JPH07325082A - Method for distingushing secretion from vagina or mucus of cervix of pregnant woman with danger of urgent premature birth - Google Patents

Abstract

PURPOSE:To detect the material as a character of the premature birth so as to distinguish a pregnant woman with a danger of urgent premature birth by measuring quantity of cytokinin in the secretion from vagina or the mucus of cervix. CONSTITUTION:Since A rise of the concentration of cytokinin in the secretion from vagina or the mucus of cervix is a special character of a pregnant woman with a danger of urgent premature birth, the secretion from vagina of a pregnant woman with a danger of urgent premature birth can be distinguished by measuring quantity of cytokinin. As a method for measuring quantity of cytokinin, any one of bioassay and immunological measuring method can be used, especially, the imunological measuring method is desirable. As the cytokinin to be measured, interleukin-6, interleukin-8 and interleukin-1 or the like is known. The measured quantity of cytokinin is compared with the quantity of cytokinin of a normal pregnant woman, and the secretion from vagina of the pregnant woman with a danger of urgent premature birth has a higher value. A pregnant woman with a danger of urgent premature birth can be distinguished from a normal pregnant woman by measuring cytokinin in the secretion from vagina or the mucus of cervix of the pregnant woman.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is useful for detecting preterm birth.
The present invention relates to a method for differentiating vaginal secretions or cervical mucus in pregnant women with urgency.

[0002]

2. Description of the Related Art Premature birth is an abnormality associated with pregnancy, and the causes thereof are various, but are mainly ascending infection, intrauterine infection, chorioamnionitis, and early placental ablation. In case of infection or chorioamnionitis, blood C-reactive protein (C
Since the RP) concentration and the number of white blood cells increase, the preterm labor test is carried out by measuring these.

However, since CRP concentration and white blood cell count are derived from blood, they are increased by factors other than preterm birth, and it is difficult to say that they are specific to preterm birth only. Further, there is a problem that CRP is only detected in a very small amount in blood and the sensitivity of measurement is low. Thus, it was difficult to diagnose preterm birth early by measuring the CRP concentration and white blood cell count in blood.

On the other hand, a method of diagnosing premature abortion by measuring interleukin-6 (IL-6) in blood has recently been reported (Japanese Patent Application No. 5-209882).
Issue). However, even with this method, since the sample is maternal blood, it was not sufficiently satisfactory in terms of sensitivity and specificity.

[0005]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to search for a substance that serves as an index for preterm birth and establish a discrimination method for the substance.

[0006]

[Means for Solving the Problems] In view of such circumstances, as a result of intensive studies by the present inventors, an increase in cytokine concentration in vaginal secretion or cervical mucus is specific to an imminent preterm pregnant woman. It was found that the vaginal secretions and the like of pregnant women with imminent preterm birth can be distinguished by measuring the amount of cytokines, and the present invention was completed.

[0007] That is, the present invention provides a method for distinguishing vaginal secretions or cervical mucus from pregnant women with impending preterm birth characterized by measuring the amount of cytokines in vaginal secretions or cervical mucus.

The specimen used in the present invention may be either vaginal fluid or cervical mucus. The method for collecting these is not particularly limited as long as it can be collected so as not to damage the vagina or cervical canal, and for example, a tube such as a silicon tube or an injection needle is attached to the tip of the injection cylinder, A method of inserting the syringe into the vagina and collecting it; a method of inserting the syringe itself into the vagina and collecting; a method of inserting an absorbent material such as a cotton swab into the posterior fornix and absorbing the cervical mucus and the like. To be Of these, the method using a cotton swab is preferred because it is simple and particularly, the use of Dacron cotton swab is more preferable because the extraction efficiency is improved. In the case of collecting a sample using a cotton swab, it is preferable to first remove impurities in the vagina with a cotton ball or the like, and confirm the contents with a colposcope.

The specimen thus collected can be directly used for the measurement, but the vaginal secretion fluid and cervical mucus stock solution have a very high viscosity and are often in the form of a sol or clot. It is preferable to use these after diluting or extracting them. It is preferable to use a buffer solution having a pH of 6 to 8 for dilution or extraction, and examples thereof include a phosphate buffer solution, a Tris buffer solution and a Good's buffer solution.
More preferably, it is in the range of 7.2 to 7.8.

