WO2000023613A1

WO2000023613A1 - A method of assessing a matter associated with parturition in a pregnant individual

Info

Publication number: WO2000023613A1
Application number: PCT/AU1999/000911
Authority: WO
Inventors: Michael Agrez
Original assignee: The University Of Newcastle Research Associates Limited
Priority date: 1998-10-19
Filing date: 1999-10-19
Publication date: 2000-04-27
Also published as: AUPP656298A0

Abstract

The invention relates to a method of assessing a matter associated with parturition in a pregnant individual. The method involves the steps of measuring the level of at least one matrix proteinase in a body fluid sample from the individual to obtain test data and utilising the test data to make the assessment. The body fluid used in the method will typically be urine obtained from the relevant individual.

Description

A METHOD OF ASSESSING A MATTER ASSOCIATED WITH

PARTURITION IN A PREGNANT INDIVIDUAL

Field Of The Invention

The present invention relates generally to a method of screening a pregnant

individual for the onset of parturition in the individual and for other matters relating to

parturition. The method has broad application and finds use both in the hospital or clinic

environment and elsewhere.

Background Of The Invention

Pre-term delivery (PTD) before 37 weeks gestation (due to premature labour,

antepartum haemorrhage and pre-term rupture of membranes) is a leading cause of

perinatal morbidity and mortality in developed countries. Premature rupture of the

membranes occurs in 10% of women at term and is also associated with 30 - 40%o of pre-

term deliveries. In most cases when a woman presents with premature symptoms of

labour, it is desirable to suppress labour and prevent delivery. Although one in seven

women develop such symptoms during the course of pregnancy, most women (up to

70%) will not progress to actual delivery. This is a significant clinical problem as

currently up to 70% of patients admitted with threatened pre-term labour will, therefore,

receive unnecessary treatment with agents that suppress labour and which may have

significant side-effects. Moreover, it is estimated that 300,000 cases of premature

rupture of the membranes occurs each year and that the treatment of the mother and the

baby account for about 2 billion dollars in health care costs every year in the United States. Premature rupture of the membranes presents, therefore, a serious management

problem because its occurrence is difficult to predict.

Although the incidence of pregnant women experiencing symptoms of premature

labour is relatively high, the early prediction of which of these women are likely to

proceed to premature deliver is still relatively in accurate.

The presence of foetal fibronectin in vaginal secretions of pregnant women with

premature uterine contractions has been suggested as a marker of imminent premature

delivery. However, testing for the presence of cerivcofoetal fibronectin while having

useful negative predictive value, at best only has moderate positive predictive value

(20 - 30%) with numerous false-positive results occurring (Lukes et al, 1997; Giles et al,

1998). Accordingly, the test is not one suitable for basing clinical decisions on in

relation to the choice of treatment for women presenting with symptoms of premature

labour.

The usefulness of markers of imminent delivery is relatively limited since there is

little time within which to assess the implications of a pre-term delivery and/or to act

upon whatever options are available to the pregnant individual. As such, there is a need

to develop methods for more readily identifying those pregnant women that are unlikely

to reach full term and in particular, for identifying which pregnant women that are

exhibiting premature symptoms of labour are likely to have a premature delivery and

those in which such symptoms are likely to resolve.

Collagen Types I and V are amongst the major extracellular components of the

foetal membrane and Type IV collagen is also the major component of the basement

membrane and is present in the spongy and reticular layers of human foetal membranes - j -

(Malak et al, 1993). Matrix metalloproteinases (MMPs) are a family of zinc-dependent

endopeptidases which degrade these extracellular matrix macromolecules. Matrix

metalloproteinase- 1 (MMP-1 ) also known as interstitial collagenase, degrades collagen

Types I, II and III, whereas degradation of Type IV collagen is carried our by the Type

IV collagenases/gelatinases matrix metalloproteinase-2 (MMP-2) and matrix

metalloproteinase-9 (MMP-9) (Matrisian, 1990; Woessner, 1991). MMP-9 additionally

degrades collagen Type V and elastin. MMP-2 also acts on collagen Type V. VII and X

and on elastin. Accordingly, the co-operative actions of several matrix

metalloproteinases are generally required for extracellular matrix catabolism.

Matrix matalloproteinases are secreted as inactive zymogens that are subsequently

cleaved to release the active form of the enzyme, and their activity is regulated by

specific tissue inhibitors. The expression of a number of matrix metalloproteinases

(MMP-1 , MMP-2, MMP-3 (stromelysin) and MMP-9) as well as tissue inhibitor of

metalloproteinase-1 (TIMP-1) and inhibitor of metalloproteinase-2 (TIMP-2) has been

demonstrated in human foetal membranes (Murphy et al, 1992; Fernandez et al, 1992;

Vadillo-Ortega et al, 1995; Rowe et al, 1997).

In recent years, elevated levels of matrix metalloproteinases in blood plasma and

urine have been reported to be associated with human malignancies and implicated in

mechanisms of tumour metastasis (Moses M.A. et al, 1998). In that study, the presence

of matrix metalloproteinase-2 (MMP-2) or matrix metalloproteinase-9 (MMP-9) in urine

was found to correlate with the presence of malignant disease in patients. Those authors

further suggested that elevated levels of matrix metalloproteinases in urine may be useful as predictors of disease status for patients with a variety of cancers not just tumours

confined within the genitourinary tract.

