Premature rupture of membranes (PROM) is a significant complicating factor in contemporary obstetric practice.
The amniotic membrane protects the foetus and intact, healthy foetal membranes encourage an optimal pregnancy outcome. Biochemical changes that occur naturally during or just prior to labour reduce the integrity and elasticity of the membranes, rendering them vulnerable to rupture and thus contributing to the initiation of labour.
Once a rupture has occurred, the mother-to-be is at risk of going into labour irrespective of gestational age, and has an increased risk of developing infection. PROM is defined as the spontaneous rupture of membranes prior to the onset of actual uterine contractions, and accurate diagnosis is crucial to allow timely and appropriate intervention.
In the UK, pre-term premature rupture of membranes (PPROM) complicates only two percent of pregnancies, but is associated with 40% of preterm deliveries and can result in significant neonatal morbidity and mortality. PPROM is associated with three causes of neonatal death: prematurity, sepsis and pulmonary hypoplasia and there are maternal risks associated with chorioamnionitis.1 Timely and accurate diagnosis allows for gestational age-specific interventions to optimise perinatal outcomes and minimise the risk of potential complications to both the mother and the foetus.
This article aims to provide an overview of the various diagnostic options available to clinicians and their comparative reliability in providing an accurate diagnosis of rupture of membranes (ROM). We are indebted to Dr Ross McQuivey, MD, for his clinical input.
Conventional Clinical Assessments
The most commonly used conventional diagnostic for PROM is the sterile speculum examination (SSE).
A speculum is used to visually determine the presence of a pool of amniotic fluid in the posterior vaginal fornix which is highly suggestive of amniorrhexis (rupture of membrane). The efficacy of this method is compromised in cases where the leak of amniotic fluid is small, and the presence of vaginal infections such as cervicitis or vaginitis can lead to high false positive diagnoses.
The Nitrazine/pH Test requires the use of a swab to collect cervicovaginal secretions. Nitrazine paper is used to confirm only a change in the pH levels of cervicovaginal secretions within the naturally acidic environment of the vagina, and is associated with high false positive rates related to cervicitis, vaginitis, alkaline urine, and contamination with blood, semen or antiseptic agents.2 As a result the sensitivity of the Nitrazine test ranges from 90-97% and the specificity from 16-70%.3
The Fern Test requires the collection of cervicovaginal secretions which are then dried and examined under a microscope. The presence of amniotic fluid is determined by the appearance of a characteristic crystal pattern. False positives have been recorded as a result of fingerprints, and contamination with blood, semen or cervical mucus. The Fern Test has a reported sensitivity of 51% for women not in labour, and a specificity of 70%.3
Although ultrasound alone is not diagnostic, it may be used to support a diagnosis as oligohydramnios (low amniotic fluid) is highly suggestive of ROM when combined with a characteristic history. Ultrasound, however, is unable to detect small reductions of amniotic fluid, and marked reductions in levels may result from other clinical factors such as severe uteroplacental insufficiency.
Once considered the gold standard for diagnosing ROM, the Amnio-Dye test requires an invasive amniocentesis and injection of a blue dye into the amniotic fluid. ROM is confirmed by placing a tampon in the vagina and observing the tampon for the presence of blue dye. Risk factors include infection, miscarriage, rupture of membranes, cramping and vaginal bleeding and the Amnio-Dye Test is seldom used in contemporary medical practice.
The Implications of False Positives and False Negatives
False positives occur when the diagnostic methodology indicates ROM when the membranes of the mother-to-be are in fact intact. The lower the specificity of the diagnostic method being utilised, the higher the likelihood of false positives. Traditional methodologies for detecting ROM have high levels of false positives and false negatives, as a result of their inability to differentiate amniotic fluid from other possible interfering components such as blood, urine, semen, vaginal mucus, and infection.
While false positives do not jeopardise the safety of either the mother-to-be or the foetus, they may lead to the mother-to-be being unnecessarily admitted for observation. As well as the potential cost implication to the NHS and the inconvenience of admission to the patient, false positives may impact the psychological well-being of the mother-to-be.
False negatives occur when the diagnostic methodology fails to detect ROM when the membranes of the mother-to-be have in fact ruptured. The lower the sensitivity of the diagnostic method being utilised, the higher the likelihood of false negatives. False negatives are clinically a much more significant issue than false positives, as they may lead to failure to implement appropriate interventions to minimise potential risk factors to both the mother-to-be and the foetus.
