http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(5): 504–508 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.926883
ORIGINAL ARTICLE
The association between maternal biomarkers and pathways to preterm birth in twin pregnancies Eric Bergh1, Andrei Rebarber1,2, Sandip Oppal2, Daniel H. Saltzman1,2, Chad K. Klauser1,2, Simi Gupta1,2, and Nathan S. Fox1,2 1
Department of Obstetrics, Gynecology, and Reproductive Science, Icahn School of Medicine at Mount Sinai, New York, NY, USA and Maternal Fetal Medicine Associates, PLLC, New York, NY, USA
2
Abstract
Keywords
Objective: We sought to estimate the association between cervical length (CL) and fetal fibronectin (fFN) and each pathway leading to preterm birth in twin pregnancies. Methods: Cohort study of 560 patients with twin pregnancies who underwent routine serial CL and fFN screening from 22 to 32 weeks in one maternal fetal medicine practice during 2005–2013. We calculated the association between a short CL (20 mm) or positive fFN with overall preterm birth 532 weeks, and then subdivided the analysis into preterm birth 532 weeks from preterm labor, preterm premature rupture of membranes (PPROM) and indicated causes. We excluded cases of monochorionic-monoamniotic placentation, vasa previa, twin–twin transfusion and patients with cerclage. Results: The overall rate of preterm birth 532 weeks was 6.9% (3.9% from preterm labor, 1.6% from PPROM and 1.4% indicated). A short cervix was associated with preterm birth 532 weeks arising from preterm labor (12.4% versus 2.0%, p50.001), but not PPROM (1.9% versus 1.3%, p ¼ 0.651). Positive fFN was associated with preterm birth 532 weeks both from preterm labor (17.0% versus 2.4%, p50.001) as well as from PPROM (5.7% versus 1.0%, p ¼ 0.034). Neither was significantly associated with preterm birth 532 weeks from indicated causes. Conclusions: The mechanism leading toward preterm influences the accuracy of screening tests chosen to assess risk in twin pregnancies. A shortened cervical length and positive fFN is associated with spontaneous preterm labor and birth 532 weeks. However, PPROM does not appear to be preceded by a short cervix, but is preceded by a positive fFN. Neither test is associated with an indicated preterm birth.
Cervical length, fetal fibronectin, PPROM, prematurity, preterm labor, twins
Introduction Preterm birth continues to be a major determinant of infant morbidity and mortality in twin pregnancies. In 2011, the twin birth rate was 33.1 per 1000 live births in the United States [1]. Among twin gestations, approximately 60% are born preterm with an average gestational age of approximately 35 weeks [2]. Despite advancements in neonatal intensive care, both short-term and long-term complications of prematurity remain significant causes of mortality in preterm infants. Of note, infant mortality rate in twins is fivefold higher than in singletons, likely due to complications of prematurity [3]. Cervical length (CL) and fetal fibronectin (fFN) have been studied extensively as independent markers of preterm delivery in multiple gestations. A large retrospective review
Address for correspondence: Nathan S. Fox, MD, Maternal Fetal Medicine Associates, PLLC, 70 East 90th Street, New York, NY 10128, USA. Tel: 212-722-7409. Fax: 212-722-7185. E-mail: [email protected]
History Received 18 February 2014 Revised 24 April 2014 Accepted 19 May 2014 Published online 19 June 2014
of CL measured in asymptomatic twin pregnancies identified both CL and gestational age at time of measurement as independent risk factors for preterm delivery [4]. These findings have been confirmed in large meta-analysis as well [5]. A positive fFN in twin pregnancies has also been shown to be significantly associated with preterm birth in a large meta-analysis [6]. Previously published data by our group has also identified fFN as a strong predictor of preterm birth 532 weeks in asymptomatic twin gestations with normal cervical length (425 mm) [7]. The combination of CL and fFN has also been studied in twin pregnancies. In the Preterm Prediction Study, both shortened cervical length (25 mm) and fFN were predictive of preterm delivery 532 weeks in twin pregnancies, the strongest association occurring when both tests resulted positive [8]. We have published similar findings as well. In our own experience following 155 asymptomatic women with twin pregnancies, a positive screen for both shortened cervical length (20 mm) and fFN had a 54.5% risk of spontaneous preterm birth532 weeks gestation in contrast to 4.2% risk among women who screened negative for both risk factors [9].