Elevated MMP-9 levels have also been identified in foetal membranes during

labour (Romero et al, 1992), and in amniotic fluid in association with normal labour

(Vadillo-Ortega et al. 1995) and premature rupture of membranes (Vadillo-Ortega et al,

1996). Recently it has been shown that women with premature symptoms of labour who

delivered prematurely had higher concentrations of MMP-9 in amniotic fluid than those

with pre-term labour who delivered at term (Vadillo-Ortega et al, 1996). It has further

been reported that MMP-9 may be involved in the process of cervical ripening and

dilatation due to matrix re-modelling (Osmers et al, 1995). MMP-2 has been shown to

be present in extracts of foetal membranes (amniochorion) from both pre- and post-

labour membranes (Vadillo-Ortega et al 1995; Fortunato et al. 1997).

In addition, the level of MMP-9 in the plasma of blood taken from pregnant

women at various stages of pregnancy has been measured (Tu. F.F et al, 1998). In that

stud)', while an increased MMP-9 level was observed in samples drawn from women in

spontaneous labour presenting for delivery, levels in samples drawn in the week prior to

delivery were found not to be significantly different to those measured earlier in

pregnancy. Moreover, no differences in prenatal MMP-9 levels between women

delivering preterm and at term were able to be demonstrated. Indeed, it was suggested

by those authors that MMP-9 is a poor candidate for an effective predictor of premature

labour and parturition due to tight regulation of its expression and in view of the results

obtained. Summary Of The Invention

It is an aim of the present invention to ameliorate one or more problems of the

prior art or to at least provide a method useful for assessing a matter relating to

parturition.

The invention is based on the recognition by the instant inventors that matrix

proteinases are present in the urine of pregnant individuals during parturition and the

finding that there is a correlation between increased levels of one or more such

proteinases in urine and parturition.

In a first aspect of the invention there is provided a method of assessing a matter

associated with parturition in a pregnant individual, comprising the steps of measuring

the level of at least one matrix proteinase in a body fluid sample from the individual to

obtain test data and utilising the test data to make the assessment.

The matter associated with parturition may be selected from the following matters:

(a) the onset of parturition in the individual in which case the assessing

comprises determining the onset of same;

(b) a likelihood of the onset of parturition in the individual;

(c) a likelihood that parturition or the onset of parturition in the individual will

resolve;

(d) a likelihood of medical induction of the onset of parturition in the individual

being successful;

(e) a likelihood of the onset of parturition in the individual resolving if

parturition is medically induced in the individual; and

(f) progress of parturition in the individual. The onset of parturition in the individual referred to in (a) above may or may not

be associated with symptoms indicating the onset of parturition. It is also to be

understood that assessing the likelihood of the onset of parturition referred to in (b)

includes the instance where the onset of parturition has not commenced and there are no -

symptoms indicating onset of parturition.

The term '^ςbody fluid" is to be taken to mean a body fluid other than amniotic fluid

and includes urine, saliva, vaginal secretions, mucus, whole blood, blood serum, and

blood plasma but with the proviso that blood plasma is excluded when used to determine

onset of parturition in the individual. Desirably, the body fluid will most usually be

urine.

Preferably, the utilising of the test data will comprise comparing the test data to

reference data. The reference data will normally indicate a reference level of the matrix

proteinase for facilitating the assessment of the matter associated with parturition.

The reference level may be a level of the matrix proteinase in urine of pregnant

individuals during gestation that is lower than that present during parturition.

Alternatively, the reference level may comprise a ratio of matrix proteinase to matrix

proteinase inhibitor in urine of pregnant individuals.

Preferably, the level of the matrix proteinase will be monitored over a period of

time by obtaining fresh samples of the body fluid at spaced apart intervals from the

individual and measuring the level of the matrix proteinase in each further sample.

The matrix proteinase may be any proteinase that is present in body fluid of

pregnant individuals during parturition at a higher level than prior to parturition. The

term "'matrix proteinase^"" is to be understood to mean a proteinase which degrades, re- models or otherwise modifies the structure or functioning of connective tissue or

extracellular matrix and includes proteinases capable of degrading one or more of fibrin,

collagens, fibronectin, laminen, elastin and proteoglycans.

Typically, the or each matrix proteinase will be a matrix metalloproteinase and

preferably, will be selected from the group comprising matrix metalloproteinase-2 and

matrix metalloproteinase-9. Most preferably, the level of at least MMP-9 will be

measured.

The method may also comprise the step of measuring the level of a matrix

proteinase inhibitor in the body fluid sample for use with the measured level of the

matrix proteinase for comparison to the reference data.

The term ''matrix proteinase inhibitor" is to be taken to mean a substance that

inhibits the action, activity or activation of a matrix proteinase through binding or

association of the matrix proteinase inhibitor with the matrix proteinase.

