The Pregnancy Lab

Laboratory testing for premature rupture of membranes

This post is by a guest author, Douglas Stickle, Ph.D.  Dr. Stickle is a professor in the Department of Pathology at Thomas Jefferson University and the director of chemistry and point-of-care testing at Jefferson University Hospitals in Philadelphia, PA.

Rupture of membranes (ROM) is the term used to describe the breaking of the amniotic sac, as normally occurs before the onset of labor. If this happens earlier than the 37^th week of pregnancy it is called preterm ROM (PROM). It’s a condition that can lead to a preterm birth, or, if very early, a preterm, premature birth.

When PROM happens, there is an increased risk of complications due to intrauterine infection, umbilical cord compression, and the neurodevelopmental disorders that are associated with a preterm delivery. Diagnosis of PROM is particularly important when the gestational age is incompatible with a viable birth, often considered to be a fetal age less than 24 weeks. In such cases, medical intervention is necessary to preserve the chances for a live birth.

Suspected cases of PROM are often investigated by laboratory analysis of fluid obtained from the vagina to detect properties or substances that should otherwise not be present unless the fluid contains amniotic fluid due to PROM. The simplest forms of testing are measurement of acidity (pH) of the fluid, or a test called "fern" testing. Fern testing refers to the fern-like appearance of amniotic fluid when it is dried on a glass slide. Both of these tests aren’t very accurate and so other tests have been developed to better identify patients with ruptured membranes.

These other tests are designed to detect molecules that are normally present in amniotic fluid but not vaginal fluid. For example, tests have been developed that detect alpha-fetoprotein (AFP) or insulin-like growth factor binding protein-1 (IGFBP-1). The presence or absence of these molecules in the specimen are determined by a lateral flow immunoassay. The assay works like commonly performed tests for human chorionic gonadotropin (hCG) (aka pregnancy tests).

These tests are highly sensitive to low concentrations of these molecules, which is both good and bad. It’s good because they can detect small amounts of the molecules and lead to a more accurate diagnosis. It’s bad because these two molecules are also present in maternal blood which means that if a sample is contaminated with blood, the certainty of a positive test to detect amniotic fluid is called into question.

From the doctor’s perspective, a practical advantage of the immunoassays is that their results are binary – the result is either positive or negative -- whereas the pH test and the fern test are more subjective and difficult to interpret definitively. However, the AFP and IGFBP-1 tests may be subject to false-positive results as the gestational age of the fetus approaches term. This suggests that, at later stages of pregnancy, these biomarkers may signify imminence of delivery.

The gold standard, or best test, to diagnose rupture of membranes is a dye test, in which a colored fluid is injected into the amniotic fluid followed by direct observation to see if the dye subsequently appears in the vaginal pool fluid. Also, ultrasound imaging of the amniotic fluid volume may also assist in diagnosis of PROM, but in individual cases such imaging may be difficult to interpret. Given the low but finite risk of complications of the dye test, the AFP and IGFBP-1 tests are often preferred as first-line tests for preterm premature rupture of membranes.

Predicting preterm birth

Preterm birth is a serious problem. Defined as delivery before 37 completed weeks, infants born preterm are at high risk of complications such as respiratory distress syndrome, bleeding into the brain, poor neurological development, and cognitive impairment. Further, preterm birth is responsible for ~75% of death in newborns without genetic anomalies.

Unfortunately, the vast majority of treatments provided to pregnant women with symptoms of preterm labor have done little to prevent preterm birth. About 13% of pregnancies end with preterm delivery and this percentage has increased steadily over the last few decades.

Risk factors for preterm birth include mother's age, socioeconomic status, and being pregnant with more than one fetus. A woman who has had a previous preterm delivery is also at risk of delivering subsequent pregnancies early as well. Preterm birth is also more common among black women compared to other ethnic groups which appears to be unrelated to socioeconomic status or other factors.

Symptoms of preterm labor are just like symptoms of term labor. However, early contractions occur frequently in pregnant women and don't always mean they are in actual preterm labor. In fact, most women with early symptoms go on to delivery term babies. This makes it difficult to diagnose true preterm labor when it occurs. A test that could accurately predict preterm labor would be helpful.

Currently there is only one lab test approved for use to predict preterm labor. Fetal fibronectin (fFN) is a protein produced by the fetal membranes during pregnancy. Although it can be detected in cervicovaginal fluid early in pregnancy it usually becomes undetectable between weeks 25 and 35. After that, it becomes detectable again after 37 weeks. In women with preterm labor, fFN can be detected in cervicovaginal fluid during the time that it is usually not present. This may occur because of a break down of the fetal membranes or in response to inflammation.

A recent study compared the ability of fFN and a interleukin-6 (IL-6), an inflammatory cytokine, to predict preterm birth. The tests were performed in 667 samples collected from 580 women with and without symptoms of preterm labor. About half of the women in the study were black.

In both groups, a positive fFN result was significantly associated with preterm birth occurring within 14 days of testing. However, the actual likelihood of delivery early was still rather low. Like other studies, this one also showed that the strength of the fFN test is it's negative predictive value. That is, the absence of fFN is a much stronger indicator that preterm birth won't occur.

IL-6 was also significantly associated with preterm birth but only in the black women. Like fFN, it also wasn't a strong predictor in this population but it was much better than fFN in either the black or non-black groups.

There's still much to learn. We don't really understand the different processes (which are multifactorial) that lead to preterm delivery. Also, it's clear that the events that lead to preterm delivery are probably not the same between women of different ethnic backgrounds. As such, a single predictor of preterm delivery is probably not going to work well. Combination testing, such as fFN and IL-6 (or other, yet to be discovered markers) may yield more promising results than any one marker alone.