OBSTETRICS

OBSTETRICS

Diagnosis of Premature Rupture of Membranes:

Inspiration From the Past and Insights for the
Future
Amira El-Messidi, MD, FRCSC, RDMS,1 Alan Cameron, MD, FRCOG, FRCP (Glas)2
1
Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal QC
2
Fetal Medicine, Department of Obstetrics and Gynaecology, The Queen Mother’s Hospital, Glasgow, Scotland

Abstract
Objective: To review the diagnostic methods described to confirm
premature rupture of membranes during pregnancy, and to assess
their effectiveness in establishing the diagnosis.
Data Sources and Extraction: The medical literature was searched
to identify all relevant studies and reviews on methods for diagnosis
of membrane rupture published in English up to January 31, 2009.
Medline and the Cochrane databases were searched, and
reference lists in identified articles were also examined. Articles
not available through journals’ online editions were retrieved by
manual search.
Study Selection: We identified 71 original studies and reviews on
diagnostic methods of chorioamniotic membrane rupture published
in English. These articles were reviewed and results were
summarized based on the diagnostic test assessed.
Conclusion: Recognition of the importance of and difficulties in
confirmation of rupture of the chorioamniotic membranes pervades
past and present obstetric publications. The subjectivity and poor
sensitivity of early diagnostic techniques for confirmation of
ruptured membranes sparked technical advancements using
biochemical markers. None of these biochemical tests have
gained popularity, although novel techniques involving placental
markers such as placental alpha microglobulin-1 may provide a
future solution to the problem of diagnosing chorioamniotic
membrane rupture.
Résumé
Objectif : Analyser les méthodes diagnostiques décrites pour
confirmer la rupture prématurée des membranes pendant la
grossesse, ainsi qu’afin d’en évaluer l’efficacité pour ce qui est
de l’établissement du diagnostic.
Sources et extraction des données : Des recherches ont été
menées dans la littérature médicale en vue d’identifier toutes
les études et analyses pertinentes portant sur les méthodes de
procéder au diagnostic de la rupture des membranes qui ont été
publiées en anglais jusqu’au 31 janvier 2009. Des recherches
ont aussi été menées dans Medline et les bases de données
Cochrane, et les listes de références des articles identifiés ont
également été examinées. Les articles n’étant pas disponibles
par l’intermédiaire des éditions électroniques des revues
spécialisées ont été récupérés par recherche manuelle.
Sélection des études : Nous avons identifié 71 études et analyses
originales portant sur les méthodes permettant de diagnostiquer
la rupture des membranes chorioamniotiques qui ont été publiées
en anglais. Ces articles ont été analysés et les résultats ont été
résumés en fonction du test diagnostic évalué.
Conclusion : La reconnaissance de l’importance de la confirmation
de la rupture des membranes chorioamniotiques et de ses
difficultés est omniprésente dans les publications passés et
présentes du domaine de l’obstétrique. La subjectivité, la faible
sensibilité et les premières techniques diagnostiques visant la
confirmation de la rupture des membranes ont donné lieu à des
percées techniques faisant appel à des marqueurs biochimiques.
Aucun de ces tests biochimiques ne s’est démarqué, et ce, malgré
le fait que des techniques novatrices faisant appel à des
marqueurs placentaires (comme l’alpha-microglobuline-1
placentaire) puissent fournir une future solution au problème
que pose le diagnostic de la rupture des membranes
chorioamniotique.
J Obstet Gynaecol Can 2010;32(6):561–569
INTRODUCTION
remature rupture of membranes may occur at term or
P immediately preceding labour, or it may be an unex-
pected complication during the preterm period, when it is
referred to as preterm premature rupture of membranes.
Chorioamniotic membrane rupture may have several
underlying causes, although in many cases PROM and
PPROM will not have recognized etiologies. The
pathophysiology leading to PROM at term has been shown
to be different from the pathophysiology leading to PPROM.
At term, weakening of the membranes may result from
physiologic changes combined with shearing forces induced
by contractions.1–4 Generalized weakness of the membranes
has been more difficult to identify with prematurely ruptured
membranes.5 PPROM may result from a focal deficit rather
than a generalized weakness of the membranes.6
Term PROM complicates approximately 8% of pregnancies.7
Among these, approximately 50% of affected women will
begin labour spontaneously within 12 hours, 70% within 24
hours, 85% within 48 hours, and 95% within 72 hours.7–9
Fetal morbidities associated with term PROM include

