KR101578526B1 - Marker IL-13 and its use to predict preterm birth risk of less than 32 weeks - Google Patents

Abstract

The present invention relates to a composition and a diagnosis kit, and a diagnosis method for diagnosing a risk of a mother of premature delivery before 32 weeks of pregnancy by measuring the concentrations of Interleukin-13 (IL-13) having a significant correlation with the risk of the mother of premature delivery before 32 weeks of pregnancy.

Description

Marker IL-13 and its Use for Predicting Preterm Delivery Risk <32 weeks Pre-delivery and use of Marker IL-13 for predicting a risk of early (<32 weeks)

The present invention relates to compositions, diagnostic kits and diagnostic methods for diagnosing the risk of premature birth of less than 32 weeks of mother by measuring the concentration of IL-13 (Interleukin-13), which has a significant correlation with the risk of premature birth of less than 32 weeks of maternal will be.

Preterm birth usually means giving birth before 37 weeks after 20 weeks. The risk of death is high enough to account for about 60% of all infant deaths, and neonatal intensive care is needed for neonatal developmental disorders, respiratory complications, postnatal growth retardation, There is a high morbidity rate for long-term or short-term diseases. In addition, about 75% of preterm birth occurs with premature labor, amniocentesis, and associated cervical incompetence and chorioamnionitis. However, the mechanism associated with preterm delivery is still unclear. Common risk factors for natural labor include genital tract infections, multiple pregnancies, a few thirds of bleeding, and previous preterm delivery history.

The minimum gestational age is 27 weeks and the birth weight is 0.9 kg. The morbidity and mortality of the newborn are known to be influenced primarily by gestational age, or maturity, rather than birth weight. Recently, preterm labor has been classified as Extremely preterm, 28 to 32 weeks (Very preterm) and 32 to 37 weeks (Moderate to Late preterm). Especially, Is known to have a devastating effect on survival and health. Thus, delaying the delivery of newborns by appropriate treatment to raise maturity when early signs of premature birth come, is a crucial issue for the health and quality of life and costs of mothers and newborns.

Until now, for the treatment of premature labor, maternal use of ant contractions, antibiotics, steroids and progesterone have been used to suppress labor and delay delivery. However, in cases of preterm labor and premature rupture of membranes, the treatment effect of antibiotics on intrauterine infection and inflammation is very limited and it is known that preterm delivery can not be prevented. In addition, women with cervical incompetence have been given cervical cerclage as a treatment and prophylaxis because of the great risk of infection and miscarriage, but this can not be treated as a fundamental treatment, It is known.

Therefore, it is important to select the preterm delivery risk mothers by predicting the risk of premature birth rather than to perform the treatment after the symptoms of premature rupture of membranes or prematurity, and to prevent premature birth or to use an effective treatment method It is necessary to develop. In addition, the earlier the preterm delivery period, the more likely it is to leave a sequel to the neonate and the degree of sequelae is so severe that it is expected that the incidence of preterm infants and children with disabilities can be significantly reduced if the preterm delivery can be predicted.

A common approach to predicting preterm birth was to identify female populations that should be given special attention in terms of obstetric, demographic, and various syndromes (Main et al., Am. J. Obste., Gynecol., 151: 892-898, 1985), this approach has the problem that the method is not sensitive or specific. To overcome these problems, many studies have been conducted to find biochemical markers for predicting sudden preterm delivery and amniotic membrane wasting. Plasma estradiol-17 beta, progesterone, C Substances such as C-reactive protein have been nominated, but this has also been shown to be less accurate. Therefore, it is necessary to develop a marker that is easier to detect and has high sensitivity and specificity.

Therefore, the present invention aims to contribute to the prevention of premature birth by predicting predictive markers of preterm delivery, which suggests the risk of premature birth, in asymptomatic pregnant women before the risk of premature labor such as premature labor and premature rupture of membranes. Particularly, in the present invention, a new marker capable of predicting the risk of premature birth of less than 32 weeks, which is known to have a life-threatening effect on the survival and health of a premature infant, was discovered and the present invention was completed.

Korean Patent Laid-Open Publication No. 2002-0039458 (May 27, 2002)

It is an object of the present invention to provide a composition for the diagnosis of maternal risk of premature birth of less than 32 weeks, which comprises an agent for measuring the concentration of IL-13 (Interleukin-13).

It is another object of the present invention to provide a kit for the diagnosis of maternal <32 weeks pregnancy risk comprising the composition.

