RU2850917C1 - OLIGONUCLEOTIDE SET FOR GENOTYPING POLYMORPHIC MARKERS OF CHRONIC ENDOMETRITIS rs1042838 OF PGR GENE IN CLINICAL MATERIAL USING POLYMERASE CHAIN REACTION WITH DETECTION OF AMPLIFICATION RESULTS IN REAL TIME - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: set of oligonucleotides is described for genotyping polymorphic markers of chronic endometritis rs1042838 of the PGR gene in clinical material using the polymerase chain reaction method with real-time detection of amplification results.

EFFECT: development of a set of oligodeoxyribonucleotide primers for detecting genetic predisposition in women to chronic endometritis by PCR with real-time hybridisation-fluorescence detection in clinical material, taking into account the possibility of using an extended list of compatible amplifiers.

1 cl, 1 ex

Description

A set of oligonucleotides for genotyping polymorphic markers of chronic endometritis rs1042838 of the PGR gene in clinical material by the polymerase chain reaction method with detection of amplification results in real time

The invention relates to the field of medicine, specifically molecular genetic diagnostics in gynecology. A reagent kit is presented for genotyping polymorphic markers of chronic endometritis in clinical samples using polymerase chain reaction and real-time detection (RT-PCR). The invention enables highly sensitive and specific detection of the chronic endometritis gene using PCR with real-time hybridization-fluorescence detection.

Chronic endometritis (CE) is a chronic inflammatory process in the functional and basal layers of the endometrium. [Savel'eva G.M., Sukhikh G.T., Manukhin I.B. Gynecology. National guidelines. Moscow: Medicine; 2017]. Chronic endometritis ranks first in the structure of pathological changes in the endometrium; its prevalence in women with infertility varies from 2.8 to 70% [Rudakova E.B., Davydov P.V., Davydov V.V. Diagnostics of intrauterine pathology in preparation for in vitro fertilization // Attending physician. 2015; (1): 119-129], [Johnston-MacAnanny E.B., Hartnett J., Engmann L.L., Nulsen J.C. et al. Chronic endometritis is a frequent finding in women with recurrent implantation failure after in vitro fertilization. Fertil Steril. 2010;93(2):437-441. DOI: 10.1016/j.fertnstert.2008.10.016]. In patients with a history of unsuccessful IVF and embryo transfer, the incidence of chronic endometritis, according to various sources, ranges from 30% to 60% [Bessmertnaya V.S., Samoilov M.V., Babichenko I.I., Serebrennikova K.G. Morphological and immunohistochemical features of the endometrium of women with primary and secondary infertility // Archives of Pathology. 2008;70(4):123-130], [Cicinelli E., Matteo M., Tinelli R., Lepera A., Alfonso R., Indraccolo U. et al. Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy. Hum Reprod. 2015;30(2):323-330. DOI: 10.1093/humrep/deu298]. Clinical manifestations of the disease are more often observed in reproductive age, the disease is characterized by a long course, menstrual and reproductive dysfunction, as well as decreased fertility in women. Infertility, habitual miscarriage, ectopic pregnancy, failures in assisted reproductive technology programs and other reproductive dysfunctions are characteristic of this pathology. Uterine factor, including chronic endometritis, remains one of the main causes of impaired fertility. [Ginter E.K. Medical Genetics. Moscow: Medicine; 2003. 445 p], [Kulakov V.I., Shurshalina A.V. Chronic Endometritis // Gynecology. 2005;7(5):7-10], [Kogan E.A., Demura T.A., Vodyanoy V.Ya., Shurshalina A.V. Molecular and Morphological Aspects of Endometrial Receptivity Disorders in Chronic Endometritis // Archives of Pathology. 2012;74(3):1-8]. Half of patients with spontaneous termination of gestation experience non-viable pregnancy [Radzinsky V.E., Morozov S.V., Kalinina E.V., Korchagina S.V. Proinflammatory cytokines and their role in the genesis of habitual miscarriage // Gynecology. 2004;6(6):48-52.].

