JP4103163B2 - Differentiation of cervical mucus in pregnant women with imminent premature delivery or asthenia gravis - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a method for differentiating cervical mucus in a pregnant woman with imminent premature labor or a pregnant woman with cervical incompetence.
[0002]
[Prior art]
There are premature births as abnormalities associated with pregnancy, and the causes are wide-ranging, but mainly ascending infections, intrauterine infections, chorioamnionitis and early placenta deprivation. In the case of infection or chorioamnionitis, the blood C-reactive protein (CRP) concentration and white blood cell count in the maternal blood increase, so these measurements are used to test preterm birth.
[0003]
However, since the CRP concentration and the white blood cell count are derived from blood, they are increased by other factors other than premature birth, and it is difficult to say that they are specific only for premature birth. In addition, CRP is detected only in trace amounts in blood, and there is a problem that the sensitivity of measurement is low. Therefore, it is difficult to diagnose premature labor at an early stage by measuring the CRP concentration or white blood cell count in the blood.
[0004]
On the other hand, recently, a method for diagnosing premature labor by measuring interleukin-6 (IL-6) in blood has been reported (Japanese Patent Laid-Open No. 5-209882). However, even in this method, since the specimen is maternal blood, it is not satisfactory in terms of sensitivity and specificity.
[0005]
Recently, a method for diagnosing premature birth by measuring cytokines in cervical mucus has been reported (Japanese Patent Laid-Open No. 7-325082). However, even in this method, cytokine production may be caused by other diseases, which is not satisfactory in terms of specificity.
[0006]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to search for a substance that serves as an index of premature birth and establish a discrimination method thereof.
[0007]
[Means for Solving the Problems]
As a result of intensive studies in view of the above circumstances, the present inventors have obtained the following knowledge. In other words, it is becoming clear that cervical ripening closely resembles the local inflammatory response. The knowledge that it is deeply involved in.
[0008]
The present invention has been completed as a result of further studies based on the above findings, and the gist of the present invention is to measure the hyaluronic acid concentration and / or hyaluronidase activity in the cervical mucus, It exists in the method for differentiating cervical mucus in imminent pregnant women with imminent or cervical incompetence.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail. In the present invention, cervical mucus is used as a specimen. The method for collecting such a specimen is not particularly limited as long as it does not damage the vagina, the cervix, or the like. A method of collecting a sample by inserting a cotton swab into the vagina or the like and absorbing the sample with the cotton swab is simple and preferable. In particular, Dacron swabs are preferably used because of their excellent absorption efficiency. When collecting a specimen with a cotton swab, it is preferable to remove the impurities in the vagina with a cotton ball or the like and then insert the cotton swab into the vagina while confirming with a colposcope to collect the specimen.
[0010]
The collected specimen can be used for measurement as it is, but the stock solution of cervical mucus is highly viscous and is often sol or clotted. It is preferable to do this. For the dilution or extraction treatment, a buffer solution having a pH of 6 to 8 (preferably 7.2 to 7.8) is suitably used. Examples of such a buffer solution include a phosphate buffer solution, a Tris buffer solution, and a good buffer solution.
[0011]
The dilution or extraction process is preferably performed as follows. That is, for example, when a sample is collected with a cotton swab, the swab on which the sample has been absorbed is immersed in a buffer solution, and the buffer solution is stirred with this cotton swab, so that hyaluronic acid (HA) and alcohol in the sol-like or clot-like sample are stirred. Ronidase is extracted into the buffer.