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OBSTETRICS
ascending infection and in utero cord compression.7 Mater-
nal risks of term PROM include chorioamnionitis and
postpartum febrile morbidity.7,9
Preterm PROM, a complication of 2% to 20% of all
deliveries,10 is a known important contributor to maternal
and perinatal morbidity and perinatal mortality. Latency in
PPROM, defined as the interval between PROM and birth,7
is known to be inversely related to gestational age at rupture,
and is also related to a multitude of other factors, including
number of fetuses,11 severity of oligohydramnios,12
myometrial thickness,13 and the existence of maternal or
obstetrical complications. The major cause of perinatal
morbidity and mortality associated with PPROM is
prematurity.7 Morbidities related to prematurity include
respiratory distress syndrome, necrotizing enterocolitis,
interventricular hemorrhage, cerebral palsy, and sepsis.7
Other complications include in utero umbilical cord com-
pression, cord prolapse and fetal distress, fetal
malpresentation, placental abruption, chorioamnionitis
with subsequent endometritis, and risk of operative delivery
from this multitude of factors.7 Maternal sepsis is a rare but
life-threatening complication reported in nearly 1% of cases.7
For over 70 years, there has been controversy among health
care professionals about the optimal approach to clinical
assessment and diagnosis of prematurely ruptured membranes.
In most cases, membrane rupture can be confirmed by doc-
umenting amniotic fluid leakage from the cervical os with
visualization of pooling in the posterior vaginal fornix.14
However, the diagnosis of PROM is difficult if there is a
slow fluid leak or any bleeding, or when the classic “gush of fluid”
does not occur.15 In addition, the relatively small amount of
amniotic fluid present early in gestation further challenges
the diagnosis of ruptured membranes.16 Ladfors et al.17
showed that even later in pregnancy (after 34 weeks), speculum
examination for visualization of amniotic fluid carries a
12% false negative rate when no fluid is seen. In 1951,
Schumann18 described the “two sac” theory of membrane
rupture: he stated that amniotic fluid can dissect between
the two layers of membranes and produce a bulge that ruptures
into the vagina, leaving one intact layer with remaining fluid
enclosed and appearing to rupture at a later time. In such
cases, women give a history of fluid leakage, and tests for
leakage should be positive, although membranes appear clin-
ically to be intact at delivery. Occasionally, women present
with a history suspicious for membrane rupture, but clinical
ABBREVIATIONS
AFI amniotic fluid index
PAMG-1 placental alpha-microglobulin-1
PROM premature rupture of membranes
PPROM preterm premature rupture of membranes
562 l JUNE JOGC JUIN 2010
examination is negative or inconclusive; they subsequently
return with clearly identifiable rupture of the membranes.
Whether these cases represent preliminary minimal
transudation of fluid across weakened membranes or minimal
leakage around a firmly applied fetal presenting part cannot
be determined. In approximately 20% to 25% of cases,
rupture of membranes is not grossly apparent.10,19 A patient’s
history may suggest membrane rupture, but test results are
non-confirmatory, creating an obstetrical dilemma. Early
and accurate diagnosis of membrane rupture would allow for
gestational age-specific interventions to optimize perinatal
outcome and minimize serious complications.20 Therefore,
the search for an ideal test to diagnose membrane rupture
definitively with no delay continues.
Although initial studies may be encouraging, diagnostic
techniques are usually found subsequently to be limited by
inaccuracies from false positives and false negatives with
poorer sensitivities and specificities than originally anticipated.
The ideal test should be simple, rapid, inexpensive, and
non-invasive. Optimally, the accuracy of the test should not
be hampered by the presence of blood, semen, infected
urine, or other contaminants. An accurate biochemical marker
for membrane rupture should have a high concentration in
the amniotic fluid, a low concentration in maternal blood,
and an extremely low background concentration in
cervicovaginal discharge with intact membranes.10
We sought to review the diagnostic methods currently used
to confirm premature rupture of membranes and to assess
their effectiveness in establishing the diagnosis.
METHODS
We searched Medline and the Cochrane databases for all
relevant studies and reviews published in English on
accuracies of diagnostic methods for the diagnosis of rupture
of the membranes up to January 31, 2009. Key words used
for the Medline search were “premature rupture of membranes,”
“amniotic fluid,” “diagnosis,” and “tests.” Articles not
available online were retrieved by manual search. We found
no Cochrane reviews of diagnostic methods of PROM, as
discussed in this report. The studies identified showed evidence
from levels II-2, II-3, and III, according to ranking of The
Canadian Task Force on Preventive Health Care.21,22
MICROSCOPIC FETAL CELL IDENTIFICATION
A literature review by Friedman et al.23 described the first
microscopic technique for identification of fetal particles in
amniotic fluid, developed by Philipp et al in 1929 and
published in the German literature. In this technique, fetal
lanugo hairs were identified in amniotic fluid. This was presumed
to be incontrovertible evidence of membrane rupture when
identified in vaginal secretions. However, because of the
 Diagnosis of Premature Rupture of Membranes: Inspiration From the Past and Insights for the Future