To dilute or extract a sample using these buffer solutions, for example, when the sample is collected using an injection cylinder, the sample is crushed so as to be cut, and this is mixed with the buffer solution. It is preferable to extract the cytokine in the sol-like or clot-like sample into a buffer solution. In this case, the amount of the buffer solution used is about 5 times the amount of the sample or more.
It is preferable for performing sufficient extraction. When a sample is collected using a cotton swab, the cotton swab absorbing the sample is immersed in a buffer solution, and the buffer solution is stirred with this cotton swab,
It is preferable to extract the sample in a buffer solution.

As a method for measuring the amount of cytokine in vaginal secretion or cervical mucus, any known method can be used as long as the amount of cytokine can be measured, such as bioassay method and immunoassay method. In particular, it is preferable to use an immunological quantification method. The cytokine measured here is interleukin-6 (IL
-6), interleukin-8 (IL-8), interleukin-1 (IL-1), and the like, and by measuring one or more of these, vaginal secretions of an imminent preterm pregnant woman, etc. Can be distinguished. Of these cytokines, IL-6 and IL-8 are particularly preferably measured.

For example, in order to measure IL-6 in a sample by an ELISA method, a conventional method may be used. For example, a 96-well microtiter plate may contain a monoclonal antibody specific to the first antibody, ie, IL-6, or By sensitizing a polyclonal antibody as a solid phase and reacting this with a sample, the antibody on the solid phase and IL-6 in the sample are reacted.
Combine. Then, after removing unreacted proteins by washing operation, a second antibody, that is, an enzyme-labeled monoclonal or polyclonal antibody specific for IL-6 is reacted to form a sandwich by an antigen-antibody reaction mediated by IL-6. Let After removing the excess second antibody by washing, a substrate solution for the labeling enzyme of the second antibody is added to develop color, and the enzymatic reaction is stopped with a stop solution. Then, the absorbance is measured with an absorptiometer, and the IL-6 concentration in the sample may be converted from the calibration curve of the standard product.

Interleukin-8 (IL-8)
Also, other cytokines can be measured in the same manner as in the method for measuring IL-6. In addition, commercially available IL-
6. A sandwich ELISA kit for measuring IL-8 concentration can also be used.

The amount of cytokine thus measured can be used to distinguish vaginal secretions or cervical mucus from an imminent premature pregnant woman, and specifically, it shows a high value in comparison with the amount of cytokines from a normal pregnant woman. Can be vaginal secretions of an imminent preterm pregnant woman. Thus, by measuring cytokines in vaginal secretions or cervical mucus of pregnant women,
Can distinguish between normal pregnant women and pregnant women with impending preterm birth.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, vaginal secretions or cervical mucus of a pregnant woman with an imminent preterm birth can be distinguished with high sensitivity, and it is useful for detection, diagnosis and treatment of preterm birth.

[0016]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 IL-6 and IL-8 levels in the vaginal secretions, CRP levels in serum and leukocytes in blood of 8 normal pregnant women of different gestational ages and 8 pregnant women diagnosed with imminent preterm birth. The numbers were measured as follows. The results are shown in Tables 1 and 2 and FIGS. 1 and 2.

Sample collection method: A Dacron swab was inserted into the vaginal dome of the posterior part of a pregnant woman, and the vaginal secretion was absorbed while rotating the swab for about 10 seconds, and 1 ml of the extract (50 mM 2-amino-2-) was used. Hydroxymethyl-1,3-propanediol, 1% BSA, 0.89% sodium chloride, 0.
2% disodium ethylenediaminetetraacetate, 1 μl /
ml aprotinin, 1 μl / ml dimethylsulfoxide, 0.17 mg / ml phenylmethylsulfonylfluoride, 1 μl / ml polyoxyethylenoctylphenyl ether, 0.5 mg / ml sodium azide; pH 7.
The vaginal secretions absorbed by the Dacron swab were extracted by soaking in 5) and stirring the extract with a Dacron swab.
This was used as a sample. In addition, it diluted with the extraction liquid as needed and used.

