The Association between Qualitative Fetal Fibronectin Test from Cervicovaginal Discharge and the Presence of Histologic Chorioamnionitis in Preterm Labor with Intact Membranes

1. Study design

A retrospective cohort study was performed on consecutive women who had been hospitalized for preterm labor with intact membranes at Seoul National University Bundang Hospital from August 2017 to May 2020. In our institution, all patients routinely underwent the qualitative fetal fibronectin test from cervicovaginal fluid on the day of admission. Because fetal fibronectin is a glycoprotein found in amniotic fluid, it was not applied to patients who presented with the evidence of ruptured membranes. Among them, 83 patients who resulted in preterm birth before 37 weeks of gestation, but delivered after 23 weeks were included for the study population (Fig. 1). In addition, the fetal fibronectin results were positive in 66.3% (55/83) for preterm births and in 46.4% (64/138) for term births with available data. Multifetal pregnancies and stillbirths were excluded. The pathologic reports of placenta were available for all cases since our institution routinely perform placental evaluation for preterm deliveries. Maternal baseline characteristics, pregnancy outcomes, and neonatal outcomes such as gestational age at delivery, the mode of delivery, birthweight of neonates, Apgar scores, meconium staining, neonatal intensive care unit (NICU) admission, respiratory distress syndrome, bronchopulmonary dysplasia, use of nasal continuous positive airway pressure (CPAP) and/or mechanical ventilator, pneumonia, intraventricular hemorrhage, necrotizing enterocolitis, early onset sepsis, and small-for-gestational age were reviewed in detail through medical records. The study protocol was checked and approved by the Institutional Review Board of Seoul National University Bundang Hospital (B-1905/543-107).

Fig. 1
The flow chart of study population.

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2. Fetal fibronectin and other evaluations to assess the risk of preterm birth

All the patients enrolled for the study underwent the commonly used commercial bedside test (Rapid fFN TLII_Q; Hologic, Sunnyvale, CA, USA) for the qualitative fetal fibronectin evaluation. During the initial speculum examination, a polyester swab was inserted into the posterior fornix of the vagina for about 10 seconds for collection of cervicovaginal fluid sampling. The swab was analyzed using the conventional qualitative bedside test according to the manufacturer’s instructions. The result of test was reported in 20 minutes. In addition to fetal fibronectin test, cervical length measured by transvaginal sonography, and C-reactive protein (CRP) from maternal serum were evaluated for the study population in 48 hours from the fetal fibronectin sampling.

Cervical length was measured through transvaginal ultrasonography by trained staff at least over 3 times and the shortest value was used in the analysis. While analyzing for diagnostic performance of cervical length, we defined short cervical length as value measured at or below 1.5 cm.14 Measurement of CRP from maternal serum was used to evaluate the inflammatory state as an indirect marker for the intrauterine infection/inflammation. The concentration of CRP was measured with a latex-enhanced turbidimetric immunoassay (Denka Seiken, Tokyo, Japan) by an automated analyzer at our laboratory department (Toshiba 200FR; Toshiba, Tokyo, Japan).

For those who underwent amniocentesis to evaluate intra-amniotic inflammation and/or infection or to determine the condition of fetal lung maturation, amniotic fluid was aseptically obtained through abdominal amniocentesis under sonographic guidance. The need for amniocentesis was dependent on the attending physician’s decision. Amniotic fluid was analyzed for WBC counts and cultured for aerobic bacteria, anaerobic bacteria, and genital mycoplasmas. In addition, Lamellar body count was evaluated routinely for all patients who underwent amniocentesis to check the maturity of fetal lung.15 Lamellar body count was quantified through the platelet channel of an automated analyzer (XN-90000 hematology Analyzer; Sysmex, Kobe, Japan) and the cut-off for maturity at out institution was over 50,000/µL.

3. Diagnosis of placental pathologic findings

Placental pathologic findings including histologic chorioamnionitis and funisitis were reported by the pathologists of our institution. For histopathologic evaluation, placental tissue samples were fixed in 10% neutral buffered formalin and embedding in paraffin was followed. Sections of blocks were stained by hematoxylin and eosin and pathologists were blinded to clinical information. Acute histologic chorioamnionitis was diagnosed in the presence of acute inflammatory changes on any of tissue samples including amnion, umbilical cord, chorion-decidua, and chorionic plate.16 Funisitis was confirmed when neutrophil infiltration was found in umbilical vessel walls or Wharton jelly.17

4. Statistical analysis

Continuous variables were compared using the Mann-Whitney U test; proportions were compared using the Fisher exact test. Multivariate logistic regression was used to determine factors associated with histologic chorioamnionitis. A P-value <0.05 was considered statistically significant. The diagnostic index of fetal fibronectin, cervical length, and CRP level was calculated to establish sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and likelihood ratios for histologic chorioamnionitis. The analysis was done with IBM SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA).