[0012]
As a method for measuring the HA concentration, the following known methods can be employed. For example, as a binding assay method using a hyaluronic acid binding protein, a sandwich method, an inhibition method (competitive method) and the like are known. In the sandwich method, HA in the sample is sandwiched between hyaluronic acid binding protein and labeled HA binding protein to form a sandwich-like conjugate, and the HA concentration is determined by measuring the labeling substance of the conjugate. As a measurement method (for example, Japanese Patent Publication No. 6-41952) and an inhibition method (competition method), hyaluronic acid binding protein is allowed to compete with HA in the sample for labeled HA, and hyaluronic acid binding protein A method of measuring either the bound labeled HA or the unbound labeled HA and measuring the HA concentration in the specimen (for example, JP-A-63-150669) can be employed. As the hyaluronic acid binding protein, hyaluronic acid binding protein (HABP), CD44, anti-hyaluronic acid antibody and the like that specifically bind to HA are preferably used. In particular, a sandwich method using solid phase HABP or CD44 and labeled HABP is preferred.
[0013]
In addition, the following method using high performance liquid chromatography (HPLC) is also preferable in that HA can be accurately measured. That is, HA in a specimen is decomposed with an HA-degrading enzyme, and the resulting hyaluronic acid oligosaccharide is separated and analyzed by HPLC. At this time, an anion exchange column or the like is used as the column. If necessary, labeling with a fluorescent labeling substance is performed to increase the sensitivity in HPLC.
[0014]
In addition to the above, a method in which separation analysis is performed by electrical permanence (capillary electrical perturbation, electrical perturbation using an acetate membrane, etc.) instead of HPLC is also preferred.
[0015]
On the other hand, hyaluronidase activity was measured by (1) Park-Johnson method in which Fe ²⁺ in which Fe ³⁺ was reduced by a reducing group of sugar under alkaline conditions was measured by a colorimetric method, and (2) Hyaluro. In addition to the turbidity reduction method, which measures the decrease in turbidity caused by the degradation of HA with a kneadase by absorbance, (3) HA labeled with a radioisotope, fluorescent substance, chemiluminescent substance, etc. is used as a substrate. In addition, a method of measuring a labeled hyaluronic acid degradation product degraded by hyaluronidase in a specimen can be employed. In particular, the above method (3) using fluoresceinamine as a fluorescent substance is preferable in that the sensitivity is excellent even when the amount of specimen is very small.
[0016]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.
[0017]
Cervical mucus was collected from 60 normal pregnant women with different gestational age, 10 pregnant women diagnosed as imminent premature delivery, and 7 pregnant women diagnosed as cervical incompetence. Concentration was measured. Further, cervical mucus was collected from the following pregnant women different from the above, that is, 56 normal pregnant women and 22 pregnant women diagnosed as imminent premature delivery, and the HA concentration was measured by the measurement method (3) below. Furthermore, cervical mucus of the following pregnant women different from the above, that is, 45 normal pregnant women with different gestational age, 7 pregnant women diagnosed with imminent premature labor, and 7 pregnant women diagnosed with cervical incompetence. The hyaluronidase activity was collected by the measurement method of (4) below. The results are summarized in (5) below.
[0018]
(1) Sample collection method:
Insert a cotton swab into the cervical canal of each pregnant woman and absorb the cervical mucus into the swab while rotating it. Absorbed cervical mucus was extracted and used as each sample. The sample was diluted with PBS as necessary.
[0019]
(2) Measurement of HA concentration by HPLC:
100 μl of each sample that was sufficiently stirred in an eppendle tube was collected, 20 μl of 5 U / ml chondroitinase ABC (Seikagaku Corporation) was added, and after light stirring, digested at 37 ° C. for 2 hours. Thereafter, 20 μl of 5 U / ml chondroitinase ACII (manufactured by Seikagaku Corporation) was added, 20 μl of 1 M sodium acetate buffer (pH 6.0) was added, lightly stirred, and digested at 37 ° C. for 2 hours. . This digestion breaks down HA into unsaturated disaccharides (ΔDi-HA). After completion of the digestion, the entire amount of the digested solution was treated with a centrifugal ultrafiltration tube having a fractional molecular weight of 10,000 under the condition of 12000 rpm for 15 minutes, and the unsaturated disaccharide was recovered on the filtrate side. And unsaturated disaccharide was quantified by HPLC and the concentration of HA was calculated. Each sample was prepared by extracting cervical mucus absorbed on a cotton swab with 1 ml of PBS. Since the dilution rate of each sample was not constant, the calcium concentration in each sample was measured, and the calcium concentration in human serum was measured. The dilution rate of each sample was calculated, and the concentration of HA was corrected. The calcium concentration was measured using “Calcium C-Test Wako” manufactured by Wako Pure Chemical Industries.