Accuracy of diagnostic tests for PROM

Discriminatory Patients, n SN, % SP, % FN, % FP, %

concentration Sampling Mean Mean Mean Mean Mean
Test used for diagnosis Mean (range) site (range) (range) (range) (range) (range)

Fetal cells (bromthymol N/A Vagina 239 90.2 98.4 7.5 1.7
blue)34,36 (176–239) (85.7–94.7) (97.4–99.3) (0.6–14.3) (0.7–2.6)
pH 19,23,31,39,40,46,54,55,57,59,62 ³ 6.5 Vagina 125 90.2 79.3 12.4 28.2
(6.0–7.0)35,36,55,57,59 (46–250) (81.3–100.0) (16.0–100.0) (0–33.3) (0–100.0)
Fetal cells (Masson stains) 24 N/A Vagina 275 94.2 99.0 5.8 1.0
Fetal cells (Papanicolaou N/A Vagina 75 92.8 92.7 4.3 7.4
stain)23,28 (50–100) (92.0-93.5) (91.3–94.0) (2.0–6.5) (6.0–8.7)
Fetal cells (Pinacyanol N/A Vagina 150 88.3 95.4 11.7 3.7
stain)23,30,31 (100–250) (71.0–98.7) (91.7–98.8) (1.3–29) (1.2–8.3)
Fetal cells (Acridene N/A Vagina 200 75.5 90 24.6 10.0
orange stain)23,32 (100–300) (61.2–89.7) (87.3–92.7) (10.3–38.8) (7.3–12.7)
Fetal cells (Nile N/A Vagina 106 89.5 98.6 10.6 1.5
blue sulphate)23,33 (100–111) (80.7–98.2) (97.1–100.0) (1.8–19.3) (0–2.9)
Crystallization23,37,38-40,43,54,74 N/A Vagina 198 90.8 95.3 4.4 4.7
(51–509) (62.0-98.5) (88.2–100.0) (1.2–12.9) (0.0–11.8)
History alone23 N/A N/A 100 90.3 88.4 9.7 11.6
23
History + pH + crystalization N/A Vagina 100 90.8 95.6 9.2 4.4
History + pH + Nile Blue stain23 N/A Vagina 100 87.1 92.7 12.9 7.3
History + Crystallization + Nile N/A Vagina 100 87.1 95.6 12.9 4.4
Blue stain23
History + ultrasound + N/A N/A 83 85.1 78.6 11.1 21.4
crytallization74
pH + Crystallization + Nile N/A Vagina 100 90.8 95.6 9.2 4.4
Blue stain23
AmnioInjection Evans blue N/A Amnio- 18 100.0 100.0 0.0 0
T–182447 centesis
DAO19,58,67 22.5 µU/L19, 67 Vagina 168 88.9 98.3 8.5 1.7
(100–272) (83.0–100.0) (95.0–100.0) (0–17.0) (0–5.0)
3.32 log glucose + log fructose49 17.7279 N/A 215 98.9 96.0 7.7 2.6

