WO1998059245A1

WO1998059245A1 - Diagnostic method and kits for detecting the rupture of foetal membranes

Info

Publication number: WO1998059245A1
Application number: PCT/EP1998/003711
Authority: WO
Inventors: Marcus Josephus Marie Koolen
Original assignee: Akzo Nobel N.V.
Priority date: 1997-06-19
Filing date: 1998-06-16
Publication date: 1998-12-30
Also published as: AU8800198A

Abstract

The invention relates to a diagnostic method for detecting the rupture of foetal membranes as well as a kit for such detection. With the present invention it has been found that, by determining the presence of inhibin B in vaginal secretions of pregnant women, a reliable method for diagnosing premature rupture of foetal membranes can be obtained.

Description

DIAGNOSTIC METHOD AND KITS FOR DETECTING THE RUPTURE OF FOETAL MEMBRANES.

The invention relates to a diagnostic method for detecting the rupture of fettle membranes as well as means for employing the method.

The occurrence of premature foetal membrane rupture (PROM) complicates 4.5% to 7.6% of pregnancies. The correct diagnosis of this disorder is crucial, because the presence of membrane rupture is associated with infectious morbidity in the mother and foetus, cord accidents and imminent term or preterm labor. Failure to identify patients with membrane rupture can result in the failure to implement salutary obstetric measures. Conversely, the false diagnosis of membrane rupture can lead to inappropriate interventions (e.g. hospitalisation or induction of labor, Lockwood et al., Am. J. Obstet. Gynecol. 171: 146-150).

Today, there are no 100% reliable techniques for the diagnosis of PROM. Invasive techniques are considered most reliable and are commonly used as "gold standard". Among those are methods in which dyes are transabdomenally injected in the amnion cavity, speculum examination, amniocentesis and amnioscopy. All of the above techniques expose the foetus and/or the mother to a considerable risk because they may induce infections.

Non-invasive techniques for the diagnosis of foetal membrane rupture have traditionally relied on the patients' report of fluid in the vagina, confirmed by the presence of gross pooling of amniotic fluid in the vagina, an alkaline vaginal pH detected by Nitrazine paper, or the presence of a characteristic arboreal or ferning pattern after microscopic examination of dried vaginal secretion. Those are still the most commonly used procedures for the detection of PROM, despite the high false positive and false negative rates (Friedman and McElin, Am. J. Obstet. Gynecol. 104: 544-550). Moreover, these methods suffer from the severe disadvantage that they are sensitive to interfering substances such as urine, semen or vaginal secretions whether or nor produced upon vaginal infection. In order to diagnose PROM in a more reliable way, it has been suggested to search for amnion-specific compounds in vaginal secretions. Such a method has been described in WO 92/12426 which method relies on the detection of insulin-like growth factor binding protein -1 (IGFBP-1 ) in vaginal secretions. Due to the fact that IGFBP-1 is specifically present in amniotic fluid (> 400-fold excess as compared with serum ) and apparently not synthesised in the chorion (Hill et al., Placenta 14: 1-12), the method is highly specific and has a good positive predictive value (92.6% and 96.7%, respectively, Lockwood et al., Am. J. Obstet. Gynecol. 171 : 146-150). However, the method has the severe disadvantage that its sensitivity and negative predictive value are very low (74.4% and 55.6% respectively, Lockwood et al., Am. J. Obstet. Gynaecol., 171 : 146-150).

Also the measurement of alpha-fetoprotein (Rochelson et al., Obstet. Gynecol. 62: 414-418) or prolactin (Koninckx et al., Br. J. Obstet. Gynaecol. 88: 607-610) has been suggested for the diagnosis of PROM. Both seem not to be useful markers because of the overlap in concentrations in women with or without ruptured membranes (Huber et al., Br. J. Obstet. Gynaecol. 90: 1183 -1185). Moreover, since both markers also appear in maternal serum in appreciable concentrations, the diagnosis becomes unreliable when the vaginal fluid samples are contaminated with blood.

Foetal fibronectin has also been suggested as a marker for PROM (US patent 5,096,830). Unfortunately, foetal fibronectin is present in vaginal secretions in approximately 50% of women with preterm labor and intact membranes (Lockwood et al., N. Engl. J. Med. 325: 669-674). It is described that the presence of foetal fibronectin in cervico-vaginal mucus has limited accuracy in predicting PROM (Chien et al., Br. J. Obstet. Gynaecol. 104: 436-444).