In instances where the level of a matrix proteinase inhibitor is also measured, the

inhibitor will generally be an inhibitor of a matrix metalloproteinase preferably selected

from the group consisting of tissue inhibitor of metalloproteinase-1 (TIMP-1) and tissue

inhibitor of metalloproteinase-2 (TIMP-2).

The individual may or may not be experiencing symptoms of labour or threatened

premature labour at the time the sample of urine is collected.

The method of the invention is particularly useful for determining which pregnant

individuals that present with symptoms of labour are actually experiencing the onset of

labour which will lead to delivery and those individuals whose symptoms will resolve

without proceeding to delivery. The matter assessed by the method may be associated with premature parturition.

Premature parturition is to be distinguished from "premature symptoms of labour" which

symptoms may or may not lead to delivery. As such, the latter term is to be taken to

encompass any symptoms that are associated with or are an indicator of labour, the onset -

of labour or the threatened onset of labour and which are exhibited prior to the end of the

normal applicable gestation period irrespective of whether the lead to delivery.

Premature uterine contractions are an example of premature of symptoms of labour.

By "resolve^" or "resolving^" and variations thereof, is meant the decrease or halting

of parturition or the onset of parturition or premature symptoms of labour. The question

of whether premature symptoms of labour or the onset of parturition will resolve may be

assessed by determining whether the measured level of the matrix proteinase is lower

than the reference level or whether there is a decrease in the level of the matrix

proteinase toward or below the reference level. On the other hand, assessment of

whether there is a likelihood of the onset of parturition in the individual or for instance

the likelihood of medical induction of the onset of parturition being successful, may be

made by determining whether the measured level of the matrix proteinase is higher than

the reference level or if there is an increase in the level of the matrix proteinase toward

or above the reference level.

The rate of change of the level of the matrix proteinase in the body fluid sample of

the individual is also an indicator of the onset of parturition or more generally, the

likelihood of the matter being assessed occurring.

The measuring may involve measuring the total relevant matrix proteinase(s) in the

sample or only the active or inactive forms of same. In the instance where the level of more than one matrix proteinase is measured, the total level or the level of the active or

inactive forms of each may be used in the assessment.

The individual will typicalh be a human being. The method, however, may

potentially be used to assess a matter relating to parturition in primates, livestock

animals such as horses, cattle, sheep and pigs, laboratory test animals including mice,

rats, rabbits and guinea pigs, companion animals such as cats and dogs, and wild animals

in captivity including for instance, kangaroos, deers and foxes.

The induction of labour in a pregnant individual is not always successful. If

successful, the labour may subsequently resolve. Accordingly, it is desirable to firstly be

able to ascertain whether medically induced labour is likely to be successful and

secondly, the likelihood that an induced labour will proceed to delivery. This knowledge

enables an informed decision to be made on whether to attempt induction of labour or

alternatively, to subject the individual to caesarean section where it is necessary for

medical reasons and the health of the patient that the pregnancy be halted. Similarly, it

is desirable to know the likelihood of the onset of parturition occurring particularly when

the individual is a human being since it allows for planning and any arrangements

personal or otherwise to be made ahead of time.

In addition, application of the method of the invention may assist the physician in

determining the proper course of action when an individual presents with premature

symptoms of labour and moreover, readily facilitates monitoring of an individual with a

history of premature parturition or whom is considered at risk of premature parturition or

premature symptoms of labour. Even if not considered at risk, an individual can still be

monitored using the method since early determination of an increased level of the matrix proteinase may enable earlier intervention then may otherwise be possible in a woman

presenting with premature symptoms of labour. Monitoring may also provide

forewarning of imminent onset of parturition and valuable information during labour.

The method is convenient given the straight forward nature of the protocol

involved, and is relatively simple to perform. Accordingly, a pregnant woman may

perform the method in the privacy and comfort of her own home if provided with a

suitable test kit or assa) system for her use.

In a second aspect of the invention there is provided a kit or assay system when

used in a method as described herein. Preferably, the kit or assay system is for

performing an enzyme linked immunosorbant assay (ELISA) or other assay on urine

from the individual to measure the level of the matrix proteinase and matrix proteinase

inhibitor if applicable. Typically, the kit or assay system will be provided with written

instructions for use and preferably, will contain all or at least the essential reagents

necessary to perform the assay. Most preferably, any disposables needed to perform the

assay will also be provided. It will be understood that the term "assay system" is to be

given a broader meaning than kit and while the term encompasses kits suitable for use in

the instant method, it is also to be taken to include apparatus capable of measuring the

level of the at least one matrix proteinase and/or matrix proteinase inhibitor irrespective

of how such apparatus functions or is used.

Unless the context clearly requires otherwise, throughout the description and the

claims, the words 'comprise', 'comprising', and the like are to be construed in an

inclusive sense as opposed to an exclusive or exhaustive sense: that is to say. in the sense

of "including, but not limited to". The invention will now be further described hereinafter with reference to a number

of preferred, non-limiting embodiments with reference to the accompanying drawings.