continued

scant amounts of fetal lanugo hair in amniotic fluid and the
fact that such hair was present in amniotic fluid only later in
pregnancy, this method never gained popularity. As this
study was not published in the English language, statistical
data are not shown in the Table.
Subsequent diagnostic tests were based on cytologic
inspection for fetal squamous cells in the vagina. Interest in
cytologic diagnosis was based on the noted absence of stain
within the cytoplasm and nucleus of vernix caseosa cells
compared with vaginal squamous cells.24 Investigators
searched for fetal cells in vaginal fluid using diverse stains
including Masson stain,24 Sudan III to demonstrate fetal fat
particles,25–27 Papanicolaou stain,28,29 pinacyanole stain,30,31
acridene orange stain,32 and Nile blue sulphate stain.33
Recognizing the challenge to diagnose PROM in clinically
equivocal cases, Averette et al.30 studied 100 patients in
whom the integrity of the chorioamniotic membrane could
not be determined by visualization of fluid on speculum
examination. Fetal cell staining with pinacyanole to identify
vernix caseosa cells in such cases showed high accuracy
rates (97%), similar to those in earlier studies among
patients with clinically confirmed membrane rupture.
Although fetal cell staining techniques were considered
rapid, simple, and durable, concerns about inaccuracies
emerged. Contamination from the powder on examination
gloves gave false positive results as anuclear granules
mimicked fetal cells,31 and hypercornified vaginal cells also
simulated anucleate fetal cells.32 False negatives resulted
from the subjectivity of inspection, having insufficient
cellular material,32 or after a prolonged time interval from
membrane rupture. Accuracy rates appear to diminish with
increased duration between presumed membrane rupture

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Table continued

Discriminatory
concentration Patients, n SN, % SP, % FN, % FP, %
Mean Sampling Mean Mean Mean Mean Mean
Test used for diagnosis (range) site (range) (range) (range) (range) (range)

Prolactin RIA50,51,52 3mU/mL,59 Vagina 57 88.0 85.0 68.0 15.0

2ng/mL,51 (20–100) (76.0–100.0)51,52 (70.0–100.0)51,52 (0–30.0)51,52
20.2µIU/mL52
AFP (RIA)60,67,76,77,80 53.3 µg/L Vagina 122 80.6 91.9 5.4 8.1
(2-125) (52–312) (17.6–100.0), (80.0–97.4), (0.0–16.0), (2.6–20.0),
NR60 NR60 NR60 NR60
AFP (RIA)57,58,59 77.5 µg/L Cervix 89 92.0 86.9 8.0 13.2
(30-125) (46–131) (84.0–100.0) (85.7–88.0) (0–16.0) (12.0–14.3)
human placental lactogen 31 ng/mL Vagina 52 NR NR NR NR
(hPL) RIA51
fFN monoclonal assay 50 ng/mL Vagina 160 93.6 65.7 13.1 17.0
(ROM check)58,59,63,64,65 (54–406) (90.0–98.2) (26.8–97.0) (3.0–25.0) (6.0–28.0)
fFN monoclonal assay 25 ng/mL Vagina 46 87.5 92.9 11.9 7.1
(ROM check)59
IGFBP-1 immunoenzyme 35 µg/L Vagina, 130 84.9 92.8 14.7 7.3
(PromTest)19, 46, 65,71,72,73,74 (3-100) cervix72 (54–174) (74.4–100.0) (71.4–98.2) (0–39.2) (1.8–28.6)
IGFBP-1 dipstick 400 µg/L Amniocentesis 20 NR NR NR NR
(ActimPROM)10
Lactate68 ³4.5 mmol/L Vagina 200 86 92 92 87
b hCG (ELCIA) 52,60,61,62
46.4 mIU/mL Vagina 137 83.9 89.5 9.1 10.5
(100–188) (68.0–100.0) (84.2–95.0) (0.0–25.0) (5.0–16.0)
Ultrasound amniotic < 80 mm N/A 151 94.0 91.0 6.2 9.0
fluid index46
Creatinine concentration 0.36 mg/dL Vagina 114 100.0 100.0 0.0 0.0
assay69,70 (88–139)
Urea concentration70 12 mg/dL Vagina 139 100.0 100.0 0.0 0
PAMG-1 5 ng/mL Vagina 194 98.8 93.8 1.2 6.3
(AmniSure™)76,77 (184–203) (98.7–98.9) (87.5–100.0) (1.1–1.3) (0.0–12.5)
PAMG-1 (AmniSure™)10 5 ng/mL Amniocentesis 20 NR NR NR NR
AmnioSense™ pH> 5.2 N/A 69 99.2 70.0 0.85 30.0
absorbent pad15,78 (34–103) (98.3–100.0) (65.0–75.0) (0–1.7) (25.0–35.0)