With the present invention it has been found that, by determining the presence of Inhibin B in vaginal secretions of pregnant women, a reliable method for diagnosing PROM can be obtained. In nature, many different forms of Inhibin exist, the whole family being referred to as Inhibins. Inhibins are part of the transforming growth factor-beta (TGF-β) superfamily, together with a class of molecules called activins.

Inhibin and activin are dimeric protein molecules composed of an α, βA - or βB - subunit: Inhibin A (α-βA), Inhibin B (α-βB), activin A (βA-βA), activin B (βB-βB), or activin AB (βA-βB, see figure 1 ). The subunits are expressed in a variety of tissues and have biological actions in many diverse organ systems (Wallace EM et al.1997. Journal of Clinical Endocrinology & Metabolism. 82:218-222. Burger HG et al.1996. Clinical Endocrinology. 44:413-418. Knight PG.1996. Frontiers in Neuroendocrinology. 17:476-509. Knight PG et al.1996. Journal of Endocrinology. 148:267-279. Mcfarlane JR et al.1996. European Journal of Endocrinology. 134:481- 489. Wallace EM and Healy DL.1996. British Journal of Obstetrics & Gynaecology. 103:945-956. Robertson DM et al.1995. Journal of Endocrinology. 144:261-269. Burger HG.1993. Journal of Clinical Endocrinology & Metabolism. 76:1391-1396.). A specific role for Inhibins and activins in human pregnancy has been hypothesised by several groups (Mayo KE et al.1986. Proc. Natl. Acad. Sci. U. S. A. 83:5849-5853. McLachlan Rl et al.1987. J. Clin. Endocrinol. Metab. 65:954-961 , McLachian et al. 1986. Biochem. Biophys. Res. Comm. 140: 485-490; Petraglia et al. 1987. Science 237: 187-189; Petraglia et al., 1992. J Clin Endocrinol. Metab. 75: 571-576). Inhibin/activin subunit mRNAs and the encoded immunoreactive proteins have been localised in placenta, foetal membranes and maternal and foetal decidua (Mayo KE et al.1986. Proc. Natl. Acad. Sci. U. S. A. 83:5849-5853. Burger HG et al.1996. Clinical Endocrinology. 44:413-418, Petraglia et al. 1987. Science 237: 187-189; Petraglia et al., 1992. J Clin Endocrinol. Metab. 75: 571-576, Petraglia et al 1993, J. Clin. Endocrinol. Metab. 77: 542-548, Merchenthaler et al, 1987, Mol. Cell. Endocrinol. 54: 239-243, Petraglia et al. Endocrine Journal 1993. 1 : 323-327; Qu J and Thomas K, 1995 Endocrine Reviews 16: 485-507).

Studies on the physiological role of the Inhibin have been hindered by the unavailability of reliable immunoassays which can specifically measure the bio^'active dimeric forms (α-βA and α-βB) of Inhibin in peripheral blood and thus distinguish these from free α-subunit forms which are also secreted into the peripheral circulation but which lack Inhibin-like biological activity. Recently, a new generation of two-site assays has been developed for both Inhibins and activins, including assays specific for Inhibin-A, Inhibin-B, activin-A, activin-B and α-subunit precursors (In Burger HG, Findlay JK, Robertson DM, de Kretser D, Petraglia F, editors. Inhibin and Inhibin-related proteins. Frontiers in Endocrinology. Rome: Ares-Serono, 1994, see Woodruff et al., p55-68; Groome N et al. p33-44; Poncelet E, Franchimont P, p45-54. See also Knight PG, et al., J Endocrinology 1996, 148: 267-279). However the detection of dimeric Inhibin levels in serum requires a pre-assay analyte oxidation step (Knight and Muttukrishna, 1994 J. Endocrinology 141: 417-425).

Clinical studies have been conducted with Inhibin-A (α-βA dimer) and Inhibin-B (α-βB dimer) specific assays in order to gain further insight into the biology of Inhibin in normal pregnancy by measuring Inhibin-A and Inhibin-B in different pregnancy compartments. Inhibin-A is present in peripheral blood throughout human gestation at concentrations up to 50 times greater than maximum values found during the spontaneous menstrual cycle (100 ng/L). The highest serum values were measured during the third trimester (Muttukrishna S et al.1995. Clinical Endocrinology. 42:391- 397.). However, Inhibin-B is undetectable in maternal serum. In amniotic fluids the levels of both Inhibin-A and Inhibin-B gradually increase during progress of the pregnancy. Typically, Inhibin-B levels rise from 216.6 (67.4-554.6) pg/ml at 14 weeks to 1078.2 (439.3-2482.2) pg/ml at 20 weeks (Wallace et al. 1997. J Clin. Endocrinol and Metabolism 82: 218-222).