Brief Description Of The Accompanying Drawings

Figure 1 illustrates a gelatin zymogram showing white bands indicating the

presence of MMP-2 and MMP-9 activity in urine samples of patients presenting with

premature symptoms of labour;

Figure 2 is a graph indicating the relationship between progression of symptoms of

premature labour to pre-term delivery and urine MMP-9 level of a first series of patients;

Figure 3 is a graph indicating the relationship between progression of symptoms of

premature labour to pre-term delivery and urine MMP-2 level;

Figure 4 is a graph indicating the relationship between progression of symptoms of

premature labour to pre-term delivery and urine MMP-9 level of another series of

patients; and

Figure 5 is a histogram showing MMP-9 levels in urine samples subjected to

centrifugation to remove cellular debri prior to being assayed, compared to

corresponding samples from the same patients that are not subjected to centrifugation

before assaying of the samples.

Best Mode Of Carrying Out The Invention

The method of the present invention is useful as a one-off test or for on going

monitoring of a pregnant individual, particularly one thought to be at risk of premature

parturition or premature symptoms of labour. The method may also be used for

monitoring the effectiveness of therapeutic or prophylactic treatments directed to

preventing premature parturition or premature symptoms of labour, or which are administered to the individual to address such symptoms. In these situations, mapping

the change in urinary matrix proteinase levels with time is a valuable indicator of the

progress of the pregnancy in issue and/or the effectiveness of a therapeutic or

prophylactic treatment in use. Accordingly, the method will be understood to extend to -

monitoring for increases or decreases in matrix proteinase levels in urine of the

individual relative to either a reference level or relative to one or more earlier matrix

proteinase levels determined in the urine of the individual. Such levels may have been

determined at any time prior to the most recent measurement.

The monitoring may be commenced quite early on in the gestation period such as

during the second trimester in human beings. Preferably, the monitoring will commence

from about 20 weeks into the gestation period of a woman thought to be at risk of

premature parturition or several weeks prior to the end of the gestation period in a

woman experiencing a normal pregnancy.

The interval of time between the collection of samples of urine from the individual

will depend on the stage of gestation and the degree of risk the individual is considered

to have for premature parturition or for experiencing premature symptoms of labour, and

may be an interval from months to quite short periods. Indeed, the level of the matrix

proteinase in an individual's urine may be measured in urine samples collected hours

and potentially fractions thereof apart. It will also be appreciated that monitoring of an

individual expecting the onset of labour or experiencing same will be at relatively close

intervals as will it be those individuals experiencing premature symptoms of labour.

A change in the ratio of MMP-9 to TIMP-1 in amniotic fluid from both women at

full term in labour and in women with premature rupture of membranes, compared to women not experiencing labour or those in relatively early stages of pregnancy, has

previously been observed (Vadillo-Ortega F et al, 1996). Accordingly, measuring the

level of TIMP-1 or other such inhibitors in urine may be beneficial in the assessment of

matters associated with parturition to which the instant invention relates.

The reference level of the matrix proteinase in the urine of pregnant individuals

may be a discreet level or comprise a range of levels. The discreet level or range of

levels may be defined by numerical values indicating concentration of the matrix

proteinase or for instance, in the case where the level of a matrix proteinase inhibitor is

also measured, a ratio of matrix proteinase to matrix proteinase inhibitor in the urine or a

range of such ratios. In order to assess whether there is either an increase or decrease in

the rate of change of the level of the matrix proteinase, values can be plotted against time

on a graph so that any difference in the rate of change may be readily visualised. This

applies both to discreet matrix proteinase concentrations or ratios of matrix proteinase to

matrix proteinase inhibitor.

Measurements of matrix proteinase or matrix proteinase inhibitor in urine of the

individual can be achieved via a number of standard techniques including functional

assays, enzymatic assays or immunological assays. Functional assays include detecting

the or each matrix proteinase by their ability to modulate extracellular matrix structural

or functional properties. Similarly, a functional test for measuring the level of matrix

proteinase inhibitor may involve testing for inhibition of modulation of extracellular

matrix structural or functional properties. Immunological tests may involve contacting a

urine sample with an antibody specific for one or more matrix proteinases or a matrix

proteinase inhibitor for a time and under conditions sufficient for an antibody-matrix proteinase or antibody-matrix proteinase inhibitor complex to form for subsequent

detection via conventional means.

Particularly preferred assays are those in which the or each matrix proteinase and

matrix proteinase inhibitor are targeted with specific antibody which may or may not be -

labelled with a reporter molecule. Depending on the concentration of target and the

strength of the reporter molecule signal, a bound target may be detectable by direct

labelling with an antibody. Alternatively, the target may be bound by a first antibody

and a second labelled antibody specific to the first antibody used to form a target-first

antibody-second antibody tertian' complex which is detected by the signal emitted by the

reporter molecule.

By "reporter molecule" is meant a molecule which, by its nature, is capable of

providing or causing the production of an analytically identifiable signal which allows

the detection of the relevant complex. Detection may be either qualitative or

quantitative. The reporter a molecule may be selected from enzymes, fluorophores,

radionuclides and chemiluminescent molecules.

In an ELISA. an enzyme is conjugated to the second antibody generally by means

of glutaraldehyde or periodate. However, as will be readily appreciated, a wide variety

of different conjugation techniques exist. Commonly used enzymes include horseradish

peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase amongst others.