Legend: SN: sensitivity; SP: specificity; FN: false negative; FP: false positive; DAO: diamine oxidase; IGFBP-1: insulin growth factor binding protein-1;
PAMG-1: placental alpha microglobulin-1; bhCG: beta subunit human chorionic gonadotropin; fFN: fetal fibronectin; AFP: alpha fetoprotein; RIA: radioimmunoassay;
NR: not reported; N/A: not applicable

and diagnostic testing. Early authors including King34 and
Bourgeois24 reported 57% and 81.8% accuracy rates
respectively, beyond the initial 24-hour period of membrane
rupture. This was later confirmed by others, and shorter
intervals to testing were shown to affect rates of diagnostic
accuracy.23
As novel methods were developed, older cytologic techniques
began losing popularity, because they were time-consuming,
required trained cytologists, were ineffective before 32 weeks’
gestation, and provided an uncertain diagnosis of membrane
rupture.29
LITMUS PAPER AND pH TESTING
Litmus paper testing began development at approximately
the same time as fetal cell staining methods. Because of the
difference in pH of vaginal secretions (4.5 to 5.5) and
amniotic fluid (7.0 to 7.5), it was rightly assumed that the
pH of vaginal secretions would rise when contaminated by
escaping amniotic fluid.35 This assumption prompted pre-
liminary experiments with bromthymol blue dye and, later,
nitrazine applicators. Although similar in principle to
bromthymol blue, a reported advantage of nitrazine is the
complete change in colour of the applicator when exposed
to amniotic fluid in vaginal secretions.36 Impregnated with