As described above, activin A (βA-βA), activin B (βB-βB), or activin AB (βA-βB) are related dimeric proteins that regulate numerous cellular activities. Activin activity is bioneutralized by follistatin, a specific and high-affinity binding protein. Specific and sensitive enzyme-linked immunosorbent activin assays have been developed (Petraglia et al 1993 Endocrine Journal 1: 323-327; Wong et al, 1993, J. Immunol. Methods 165: 1-10; Woodruff et al., 1993, Human Reproduction 8: 133-137). The specificity and sensitivity of these activin assays is shown by the fact that these assays do not detect either activin isoform when bound to follistatin, therefore, the assays are specific for the biologically relevant ligands. Studies have shown that activin A is measurable in the serum of pregnant women, while activin B is not detected in maternal serum (Woodruff et al 1997, J. Endocrinology, 152: 167-174.). However, activin B is measurable in amniotic fluid and cord blood sera (Woodruff et al 1997, J. Endocrinology 152: 167-174).

With the present invention it has been found that, by determining the presence of Inhibin B in vaginal secretions of pregnant women, a reliable method for diagnosing PROM can be obtained.

An embodiment of the present invention provides a sensitive and specific diagnostic method for PROM. Thus, the present invention is directed to a diagnostic method for detecting the rupture of foetal membranes, said method comprising a) obtaining a sample of vaginal secretion fluid from a pregnant patient and b) determining the presence of Inhibin-B in said sample.

Methods for obtaining samples of vaginal fluid are well known in the art. It may be performed by sampling the vagina with a cotton-wool tip which is then placed in an appropriate small volume of extraction fluid such as phosphate buffered saline. In cases where Inhibin B is detected using a rapid manual assay, the sample taking device may be an integral part of the detection device or alternatively be fitted to the detection device after obtaining a sample.

Determining the presence of Inhibin B may be performed in many ways using specific binding substances for Inhibin B. Such binding substances may be a specific receptor, a substrate for Inhibin B or the like. In a preferred embodiment , detection of Inhibin B is performed immunologically, in which case a specific antibody is used to allow specific detection. Such antibodies may be polyclonal or monoclonal. Methods of making such antibodies are well known in the art, monoclonal antibodies against Inhibin B are described in Groome et al. 1996, J. Clin. Endocrinol. Metab. 81: 1401-1405. Such antibodies may be attached to a solid support. Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an aldehyde particle (such as a ceramic magnetizable particle with active aldehyde surface groups), an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.

Binding of Inhibin B to the specific antibody may be reported by a labelling substance either or not attached to a second antibody. Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.

In an especially preferred method for the detection of Inhibin B in a sample, Inhibin B is determined in an ELISA assay that comprises a monoclonal antibody against the β-B subunit of Inhibin B attached to the wells of a microtiter plate which is then brought into contact with the sample. After which, the presence of immune complexes formed between the antibody and Inhibin B in the sample is detected using a second monoclonal antibody against the α subunit of Inhibin B labelled with alkaline phosphatase. Any unreacted material is then removed by washing before the detection of alkaline phosphatase using a sensitive amplified substrate.

Yet another particularly suitable method for the detection of Inhibin B in a sample is based on a competition reaction between a labelled peptide with affinity for the specific monoclonal antibody and Inhibin B from the sample whereby the peptide and Inhibin B are competing with the antibody directed against Inhibin B whether or not attached to a solid support.

The present invention also provides a diagnostic method for the rapid diagnosis of

PROM that can be performed while the patient is waiting (a so-called bed-side test) or can be performed by a lay person at home. Rapid diagnosis of PROM can be achieved by implementing the diagnostic method in a rapid manual assay. Such assays are well-known in the art and a particularly suited format may be a dipstick format, or alternatively a filter-based assay.

In a preferred embodiment, the rapid manual assay device comprises a porous carrier through which a sample liquid is to be transported. The device also incorporates a labelled specific binding substance which is freely mobile in said porous carrier when in the moist state. The device also comprises an unlabeled binding substance which is immobilised in a detection zone on said porous carrier, the positioning of the labelled and immobilised antibody being such that test sample, when applied to the porous carrier, will first pick up the labelled antibody and thereafter will migrate to the detection zone.

