Raised Vaginal Fluid Fibronectin Level Indicates Premature Rupture of Membrane

Background: Premature rupture of membrane (PROM) is one of the common complications of pregnancy that has major impact on fetal and neonatal outcome. It is the commonest clinical event where a normal pregnancy becomes suddenly a high-risk one for mother and fetus or neonate. Objective: The study was undertaken to investigate whether raised fibronectin level in vaginal fluid may indicate premature rupture of membrane. Materials and Methods: This cross sectional study was conducted in the department of Obstetrics and Gynecology in Sir Salimullah Medical College & Mitford Hospital, Dhaka during the period of January 2006 to December 2007. A total of 114 pregnant women with gestational age 28 week up to 40 week were included. Sixty were PROM (Group I) and 54 were non-PROM (Group II) subjects. Fibronectin in vaginal fluid was measured by an immunochemical reaction by nephelometer. Statistical analysis was done by SPSS version 10.0. Results: The PROM patients had significantly higher concentration of fibronectin (225.77 ± 115.18 ng/mL) compared to that in non-PROM subjects (8.04 ± 16.17 ng/mL) (p < 0.001). Conclusion: It can be concluded that in cases of unequivocal rupture or intactness of the membranes, the result of the fibronectin test corresponds well with the clinical situation. So fibronectin is a sensitive test for detection of amniotic fluid in the vagina.


Introduction
occurs markedly at higher rate than in non-PROM patients. 3So it is obvious that PROM is one of the obstetric conditions which lead to significant infant mortality and morbidity and maternal ill health.
Early diagnosis of PROM has a great impact on maternal and child health.Diagnosis of PROM requires a thorough history, physical examination and selected laboratory studies. 4Commonly used methods for detection of amniotic fluid in the vagina include pooling, ferning, nitrazine paper test, ultrasound measurement of amniotic sac dimensions. 5This study was undertaken to investigate whether raised fibronectin level in vaginal fluid may indicate premature rupture of membrane, to measure the concentrations of fibronectin in vaginal fluid of PROM and non-PROM patients and to assess the performance of fibronectin test for diagnosis of PROM by sensitivity, specificity, positive predictive value, negative predictive value and accuracy test.

Materials and Methods
This cross sectional study was conducted in the department of Obstetrics and Gynecology in Sir Salimullah Medical College & Mitford Hospital, Dhaka from January, 2006 to December, 2007.A total of 114 pregnant ladies with gestational age 28 th week up to 40 th week were included.Sixty were PROM (Group I) and 54 were non-PROM (Group II) patients.Both primipara and multipara patients with gestational age from 28 th week up to 40 th week pregnancy with history of spontaneous rupture of membrane within 72 hours and positive per speculum test for pooling were included in Group I.Both primipara and multipara women with gestational age from 28 th week up to 40 th week pregnancy with no history of spontaneous rupture of membrane and negative per speculum test for pooling were included in Group II.Verbal and written consent to participate in the study was taken from the patients.Patients with antepartum hemorrhage, induced or accidental rupture of membrane, presence of any blood on speculum examination or having sexual intercourse during last 24 hours were excluded from the study.Purposive (non probability) sampling was applied here for the convenience.The researcher interviewed the respondents according to their convenience after fulfillment of study criteria.

Confirmation of PROM
In this study, positive result of both nitrazine test and fern test (double positive) was taken as the gold standard for confirmation of diagnosis of PROM.

Specimen collection and preservation
For Group I patients Patients were asked to lie in lithotomy position.After maintaining all aseptic precautions, a sterile speculum was introduced into patient's vagina.A sterile cotton tipped swab was used to collect fluid from the vagina and applied it to nitrazine paper.Nitrazine paper turned blue demonstrating an alkaline pH (7.0-7.25) if amniotic fluid was present in vaginal secretion.Another drop of fluid was placed on a slide and allowed to dry in air and was examined under low power (10×) microscope for fern pattern due to crystallization of sodium chloride derived from amniotic fluid.
Then sterile cotton tipped swab was inserted into posterior fornix of vagina, gently rotated across posterior fornix and around ectocervix for at least 10 seconds, and then extracted in 750 microliter of phosphate buffer.Specimens were stored at --20 degree centigrade.

For Group II patients
Only vaginal swab was collected for fibronectin concentration. 6ibronectin is measured by an immunochemical reaction by nephelometer.Fibronectin in the amniotic fluid sample form immune complexes with specific antibodies.These complexes scatter a beam of light passing through the sample.The intensity of the scattered light is proportional to the concentration of the relevant protein in the sample.The result is evaluated by comparison with a standard of known concentration.Taking the standard from a number of similar international studies conducted in Germany, Italy, Belgium and US, the fibronectin test was considered positive at the concentration of 50 ng/mL.

Ethical consideration
Before starting the research work permission was taken from the hospital authorities.All women enrolled in the study were explained about the nature 75 and purpose of the study and only those who gave written consent were included in the study.

Results
One hundred and fourteen pregnant patients with gestational age of 28 th to 40 th weeks who attended the outdoor department or were admitted in the hospital were included in the study.For Group I (PROM), 60 pregnant patients presenting with the clinical history of PROM within 72 hours and positive per speculum examination for pooling were selected.Then they underwent nitrazine paper test and fern test.In this study, positive result of both nitrazine test and fern test (double positive) was taken as the gold standard for confirmation of diagnosis of PROM.Of the 60 patients, 47 were found double positive in the test.
For Group II, 54 pregnant patients presenting with no history of PROM and negative per speculum examination for pooling were selected.Then both groups underwent fibronectin test.The findings of the study are presented below.

