RU2477861C1 - Early diagnostic technique in postpartum endometritis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is an early diagnostic technique in postpartum endometritis involving RNA recovery from epithelial cells of a cervical canal of a woman on her late pregnancy or on 3-4^th postpartum day, reverse transcription to produce cDNA and evaluation of iRNA TLR5 expression by quantitative polymerase chain reaction. Decrease of the iRNA TLR5 expression below 0.01202 in the women on their late pregnancy and below 0.16938 on the 3-4^th postpartum day testifies to developing postpartum endometritis.

EFFECT: diagnosis of postpartum endometritis of late pregnancy enables initiating the measures on endometritis prevention immediately after the childbirth.

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Description

The invention relates to the field of medical diagnosis, can be used for early diagnosis of postpartum endometritis.

Postpartum suppurative inflammatory diseases currently occupy one of the first places in the structure of maternal morbidity and mortality [1]. The current methods of diagnosis, prevention and treatment of postpartum inflammatory complications are not effective enough, the number of purulent-septic complications remains stably high (5-26%), accounting for 26% in the structure of maternal mortality. In addition, postpartum infectious diseases directly affect the reproductive health of women. Postpartum endometritis often leads to infertility and miscarriage. Obstetric peritonitis, which developed against the background of postpartum endometritis, is an indication for the extirpation of the uterus, which makes it impossible for a woman to further perform her reproductive function. All this makes timely diagnosis, prevention and treatment of postpartum purulent-septic diseases one of the leading tasks in maintaining the reproductive health of the population.

The reason for the high frequency of postpartum purulent-inflammatory diseases (GVZ) is also a constant increase in the number of caesarean sections, on average by 1% per year. The probability of developing postpartum endometritis after cesarean section is 5-10 times higher than after delivery through the natural birth canal, and after repeated cesarean section, the risk of purulent-inflammatory diseases increases by 2.5 times. At the same time, the frequency of erased and abortive forms of endometritis increases (up to 40% of the total), the diagnosis of which is often difficult due to the mismatch of the general reaction of the body and the severity of the local pathological process [2, 3]. The method of ultrasound research, which is traditionally used to diagnose signs of postpartum endometritis (subinvolution of the uterus), is not always informative. There is the so-called "second option" of the ultrasound picture of postpartum endometritis. This is basal endometritis with the presence of hyperechoic deposits in the uterine wall and unexpressed subinvolution without the formation of lohiometers, which occurs in 28% of cases and most often is not diagnosed in a timely manner [4]. The bacteriological study, which is most often used in the clinic, has a significant drawback - the time factor, since the doctor usually receives its results for 5-7 days, that is, after the patient’s discharge from the maternity hospital. Moreover, the degree of contamination does not always correlate with the risk of developing an infectious process. So, in one patient, endometritis develops with bacterial contamination of 10 ⁴ CFU / ml, while in the other, 10 ² CFU / ml of the pathogen is sufficient for its development. Therefore, to assess the risk of HBV, it was proposed to determine the indicators of the body's immunoreactivity.

A known method for the early diagnosis of endometritis after cesarean section (RU No. 98103142/14, publ. 02.24.1998), based on the determination in the venous blood of the woman in the first 3 days after delivery the level of cytokines IL-1α, IL-1β, IL-8. An increase in pro-inflammatory cytokines above normal indicates postpartum endometritis. Using the method allows to identify the development of postpartum endometritis in puerperas of the group of high infectious risk at an early stage.

A known method for the diagnosis of subclinical forms of postpartum endometritis (RU No. 2007105844/15, publ. 02.16.2007), comprising determining the level of pro-inflammatory interleukins in the venous blood of the puerperal, in the first 3 days after delivery determine the level of TNF-α and IL-6 induced and with TNF -α more than 1024 pg / ml, IL-6 more than 4077 pg / ml diagnose postpartum endometritis, as well as spontaneous and induced IL-4 and with a serum IL-4 of more than 837 pg / ml, spontaneous IL-4 51 induced by IL-4 more than 46 pg / ml diagnose postpartum endometritis.

However, these methods are nonspecific for postpartum endometritis, since an increase in cytokine levels is possible with any inflammatory processes of a different localization.

A known method for the diagnosis of postpartum endometritis (RU 2004118386/15, publ. 06/17/2004), which includes the study of indicators kinin-kallikreinovoy blood plasma system. With an increase in spontaneous esterase activity and with a decrease in prekallikrein and a kallikrein inhibitor, postpartum endometritis is diagnosed. The method provides early diagnosis of complications of the postpartum period on average 4 days before changes determined by ultrasound. This method can only be used in the postpartum period, which does not provide sufficient time for preventive measures. In addition, it is also not specific for postpartum endometritis.