Although various types of devices can be used, in an even more preferred embodiment of the present invention an apparatus is used as described in WO 94/22011. Said apparatus comprises a housing in which a sample collector can be inserted, and a holding device attached to the housing, which holding device holds a porous carrier such as a test strip.

Yet another embodiment of the invention provides a test kit characterised in that it contains at least one binding substance with specific binding for the beta-subunit of Inhibin-B as well as means for vaginal fluid sample collection as well as means for detecting complexes formed between the specific binding substance and Inhibin B.

Brief description of the figures.

Figure 1 : Inhibin B concentration is determined in serum, urine, and amniotic fluid samples collected of 16 normal healthy pregnant subjects of Group C from example 1.

The present invention is further exemplified by the following examples: EXAMPLES:

EXAMPLE 1 Selection of patient population

The following groups of subjects were selected to take part in the study:

Group A. Control subjects. Low-risk pregnant women divided in to three gestational ages at the time of sampling i.e. 16-28 weeks, 28-37 weeks, 37-42 weeks. A total of 20 subjects per gestational age were selected. All women had singleton pregnancies and no complications. Specifically, all had normal urine tests and blood pressure, and normal foetal development.

Group B. Samples collected from one hundred patients with a questionable status of the foetal membranes were included in the study. The following criteria were applied as the gold standard in order to classify the clinical status of the foetal membranes of the selected subjects:

Intact (non-ruptured) membranes were defined if no complications were encountered during at least one week after the first complaint, no loss of amniotic fluid, and positive internal examination of foetal membranes. Premature rupture of membranes were defined when irregular loss of amniotic fluid was observed (speculum examination), and a negative internal examination of the foetal membranes.

Group C. Vaginal samples were collected from fifty patients who attended the clinic for an amniocentesis in the framework of prenatal diagnosis. A vaginal secretion sample was collected before and one hour after amniocentesis. In addition, maternal serum samples as well as urine samples were collected and stored frozen at -20 °C. EXAMPLE 2 Collection of samples from the patient group of example 1

Amniotic fluid, urine, maternal serum, fluor vaginalis and cervical mucus samples were collected of each patient. Fluor vaginalis samples were obtained by speculum examination using a syringe as well as by using a Dacron swab without visual examination. Cervical mucus samples were collected by using a Dacron swab in combination with speculum examination. The collected samples were eluted in 1 ml phosphate buffered saline (pH 7.2) and stored at -20 °C. All subjects provided informed consent to undergo vaginal fluid sampling during their pregnancies. Samples collected of women who did not meet the requirements for a variety of reasons i.e. unable to perform follow-up assessments, who delivered before 37 weeks' gestation, or gross blood in vaginal secretions were excluded from the study.

EXAMPLE 3 Inhibin B is preferentially present in amniotic fluid

In order to determine whether the antigen Inhibin B is preferentially localised in the amniotic fluid, paired samples of amniotic fluid, urine and maternal serum of 16 subjects of Group C were analysed in the Inhibin B specific assay (Serotec, UK). As depicted in Figure 1 , 14 out of 16 amniotic fluid samples exhibit a high Inhibin B concentration (>100 pg/ml). The calculated Inhibin B values for the remaining two samples nrs 30C and 37C were 47 and 25 pg/ml, respectively. In contrast the majority (13 out of 16) of the paired maternal serum samples tested showed low Inhibin B levels (<15 pg/ml). The values calculated for the three outliers i.e. samples 9C, 33C, and 44C were 16.3, 28 and 48 pg/ml, respectively. Five urine samples tested for the presence of Inhibin B were all negative. It can be concluded that there is a significant difference in the presence of Inhibin B between maternal serum, urine and amniotic fluid. Therefore, antigen Inhibin B is predominantly present in amniotic fluid. EXAMPLE 4 Inhibin B is a marker for premature rupture of membranes

Studies were performed to address the question whether Inhibin B can be detected in the vaginal fluids and in consequence can be used as marker for the diagnosis of premature rupture of foetal membranes of subjects at high risk (Group B in example 1). Vaginal samples were collected from a total of 35 women of Group B (nrs 1B- 35B) who's foetal membranes were retrospectively clinically diagnosed to be intact. Samples were collected by means of a Dacron swab and extracted in 1 ml phosphate buffered saline (pH 7.2). Collected samples were tested for the presence of the Inhibin B. In addition, vaginal samples collected from a group consisting of 17 pregnant women from group B (nrs 101B-117B) who were retrospectively clinically diagnosed to have premature rupture of foetal membranes were evaluated in parallel. Table 1 shows that the Inhibin B concentration in samples collected from women clinically diagnosed of having intact foetal membranes varied between 0 and 75.2 pg/ml. Twenty out of 35 samples revealed an Inhibin B concentration below the detection limit of the assay, i.e. <15 pg/ml. Inhibin B values in the group of intact foetal membranes averaged on 26.8 pg/ml (S.D. 18.9).