The substrates used with a specific enzyme are generally chosen for the production, upon

hydrolysis by the corresponding enzyme, of a detectable colour change. It is also

possible to employ flurogenic substrates which yield a fluorescent product rather than a

chromogen as indicated above. In each instance the enzyme-labelled second antibody is added to the matrix proteinase-antibody or matrix proteinase inhibitor-antibody complex

and allowed to bind prior to excess reagent being washed away. A solution containing

the appropriate substrate is then added to the complex resulting from the binding of the

second antibody, which reacts with the enzyme to provide the visual signal which can be -

quantitated, usually spectrophotometrically, to give an indication of the amount of the

matrix proteinase or matrix proteinase inhibitor in the urine sample.

Alternatively, fluorescent compounds such as fluorescein and rhodamine may

potentially be conjugated to antibodies without altering their binding capacity for use in

assays of the above type. When activated by illumination with light of a particular

wavelength, the fluorochrome-labelled antibody adsorbs the radiation, inducing a state of

excitability in the fluorescent compound, resulting in emission of light at a characteristic

colour visually detectible with a light microscope. As in an ELISA, the fluorescently

labelled antibody is allowed to bind to its target before unbound reagent is washed off

and the remaining complex exposed to light of the appropriate wavelength.

Immunofluorescence and ELISA techniques are both very well established in the art and

the skilled addressee will be readily able to employ such assays in the method of the

present invention.

Preferably, mid-stream urine is collected from the pregnant individual and is

centrifuged so that any cells or cell debris present in the urine are pelleted prior to the

supernatant being collected for use in the method of the invention.

The invention will now be described with reference to the following non-limiting

examples. Example 1

The level of MMP-9 in urine may be measured using a Biotrak Activity Assay

System commercially available from Amersham Pharmacia Biotech, Buckinghamshire,

England. The assay detects total MMP-9 activity (that is the inactive zymogen form of -

MMP-9 as well as its activated enzyme form) through activation of a modified pro-

enzyme and subsequent cleavage of its chromogenic peptide substrate.

Briefly, the assay system involves aliquoting 100/// of urine sample into an anti-

MMP-9 coated 96 well microtitre plate which is then incubated over night at a

temperature of between 2 to 8°C. Following incubation, the wells are washed and 50///

of IM p-Aminophenylmercuric acetate in DMSO is added. Subsequently, 50/// of assay

buffer (50mM Tris-HCl pFI 7.6. 150mM sodium chloride, 5mM calcium chloride, 1//M

zinc chloride plus 0.01% v/v BRIJ ^' 35) is added. The plate is then incubated at 37°C

for two hours. Following the incubation, 50/// of detection reagent (25/// detection

enzyme [modified urokinase in assay buffer] plus 25/// substrate [S-2444 peptide in

assay buffer]) is added to each well. The plate is read at 405nm to obtain a t₀ value and

then incubated at 37°C for approximately six hours prior to being read again.

MMP-9 levels are graphically represented as the rate of change of adsorbence at

405nm, ie δ Abs₄₅₀/H₂ 1000. The results for the test sample of urine are then compared

with a standard curve generated using samples of known MMP-9 concentration.

Example 2

Urine from patients at risk of pre-term delivery was tested for the presence of

MMP-9 and MMP-2 by means of gelatin zymography. As shown in Figure 1, the white

bands reflecting gelatin degrading activity are shown for the purified gelatinases MMP-9 (at 92kD) and MMP-2 (at 72kD). All patients tested present with premature symptoms

of labour and in the three patients who proceeded to pre-term delivery within one week

of admission, large amounts of MMP-9 were observed as evidenced by the heavy white

bands at the 92kD position. Increased levels of MMP-2 are also indicated in at least two -

of the patients that proceeded to pre-term deliver.

Example 3

Mid-stream urine collection and cervico-vaginal fluid fibronectin testing was

routinely performed on all patients admitted with symptoms of premature labour to the

John Hunter Flospital. Newcastle. Australia. Institutional ethics approval was not

required to obtain aliquots from collected urine specimens from a case series of 15

patients nor for fibronectin testing. Urine samples were cleared of cells and cell debris

by means of centrifugation at 4.000rpm for 10 mins. and stored at -20°C in preparation

for testing as a batch in the MMP-9 assay described in Example 1. Duplicate samples

were averaged and urine creatinine levels of all samples were in the normal range (6-

22mmol/L). Eight of the 15 patients also underwent cervico-vaginal fluid testing using a

commercially available firbronectin test (Fetal Fibronectin Membrane Immunoassay Kit,

Adeza Biomedical, Sunnyvale, California, USA).

Example 4

The gestation in weeks at the time of admission of the 15 patients of Example 3

was in the range of 27- 34 weeks. Among the 15 patients, 6 pregnancies resulted in

premature delivery (classified positive) during the week of admission to hospital, and the

remaining 9 patients proceeded to full-term delivery (classified negative). Two of the 15

patients presented with rupture of membranes (ROM), one patient with placental abruption (PA), and the remainder with symptoms of premature labour (SPL) as detailed

in Table 1. Urine MMP-9 levels were estimated from mid-stream urine samples for all

15 patients and were in the range of 0.1 - 26.6ng/ml with the highest value recorded in

the patient presenting the placental abruption.