DOI: 10.3109/14767058.2014.926883
Cervical length and fetal fibronectin in twins with preterm labor and PPROM
Despite the known association between CL, fFN and preterm birth in twins, studies typically only examine the outcome of preterm birth overall or spontaneous preterm birth. However, preterm birth can arise through three primary pathways: preterm labor, preterm premature rupture of membranes (PPROM) or medically-indicated [10]. The objective of this study was to estimate the association between a short CL, positive fFN and preterm birth in twin pregnancies, separately examining each of the three pathways of preterm birth in twin pregnancies: preterm labor, PPROM and indicated preterm birth. This information may also add to our overall understanding of the pathways of preterm birth in twin pregnancies.
Methods We reviewed the charts of all patients with twin pregnancies 422 weeks delivered by a single maternal-fetal medicine practice between June 2005 (when our electronic medical record was established) and June 2013. We excluded patients with monochorionic-monoamniotic placentation, vasa previa, twin–twin transfusion syndrome and patients with cerclage. We obtained baseline characteristics and pregnancy outcomes from our computerized medical record. Gestational age was determined by last menstrual period and confirmed by ultrasound in all patients. The pregnancy was redated if there was a 45 day discrepancy up to 14 weeks or a 47 day discrepancy after 14 weeks. If the pregnancy was the result of IVF, gestational age was determined from IVF dating. The Biomedical Research Alliance of New York Institutional Review Board approval was obtained before reviewing the charts. In our practice, patients with twin pregnancies are followed with serial CL and fFN testing every 2–4 weeks until 32 weeks [9]. fFN testing is done from 22 0/7 to 31 6/7 weeks, and CL screening is performed from 16 0/7 to 31 6/7 weeks; however, for the purpose of this study we only included CL results from 22 to 31 6/7 weeks as this was the window of fFN testing. All CL assessments and fFN testing were done in an outpatient setting on asymptomatic patients. All tests done on labor and delivery were excluded, as they were done on symptomatic patients as part of a preterm labor evaluation. Measurements of CL were performed using a 4- to 8-MHz transvaginal probe with an empty bladder according to criteria established by Iams et al. [11]. The shortest functional CL was used as this has been found to be the most reproducible measurement [12]. A short CL was defined a priori as a measurement 20 mm [9]. Fetal fibronectin testing was performed without the use of a speculum using a published protocol in singleton pregnancies [13] at least 24 h from the last reported intercourse or endovaginal ultrasound. Testing was not performed in the setting of vaginal bleeding. Swabs were sent for evaluation using a fetal fibronectin assay, and a concentration of 50 ng/mL or greater was considered positive. Physicians were not blinded to CL or fFN results. The primary outcome was preterm birth 532 weeks. We calculated the association between a short CL and a positive fFN with preterm birth 532 weeks. We then calculated the association between a short CL and positive fFN with each of
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the three pathways of preterm birth532 weeks: preterm labor, PPROM and indicated preterm birth. Preterm labor was defined as the onset of labor (contractions, cervical change) prior to rupture of membranes. PPROM was defined as ruptured membranes prior to the onset of labor. Indicated preterm birth included any indication for preterm birth due to fetal or maternal indications (e.g. preeclampsia or fetal growth restriction). If a patient had preterm labor or PPROM prior to 32 weeks, but delivered after 32 weeks, she was not considered as having a preterm birth 532 weeks (i.e. the outcome measured was preterm birth from each cause, and not the cause itself). Chi square and Fisher’s exact tests were used, as appropriate (SPSS for Windows 16.0, SPSS Inc., Chicago, IL). A p value of 0.05 was considered statistically significant.