It is another object of the present invention to provide a method for providing information necessary for the diagnosis of premature birth risk of a mother, comprising measuring the concentration of IL-13 (Interleukin-13) from a maternal sample .

It is another object of the present invention to provide a method for diagnosing a maternal risk of premature birth of less than 32 weeks, comprising the step of measuring the concentration of IL-13 (Interleukin-13) from a maternal sample.

In order to achieve the above object, the present invention relates to a composition for the diagnosis of maternal risk of premature birth of less than 32 weeks, comprising an agent for measuring the concentration of IL-13 (Interleukin-13).

In a preferred embodiment, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the composition comprises at least one of IL-1β, IL-6, IL- A concentration of at least one selected from the group consisting of granulocyte-colony stimulating factor (CSF), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), macrophage inflammatory protein 1α and MIP1β The present invention relates to a composition for the diagnosis of a risk of premature birth of a child under 32 weeks.

The term "diagnosis" or "prediction" in the present invention means that there is a possibility of premature birth at less than 32 weeks of gestation, a relatively high possibility of premature birth at less than 32 weeks, And the like. The present invention can be used to delay or prevent premature delivery of maternal mothers at risk of premature birth of less than 32 weeks through special and proper care. In addition, the present invention can be used clinically to make treatment decisions by early diagnosing premature births less than 32 weeks and selecting the most appropriate treatment regimen.

The term "diagnostic marker, diagnostic marker" or "marker" in the present invention refers to a substance that can predict and distinguish mothers delivering at least 32 weeks in advance, , Which refers to bioorganic molecules that show significant increases or decreases in maternal samples predicted by preterm delivery of less than 32 weeks compared to normal maternal maternal samples. IL-1, IL-6, IL-7, IL-8, IL-10, and IL-6, which specifically predict increased concentrations in maternal samples predicted by preterm delivery of less than 32 weeks of diagnosis -13, IL-15, IL-17 alpha, TNF alpha, G-CSF, MMP8, MMP9, MIP1 alpha and / or MIP1 beta.

More specifically, the present invention provides IL-13 as a diagnostic marker of preterm birth risk. IL-1β, IL-6, IL-7, IL-8, IL-10, IL-15, IL-17α, TNFα, G-CSF, MMP8 and MMP9 as diagnostic markers of preterm birth risk , MIP1? And / or MIP1?.

In a specific embodiment of the present invention, the ultrasonography or physical examination performed between 16 and 24 weeks of gestation showed a cervical incompetence of more than 1 cm. In amniotic samples of normal delivery mothers (n = 41), cytokines were screened by the Membrane method, and 15 cytokines were selected that differed significantly in the two groups. In addition, for these 15 cytokines, the women who underwent cervical encasement were divided into two groups according to the delivery frequency: early preterm birth (32 weeks), late preterm birth (32-37 weeks), and 37 weeks Term deliveries, and quantitative analysis of the 15 species of cytokines was carried out using a multiplex bead array using amniotic fluid samples of these mothers.

In addition, receiver-operation chracteristic curves were obtained for sensitivity and specificity for 15 cytokines and clinical features to determine predictors of preterm delivery below 32 weeks. As a result, IL-13, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-15, IL-17α, TNFα, G-CSF, MMP8 and MMP9 , MIP1α and MIP1β were found to be significantly associated with preterm birth risk of less than 32 weeks, and sensitivity, specificity, and cut-off were confirmed by ROC curve. Simplified logistic regression analysis again confirmed that the markers were significant for predicting preterm birth risk of less than 32 weeks.

Therefore, the risk of premature birth of less than 32 weeks can be predicted and diagnosed by measuring the concentration of IL-13 in maternal samples. Further, , IL-15, IL-17α, TNFα, G-CSF, MMP8, MMP9, MIP1α and / or MIP1β as indicators for predicting prematurity risk of less than 32 weeks.

IL-10, IL-15, IL-17 alpha, TNF alpha, G-CSF, MMP8, MMP9, IL- The agent for measuring the concentration of MIP1? And / or MIP1? May be an antibody specific for each of these.

In the present invention, "antibody" means a protein molecule specific for an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to the marker, and includes both a monoclonal antibody, a polyclonal antibody, and a recombinant antibody.