TABLE 1 : Detected MMP-9 Levels in Patients

IT¹ = tnie positiN e 1^'N = true negative. TP = false posim e TN = false negative. NA = not assayed The linear regression co-efficient for the standard curve for the MMP-9 assay was

1-0.997. The mean MMP-9 level for all 15 patients was 5.0ng/ml. A receiver-operator

characteristic curve confirmed that 5.0ng/ml was the appropriate cut-off point for MMP-

9 analysis. Urinary MMP-9 levels <5ng/ml and >5ng/ml were compared with clinical

outcome (premature delivery versus full-term delivery). The ability of method of the

invention to accurately predict outcomes correlated as shown in Table 1 and indicated in

Figure 2.

Of the 5 positive test results ( MMP-9 ≥5ng/ml). 4 were from patients who

delivered within 7 days of admission (positive predictive value 80%). Of 10 negative

results (MMP-9 ≤5ng/ml), 8 were from patients who proceeded to a full-term delivery

(negative predictive value 80%). As indicated above, of the 15 patients in this study, 8

underwent testing of cervico-vaginal fluid for the presence of fibronectin. As shown in

Table 1. the test was positive in 7 of the 8 patients (4 false positives) one of whom was

also the only false positive result as identified by the MMP-9 assay.

The presence of fetal fibronectin in vaginal secretions of pregnant women

experiencing premature uterine contractions has been used as a marker of imminent

premature delivery (Lukes et al, 1997). On the basis of a cut off MMP-9 activity level of

5.0ng/mL. both the positive and negative predictive values of the method of the

invention were 80%. This finding compares favourably with those for the fetal

fibronectin test, which has a positive predictive value in the range of only 35% to 50%o.

Of the four patients with false-positive fibronectin results, urinary MMP-9 levels were

extremely low in three of them and were elevated in the fourth leading to the only false-

positive result as identified using the MMP-9 assay. Example 5

The relationship between urinary MMP-2 levels and clinical outcome (delivery

within one week of admission) for the 15 patients listed in Table 1. Urinary MMP-2 was

measured using the commercially available Biotrak MMP-2 Activity Assay System

(Amersham Pharmacia Biotech.). While not as sensitive a test as the MMP-9 activity

assay system, similar results for patients who proceeded to a pre-term delivery were

observed in this series of patients with higher urinary levels of MMP-2 (see Figure 3).

The presence of significant levels of either interstitial collagenase (MMP-1) or

stromelysin-1 (MMP-3) when evaluating urine from patients presenting with symptoms

of premature labour was not detected, irrespective of whether they did or did not proceed

to a pre-term delivery.

Example 6

Re-test reliability of urinary MMP-9 levels was assessed in 3 true positive and 3

true negative patients assayed in Example 4. Specifically, urine samples were stored at

-20°C and re-assayed approximately one year later. Although there is variability in

absolute levels of MMP-9 (ng/ml) for the same urine samples tested by different batches

of the commercially available assay kit (see Table 2) the trend remained unchanged for

patients presenting with threatened premature labour who did not proceed to a pre-term

delivery compared with those who did. Table 2: Urinary MMP-9 Levels In Urine Samples Reassayed After 1 Year

MMP-9 ng/ml

Patient No. Diagnostic Initial Assay* Reassayed 1 Discrimination Year Later

1 True positive 14.4 2.7

6 True positive 26.6 14.0

12 True positive 5.0 7.5

2 True negati \ e 0.1 0.0

13 True negative 0.1 0.0

15 True negative 3.6 0.0

* Patient Number as Shown in Table 1

Reasons for variation in absolute amounts of MMP-9 between different kit batches

include ageing of reagents, variability in the amount of antibody coating and variability

in specific activities of the enzymes (per weight) from batch to batch.

Example 7

Urine samples from another group of 10 patients presenting with premature

symptoms of labour were assayed for MMP-9 levels using the Biotrak MMP-9 Activity

Assay System. Five of the 10 patients proceeded to a pre-term delivery within one week

of admission with threatened premature labour and 5 did not. Clean-catch mid-stream

urine samples were centrifuged to remove cells and cell debris and triplicate aliquots

from each of the 10 urine samples assayed for MMP-9. The urine samples were

identifiable only by a number and clinical outcome information was not available to the

technician performing the MMP-9 activity assay. Details of admission, gestation, presenting diagnosis, diagnostic discrimination and results of cervico-vaginal fluid

testing for fibronectin are given in Table 3. Using a cut-off MMP-9 level of >1 ng/ml, all

patients with values < 1 ng/ml did not proceed to a pre-term delivery whereas all 5

patients who had values exceeding 1 ng/ml went on to pre-term delivery as indicated in

Table 3 and Figure 4.

Table 3: Relation between admission gestation, urinary MMP-9 level, cervico-vaginal

fluid fibronectin assay and clinical outcome (delivery within one week of

admission) for 10 patients presenting with premature symptoms of labour.