The Inhibin B concentration in samples collected from women who were clinically diagnosed of having premature rupture of foetal membranes (nrs. 101B-117B) varied between 47.6- 954 pg/ml with an average of 273.8 pg/ml (S.D. 225.5). These data show that there is a significant difference in Inhibin B concentration between the two groups of subjects. Therefore, premature rupture of foetal membranes can be diagnosed by measuring the Inhibin B concentration in the vaginal fluid of pregnant women.

Table 1 : Inhibin B concentration determined in vaginal swabs collected from individuals from group B in example 1 , clinically diagnosed as having either intact or premature ruptured foetal membranes.

Patient Clinical diagnosis Inhibin B concentration*

1B Intact membranes 16.6

2B Intact membranes <15

3B Intact membranes 27.2

4B Intact membranes <15

5B Intact membranes <15

6B Intact membranes 34.7

7B Intact membranes <15

8B Intact membranes 21.1

9B Intact membranes 24.7

10B Intact membranes <15

11B Intact membranes <15

12B Intact membranes <15

13B Intact membranes <15

14B Intact membranes <15

15B Intact membranes <15

16B Intact membranes 39.1

17B Intact membranes <15

18B Intact membranes 31.9

19B Intact membranes 19.1

20B Intact membranes 51.5

21B Intact membranes <15

22B Intact membranes <15

23B Intact membranes <15

24B Intact membranes <15

25B Intact membranes 56.4

26B Intact membranes 67.6 7B Intact membranes <15 8B Intact membranes 75.2 9B Intact membranes <15 0B Intact membranes <15 1 B Intact membranes 69.1 2B Intact membranes <15 3B Intact membranes <15 4B Intact membranes 64 5B Intact membranes 38.4

01 B ruptured membranes 204 02B ruptured membranes 954 03B ruptured membranes 140 04B ruptured membranes 247 05B ruptured membranes 438 06B ruptured membranes 133 07B ruptured membranes 317.4 08B ruptured membranes 47.6

109B ruptured membranes 175.7 110B ruptured membranes 63.1

111B ruptured membranes 118.1

112B ruptured membranes 185.9

113B ruptured membranes 549

114B ruptured membranes 216.2 115B ruptured membranes 607.8

116B ruptured membranes 60.6

117B ruptured membranes 196.8

* Inhibin B concentration was determined by a commercially available Inhibin B specific ELISA (Product code: MCA 1312KZZ; Serotec, UK; Groome NP et al., 1996 J. Clin. Endocrinol. Metab. 81: 1401-1405). Values are expressed as pg/ml.

Claims

1 A diagnostic method for detecting the rupture of foetal membranes, said method comprising a) obtaining a sample of vaginal secretion fluid from a pregnant patient and b) determining the presence of Inhibin-B in said sample.

2 The method of claim 1 in which Inhibin B is determined immunologically.

3 The method of claimsl and 2 in which Inhibin B is determined in a sandwich immunoassay that comprises a monoclonal antibody against the β subunit of Inhibin B attached to a solid support and an antibody against the α subunit attached to a label, or vice-versa.

4 The method of any of claims 1 - 3 in which Inhibin B is detected in a rapid manual assay device, said device comprising a porous carrier, a labelled antibody specific for Inhibin B which is freely mobile through said porous carrier and an immobilised unlabeled antibody able to capture the complex formed between Inhibin B and said labelled antibody.

5 A kit for detecting rupture of foetal membranes characterised in that it contains at least one binding substance with specific binding for the beta-subunit of Inhibin-B as well as means for vaginal fluid sample collection and means for detecting the complex formed between Inhibin B and the specific binding substance.

fi The kit of claim 5 wherein said binding substance is an immobilised antibody and said means for detecting the complex comprises a labelled antibody.

Z Use of inhibin B for detecting the rupture of foetal membranes