It is evident that diagnostic accuracy is critical in determining timely and appropriate gestational age-specific interventions. Traditional diagnostic methodologies may lead to misdiagnosis as they are complicated by factors beyond the simple presence or absence of amniotic fluid in the vaginal vault, which has led to considerable variances in the reported efficacy of traditional methodologies in terms of sensitivity and specificity.
Immunoassay Tests – technological advances in the detection of ROM
The introduction of non-invasive immunoassay tests has provided clinicians with a new method of diagnosing ROM, and comparative studies with traditional methodologies have confirmed that immunoassay tests offer greater diagnostic accuracy.4 Immunoassay tests utilise lateral flow technology to detect the presence of proteins recognised as being present in high levels in amniotic fluid, and are designed to provide a rapid point of care diagnosis.
Immunoassay tests such as AmniSure and Actim PROM utilise a single protein marker and monoclonal antibodies to detect ROM. AmniSure detects the presence of placental alpha-microglobulin-1 (PAMG-1) whilst Actim PROM detects placental protein 12 (PP12) otherwise known as Insulin-like Growth Factor Binding Protein-1 (IGFBP-1). Both proteins are well documented in the literature as being excellent markers for the detection of ROM. PAMG-1 is abundant in amniotic fluid (2000-25000ng/mL) whilst concentrations in cervicovaginal secretions in the absence of ruptured membranes are 1000-10,000 times lower (0.05-0.2ng/mL).5 The concentration of IGFBP-1 is even more abundant in amniotic fluid (10500-35000ng/mL), whilst concentrations in cervicovaginal secretions in the absence of ruptured membranes are extremely low. In addition, the concentration of IGFBP-1 is 100-1000 times lower in maternal serum.6,7
AmniSure’s reported sensitivity and specificity ranges between 93-99% and 69-99% respectively.2,4,5,8 Actim PROM’s reported sensitivity and specificity ranges between 74-97%.4 Chen and Dudenhausen (2008) concluded in a comparative study of AmniSure and Actim PROM that the PAMG-1 test (AmniSure) seemed to be a more sensitive immunoassay test when compared with the IGFBP-1 test (Actim PROM).9 Tagore and Kwek (2010), however, in a later study have found no significant statistical difference in the respective sensitivity of PAMG-1 and IGFBP-1.6 ROM Plus, a new foetal membrane rupture test now available in the UK, is unique in its detection of two protein markers: PP12 and alpha-fetoprotein (AFP). The utility of PP12 aka IGFBP-1 as an attractive marker for ROM has been established above. Whilst PP12 provides a good marker for ROM across the entire gestational age range, the level of AFP increases with advancing gestation peaking late in the second trimester/early third trimester. The concentration of AFP in amniotic fluid has been determined as being between 2800-26000ng/mL.10 While utilising AFP, Kishida et al (1996) reported a 97.9% sensitivity and a specificity of 97.8% when diagnosing PROM in patients between 11 and 40 weeks gestation.11 Several others have shown AFP to be an excellent marker for the detection of PPROM where accurate diagnosis is most critical.12,13
ROM Plus is also unique in utilising a combination monoclonal and polyclonal antibody approach. Monoclonal antibodies will combine only to a single amino acid epitope, while polyclonal antibodies will combine to multiple epitopes. Proteins break down (denature) over time and monoclonal antibodies do not recognise denatured proteins. A polyclonal approach therefore improves the test’s sensitivity over time, whilst a monoclonal approach alone may fail to detect ROM.
In a multi-centre prospective observational study, ROM Plus was shown to have a sensitivity of 99.5% and a specificity of 90.7%.14 In pre-term patients, where diagnosis is most critical to allow timely and appropriate intervention, both sensitivity and specificity were found to be 100%.
Performing an Immunoassay Test
Immunoassay tests provide a convenient point of care alternative to traditional diagnostic methodologies and are easy to use and read.
A swab is taken from the vagina, mixed with a buffer solution, and applied to a test strip. Depending on the test being used, results can be read within five to 20 minutes.