Monoclonal antibodies can be generated using hybridoma methods well known in the art (Kohler and Milstein (1976) European journal og Immunology 6: 511-519), or phage antibody libraries (Clarkson et al, Nature, 352: 624-628, 1991; Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

Polyclonal antibodies can be produced by methods well known in the art for injecting the above protein antigens into animals and obtaining blood from animals to obtain sera containing antibodies. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, and the like. The antibody of the present invention also includes a special antibody such as a chimeric antibody, a humanized antibody, and a human antibody.

Further, the antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and examples thereof include Fab, F (ab ') 2, F (ab') 2 and Fv.

As a method for measuring the concentration of IL-13 and the like in the sample using such an antibody, any method can be used as long as it can confirm the degree of production of the antigen-antibody complex by treating the antibody in the sample. The above-mentioned "antigen-antibody complex" means a conjugate of IL-13 and an antibody specific thereto, and the formation amount of the antigen-antibody complex can be quantitatively measured through a signal label of a detection label.

For example, Western blotting, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, Complement Fixation Assay, Fluorecence Activated Cell Sorter (FACS), protein chip protein chip, radioimmunoassay (RIA), enzyme immunoassays (EIA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA) but are not limited to, nephelometric inhibition immunoassays (NIA), microparticle enzyme immunoassays (MEIA), and chemiluminescent magnetic immunoassay (CMIA).

In another embodiment, the concentration of white blood cells in the blood of the present invention can be measured by a general method known in the art. For example, it can be quantitated by automated hematology, manual blood count, flow cytometry, PCR, and microfluorometry.

As another example, the present invention relates to a diagnostic kit comprising the composition for the diagnosis of less than 32 weeks pregnancy risk.

The kit of the present invention may include not only agents capable of measuring concentrations of IL-13 and the like in a maternal sample but also one or more compositions, solutions or devices suitable for concentration analysis. For example, the kit may comprise a support or suitable carrier, substrate, detection label (or secondary antibody labeled with a detection label), buffer solution, quencher, solubilizer, wash solution and / or stabilizer And the like.

Examples of suitable carriers or supports include polymers, glasses, metals, silicones, membranes, and the like. Specific examples thereof include polybutylene succinate, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose , Polyacrylamide, polysterine, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass , Glass beads, or magnetic particles. Other solid substrates include culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).

As a specific example, the above-mentioned 32-week-old preterm birth risk diagnostic kit may be a kit including necessary elements for performing ELISA in order to implement various ELISA methods such as an ELISA kit, a sandwich ELISA, and the like. ELISA kits include antibodies specific for these proteins. Antibodies can be monoclonal, polyclonal, or recombinant antibodies, with high specificity and affinity for each marker and little cross-reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes and other substrates capable of binding to the substrate or antibody.

In addition, the kit may be a kit for implementing Western blots, immunoprecipitation assays, complement fixation assays, flow cytometry, or protein chips, and may further include additional structures suitable for each assay method. Through these analytical methods, the risk of premature birth of less than 32 weeks can be diagnosed by quantifying the amount of antigen-antibody complex formation, thereby preventing the premature birth of less than 32 weeks, thereby causing serious complications and sequelae of newborns such as neonatal morbidity, cerebral palsy .

The amount of the antigen-antibody complex formed is quantitatively measurable through the size of the signal of the detection label. The detection label may be labeled with an antibody specifically binding to IL-13 or the like, or may be detected by reacting a secondary antibody labeled with a detection label. For example, the amount of antigen-antibody complex formed can be measured by fluorescence, luminescence, chemiluminescence, absorbance, reflection or transmission of the detection label. To this end, the detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, emitters, microparticles, colloids, redox molecules and radioisotopes, but is not necessarily limited thereto.

As a quantitative measure of detection labels, a high throughput screening (HTS) system can be used. The fluorescence method is carried out by detecting fluorescence with the fluorescent substance attached to the detection body, the radiation method in which the radioactive isotope attached to the detection body is detected by the detection body, the SPR a surface plasmon resonance (SPRI) method in which an SPR system is imaged and confirmed, but the present invention is not limited thereto.

As another example, the present invention relates to a method for providing information necessary for the diagnosis of a maternal risk of premature birth less than 32 weeks, comprising the step of measuring the concentration of IL-13 from a maternal sample.

In a preferred embodiment, the present invention provides a method of detecting IL-13 in a sample comprising the steps of: measuring the concentration of IL-13 from a maternal sample; and, if the concentration of IL-13 is higher than normal mothers or higher than a cut- And a method for providing information necessary for the diagnosis of a pregnancy risk of less than 32 weeks of pregnancy.

