An example of using the invention

On an outpatient basis, using an aspiration curette “Pipelle de Cornier” (Laboratorie C.C.D. ”, France), an endometrial aspiration pipeline biopsy was performed. Two groups of women of reproductive age were examined. The first group consisted of 47 patients with tubal-peritoneal infertility factor who applied for the IVF and PE program, with a successful outcome of this program. The criteria for inclusion of patients in the study were: the age of women under 39; regular menstrual cycle; tubal-peritoneal factor of infertility; stored ovarian reserve; history of no more than 3 unsuccessful IVF attempts; the absence of endometrial pathology according to ultrasound; the absence of contraindications for IVF and gestation; spouse fertile or subfertile sperm; normal karyotype of both spouses. Exclusion criteria: infertility due to an immunological factor; infertility associated with a lack of ovulation; uterine fibroids of any size; acute inflammatory diseases of the pelvic organs; chronic diseases in the acute stage; oligoastenoteratozoospermia (according to WHO guidelines, 2010); malformations of the internal genital organs; somatic and mental diseases, which are a contraindication for gestation of pregnancy and childbirth. An endometrial biopsy was obtained 7-8 days after the peak of luteinizing hormone (LH + 7-8 dm.ts.), diagnosed by ultrasound and / or urine test Clear Blue (Unipath Ltd, UK), based on the determination of LH level in urine. Pipel biopsy was performed in a cycle prior to stimulation of superovulation.

The second group consisted of 24 conditionally healthy women (comparison group) who applied for the purpose of a routine examination or selection of contraception. Inclusion criteria: women age up to 39 years; regular 28-day menstrual cycle; the presence of children; the absence of endometrial pathology according to ultrasound, the correspondence of the endometrium to 19-21 days of the menstrual cycle. Exclusion criteria: uterine fibroids of any size; acute inflammatory diseases of the pelvic organs; chronic diseases in the acute stage.

In addition to studying the transcriptional profile of genes, a histological study was performed. Endometrial biopsies were fixed in 10% neutral formalin for 24 hours, after appropriate standard processing they were enclosed in paraffin. Sections 5 microns thick were stained with hematoxylin and eosin. The study of histological preparations was carried out under a light microscope at magnifications from × 50 to × 400.

Cluster analysis of the results of the study of transcriptional gene profiles allowed us to distinguish 2 groups of samples: prereceptive and receptive endometrium (Fig. 1). The figure shows heat maps of a tree of hierarchical classification of 71 tissue samples of endometrial biopsies based on a study of the levels of mRNA expression of functional genes using quantitative RT-PCR. Two groups of samples were identified that can be described as prereceptive and receptive endometrium. According to the gradient of the transcript content change, samples with a high level of expression are marked with dark gray, light gray with low.

The division into groups was well correlated, but not in all cases it coincided with the data of a histological examination of endometrial biopsies. So, only 27 (77.1%) of 35 samples of the stage of medium secretion according to histological data were determined by RT-PCR as “receptive endometrium”. Eight samples identified by the histological method as the stage of medium secretion were functionally immature and not ready for implantation of the embryo; all of them belonged to the group of women with infertility.

According to the histological data, 32 out of 36 samples of the stage of early secretion were determined by RT-qPCR as “prereceptive endometrium” according to histological data. Four samples identified by the histological method as the stage of early secretion were functionally ready for nidation of the embryo, all of them also belonged to the group of women with infertility.

Separation of samples into separate clusters was not observed depending on the group of women examined (comparison group or women with infertility).

In the receptive endometrium, as compared with the preceptive one, the expression of mRNA of the RAEP genes was increased by 144 times (53-663, p = 5.8 × 10 ^-14 ), GPX3 by 143 (61-299, p = 1.5 × 10 ^-12 ) IGFBP1 52 times (3.7-3395, p = 2.8 × 10 ^-7 ), DPP4 46 (16-138, p = 1.7 × 10 ^-12 ), NAVP2 26 (14-54, p = 4.0 × 10 ^-11 ), AQP3 at 5.9 (1.7-8.5, p = 1.0 × 10 ^-7 ), IMPA2 at 4.7 (3.6-7.8, p = 2.5 × 10 ^-11 ), TAGLN at 3.3 (2.7-8.1, p = 1.5 × 10 ^-9 ), MMP7 at 2.4 (1.2-6.4, p = 0.03) times, decreased mRNA expression of HLA-DOB genes by 17.5 (9.5-26.5, p = 8.2 × 10 ^-12 ), MSX1 by 2.8 (1.4-4 , 2, p = 3.4 × 10 ^-7 ), POSTN 1.6 (0.9-3.3, p = 0.002) times (Fig. 2). The figure shows the change in the level of expression of mRNA of functional genes in biopsy samples of endometrial tissues of women in preceptive and receptive endometrium. The x-axis lists genes. The median value of the mRNA expression level in the preceptive endometrium is taken as 1 p.u. The y axis shows the median of the mRNA expression level in the tissues of the receptive endometrium (this value shows how many times the amount of mRNA is higher compared with prereceptive endometrium). Columns directed upwards indicate an increase in expression, downwards indicate a decrease.

Based on multivariate analysis, a discriminant function was selected that allows us to classify samples according to the expression level of 4 genes: PAEP, DPP4, MSX1 and HLA-DOB. The equation of the linear function was:

(формула 3), где

(formula 3), where

z - classifying discriminant function;

- соотношение уровней экспрессии мРНК соответствующих генов.

- the ratio of the levels of mRNA expression of the corresponding genes.

The distribution of the IRE value in the samples is shown in the figure (Fig. 3). The optimal value of the cutoff threshold (cut off points) was determined using ROC analysis. The area under the ROC curve was AUC = 1.0 (p = 6.6 × 10 ^-13 ). The cut-off value of the IRE was 62. The cut off values above 62 classify the samples as “receptive endometrium”. The sensitivity and specificity of the proposed method for assessing endometrial receptivity in the region of the threshold value was 100% compared with the cluster analysis.

LIERATURE

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Description of figures

FIG. 1. Heat maps of the hierarchical classification tree

FIG. 2. Change in the level of mRNA expression of functional genes in biopsy specimens of endometrial tissues of women in prereceptive (stage of middle secretion) and receptive endometrium (stage of middle secretion - implantation window).

FIG. 3. Distribution of the endometrial receptivity index in prereceptive and receptive endometrial clusters.

Claims (6)

A method for determining a personal “implantation window” in women implementing reproductive function in the assisted reproductive technology program, characterized in that the level of mRNA expression of human functional genes is measured in endometrial tissue samples obtained by pipe-biopsy 7-8 days after the peak of luteinizing hormone RAEP, DPP4, HLA-DOB, MSX1; mRNA expression levels obtained during the reaction are used to group samples by multivariate analysis; when using the method of binary logistic regression, the values of mRNA expression levels are substituted into the formula:

where p is the endometrial receptivity index; z is the value of the regression function, is determined by the formula:

, Where

- the ratio of mRNA expression levels of the corresponding genes; moreover, if the value of the function p is less than or equal to the threshold value, a conclusion is made about the mismatch between the endometrium and the stage of the “implantation window”; if the p value is greater than the threshold value, make a conclusion about the good receptivity of the endometrium and its compliance with the stage of the "implantation window"; the threshold value is determined on the basis of experimental data using ROC analysis.

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