There is also a method for the early diagnosis of postpartum endometritis (RU 96101677/14, publ. 30.01.1996), including laboratory analysis of metaspirate one day after delivery, the level of DNAase is determined in the contents of the uterine cavity and, after an increase in this indicator compared with the control, postpartum is diagnosed endometritis. This method is invasive and technically complex.

A known method for the diagnosis of chronic endometritis and the nature of inflammation (RU 2003104391/15, publ. 02/10/2003), including a clinical and morphological examination by the immunohistocytochemical method, measures local immunity in endometrial tissue: the number of lymphocytes expressing markers of natural killer cells CD56 +, CD16 + and markers activation of HLA-DR (II) +, while the calculation is carried out in a light microscope with an increase in the lens × 40, eyepiece × 10 in three fields of view. When the number of cells expressing CD56 +, CD16 +, HLA-DR (II) + from 0 to 10 in the field of view, the absence of endometritis is diagnosed, when the number of cells expressing CD56 + is above 10, and when the number of cells expressing CD16 + and HLA-DR (II ) + from 0 to 10 in the field of view, diagnose autoimmune chronic endometritis, with the number of cells expressing CD56 +, CD16 +, HLA-DR (II) + above 10 in the field of vision, diagnose an exacerbation of autoimmune chronic endometritis, with the number of cells expressing CD16 + and HLA-DR (II) + above 10, and when the number of cells expressing CD56 + from 0 to 10 in the field of view, chronic endometritis with exacerbation or acute endometritis is diagnosed, with the number of cells expressing CD16 +, CD56 + above 10, and with the number of cells expressing HLA-DR (II) + from 0 to 10 in the field of view, chronic inflammation is diagnosed with autoimmune component, but without activation of the process, with the number of cells expressing CD16 + above 10, and with the number of cells expressing CD56 + and HLA-DR (II) + from 0 to 10 in the field of view, chronic endometritis is diagnosed.

The disadvantage of this method is its invasiveness and the difficulty of obtaining endometrial tissue, in connection with which the doctor must collect the material, as well as its complexity (it is possible to use only fresh material for work, the lack of storage capacity).

In addition, the well-known sources of information suggest using the measurement of the partial pressure of oxygen and carbon dioxide, the pH of the gas [5]. These methods also have low specificity. The use of gas chromatography and mass spectrometry to detect the quorum sensing signaling compounds (quorum feelings) of microorganisms is considered a highly informative method for screening at the GVZ [6], however, the use of these methods is not available even in most regional clinics due to the lack of expensive equipment and high research costs.

The choice of determining the expression of Toll-like receptor 5 (TLR5) as a diagnostic method for postpartum purulent-septic diseases is associated with a high specificity of the method, since it recognizes one of the bacterial ligands - flagellin bacteria.

Female genital tract epithelial cells express 10 types of TLRs, each of which binds only to specific ligands, which are parts of the cell wall of bacteria, fungi, DNA or RNA viruses. The recognition of bacterial structures (lipopolysaccharides, lipoprotein, flagellin, etc.) occurs through the activation of TLRs 1, 2, 4, 5, and 6. Four Toll-like receptors are able to recognize nucleic acids - these are TLRs 3, 7, 8, and 9. TLR7 and TLR 8 recognize intrinsic and viral single-stranded RNA, and TLR 9 binds unmethylated bacterial DNA. TLR 3 is able to recognize double-stranded RNA of viruses, therefore, this receptor plays a key role in the antiviral immune response [7, 8, 9, 10, 11].

The binding of TLR to a ligand leads to the production of cytokines and antimicrobial peptides [12].

The objective of the invention is to develop a method for the early diagnosis of postpartum endometritis based on the determination of TLR5 mRNA expression.

EFFECT: obtaining criteria for the diagnosis of postpartum endometritis, which allow to start antibiotic therapy as early as possible and thereby reduce economic costs and reduce the number of patient days in the postpartum department.

In accordance with the task, a method for the early diagnosis of postpartum endometritis was developed, based on the determination of TLR5 mRNA expression, including:

- the allocation of ribonucleic acid (RNA) from the epithelial cells of the cervical canal in women in late pregnancy or 3-4 days after birth;

- reverse transcription to obtain complementary deoxyribonucleic acid (cDNA);

- determination of TLR5 mRNA expression using a quantitative polymerase chain reaction;

- diagnosis based on a decrease in the expression of TLR5 mRNA below 0.01202 in women in late pregnancy and below 0.16938 3-4 days after birth, the development of postpartum endometritis.

The novelty and inventive step is that the possibility of early diagnosis of postpartum endometritis based on the determination of TLR5 mRNA expression is not currently known.