IL-10, IL-15, IL-17 alpha, TNF alpha, G-CSF, IL-6, IL-7, IL-8, Wherein the method further comprises the step of measuring the concentration of at least one selected from the group consisting of MMP8, MMP9, MIP1? And / or MIP1 ?.

In the present invention, the IL-13, IL-1, IL-6, IL-7, IL-8, IL-10, IL-15, IL-17 ?, TNF ?, G- CSF, MMP8, MMP9 , MIP1α and / or MIP1β can be measured from maternal amniotic fluid samples and white blood cells can be measured from maternal blood samples. The mid-trimester amniocentesis is widely used to identify fetal chromosomal anomalies, but it is not widely used for predicting high-risk pregnancies in the future. The markers of the present invention can be measured in the amniotic fluid sample obtained in the amniotic fluid examination of the middle pregnancy. Alternatively, the concentration of the amniotic fluid leaking into the vagina through the cervix can be measured by the amniotic fluid collection device to obtain the concentration in the amniotic fluid sample.

The sample can also be prepared to increase the detection sensitivity of the marker. For example, the amniotic fluid sample extracted from the mother can be analyzed by anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, Such as, but not limited to, sequential extraction or gel electrophoresis.

The process of measuring the concentration of the markers in a maternal sample can be carried out using a known process, for example, by the above-mentioned various assay methods of antigen-antibody complexes.

In one embodiment, the present invention can be carried out by measuring the concentration of the markers using an antibody specific for each marker. The antibody may be a monoclonal antibody or a polyclonal antibody specific for each marker, but may further be a functional fragment of an antibody having antigen binding activity.

Considering both sensitivity and specificity of predicting preterm birth, the threshold of IL-13 was about 50 pg / ml (sensitivity 78%, specificity 65%), IL (Sensitivity: 78%, specificity: 71%), the threshold of IL-1β was at least 22 pg / ml (sensitivity: 65%, specificity: 87%), IL (Sensitivity: 78%, specificity: 84%), the threshold of IL-8 was more than 854 pg / ml (sensitivity: 83%, specificity: 71% (Sensitivity: 78%, specificity: 71%), the threshold value of IL-15 is about 25 pg / ml or higher (sensitivity: 83%, specificity: 77% (Sensitivity: 74%, specificity: 84%). The threshold value of G-CSF was about 3246 (sensitivity: 61%, specificity: 94% (sensitivity: 70%, specificity: 87%). The threshold value of MMP8 is about 214 pg / ml (sensitivity: 96%, specificity: 61%) and the threshold value of MMP9 is about 3 pg / ml (Sensitivity: 52%, specificity: 90%). The threshold of MIP1α was about 411 pg / ml (sensitivity: 70%, specificity: 94%) and the threshold value of MIP1β was about 214 pg / Specificity: 90%) can be exemplified. However, the threshold value may be appropriately changed within a range that does not significantly impair the sensitivity and the specificity.

By using the present invention, it is possible to early diagnose the risk of premature birth below 32 weeks and to prevent premature birth, thereby preventing serious complications and sequelae of newborn infants such as neonatal morbidity and cerebral palsy.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Research subjects

Cervical length of the uterine cervix and maternal cervix protruding out of the uterine cervix are known to be at high risk of infection and this may be the cause of premature birth. Therefore, the uterine cervical cerclage is performed for these women do. Therefore, in this example, if ultrasonography or physical examination performed between 16 and 24 weeks of gestation has a uterine cervical dilatation of more than 1 cm, ) Were studied. Cervical cerclage was not performed when there was vaginal bleeding, regular uterine contractions, and clinical suspicion of chorioamnion before the procedure. Amniocentesis was performed to confirm the decompression and amniotic fluid infiltration before the procedure. In the subsequent cytokine analysis of the membrane type (cytokine array), amniocentesis was performed to determine the presence of chromosomal abnormality between 16 and 20 weeks in order to compare cytokines in amniotic fluid, and normal pregnant women (n = 41) Were also included.