It has been seen that traditional methodologies for detecting ROM have high levels of false positives and false negatives, as a result of their inability to differentiate amniotic fluid from other possible interfering components such as blood, urine, semen, vaginal mucus, and infection. The determination of the presence or absence of amniotic fluid is largely subjective, and may lead to inappropriate intervention.
False negatives are clinically a much more significant issue than false positives, as they may lead to failure to implement appropriate interventions to minimise potential risk factors to both the mother-to-be and the foetus.
It is evident that diagnostic accuracy is critical in determining timely and appropriate gestational age-specific interventions, and this review suggests that the increased sensitivity and specificity of immunoassay technology allows a more confident diagnosis of ROM than traditional diagnostic methodologies.
- RCOG, 2006 (minor amendment October 2010). Green-top Guideline No. 44: Preterm Prelabour Rupture of Membranes. London: Royal College of Obstetricians & Gynaecologists.
- Lee, S.E., Park, J.S., Norwitz, E.R., Kim, K.W., Park, H.S. and Jun, J.K., (2007). Measurement of Placental Alpha-Microglobulin-1 in Cervicovaginal Discharge to Diagnose Rupture of Membranes. Obstet Gynecol, 109, pp.634-640.
- Mead, P.B. (1980). Management of the patient with premature rupture of the membrane. Clin Perinatol, 7:243-255.
- Caughey, A.B., Robinson, J.N., and Norwitz, E.R., (2008). Contemporary Diagnosis and Management of Preterm Premature Rupture of Membranes. Rev Obstet Gynecol,1(1), pp.11-22.
- Phupong, V. and Sonthirathi, V., (2012). Placental alpha-microglobulin-1 rapid immunoassay for detection of premature rupture of membranes. J Obstet Gynaecol Res, 38(1), pp.226-230.
- Tagore, S. and Kwek, K., (2010). Comparative analysis of insulin-like growth factor binding protein-1 (IGFBP-1), placental alpha-microglobulin-1 (PAMG-1) and nitrazine test to diagnose premature rupture of membranes in pregnancy. J Perinat Med, 38, pp.609-612.
- Rutanen, E.M., Pekonen, F., Karkkainen, T., (1993). Measurement of insulin-like growth factor binding protein-1 in cervical/vaginal secretions: comparison with the ROM-check Membrane Immunoassay in the diagnosis of ruptured fetal membranes. Clin Chim Acta, 214(1), pp.73-81.
- Lee, S.M., Lee, J., Seong, H.S., Lee, S.E., Park, J.S, Romero, R. and Yoon, B.H., (2009). The clinical significance of a positive Amnisure testTM in women with term labor with intact membranes. J Matern Fetal Neonatal Med, 22(4), pp.305-310.
- Chen, F.C. and Dudenhausen, J.W., (2008). Comparison of Two Rapid Strip Tests Based on IGFBP-1 and PAMG-1 for the Detection of Amniotic Fluid. Am J Perinatol, 25(4), pp.243-246.
- Seppala, M., Ruoslahti, E., (1972). Alpha fetoprotein in amniotic fluid: an index of gestational age. Am J Obstet Gynecol, 114, pp.595-8.
- Kishida, T., Yamada, H., Negishi, H., Sagawa, T., Makinoda, S., Fujimoto, S., (1996). Diagnosis of premature rupture of the membranes in preterm patients, using an improved AFP kit: comparison with ROM-check and/or nitrazine test. Euro J Obstet Gynecol Reprod Bio, 69(2), Nov, pp.77-82.
- Rochelson, B.L., Richardson, D.A., Macri, J.N., (1983). Rapid assay – possible application in the diagnosis of premature rupture of the membranes. Obstet Gynecol, 62(4), pp.414-8.
- Yamada, H., Kishida, T., Negishi, H., Sagawa, T., Yamaguchi, M., Sato, C., Nakamura, I., Sato, H., Sakai, K., Yamaguchi, T., Fujimoto, S., (1997). Comparison of an Improved AFP Kit with the Intra-Amniotic PSP Dye-Injection Method in Equivocal Cases of Preterm Premature Rupture of the Fetal Membranes, J Obstet Gynaecol Res, 23(3), pp.307-311.
- Thomasino, T., Levi, C., Draper, M., Neubert, G., (2012). Diagnosing Rupture of Membranes Using Combination Monoclonal/Polyclonal Immunological Protein Detection. (Under review).