The method is as follows.

The material for determining the expression of Toll-like receptor 5 for the diagnosis of postpartum purulent-septic diseases are cervical canal epithelium cells. The method was developed taking into account the MIQE standard (Bustin S.A. et al., 2009) [13].

Taking material from pregnant women at full term gestation or 3-4 days after the postpartum period. The cervix is exposed in the mirrors, mucus is removed from the cervical canal with a sterile swab. Then, using a cytobrush, the material is collected in a 1.5 ml Eppendorf tube containing 500 μl of RNAlater preservation medium (Ambion).

If there is a need for storage of the material, after placing the material in the RNAlater solution, the tubes are left in the refrigerator at a temperature of + 4 ° C for 1 day, then placed in a freezer and stored at a temperature of - 28 ° C. After thawing the samples, the supernatant is drained and RNA isolation proceeds.

RNA is isolated by phenol-chloroform extraction using the Trizol reagent (“Invitrogen”) described by the manufacturer.

1. To the precipitate obtained by centrifugation, add 10 parts of Trizole (1000 μl), incubate for 5 minutes at 25 ° C, during which cell lysis and complete dissociation of the nucleoprotein complex occur. After that, the samples are centrifuged at 12000 rpm for 10 minutes at a temperature of +2 to + 8 ° C.

2. 200 μl of chloroform is added to the tube, after which the mixture is shaken for 15 seconds and centrifuged at 12000 rpm for 15 minutes at a temperature of +2 to + 8 ° C. After centrifugation in a test tube, the lower phenol-chloroform phase (colored in crimson) is formed, containing DNA, an interphase composed of proteins, and a colorless aqueous phase containing RNA. The volume of the aqueous phase is about 60% of the volume of Trizol.

3. Precipitation of RNA. The aqueous phase is transferred into sterile tubes, RNA precipitation is done by adding 500 μl of isopropyl alcohol, then incubated at a temperature of (+15) - (+30) ° C for 10 min, centrifuged at 12000 rpm for 10 min at a temperature of +2 up to + 8 ° C. After centrifugation, RNA forms a colorless gel-like pellet at the bottom of the tube.

4. After removing the supernatant, the RNA is washed by adding 1000 μl of cold 75% ethanol, centrifuged at 7500 rpm for 5 minutes at a temperature of +2 to + 8 ° C.

5. The resulting RNA was dried in air at room temperature for 5-10 minutes, then diluted with sterile RNase-free water (water supplemented with DEPC), incubated at + 55 ° C for 10 minutes.

The resulting RNA can be frozen in 75% ethanol at a temperature of -80 ° C for a long time.

The quality of the RNA is checked by electrophoresis for 15 minutes at a voltage of 10 V / cm by the intensity of the luminescence of ribosomal RNA bands in a 1.1% standard ethidium bromide agarose gel.

To remove genomic DNA, DNAse I RNAse free ("Fermentas") samples are treated.

In a sterile tube add:

1) 1 μg RNA

2) 1 μl 10Hbuffer with magnesium chloride

3) DNase I (1 μl = 1 unit)

4) RNase-free water (treated with DEPC) - up to 10 μl.

The mixture was incubated at + 37 ° C for 30 minutes, then 1 μl of 50 mM EDTA was added and incubated at + 65 ° C for 10 minutes. The RNA preparation is used for reverse transcription.

For reverse transcription in a test tube with a volume of 500 μl mix:

1) 500 ng of RNA preheated to 55 degrees (mRNA concentration was checked on a Nanodrop spectrophotometer (ThermoScientific), then the volume of the mixture needed to add to the reaction was calculated);

2) 4 μl of oligoDT;

3) water free of RNases, up to 10 μl.

The mixture is heated at +70 degrees for 2 minutes, then transferred to ice.

In each tube, add 8 μl of the prepared mixture:

1) 4 μl of 5x MINT buffer for the first chain,

2) 2 μl dNTP,

3) 2 μl of a mixture of DTT (20 mm).

After vortexing, 2 μl of Mint revertase is added.

On top of the mixture lay 1 drop (15-20 μl) of mineral oil so that the volume of the mixture does not change during evaporation.

The mixture is incubated at +42 degrees in a thermocycler for 1 hour 40 minutes, after which the tubes are frozen.

For further PCR, dilution of the obtained cDNA with water in a volume of 1 to 25 was used.

The quality of the obtained cDNA was assessed by gel electrophoresis in a 1.1% ethidium bromide agarose gel.

The first cDNA strand can be stored for up to 3 months at - 20 ° C, for longer storage it is recommended to use - 70 ° C.