1. Principle findings of the study

(1) In this cohort study diagnosed as preterm labor without ruptured membranes, the proportion of histologic chorioamnionitis was higher in the group with positive fetal fibronectin results than that with negative results (62% vs. 18%, P<0.001). (2) The positive fetal fibronectin was significantly associated with histologic chorioamnionitis with relative risk 4.6 (95% confidence interval, 1.3-16.0; P=0.017) in multivariate logistic regression analysis. (3) Through diagnostic indices analysis, the sensitivity of positive fetal fibronectin was 87% for the prediction of histologic chorioamnionitis after birth.

2. Literature review for fetal fibronectin in the field of obstetrics

A number of tests have been studied to predict the preterm birth before active symptoms develop. The representative tests include cervical length measured by transvaginal ultrasonography and fetal fibronectin in cervicovaginal fluid.18, 19, 20, 21 While short cervical length has been widely used as universal screening tool for preterm birth, fetal fibronectin, both alone or in combination with other markers, has not proved the ability to predict preterm birth. Because of poor positive predictive value (30%-50%) in several observational studies, it is not recommended to apply the result of fibronectin into the clinical management.22, 23

In a meta-analysis of fetal fibronectin as a predictor of preterm birth in symptomatic women, the sensitivity and specificity to predict the preterm delivery within 7 days were 33-100% and 62% to 91%, respectively.24 Interestingly, more recent studies had less accuracy than those published later.13 The authors assumed that recent studies employed the qualitative point-of-care test compared to the quantitative methods such as the immunoassay which has been used more commonly before early 21st century. Therefore, interpretation of fetal fibronectin through quantified method might show higher ability to predict the impending preterm birth rather than the rapid bedside test. More researches are needed to find the usefulness of fetal fibronectin as a marker for remote delivery.

3. The importance of antenatal prediction for histologic chorioamnionitis

Intra-amniotic infection has been known as an important etiology of preterm labor, especially at less than 32 weeks of gestational age.25, 26 The pathologic evidence of maternal inflammatory response to intra-amniotic inflammation and/or infection remained in placenta, histologic chorioamnionitis, is found in more than 50% of preterm births.27, 28 Histologic chorioamnionitis has been reported to be associated with adverse neonatal outcomes such as early-onset neonatal sepsis,29 small-for-gestational age,30 necrotizing enterocolitis,31 bronchopulmonary dysplasia,3, 32 and cerebral palsy33 although the relation to several neonatal outcomes, especially neurodevelopmental development, are controversial or conflicting.34, 35 Even in term babies, the ones with histologic chorioamnionitis revealed higher prevalence of complications including meconium staining and pulmonary hypertension.36, 37

Clinical chorioamnionitis is an indirect way to diagnose the presence of placental inflammation antenatally through specific clinical symptoms such as maternal fever, maternal leukocytosis, uterine tenderness, maternal and fetal tachycardia.38 Nevertheless, clinical chorioamnionitis and the underlying pathology of placenta evaluated through histologic examination postnatally do not always coincide since the presentation is often subclinical.39 Catano Sabogal et al.40, performed the validation of numerous diagnostic modalities to predict histologic chorioamnionitis antenatally and concluded that no specific tool showed enough sensitivity or specificity. In this systemic review, the diagnostic indices of maternal clinical signs and laboratory tests such as CRP, leukocytes, and various cytokines from maternal blood or vaginal discharge, and ultrasonographic characteristics such as the area of fetal thymus were summarized, however fetal fibronectin was not included.

4. Clinical implications

As mentioned above, histologic chorioamnionitis can be diagnosed after birth since placenta needs to be examined by pathologists. Expecting the presence of histologic chorioamnionitis in high-risk pregnancies for preterm birth is important to decide the timing of delivery and to predict the neonatal outcomes. This cohort study demonstrates the possibility of fetal fibronectin as the predictive method for histologic chorioamnionitis in antenatal period. The hypothesis to explain the association between positive result of fetal fibronectin and histologic chorioamnionitis is that placental inflammation originated from microscopic rupture of membranes or leakage of amniotic fluid might be caught by the detection of fetal fibronectin. Since funisitis implicates the inflammatory reaction of fetal part, was not associated with fetal fibronectin result.

Because the timing of fetal fibronectin sampling for few cases in this study was approximately 100 days before giving birth (the median value of interval from sampling to delivery was 10 with the range from 1 to 118 days), there is certain limitation implying the indirect and low association between cervical fetal fibronectin result and obstetric outcomes such as histologic chorioamnionitis or impending preterm birth. Nevertheless, it will be remarkably helpful in clinical settings if the effectiveness of fetal fibronectin test to predict histologic chorioamnionitis is investigated more since the qualitative test of fetal fibronectin is easy to perform and the result can be identified in a very short time. Further future researches containing larger sample size prospective study population are needed to determine the usefulness of fetal fibronectin to predict placental pathologies.