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 Diagnosis of Premature Rupture of Membranes: Inspiration From the Past and Insights for the Future
sodium-dinitro-phenylozonapthol-disulphonate, nitrazine
paper showed promising early results in detecting membrane
rupture, with accuracies of 100%35 and 98.9%36 in clinically
ruptured cases, and similarly high rates of accuracy among
intact cases. This preliminary high optimism regarding
nitrazine testing decreased by the 1960s,31,37 because of false
positive results from vaginal infections, blood, semen, alka-
line urine or alkaline antiseptics, and false negatives in cases
of minimal leakage from chronic membrane rupture or
“high leak” of the membranes.15
AMNIOTIC FLUID CRYSTALLIZATION
Amniotic fluid crystallization, created primarily by the
sodium chloride and protein content, began to dominate
cytologic stains, with reported accuracies in clinically
ruptured cases ranging from 73.0% to 98.5%.26,27,32,37–40
Screening for arborization provided an accuracy of 97.8%
compared with 87.3% for litmus paper testing.37 Interest-
ingly, Tricomi et al.37 suggested that fluid for examination
should be aspirated from the vagina no further than 3 cm
from the introitus to avoid contamination and false positive
ferning results from cervical mucus lying in the posterior
fornix. Initial optimism with low false negative rates
changed as missed diagnoses in the presence of blood,
meconium, or heavy leukorrhea became recognized.23 False
positive results were subsequently attributed to fingerprints,
semen, or cervical mucus.23,41,42
In most ferning specimens tested in early studies,23,26,37–40
sensitivity and specificity analyses were performed on
women in labour, in whom there was certainty about the
diagnosis of membrane rupture.43 In most clinical situa-
tions, however, the test is intended for patients who have
equivocal rupture. De Haan et al.43 showed that the modest
diagnostic reliability of the fern test compared with earlier
published data is at least partly due to differences in study
populations (i.e., whether the women were in labour or
not). These authors showed false positive and false negative
rates of 11.8% and 2.0%, respectively, in labouring women
tested for amniotic fluid crystallization, compared with
rates of 21.2.% and 40.6%, respectively, in women not in
labour.43 Thus, the result of the fern test became viewed as
supportive rather than conclusive for non-labouring
women with non-specific vaginal fluid loss.
COMBINATION OF DIAGNOSTIC TESTS
Many early studies either tested one method for diagnosis
of membrane rupture or compared two methods. Friedman
et al.23 were the first to test individual methods as well as dif-
ferent combinations of tests, which more commonly
reflects true clinical practice. They also attempted to corre-
late the time of testing with the time of presumed rupture.
Indeed, these authors showed a precipitous increase in error
rate when the time interval between membrane rupture and
sampling exceeded two hours.23 False negative tests were
more commonly associated with prolonged rupture. None
of the traditional procedures proved entirely satisfactory in
isolation, but combinations of any three of the following
produced a diagnostic accuracy of 93.1%23:
1. a positive history,
2. a positive nitrazine test,
3. fluid crystallization, or
4. Nile blue sulphate staining.
ULTRASOUND ASSESSMENT OF
AMNIOTIC FLUID VOLUME
In the late 20th century, it was theorized that the
sonographic identification of oligohydramnios would
develop after membrane rupture,44 thereby facilitating diag-
nosis and subsequent management. Manning et al.45 initially
described a technique for measuring by ultrasound the
deepest vertical pool of amniotic fluid in patients with
intrauterine growth restriction. This method was later used
to assess membrane rupture; it was shown that ultrasound
quantification of the deepest amniotic fluid pocket is of
poor quality in confirming membrane rupture.44 No signifi-
cant difference was found in the mean depth of amniotic
fluid pocket between 100 patients with confirmed term
PROM and 51 patients with intact membranes.44
Oligohydramnios may not be detected in patients with
confirmed PROM, possibly because drainage may become
intermittent or even stop once the presenting part descends
and acts as a plug, preventing further drainage.44 Robson et
al. suggested that a significant amount of amniotic fluid
needs to drain rapidly and continuously for
oligohydramnios to occur, especially because the fluid is
replaced to varying degrees by the fetus.44 Erdemoglu et
al.46 showed that a reduction in the four-quadrant AFI
below 80 mm did not reliably identify cases of suspected
membrane rupture by history with negative visualization of
fluid by speculum examination. The measurement of AFI
offers no advantage over measurement of a single vertical
pocket of fluid in cases where ultrasound is used to evaluate