Example  2. Positive numbers cytokine  array and quantitative analysis

To screen for cytokines showing differences in the amniotic fluid between normal (n = 41) and cesarean section (n = 71), 39 cytokines, chemokines, and growth factors in amniotic fluid Membrane-based cytokine assays were performed using RayBio® Membrane-Based Antibody Arrays (C-Series) (RayBiotech, USA). The cytokine array membrane was blocked according to the manufacturer's protocol, and 1 ml of amniotic fluid was added thereto, followed by overnight incubation at 4 ° C. The cells were incubated with biotin-conjugated antibody for 1 hour, washed with streptavidin-binding peroxidase diluted 1: 40,000, and then washed. Membranes were transferred to blotting paper and then detected by LAS-3000 (FUJIFILM Life Sci.Tokyo, Japan). The intensity of the signal was measured using Multi Gauge V 3.0 (FUJIFILM Life Sci.Tokyo, Japan) image analyzer software. The difference in image intensities between the two groups was more than three fold Four species of cytokines of interest were selected from among the cytokines showing about twice the expression difference, and a total of 15 cytokines were selected.

Quantitative analysis of these 15 cytokines was performed using a multiplex bead array using positive amniotic fluid samples of cervical cerclage. Multiplex bead array is an immune response analysis of various cytokines using several beads in one well. Quantitative analysis of various cytokines in a single well was performed and the concentration characteristics of cytokines in preterm infants less than 32 weeks were investigated using this method.

Example  3. Statistical analysis

To determine the predictive index of preterm birth less than 32 weeks, the preterm delivery was divided into two groups: prematurity less than 32 weeks, preterm delivery less than 32 weeks to 37 weeks, and term delivery. The characteristics were analyzed. We analyzed the correlation between leukocyte and 15 cytokines and Spearman 's blood among the clinical features. Sensitivity and specificity of leukocyte and cytokine were measured by receiver-operation chracteristic curve for less than 32 weeks of preterm delivery and were significantly correlated with less than 32 weeks using simple logistic regression analysis. Respectively.

Experiment result

1. Clinical characteristics of patient

(N = 71), who underwent cervical cerclage between 16 and 25 weeks of age, underwent 32 weeks of preterm birth (n = 26) and 32 to 37 weeks of preterm birth , n = 12), and term deliveries (n = 29) over 37 weeks. There were no significant differences in the WBC, neonatal weight, and Apgar score between the three groups at the time of uterine anastomosis (Table 1). Data were not available for the four delivery mothers and were excluded from Table 1, but the amniotic fluid samples of these mothers were also included in the subsequent cytokine array of Membrane type.

Clinical characteristics of study subjects Early preterm birth
(n = 26) Late preterm birth
(n = 12) Term deliveries
(n = 29) P-value 1 vs 2 1 vs 3 2 vs 3 Age (years) 35
(28-41) 35.5
(31-39) 34
(25-39) 0.367 0.568 0.281 WBC number
(cell / mL) 9830
(5760-16740) 8010
(6430-12980) 7600
(5400-14110) 0.043 0.012 0.989 Cervical incompetence 4
(1-10) One
(1-10) One
(1-4) 0.116 0.001 0.259 During pregnancy, 22.2
(18.6-24.5) 22.3
(17.3-25.0) 21.4
(18.3-24.2) 0.329 0.283 0.666 When amniocentesis is performed, 22.2
(18.6-24.5) 22
(17.3-25) 22
(18.3-24.2) 0.281 0.829 0.500 Positive WBC count
(cell / mL) 8
(0-1120) 3.5
(0-100) 3.5
(0-150) 0.199 0.143 0.818 Delivery time (week) 25.2
(19.3-30.5) 35.3
(32.1-36.4) 38.4
(37-41.2) <0.001 <0.001 <0.001 Newborn body weight (g) 780
(360-1570) 2235
(1690-3220) 3430
(2500-4100) <0.001 <0.001 <0.001 1 minute Apgar score 2
(1-10) 7.5
(6-8) 8.5
(6-10) 0.005 <0.001 0.106 5 minute Apgar score 5.5
(1-8) 9
(7-9) 10
(8-10) 0.003 <0.001 0.052

The values shown in the table above mean the median (inter-quartile range)

2. Analysis results of C-series membrane

The relative expression levels of 39 cytokines, chemokines, and growth factors were analyzed using a C-series membrane to screen for cytokines that differ in the amniotic fluid between the mothers who were treated with cervical cerclage and the normal mothers. Eleven species of cytokines expressed more than three times were selected from mothers who underwent uterine artery occlusion (n = 71), and four of the four cytokines Cain was further selected and a total of 15 cytokines were selected (Table 2).