[0020]
(3) Measurement of HA concentration by sandwich method:
Using a commercially available HA measurement kit, the HA concentration of each sample diluted with PBS was measured by the sandwich protein binding method. As the kit, hyaluronic acid “Chugai” (trade name) manufactured by Chugai Pharmaceutical Co., Ltd., which is commercially available as a kit for measuring high molecular weight HA by the sandwich method, was used.
[0021]
(4) Measurement of hyaluronidase activity:
20 μl of each well-stirred sample was taken in each of two eppendle tubes. One sample was treated at 100 ° C. for 5 minutes to inactivate hyaluronidase in the sample. 20 μl of 100 mM sodium acetate buffer (pH 4.0) and 20 μl of hyaluronic acid labeled with fluoresceinamine (FA-HA) (100 μg / ml) were added to each sample treated at 100 ° C. and untreated samples. It was allowed to stand for 24 hours at ° C. Thereafter, each sample was treated with a 0.45 μm centrifugal ultrafiltration tube at 12000 rpm for 15 minutes. And gel filtration was performed by HPLC, FA-HA was detected with the fluorescence detector, and the difference ((DELTA) Time) of retention time of FA-HA of each sample was computed. Since the hyaluronidase activity is stronger, FA-HA is decomposed and the molecular weight of HA becomes smaller, so ΔTime increases.
[0022]
(5) Results:
<About imminent pregnant women>
(I) The measurement result of HA concentration by the method of (2) is 2.4 ± 1.2 μg / ml in normal pregnant women (12 to 36 weeks), and 3.9 ± in imminent pregnant women (21 to 30 weeks). 3.7 μg / ml. Moreover, the measurement result of the hyaluronidase activity by the method of (4) was 1.8 ± 1.6 min. , 2.6min. For imminent pregnant women. Met. From these results, the following can be understood. That is, in the case of a pregnant woman diagnosed as imminent premature delivery, the HA concentration and hyaluronidase activity are higher than those in normal pregnant women at the same time (12 to 36 weeks), so the HA concentration or hyaluronidase in cervical mucus By measuring the activity, it is possible to differentiate cervical mucus from a pregnant woman with imminent premature birth and diagnosis of imminent premature birth is possible. Such a fact is clear also from the measurement result (FIG. 1) of the HA concentration by the method (3).
[0023]
<About pregnant women with cervical incompetence>
(Ii) In the case of a pregnant woman with cervical incompetence, the measurement result of HA concentration by the method of (2) is 1.8 ± 0.6 μg / ml, and the measurement result of hyaluronidase activity by the method of (4) is 0. .9 ± 1.0 min. Met. From these results, the following can be understood. That is, in the case of a pregnant woman diagnosed with cervical incompetence, the HA concentration and hyaluronidase activity are lower than in a normal pregnant woman at the same time (12 to 36 weeks). By measuring the hyaluronidase activity, the cervical mucus of pregnant women with cervical asthenia can be differentiated, and cervical asthenia can be diagnosed.
[0024]
【The invention's effect】
According to the present invention described above, the hyaluronic acid concentration and / or hyaluronidase activity in cervical mucus is measured and compared with the normal value, so that Identification can be performed.
[Brief description of the drawings]
FIG. 1 is a graph comparing hyaluronic acid concentrations in cervical mucus of normal and imminent pregnant women

Claims (1)

A method for distinguishing cervical mucus from an impending premature pregnant woman or a cervical incompetent pregnant woman characterized by measuring hyaluronic acid concentration and / or hyaluronidase activity in cervical mucus and comparing with normal values.

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