IL-6 measuring method: Quantik by R & D
IL-6 in the vaginal fluid was measured as follows according to the method of measuring ine. That is, first, mouse anti-IL-6
After 100 μl of the assay diluent was dispensed into each well of the microtiter plate sensitized with the monoclonal antibody, 100 μl of the sample was added. Then, after allowing to stand at room temperature for 2 hours, 400 μl of each well of the washing solution
Each was washed 3 times. After removing the washing solution, 200 μl of horseradish peroxidase (HRP) -labeled anti-IL-6 polyclonal antibody solution was added to each well, and the reaction was allowed to stand at room temperature for 2 hours. 3 with 400 μl of washing solution
After washing twice, 200 μl of a coloring solution using tetramethylbenzidine as a substrate was added to each well, and the reaction was allowed to stand at room temperature for 20 minutes.
The enzyme reaction was stopped by adding 0 μl each. The measurement wavelength of the microtiter plate reader was set to two wavelengths of 450 nm and 550 nm, and the absorbance was measured. IL-6 in the sample
The concentration was converted from the standard curve.

IL-8 measuring method: Quantik by R & D
IL-8 in the vaginal fluid was measured as follows according to the method for measuring ine. That is, first, mouse anti-IL-8
After 100 μl of the assay diluent was dispensed into each well of the microtiter plate sensitized with the monoclonal antibody, 100 μl of the sample was added. Then, after allowing to stand at room temperature for 2 hours, 400 μl of each well of the washing solution
Each was washed 3 times. After removing the washing solution, 200 μl of horseradish peroxidase (HRP) -labeled anti-IL-8 polyclonal antibody solution was added to each well, and the reaction was allowed to stand at room temperature for 2 hours. 3 with 400 μl of washing solution
After washing twice, 200 μl of a coloring solution using tetramethylbenzidine as a substrate was added to each well, and the reaction was allowed to stand at room temperature for 20 minutes.
The enzyme reaction was stopped by adding 0 μl each. The measurement wavelength of the microtiter plate reader was set to two wavelengths of 450 nm and 550 nm, and the absorbance was measured. IL-8 in the sample
The concentration was converted from the standard curve.

On the other hand, the CRP concentration is a commercially available kit using the serum of a pregnant woman as a sample (Yatron, latex agglutination method).
It was measured at. The white blood cell count was measured by an automatic hemocytometer manufactured by Sysmex Co. using pregnant woman blood.

[0022]

[Table 1]

[0023]

[Table 2]

From the above results, the concentration of IL-6 in the vaginal fluid in the normal pregnant woman was 6 or less in the 8 cases, which was less than the detection limit (0.03 ng / ml), but it was 0 in the imminent premature birth group. ．
42 to 13.0 ng / ml, which is significantly (p <0.001) high by Mann-Whitney test, and differentiates vaginal fluid in imminent preterm pregnant women by measuring the amount of IL-6 in vaginal fluid Therefore, it is found that preterm birth can be detected (Table 1, Table 2, FIG. 1). In addition, the concentration of IL-8 in the vaginal fluid is 1.5 to 12.1 ng / ml in a normal pregnant woman, but is 41.5 to 1680 ng / ml in the imminent preterm birth group.
Significant by nn-Whitney test (p <0.001)
It can be seen that the vaginal secretions of imminent preterm pregnant women can be differentiated by measuring the amount of IL-8 in the vaginal secretions, and that preterm birth can be detected. (Table 1, Table 2, FIG. 2).

In addition, the CRP concentration in serum was abnormal (0.8
6 out of 8 in the threatened preterm labor group.
Existence was present and no effective preterm birth was detected at CRP levels in the blood. Normal white blood cell count (4000-80
00 / mm ³ ) was shown in 3 out of 8 cases of imminent premature birth group, and the examination of white blood cell count could not detect all imminent preterm birth.

Example 2 FIG. 3 shows a case in which a preterm birth was diagnosed at 25 weeks of gestation and tocolysis treatment was performed, and the preterm birth was reached on the 5th day. On the vertical axis, IL-6 and IL-8 concentrations in vaginal secretions and serum C
The RP concentration is shown, and the horizontal axis is the number of days from the first medical examination day. The IL-6 and IL-8 concentrations in the vaginal secretions of pregnant women were measured by extracting a sample, extracting the sample in the same manner as in Example 1. The IL-6 concentration was measured by using an IL-6 measuring ELISA kit (manufactured by R & D)
L-8 concentration is the ELISA kit for IL-8 measurement (R & D
Manufactured by the company). As a result, IL-6 and IL-8
Both the concentrations continued to rise, leading to preterm birth, and an increase in the concentrations of IL-6 and IL-8 in the vaginal secretion fluid was consistent with clinical symptoms. However, the CRP concentration in serum, which is a conventional method, did not become an abnormal value until immediately before preterm birth.