Cytokine analysis using C-series membrane Cytokine Percentage of immune substances in pregnant women who are undergoing normal uterine and uterine anesthesia First choice term (normal mother) preterm O IL-7 One 219.0 O GCSF One 166.0 O IL-6 One 59.4 O MMP9 One 6.1 O MMP8 One 5.7 O MIP-1? One 4.9 O IL-13 One 4.7 O MIP-1? One 4.4 O IL-10 One 4.3 O IL-1? One 4.1 O IL-8 One 3.7 O IL-5 One 2.7 MCP3 One 2.5 IL-17 One 2.3 O IL1-ra One 2.1 IL-15 One 1.9 O TNFα One 1.9 O IL-12 One 1.8 IFN-gamma One 1.7 MMP3 One 1.7 O MCP1 One 1.7 IGF1 One 1.6 IL-2 One 1.6 TGFb3 One 1.6 IL-18BPa One 1.3 Angiopoietin2 One 1.3 Eotaxin One 1.3 IL-9 One 1.2 IL-4 One 1.2 TNFR1 One 1.2 TNFR2 One 1.1 IP-10 One 1.1 MMP2 One 1.0 VRGF One 1.0 IL-18Rb One 1.0 RANTES One 1.0 PDGF-BB One 0.9 FasL One 0.8 MMP1 One 0.8

3. Quantitative analysis results

15 cytokines selected in the C-series membrane analysis were quantitatively analyzed and their concentrations were analyzed. As a result, IL-1β IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-17α, TNFα, G-CSF, MMP8, MMP9, MIP1α, (P <0.05). There was no significant difference in the amniotic fluid concentration of mothers who delivered during term delivery among the mothers who underwent cervical canal harvesting (p <0.05).

The concentration of cytokine (pg / ml) in the amniotic fluid of the mothers who underwent cervical canalization Early preterm birth
(n = 26) Late preterm birth
(n = 12) Term deliveries
(n = 29) P-value 1 vs 2 1 vs 3 2 vs 3 IL-1? 37.9
(0-1125.6) 10.19
(2.2-31.8) 8.9
(0.6-214.7) 0.008 <0.001 0.656 IL-6 28975.8
(500.1-110243.5) 1907.6
(345.7-55065.2) 1503.5
(293.3-109157.8) 0.026 <0.001 0.616 IL-7 132.6
(30.3-348.4) 70.7
(14.7-178.2) 65.2
(19.9-150.7) 0.028 0.001 0.749 IL-8 2187.2
(0-10629.8) 590.1
(135.9-5086.8) 364.1
(0-3232.9) 0.087 <0.001 0.191 IL-10 242.7
(38.9-3973.6) 78.6
(30.6-296.1) 72.6
(24.3-1481.1) 0.067 0.001 0.495 IL-13 86.6
(18.3-251.8) 38.4
(8.3-101.7) 35.4
(10.5-125.4) 0.064 0.006 0.770 IL-15 33.5
(5.1-136.4) 19.1
(1.1-47.1) 16.5
(1.1-63.2) 0.043 0.003 0.845 IL-17a 9.3
(1.5-55.7) 2.4
(0.1-6.6) 1.3
(0.0-20.8) 0.018 <0.001 0.707 TNFα 165.3
(30.5-2423.7) 55.9
(31.9-150.3) 59.16
(35.1-522.2) 0.014 0.003 0.824 G-CSF 5238.5
(837.9-32296.2) 1470.5
(262.6-32296.2) 933.7
(153.6-11682.5) 0.052 <0.001 0.221 MMP3 365.1
(133.6-1287.0) 324.6
(90.2-660.2) 244.6
(152.3-867.2) 0.522 0.115 0.360 MMP8 1213.3
(158.1-5434.2) 183.2
(27.0-2007.7) 138.1
(40.7-3251.9) 0.101 0.001 0.578 MMP9 3.9
(0.0-157.3) 1.2
(0.2-5.6) 0.5
(0.0-8.8) 0.222 0.028 0.531 MIP1α 605.0
(78.1-2001.0) 136.4
(57.0-579.0) 136.2
(47.6-972.8) 0.014 <0.001 0.636 MIP1? 445.1
(79.0-11610.2) 81.1
(63.4-280.8) 88.9
(40.4-2614.8) 0.005 <0.001 0.900

The values shown in the table above mean the median (inter-quartile range)

4. The ROC curve (receiver- operation chracteristic  curve analysis results

Table 4 shows receiver-operation chracteristic curves for sensitivity and specificity for 15 cytokines and clinical features in order to determine predictors of preterm delivery under 32 weeks. In particular, the relationship between white blood cell (WBC) detected in maternal blood and maternal perinatal outcome in 32 weeks of pregnancy was included in the ROC curve. IL-10, IL-13, IL-15, IL-17α, TNFα and G-CSF in the maternal blood leukocyte, cervical incontinence and amniotic fluid , MMP8, MMP9, MIP1α and MIP1β predicted preterm birth of less than 32 weeks (Table 4, p <0.05).