Given that the kit used to assess MMP-9 levels in these 10 patients cam from a

different batch from those used previously, it was not unexpected that different absolute

values for MMP-9 were observed in this group of patients compared with those listed in

Table 1. In the present group of 10 patients, a cut-off of > 1 ng/ml correctly identified

patients who proceeded to a pre-term delivery with 100% sensitivity and specificity.

Comparison with the cervico-vaginal fibronectin assay for these 10 patients is also

shown in Table 3. Six out of the 10 patients could not be assayed for the presence of

cervico-vaginal fibronectin because of prior vaginal examination, abruptio placentae or

rupture of membranes. Example 8

Centrifuged (4000 rpm for 10 minutes) and non-centrifuged urine samples

(standing at room temperature for 30 minutes) were compared for MMP-9 activity using

the same assay kit as that used to obtain MMP-9 levels shown in Table 3. Similar

urinary MMP-9 levels were observed from the same 5 patients irrespective of whether

the samples were centrifuged or not prior to being assayed as indicated in Figure 5.

Example 9

The urinary MMP-9 activity of one patient undergoing medical induction of labour

was found to have elevated MMP-9 level (3.8ng/ml as tested with the same assay kit

used to obtain the results listed in Table 3). This was consistent with successful

induction of labour.

Although the present invention has been described above with reference to a

number of preferred embodiments, the skilled addressee will appreciate that numerous

variations and modifications are possible without a departing from the scope of the invention. References Cited

1. Lukes AS. Thorp JM Jr., Euker B, Pahel-Short L. (1997). Predictors ofpositivity

for fetal fibronectin in patients with symptoms ofpreterm labour. Am J Obstet

Gynecol 176: 639-641.

2. Giles W, Knox M. Madsen G. Bisitis A, Smith R (1998). The effect of fetal

fibronectin usage on admissions to a tertiary maternal fetal medicne unit and costs

savings. Am J Obstet Gynecol 178:S 121.

3. Malak TM, Ockleford C, Bell S. Dalgleish R. Bright N, Macvicar J (1993).

Confocal immunofluorescence localization of collagen types I, 111, IV, V and VI

and their ultrastructural organization in term human fetal membranes.

Placenta. 14:385-406.

4. Matrisian L ( 1990). Metalloproteinases and their inhibitors in matrix remodeling.

Trends Genet 6: 121-125.

5. Woessner JF ( 1991 ). Matrix metalloproteinases and their inhibitors in connective

tissue remodeling. FASEB J 5:2145-2154.

6. Murphy G, Ward R. Gavrilovic J, Atkinson S (1992). Physiological mechanisms

for metalloproteinase activation. Matrix Suppl. 1 :224-230.

7. Fernandex P, Merino M. Nogales F, Charonis A, Stetler-Stevenson W, Liotta L

(1992). Immunohistochemical profile of basement membrane proteins and 72

kilodalton type IV collagenase in implantation placenta! site. Lab. Invest

66:572-579.

8. Vadillo-Ortega F. Gonzalez-Avila G, Furth EE. Lei H. Muschel RJ, Stetler-

Stevenson WG and Strauss JF 3^r (1995). 92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labour. Am J

Pathol 146: 148-56.

9. Rowe TF. King LA. MacDonald PC, Casey ML (1997). Tissue inhibitor of

metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 expression in

human amnion mesenchymal and epithelial cells. Am J. Obstet. Gynecol.

176:915-912.

10. Moses MA, Wiederschain D. Loughlin KR, Zurakowski D, Lamb CC and

Freeman MR (1998). Increased incidence of matrix metalloproteinases in urine of

cancer patients . Cancer Research. 58: 1395-1399.

1 1. Romero R, Ray JM, Sepulveda W, Zimmerman M, Stetler-Stevenson WA.

Evidence of active forms of the 72kDa and 92kDa type IV collagenase in human

parturition [abstract 228]. In: Proceedings of the Thirty-ninth Annual Meeting of

the Society for Gynecologic Investigation: 1992 March 18-21 , San Antonio, Texas

p. 222.

12. Vadillo-Ortega. Hernandez A, Gonzalez-Avila G, Bermejo L. Iwata K, and Strauss

JF III ( 1996). Increased matrix metalloproteinase activity and reduced tissue

inhibitor of metaUoproleinases-1 levels in amniotic fluids from pregnancies

complicated by premature rupture of membranes. Am J Obstet Gynecol 174: 1371-

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13. Osmers RG, Adelmann-Grill BC, Rath W, Stuhlsatz HW, Tschesche H and Kuhn

W (1995). Biochemical events in cervical ripening dilatation during pregnancy

and parturition. J. Obstet. Gynaec. 21(2): 185-194. 14. Fortunato SJ, Menon R and Lombardi SJ (1997). Collagenolytic enzymes

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Claims

The Claims Defining The Invention Are As Follows:-

1 . A method of assessing a matter associated with parturition in a pregnant

individual, comprising the steps of measuring the level of at least one matrix

proteinase in a body fluid sample from the individual to obtain test data and

utilising the test data to make the assessment.

2. A method according to claim 1 wherein the matter is the onset of parturition in the

individual and the assessing comprises determining said onset.

3. A method according to claim 1 wherein the matter is a likelihood of the onset of

parturition in the individual.