possible membrane rupture.
INTRA-AMNIOTIC DYE INJECTION
By 1970, amniocentesis with injection of dye to confirm
amniotic membrane rupture had become a commonplace
procedure; it was thought to be safe and had high patient
acceptability rates.47 Prior to amniocentesis, intravenous
injection of radioisotope was performed for placental local-
ization, and the amnio-injection was performed under local
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OBSTETRICS
anaesthesia. Interestingly, the two reported disadvantages
of the procedure at that time were related to difficulty in
diagnosis in the presence of meconium-stained fluid and
the possibility of neonatal skin staining for 48 hours after
dye injection.47 Several types of stains have been used for
amnio-injections, with safety hazards reported only for
methylene blue (see below).
Although ultrasonographically guided transabdominal
instillation of indigo carmine dye (1 mL of dye in 9 mL of
sterile normal saline) and observation for fluid passage
transvaginally is designated an “unequivocal” diagnostic
method for confirmation of membrane rupture,14 this inva-
sive test carries increased maternal and fetal risk. Inherent
risks of intra-amniotic dye injection include trauma, bleeding,
infection, and preterm labour.48 While strengthening
diagnostic certainty, a “negative dye test” may occur if the
membranes seal after previous amniotic fluid leakage.
GLUCOSE AND FRUCTOSE MEASUREMENTS
Gorodeski et al.49 examined the difference between
amniotic fluid and cervicovaginal mucus in their concentra-
tions of solutes. During pregnancy, glucose and fructose are
present in high concentrations in cervical mucus, with
reported means of 240 mg/100 mL and 30.4 mg/100 mL,
respectively.49 Amniotic fluid concentrations are noticeably
lower, with mean concentrations of 39 mg/100 mL and
3.3mg/100 mL, respectively. Gorodeski et al.49 showed that
low values of glucose and fructose are found in amniotic
fluid aspirated in a true case of membrane rupture. Best
results are obtained with a calculated linear sum of the values
(3.32 log glucose + log fructose). These authors reported no
confounding by meconium or vaginal discharge.
This technique is fairly impractical in testing for membrane
rupture, and further studies have not expanded upon differ-
ential carbohydrate gradients.
MODERN METHODS
Because of the limitations of available testing methods,
investigators have sought alternative markers in vaginal
amniotic fluid, such as prolactin50–52, alpha-fetoprotein,51–59
b-subunit of human chorionic gonadotropin,52,60–62 fetal
fibronectin,58,59,63–66 diamine oxidase,19,58,67 lactate,68
creatinine,69,70 urea,70 and insulin growth factor binding
protein-1, previously called placental protein
12.10,19,46,65,66,71–74 Interest in assessment of these markers
stems from their high concentrations in amniotic fluid com-
pared with normal vaginal secretions, but all require special
laboratory equipment and training.14 Although these markers
are useful for patients with intact membranes or unequivocal
membrane rupture, they remain unpopular due to cost,
566 l JUNE JOGC JUIN 2010
testing complexity, and low test sensitivities in cases of
equivocal rupture.
PLACENTAL ALPHA-MICROGLOBULIN-1
Initially isolated in Moscow in 1975,75 PAMG-1 has under-
gone recent evaluation for diagnostic testing in PPROM.
This 34kDa placental glycoprotein is abundant in amniotic
fluid (2000–25 000 ng/mL), with much lower concentra-
tions in maternal blood (5–25 ng/mL).20 The protein is
present in negligible amounts in cervicovaginal secretions
with intact membranes (0.05–0.2ng/mL).76 The 1000- to
10 000-fold difference in concentration between amniotic
fluid and cervicovaginal secretions stimulated interest in a
PAMG-1 immunoassay. Marketed as AmniSure (AmniSure
International, Cambridge, MA), the assay’s minimum detec-
tion threshold for PAMG-1 is 5 ng/mL, sufficient for 99%
accuracy with minimal false negatives. PAMG-1 can be
detected with as little as 0.25 µL of amniotic fluid in 1 mL of
vaginal secretions.76 In the presence of blood or vaginitis,
the background level of PAMG-1 can occasionally reach a
maximum of 3 ng/mL.76 False-positive results with use of
the AmniSure assay seem very unlikely, although these may
appear with increased use.77 Further, assay of PAMG-1
appears to be reliable over a wide range of gestational ages
(11 to 42 weeks), and proved superior to conventional com-
bined clinical tests involving visualization of fluid pooling in
the posterior fornix, arborization, and nitrazine testing.77
This test is currently available in Europe and was recently
approved by the Food and Drug Administration for use in
the United States. AmniSure is a novel rapid, non-invasive
bedside test that may be very helpful in diagnosis of difficult
cases without visible leakage. Further studies are needed to
assess the reliability of the test according to the time from