Today, pelvic inflammatory diseases (PID) are characterized by polymicrobial etiology and are the main cause of chronic pathology of the reproductive system [WHO. Global strategy for the prevention and control of sexually transmitted infections: WHO document. Geneva; 2006-2015]. Chronic endometritis is actively studied in obstetrics and gynecology; however, many issues of diagnosis and therapy remain unresolved to date. Although the standard for diagnosing chronic endometritis is the determination of CD-138 cells by endometrial biopsy, timely diagnosis of the etiologic agent of pelvic inflammatory diseases of non-specific etiology is difficult due to the variety of clinical manifestations and the difficulty of determining the infective agent. [Peipert J.F., Ness R.B., Blume J. et al. Clinical predictors of endometritis in women with symptoms and signs of pelvic inflammatory disease // Am J Obstet Gynecol. 2001;184(5):856-863. DOI: 10.1067/mob.2001.111550], [Wiesenfeld H.C., Hillier S.L., Meyn L.A. et al. Subclinical pelvic inflammatory disease and infertility // Obstet Gynecol. 2012;120(1):37-43. DOI: 10.1097/AOG.0b013e3182594c6b]. The morphological features of chronic endometritis, immunological disorders, the role of gene expression in physiological and pathological processes in the endometrium are studied, the role of infection in the recurrent course of the chronic inflammatory process and the possibilities of its elimination are discussed [Maltseva L.I., Sharipova R.I., Zhelezova M.E. Chronic endometritis - a change in habitual ideas // Practical medicine. 2018; (6): 99-105].

Today, one of the reasons for the spread of antibiotic resistance is the irrational and uncontrolled use of antimicrobials in healthcare, which leads to disruption of the qualitative and quantitative composition of the normal human microbiota. Therefore, indications for antibiotic therapy in patients with chronic endometritis should be based on modern diagnostic methods and interpretation of examination results, allowing for the development of a personalized treatment and preventive program aimed at improving the patient's health and quality of life.

Effective diagnosis of chronic endometritis requires a combination of various methods, such as microbiological, histological, and immunological. Histological verification of the diagnosis is the presence of plasma cells in endometrial biopsies in the first phase of the menstrual cycle. Plasma cells may not always be detected due to reasons such as poor staining or operator error, as well as difficulties in visually identifying cells using simple light microscopy [Adegboyega P.A., Pei Y., McLarty J. Relationship between eosinophils and chronic endometritis // Hum Pathol. 2010;41(1):33-37. DOI: 10.1016/j.humpath.2009.06.008], [Greenwood S.M., Moran J.J. Chronic endometritis: morphologic and clinical observations // Obstet Gynecol. 1981;58(2):176–184]. Modern molecular genetic methods, such as real-time polymerase chain reaction (RT-PCR), play a key role in the diagnosis and risk assessment of chronic endometritis. RT-PCR allows us to identify the presence of pathogens and genetic markers associated with inflammatory processes in the endometrium, which significantly improves the accuracy of diagnosis and facilitates timely intervention. Recent studies demonstrate the effectiveness of RT-PCR in detecting microbial pathogens in women with menstrual irregularities, which emphasizes the importance of using this method for the prediction and prevention of chronic endometritis [Nandagopal M., Padhiar C., Abhaya M., Bansal U. Revolutionizing chronic endometritis diagnosis: real-time polymerase chain reaction unveils microbial pathogens in Indian women with abnormal bleeding // AJOG Global Reports. 2024; DOI: 10.1016/j.ajog.2024.02.004]. Molecular genetic methods, including real-time PCR, are also used to detect chronic endometritis. This method demonstrates sensitivity and specificity of up to 95-100%.

The aim of the present invention is to develop a set of unique, highly specific oligodeoxyribonucleotide primers for identifying genetic predisposition in women to chronic endometritis using PCR with hybridization-fluorescence detection in real time in clinical material, taking into account the possibility of using an extended list of compatible amplifiers.

The kit contains one pair of oligonucleotides that function as forward and reverse primers in real-time PCR. The oligonucleotide structures in this kit are: forward primer PGR-rs1042838-FJ (SEQ ID NO: 1), GAGGTGTCAGGTTTTGTG; reverse primer PGR-rs1042838-RJ (SEQ ID NO: 2), GTCAGAGTTGTGAGAGCA; fluorescently labeled DNA probes PGR-rs1042838-FAM (SEQ ID NO: 3), FAM-cgc+Cca+Atg+Gctg-BHQ-1; PGR-rs1042838-VIC (SEQ ID NO: 4), VIC-cgc+Cca+Ctg+Gctg-BHQ-2. The oligonucleotide set uses the rs1042838 fragment of the PGR gene as a target.

This kit detects genotyping of polymorphic markers using real-time PCR, with a sensitivity of at least 10 GE/μL. Polymerase chain reaction (PCR) testing using this oligonucleotide array allows for the identification of genetic predisposition to chronic endometritis in clinical specimens.

To use the oligonucleotide set, it is necessary to prepare a PCR mixture (total volume - 10 µl), including:

- Mixture for PCR PGR-rs1042838 in a volume of 4 µl

- Polymerase without 5`-3`-exonuclease activity in a volume of 2 µl

- Deionized water in a volume of 3 µl

- The test sample in volume (~30 ng DNA/µl) - 1 µl

To monitor the progress of reactions, a negative control sample (NCS) is used. The NCS consists of DNase-free deionized water.