Age distribution of the subjects
Table I compares the age distribution between groups.In Group I 36.7%subjects were between 31-35 years, 35% between 26-30 years, 20% between 21-25 years, 5% were of 20 or below 20 years and 3.3% above 35 years of age.In the Group II nearly half (46.3%) of the subjects ranged from 26-30 years followed by 27.8% between 31-35 years, 22.2% between 21-25 years and 3.7% were of 20 or below 20 years.The groups were almost identical with respect to age (28.87 ± 4.15 vs 27.87 ± 3.82 years, p = 0.187).

Occupation of the subjects
About 57% of the Group I and 56% of the Group II were housewives.The service-holders formed the second majority in both the groups (28.3% in Group I and 29.6% in Group II) followed by day laborers (11.7% in Group I and 3.7% in Group II).

Income distribution of the subjects
Income distribution shows that 30% subjects of the Group I had monthly family income of Taka 5000 or below, 48.3% between Taka 5,000-10,000, 20% between Taka 10,000-15,000 and very few had Taka 15,000 or above.In Group II, majority (92.5%) had income of Taka 5,000-15,000 (44.4% between 5000-10,000 and 48.1% between 10,000-15,000).Very few subjects had income below 5000 or above 15000 taka.

Distribution of subjects by parity
Distribution of subjects by parity demonstrates that majority of the Group I (81.7%) and Group II (61.1%) was multipara.Multiparity was found to be significantly associated with premature rupture of membrane (Table II).

Gestational age
Data pertaining to gestational age shows that 57% subjects of the Group I had preterm PROM and the 43% had term PROM.

Pattern of antenatal care
Table III shows that 48.3% subjects of the Group I did not receive any antenatal care (ANC) and only 28.3% received ANC regularly.In Group II, majority (83.3%) had the history of receiving regular antenatal care indicating that PROM is associated with no or irregular antenatal care (p < 0.001).

Infections during current pregnancy
Diseases acquired during current pregnancy demonstrate that approximately 36.7% subjects of the Group I had history of vaginal infection whereas it was 14.8% in Group II (p = <0.001).The incidence of urinary tract infection was found to be significantly higher in Group I (50.0%) than that in the Group II (24.1%) (p = 0.004).

Past obstetric history
Table V shows the association of PROM in previous pregnancy with that in current pregnancy.The Group I had a significantly higher frequency of PROM in previous pregnancy (31.7%) compared to Group II (1.9%) (p = <0.001).The past history of MR was 33.3% in Group I and 14.8% in Group II (p = 0.022).Regarding D & C there was no difference between the groups (p = 0.138).
TableV: Comparison of past obstetric history between groups (N = 114)

Diagnostic tests for PROM
A preset self-administered questionnaire was used for data collection.Socio-demographic (age, occupation, monthly income), etiological (vaginal infection, urinary tract infection, antenatal care), present pregnancy related (parity, gestational age, watery discharge per vagina), past obstetric (history of PROM in previous pregnancy, history of MR, history of D & C) and diagnostic (nitrazine paper test, fern test, fibronectin test) variables were included.The raw data were edited properly.All edited data were consolidated, processed statistically and analyzed with SPSS (Statistical Program for Social Science) Version 10.0.Statistical analyses were done by Unpaired Student's t test, Chi-square Test, Sensitivity, Specificity, Positive predictive value, Negative predictive value and Accuracy test.

Table I :
Comparison of age between Group-I and Group-II (N = 114) Figures in the parentheses denote corresponding percentage.Data were analyzed using Student's t test and level of significance was 0.05.

Table II :
Distribution of parity between groups (N = 114)

Table IV :
History of infections during current pregnancy (N = 114)

Table VII :
Table VII summarizes the findings of different diagnostic tests for PROM.In nitrazine paper test, out of 60 patients in Group I, 49 (81.65%) were diagnosed of having PROM and 11 patients were negative for the test.In fern test, 53 (88.30%) patients out of 60 in Group I demonstrate positive results and 7 demonstrate negative results.In this study, positive results of both nitrazine test and fern test (double positive) were taken as the gold standard for confirmation of diagnosis of PROM.Out of the 60 patients, 47 were found double positive in the test and 13 were found double or single negative for nitrazine and fern tests.Comparison of diagnostic tests for PROM 93% and 82% for fetal cells; 100%, 90% and 93% for fibronectin test.Their sensitivity is a bit higher than the present study.They made conclusion that fibronectin test appeared to be the most sensitive and accurate.Salfelder et al 9 conducted a study on 133 pregnancies.Their observation had similar picture like present study regarding the value of fibronectin test to confirm the diagnosis of PROM.Finally, the diagnostic accuracy of the test in our study was 95%.One patient not diagnosed as ruptured membranes based on standerd tests was positive in fibronectin test.It can be explained by prerupture stretching and consequent leaking of the membranes.The clinical significance of these 'false positives' requires further evaluation.Two patients diagnosed as ruptured membranes based on standard tests were negative for fibronectin test.This small number of false negative results may be due to flaws during sampling or technical difficulties in running the assay.From the findings of this study it can be concluded that in cases of unequivocal rupture of t h e membranes, the result of the fibronectin test corresponds well with the clinical situation.So measurement of fibronectin in vaginal fluid is a sensitive test for detection of amniotic fluid in the vagina.However, further large scale studies are recommended to determine the usefulness of fetal fibronectin in the diagnosis of rupture of the membranes.