Example 3 FIG. 4 shows an example in which the patient was diagnosed as having an impending preterm birth at 30 weeks of gestation and reached to term delivery by treatment with tocolysis. The vertical axis shows IL-6 and IL-8 concentrations in vaginal secretions and CRP in serum,
The horizontal axis is the number of days from the first visit. IL in vaginal secretions of pregnant women
For -6 and IL-8 concentrations, samples were collected in the same manner as in Example 1,
Extracted and measured. As the symptoms stabilize, IL-6,
The IL-8 concentration showed a decreasing tendency. Measurement of IL-6 and IL-8 concentrations in vaginal secretions was effective in monitoring the efficacy of treatment.

Example 4 The changes in the concentrations of IL-6 and IL-8 in the serum of the imminent preterm labor group and IL-6 and IL-8 in the vaginal secretion were measured according to Example 1. The results are shown in Table 3.

[0029]

[Table 3]

The amounts of IL-6 and IL-8 in serum are small, which is near the lower limit of the measurement range, but IL-
6, IL-8 is present in a sufficient amount for measurement. Therefore, by using a vaginal secretion that is close to the local area as a sample, it is possible to distinguish vaginal secretions of a pregnant woman with imminent preterm birth with a small amount of sample, and premature birth can be detected with higher sensitivity than serum, plasma and blood.

Example 5 At the 30th week of pregnancy, the patient was diagnosed as having an impending preterm birth and treated by tocolysis. However, IL-6 and IL-8 were treated in the same manner as in Example 1 in the case where the preterm birth was performed by Caesarean section on the 20th day. Was measured. The results are shown in Table 4.

[0032]

[Table 4]

From the test results of the first examination day, IL in vaginal secretions
-6 and IL-8 showed high concentrations that can be judged to be abnormal,
Serum CRP concentration and blood leukocyte count were in the normal range. After that, the levels of IL-6 and IL-8 remained high, leading to preterm birth, but the white blood cell count showed abnormalities on the 10th day after the initial diagnosis, and the CRP concentration became abnormal immediately before preterm birth. As described above, by measuring the IL-6 and IL-8 concentrations in the vaginal secretion, the vaginal secretion of a pregnant woman with an imminent preterm birth can be distinguished, and the CRP concentration in serum and the white blood cell count in blood can be measured. Premature birth can be detected earlier.

[Brief description of drawings]

FIG. 1 is a diagram comparing the IL-6 concentration in vaginal secretion between a normal pregnant woman group (left) and an imminent preterm birth group (right).

FIG. 2 is a diagram comparing the IL-8 concentration in vaginal secretions between a group of normal pregnant women (left) and a group of preterm labor (right).

FIG. 3 is a diagram showing changes in IL-6 and IL-8 concentrations in vaginal secretions and CRP concentrations in serum of a pregnant woman who was diagnosed as having an impending preterm birth and was treated, but who reached the preterm birth.

FIG. 4 is a diagram showing changes in IL-6 and IL-8 levels in vaginal secretions and CRP levels in serum of a pregnant woman who was diagnosed as having an impending preterm birth and recovered by treatment and delivered normally at term.

Claims (4)

[Claims] 1. A method for differentiating vaginal secretions or cervical mucus in an imminent preterm pregnant woman, which comprises measuring the amount of cytokines in vaginal secretions or cervical mucus. 2. A method for differentiating vaginal secretions or cervical mucus in an imminent preterm pregnant woman, which comprises measuring the amount of cytokines in vaginal secretions or cervical mucus and comparing it with that of normal pregnant women. 3. The collected vaginal secretion or cervical mucus is collected at a pH of 6 to 6.
The method according to claim 1 or 2, wherein the amount of cytokine is measured after diluting or extracting with the buffer solution of 8. 4. The discrimination method according to claim 1, wherein the cytokine is interleukin-6 or interleukin-8.