This set is used as follows.

1) Carry out DNA extraction from biological material (whole blood) using specialized DNA extraction kits (sample preparation reagent kits, not the subject of this patent); during this procedure, DNA is extracted from the biological material, which in turn is used for PCR;

2) Prepare the required number of 0.1-0.2 ml test tubes for PCR (the number of test tubes is determined based on the number of samples being tested;

3) In a separate disposable sterile Eppendorf tube with a volume of 1.5-2.0 ml, prepare a reaction mixture consisting of oligonucleotides, polymerase without 5`-3`-exonuclease activity and deionized water;

4) Add 10 µl of the prepared reaction mixture to each PCR tube;

5) Add 1 µl of the isolated DNA to the appropriate test tubes for the samples being studied;

6) Mix the contents of the test tubes, then discard the drops from the walls by briefly centrifuging for 1-3 seconds in a microcentrifuge-vortex;

7) Place the tubes in the reaction module of the real-time PCR instrument. Amplification is performed according to the following program: 1 cycle: 95°C, 2 minutes; 50 cycles: 94°C, 15 seconds, 67°C, 30 seconds (at this stage, fluorescent signal detection is required in the FAM/HEX channels).

After amplification is complete, a melting curve analysis should be performed with the temperature increasing by 1°C, starting at 25°C and ending at 75°C, to obtain melting temperatures. The following PCR instruments can be used: CFX96 (BioRad, USA), DTprime (DNA-Technology Research and Production Association, Russia), Rotor-Gene Q (Qiagen, Germany), QuantStudio 5 (Thermo Fisher Scientific, USA), and Gentier 96 (Tianlong, China).

After amplification and real-time detection of amplification products, the results are interpreted. A positive result is considered if the rs3020450 polymorphism of the PGR gene is detected in the sample. This is determined by the presence of characteristic melting temperatures:

FAM 55°C, HEX 43°C - for genotype GG;

FAM 55°C, HEX 54°C - for GT genotype;

FAM 47°C, HEX 53.5°C - for genotype TT.

Fluorescence growth curves should be S-shaped and cross the threshold line before the 50th amplification cycle.

It is recommended that DNA extraction be performed on the day of clinical specimen collection. Whole venous blood samples may be transported and stored at 2°C to 8°C for no more than 48 hours. If delivery to the laboratory within 24 hours is not possible, a single freezing of the blood is permitted.

It should be understood that one skilled in the art, given the present description, can practice the present invention to its fullest extent without further experimentation. Therefore, the description of the invention provided in this context is provided for illustrative purposes only and does not diminish the scope of the claims.

Clinical case:

Patient N., 36, came to the doctor for a consultation to plan a pregnancy, as she does not have a sexual partner. During the examination and anamnesis, it was established that the patient has several chronic diseases. She was examined and referred to specialized specialists such as an endocrinologist, hematologist, reproductive specialist, and geneticist. An examination was conducted to identify a genetic predisposition rs1042838 of the PGR gene to chronic endometritis in this patient using PCR with real-time fluorescence hybridization detection. After additional examination, the following diagnosis was made: Infertility II. Age factor. Lack of sexual partner. Multiple uterine fibroids. Chronic endometritis. Euthyroidism. Chronic endometritis. Congenital uterine fibroids, non-classical form. NJO stage I. Circulation of lupus anticoagulant. A CYP 21 A 2*15 mutation (homozygous) was detected. IVF was recommended under the compulsory medical insurance policy with the sperm donor being tested for the mutation. In 2018, pregnancy occurred on the second IVF attempt. The pregnant woman was monitored and examined in accordance with the Order of the Ministry of Health of the Russian Federation dated October 20, 2020, No. 1130n "On approval of the Procedure for the provision of medical care in the profile of "obstetrics and gynecology". The pregnant woman was classified as being in a high-risk group. The pregnancy ended on September 11, 2024, with an urgent surgical delivery at 39 weeks. Laparotomy. Retrovesical cesarean section in the lower uterine segment. The fetus is female, weighing 3550 g, 55 cm. The woman and the child were discharged home in satisfactory condition.

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Claims (1)

A set of oligonucleotides for genotyping polymorphic markers of chronic endometritis rs1042838 of the PGR gene in clinical material by the polymerase chain reaction method with detection of amplification results in real time, including a forward primer SEQ ID NO: 1, a reverse primer SEQ ID NO: 2, fluorescently labeled DNA probes SEQ ID NO: 3 and SEQ ID NO: 4.