ROC curve analysis results AUC 95% CI P-value Cut-off
(pg / ml) responsiveness
(Sensitivity) Specificity
(Specificity) Serum WBC 0.73 (0.59-0.87) 0.004 13155 44% 97% Cervical incompetence 0.79 (0.66-0.92) <0.001 1.25 78% 77% IL-1? 0.78 (0.65-0.92) <0.001 22.22 65% 87% IL-6 0.84 (0.72-0.95) <0.001 6918.20 78% 84% IL-7 0.80 (0.68-0.92) <0.001 82.15 78% 71% IL-8 0.82 (0.72-0.94) <0.001 854.67 83% 71% IL-10 0.79 (0.67-0.92) <0.001 110.15 78% 71% IL-13 0.75 (0.61-0.88) 0.002 50.39 78% 65% IL-15 0.80 (0.68-0.92) <0.001 25.67 83% 77% IL-17? 0.84 (0.73-0.94) <0.001 6.32 61% 94% TNFα 0.79 (0.66-0.93) <0.001 79.94 74% 84% G-CSF 0.83 (0.72-0.94) <0.001 3246.73 70% 87% MMP3 0.63 (0.47-0.78) 0.113 251.22 83% 52% MMP8 0.82 (0.71-0.93) <0.001 214.36 96% 61% MMP9 0.72 (0.57-0.87) 0.06 3.69 52% 90% MIP1α 0.83 (0.71-0.95) <0.001 411.50 70% 94% MIP1? 0.87 (0.78-0.97) <0.001 214.93 74% 90%

5. Simple Logistics  Regression analysis result

In simple logistic regression analysis, maternal blood leukocyte (WBC), cervical incompetence and amniotic fluid IL-1β IL-6, IL-7, IL-8, IL-10, , IL-17α, TNFα, G-CSF, MMP8, MMP9, MIP1α and MIP1β predicted preterm birth of less than 32 weeks (Table 5, p <0.05).

Simple logistic regression analysis Simple logistic regression analysis Odd Ratio 95% confidence interval (CI) p value Serum WBC 24.38 (2.88-206.43) 0.003 Cervical incompetence 7.18 (2.33-22.11) 0.001 IL-1? 9.28 (2.98-28.90) <0.001 IL-6 13.02 (3.89-43.58) <0.001 IL-7 7.28 (2.27-23.32) 0.001 IL-8 10.61 (3.05-36.86) <0.001 IL-10 6.43 (2.10-19.66) 0.001 IL-13 6.56 (2.06-20.93) 0.001 IL-15 10.15 (3.10-33.19) <0.001 IL-17? 11.52 (3.39-39.19) <0.001 TNFα 10.33 (3.25-32.89) <0.001 G-CSF 9.65 (3.09-30.15) <0.001 MMP3 0.23 (0.0-0.72) 0.012 MMP8 28.94 (3.58-234.25) 0.002 MMP9 5.83 (1.73-18.57) 0.003 MIP1α 20.81 (5.53-78.36) <0.001 MIP1? 13.18 (4.02-43.27) <0.001

Claims (10)

delete delete delete delete delete Measuring the concentration of IL-13 (Interleukin-13) from the amniotic fluid sample of the mother, and
Predicting that there is a risk of premature birth of less than 32 weeks if the concentration of IL-13 is higher than a normal mother or higher than a cut-off.
Methods of providing maternal information for the diagnosis of premature births below 32 weeks.
delete 7. The method of claim 6, wherein the measurement of the concentration of IL-13 is performed using an antibody specific for IL-13.
9. The method of claim 8, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
The method according to claim 6, wherein the amniotic fluid sample of the mother is composed of IL-1 ?, IL-6, IL-8, IL-10, IL-15, IL-17 ?, TNF ?, G- CSF, MMP8, MMP9, MIP? Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;