4. A method according to claim 1 wherein the matter is a likelihood that parturition

or the onset of parturition in the individual will resolve.

5. A method according to claim 1 wherein the matter is a likelihood of medical

induction of the onset of parturition in the individual being successful.

6. A method according to claim 1 wherein the matter is a likelihood of parturition in

the individual resolving if parturition is medically induced in the individual.

7. A method according to claim 1 wherein the matter is progress of parturition in the

individual.

8. A method according to any one of claims 1 to 7 wherein said parturition is

premature parturition in the individual.

9. A matter according to any one of claims 1 to 8 wherein the utilising comprises

comparing the test data to reference data.

10. A method according to claim 9 wherein the reference data indicates a reference

level of the matrix proteinase in said body fluid and the assessing comprises determining whether the measured level of the matrix proteinase in the body fluid

sample from the individual differs from the reference level.

1 1. A method according to claim 10 wherein the reference level of the matrix

proteinase in said body fluid is determined from levels of the matrix proteinase in -

samples of the body fluid from a number of pregnant individuals.

12. A method according to claim 10 or 1 1 wherein the utilising comprises determining

whether the measured level of the matrix proteinase in the body fluid sample from

the individual is above the reference level.

13. A method according to claim 9 further comprising the step of measuring the level

of a matrix proteinase inhibitor in the body fluid sample from the individual and

wherein the utilising comprises determining a test ratio of matrix proteinase to

matrix proteinase inhibitor for comparison to the reference data.

14. A method according to claim 13 wherein the reference data comprises a reference

ratio of matrix proteinase to matrix proteinase inhibitor in said body fluid.

15. A method according to claim 13 or 14 wherein the assessing comprises

determining whether the test ratio differs from the reference ratio.

16. A method according to claim 14 or 15 wherein the utilising comprises determining

whether the ratio of matrix proteinase to matrix proteinase inhibitor in the body

fluid sample from the individual is greater than the reference ratio.

17. A method according to claim 16 wherein the reference ratio of matrix proteinase to

matrix proteinase inhibitor is determined using samples of the body fluid from a

number of pregnant individuals.

18. A method according to any one of claims 13 to 17 wherein the matrix proteinase

inhibitor is a tissue inhibitor of metalloproteinase.

19. A method according to any one of claims 1 to 18 further comprising monitoring

the level of the matrix proteinase in said body fluid of the individual over a period -

of time utilising one or more further samples of the body fluid from the individual.

20. A method according to claim 19 wherein the monitoring comprises monitoring a

rate of change in the level of the matrix proteinase in the body fluid sample from

the individual over the period of time.

21. A method according to claim 19 wherein the monitoring involves monitoring for a

marked increase or decrease in the rate of change in the level of the matrix

proteinase.

22. A method according to any one or claims 1 to 21 wherein the measuring comprises

measuring the level of only one matrix proteinase in the body fluid sample from

the individual.

23. A method according to any one of claims 1 to 21 wherein the measuring comprises

measuring the level of more than one matrix proteinase in the body fluid sample

from the individual.

24. A method according to any one of claims 1 to 23 wherein the at least one matrix

proteinase is a matrix metalloproteinase.

25. A method according to claim 22 wherein the matrix proteinase is matrix

metalloproteinase-9 (MMP-9).

26. A method according to claim 23 wherein the measuring comprises measuring the

level of one or both of matrix metalloproteinase-9 (MMP-9) and matrix

metalloprotenase-2 (MMP-2).

27. A method according to claim 23 wherein the measuring comprises measuring the

level of the matrix metalloproteinase-9 and one or more other matrix

metalloproteinases.

28. A method according to any one of claims 1 to 27 wherein the body fluid assessed

is urine.

29. A test kit or assay system in a method as defined in any one of claims 1 to 27 to

measure the level of the at least one matrix proteinase in the sample of urine from

the individual.

30. A test kit or assay system accordingly to claim 28 comprising an enzyme linked

immunosorbant assay (ELISA) kit or kit for preforming an activity assay.

31. Use of a test kit or assay system in a method as defined in any one of claims 1 to

27 to measure the level of the at least one matrix proteinase in the sample of urine

from the individual.

AMENDED CLAIMS

[received by the International Bureau on 31 January 2000 (31.01.00); original claims 29-31 cancelled; new claims ^"29-32 added; remaining claims unchanged Opage )]

level of one or both of matrix metalloproteinase-9 (MMP-9) and matrix

metalloprotenase-2 (MMP-2).

level of the matrix metalloproteinase-9 and one or more other matrix

metalloproteinases.

is urine.

29. A method according to any one of claims 1 to 28 wherein the measuring comprises

measuring total active and activatable said at least one matrix proteinase to thereby

obtain the test data.

30. A test kit or assay system when used in a method as defined in any one of claims 1

to 29 to measure the level of the at least one matrix proteinase in the sample of

urine from the individual.

31. A test kit or assay system accordingly to claim 30 comprising a test kit or assay

system for performing an enzyme linked immunosorbant assay (ELISA) or an

activity assay.

32. Use of a test kit or assay system in a method as defined in any one of claims 1 to

29 to measure the level of the at least one matrix proteinase in the sample of urine

from the individual.