For real-time PCR, specific primers were selected in the Blast database (www.ncbi.nlm.nih.gov). One of the conditions for the search for primers was the difference between the amplification temperature of the forward and reverse primers of not more than 2 ° C.

For quantitative real-time PCR, the SYBR GREEN I intercalation dye was used.

All primer pairs obtained were tested for the possibility of the formation of hairpins and dimers using the Beacon Designer Free Edition program (http://www.premierbiosoft.com/qOligo.jsp?PID=1). In addition, in order to test for primer dimers, a step for analyzing the melting curve (melt curve) was added to the PCR program. Peptidylpropylisomerase A (PPIA) and beta-actin were used as normalizing genes.

The following primers were selected:

Gene name Direct primer 5'-3 ' Reverse Primer 5'-3 ' Annealing temperature, ° C TLR5 GGGTCAGTTCTGGACTTCAGAG GGCTTCAAGGCACCAGCCATCTC 58 β-actin CAGGCACCAGGGCGTGATGG GATGGAGGGGCCGGACTCGT 64 PPIA CCGCCGAGGAAAACCGTGTACT TGGACAAGATGCCAGGACCCGT 64

For real-time PCR, a mixture of qPCRmix-HS SYBR with an intercalating dye SYBR I (Eurogen) was used, which included highly processive Taq polymerase with a hot start, a mixture of nucleotide triphosphates, magnesium, and buffer solution.

To prepare the mixture for one tube, you need:

2 μl of DNA

2 μl direct primer

2 μl reverse primer

5 μl qPCRmix-HS SYBR mixture

14 μl of sterile water

Amplification was performed on an I-cycler IQ5 thermocycler (Biorad).

Amplification mode for PPIA and TLR5:

1. Pre-denaturation - 1 cycle 95 ° C 5 min.

2. Denaturation - 1 cycle 95 ° C 30 sec.

3. Annealing at a temperature corresponding to each primer (see table) - 30 sec.

4. Elongation at 68 ° C - 30 sec.

Amplification mode for beta actin:

1. Pre-denaturation - 1 cycle 95 ° C 3 min.

2. Denaturation - 1 cycle 95 ° C 15 sec.

3. Annealing at 64 ° C - 30 sec.

4. Elongation at 70 ° C - 30 sec.

The number of cycles is 50.

The results were expressed in relative units, calculating them according to the formula

R = 2 ^{((Cq ref} ₁ ^{+ Cq ref} ₂ ^{) / 2-Cq target)} ,

where R is the level of normalized expression of TLR5,

cq ref ₁ and cq ref ₂ - level of expression of normalization genes (beta-actin and PPIA),

cq target - expression level of TLR5.

The possibility of using the proposed method for the early diagnosis of postpartum endometritis is confirmed by an analysis of the results of observations of 107 pregnant women in the late stages of gestation and 104 patients in the early postpartum period (48 of them with signs of postpartum endometritis, 56 - with a normal postpartum period). The diagnosis of postpartum endometritis was confirmed by clinical and laboratory data, the results of an ultrasound scan and histological examination of the placenta.

The following levels of TLR5 mRNA expression were identified (Table 1).

Table 1 Expression of TLR5 mRNA in late pregnancy and the early postpartum period TLR5 mRNA expression Postpartum Endometritis (M ± m) Normal postpartum period (M ± m) In late pregnancy 0.00862 ± 0.0034 ^∗ 0,092 ± 0,037 3-4 days after birth 0.06358 ± 0.1058 ^∗ 0.2941 ± 0.0445

Thus, the level of normal expression of TLR5 mRNA in late pregnancy is from 0.0550 to 0.1290 relative units, and in the risk group for the development of postpartum endometritis is from 0.00522 to 0.01202 relative units.

In the early postpartum period, the expression level of TLR5 mRNA is normally from 0.2496 to 0.3386 relative units, and in patients with postpartum endometritis, from 0 to 0.16938 relative units (p <0.05).

Thus, the data obtained indicate that a decrease in TLR5 mRNA expression can be considered a risk factor for the development of postpartum endometritis.

The advantage of the proposed method is the ability to determine TLR5 mRNA in late pregnancy, which allows you to start the prevention of endometritis immediately after the birth of a child, in addition, the method does not require a large investment of time, is simple to execute.

Bibliography

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Claims (1)

A method for early diagnosis of postpartum endometritis, including the isolation of RNA from cervical canal epithelial cells in women in late pregnancy or 3-4 days after delivery, reverse transcription to obtain cDNA, determination of TLR5 mRNA expression by quantitative polymerase chain reaction and based on reduction the expression of TLR5 mRNA below 0.01202 in women in late pregnancy and below 0.16938 3-4 days after delivery, the development of postpartum endometritis is diagnosed.