membrane rupture.
NON-INVASIVE ABSORBENT PAD
Efforts to be able to confirm chorioamniotic membrane
rupture with minute amounts of amniotic fluid have
recently led to the development of the absorbent pad,
AmnioSense. This 12 ´ 4 cm pad has a central strip that
changes colour on contact with fluid with a pH > 5.2.15,78
After contact with urine, the strip reverts to its original col-
our when dry. This is due to the detachment of conju-
gate-based nitrazine molecules by the urine ammonium
ions.15 AmnioSense has undergone cytotoxicity and skin
irritation and sensitization testing, and it complies with the
US Pharmacopoeia Guidelines.15 In a study of 34 women
presenting with suspected membrane rupture, the
AmnioSense pad initially showed 100% sensitivity; overall
specificity was 75%, but when women with bacterial
 Diagnosis of Premature Rupture of Membranes: Inspiration From the Past and Insights for the Future
vaginosis or Trichomonas vaginalis were excluded from
analysis, the specificity increased to 90%.15
In a recent study, Mulhair et al.78 compared the reliability of
the absorbent pad test with a standard of amniotic fluid
pooling in the posterior fornix on speculum examination in
a cohort of 139 women. They found a specificity of 65.0%
and a sensitivity of 98.3% for the AmnioSense pad. The two
studies of the absorbent pad currently available15,78 suggest
that a negative AmnioSense result indicates intact
membranes in term and preterm gestations in 99% of cases.
A positive result, however, suggests only a 70% chance of
ruptured membranes, and thereby warrants confirmation or
further investigations to identify infections (Table).15,78
It remains unknown whether potential confounding
substances such as semen, blood, or meconium may be
distinguished from amniotic fluid by the AmnioSense pad
test. As women with negative pad checks are unlikely to
have ruptured membranes, this would imply decreased
need for an uncomfortable and intrusive speculum
examination.
SAFETY
Safety data on diagnostic testing for rupture of
chorioamniotic membranes show potential adverse
neonatal effects related to intra-amniotic administration of
methylene blue.79,80 Several cases of neonatal hemolytic
anemia and hyperbilirubinemia, including sepsis and
neonatal death, have been reported after intra-amniotic
injection of methylene blue.80 These cases involved injection
of 3.5 mL of 1% methylene blue solution, resulting in doses of
30 to 5mg.80 Even at a lower dose of 10 mg, Cowett et al.79
reported a case of hemolytic anemia and hyperbilirubinemia
requiring two exchange transfusions and phototherapy.
These adverse effects could not be attributed to other
causes of neonatal jaundice. The neonate’s skin was discol-
oured for the first few days of life and methylene blue was
identified in the urine during the first day of life.
Intra-amniotic dye as a marker for confirmation of PROM
also poses risk to the mother, including bleeding, infection,
and preterm labour.48 It is also an invasive test used to
assess a common complaint.
We were unable to identify any safety data related to the use
of vaginal swabs, either with or without use of a speculum,
in screening for chorioamniotic membrane rupture. Potential
risks of swabs might include ascending infection. A
non-intrusive method, the AmnioSense absorbent pad, may
conceivably lead to negligible rates of infections and skin
irritation. Neither of the two published studies testing
absorbent pads15,78 report any complications from use of
the product.
CONCLUSION
Early techniques for establishing the diagnosis of ruptured
membranes included fetal cell staining, vaginal pH determi-
nation and amniotic fluid crystallization. Criticism of such
methods emphasized subjectivity in interpretation, poor
sensitivity, and numerous confounding factors cross-reacting
with substances in the vaginal reservoir.
The absence of a non-invasive “gold standard” for the diag-
nosis of PROM has led to technically advanced biochemical
markers. Despite improved diagnostic value in cases of
unequivocal membrane rupture or intactness, biochemical
markers lack the sensitivity and specificity required in
equivocal cases. They have also not become popular, in
part, because of cost and complexity in testing.
In the common clinical situation where a health care provider
encounters a patient with possible ruptured membranes,
diagnostic accuracy is the key to successful management
and improved perinatal outcome. Nearly one quarter of
patients do not present with overt clinical evidence of ruptured
membranes. We believe that recent techniques involving
placental markers such as PAMG-1 and the AmnioSense
absorbent pad, with minute sampling required,
non-invasiveness, rapidity of testing, and high accuracy
rates, may provide a solution to the clinical challenge of
diagnosing ruptured membranes.
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