Results During the study period, 611 patient with twin pregnancies 422 weeks were delivered in our practice. 51 were excluded from analysis (25 with cerclage, 10 monochorionicmonoamniotic, nine late transfers with no CL/fFN testing, four with twin–twin transfusion, and three with vasa previa) leaving 560 patients for analysis. The characteristics of the population are listed in Table 1. The incidence of preterm birth 532 weeks overall was 6.9%. Subdivided by cause of preterm birth 532 weeks, the incidence of preterm birth 532 weeks from preterm labor was 3.9%, from PPROM 1.6%, and from an indicated cause 1.4%. Three patients (0.5%) had PPROM less than 32 weeks and delivered after 32 weeks. 552 patients had CL measurements, 105 (19.0%) of whom had a short CL 20 mm prior to 32 weeks. 544 patients underwent fFN testing, 53 (9.7%) of whom had a positive fFN prior to 32 weeks. The association between a short CL and preterm birth 532 weeks is shown in Table 2. A short CL was associated with overall preterm birth 532 weeks (16.2% versus 4.4%, p50.001) and was associated with preterm birth 532 weeks from preterm labor (12.4% versus 2.0%, p50.001). However, a short CL was not associated with preterm birth 532 weeks from PPROM (1.9% versus 1.3%, p ¼ 0.651) or indicated preterm birth 532 weeks (1.9% versus 1.1%, p ¼ 0.623). The association between a positive fFN and preterm birth 532 weeks is shown in Table 3. A positive fFN was associated Table 1. Characteristics of the study population. Number of pregnancies Total cervical length measurements Total fetal fibronectin tests Maternal age (years) Conception Spontaneous Ovulation induction In-vitro fertilization Multifetal pregnancy reduction Caucasian Prepregnancy BMI (kg/m2) Prior term birth Prior preterm birth Prior LEEP or cone biopsy Chorionicity Monochorionic Dichorionic
560 2247 2140 34.2 ± 7.5 years 23.0% 11.6% 65.2% 7.3% 87.4% 23.5 ± 4.5 33.9% 6.2% 2.8% 12.9% 87.1%
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J Matern Fetal Neonatal Med, 2015; 28(5): 504–508
Table 2. Likelihood of preterm birth 532 weeks from all causes in twin pregnancies, based on serial cervical length testing from 22 to 32 weeks. CL 20 mm CL420 mm 22–32 weeks 22–32 weeks N ¼ 447 N ¼ 105 Any preterm birth 532weeks Preterm birth 532 weeks from: Preterm labor PROM Indicated
p
16.2%
4.4%
50.001
12.4% 1.9% 1.9%
2.0% 1.3% 1.1%
50.001 0.651 0.623
CL, cervical length; PROM, premature rupture of membranes. Table 3. Likelihood of preterm birth 532 weeks from all causes in twin pregnancies, based on serial fetal fibronectin testing from 22 to 32 weeks. fFN positive fFN negative 22–32 weeks 22–32 weeks N ¼ 491 N¼53 Any preterm birth 532weeks Preterm birth 532 weeks from: Preterm labor PROM Indicated
p
24.6%
4.6%
50.001
17.0% 5.7% 1.9%
2.4% 1.0% 1.2%
50.001 0.034 0.514
fFN, fetal fibronectin; PROM, premature rupture of membranes. Table 4. Prediction of preterm birth 532 weeks from preterm labor in twin pregnancies, based on serial fetal fibronectin and cervical length testing from 22 to 32 weeks.
fFN positive CL 20 mm
Sensitivity
Specificity
PPV
NPV
+LR
LR
42.9% 59.1%
91.6% 82.6%
17.0% 12.4%
97.6% 98.0%
5.1 3.4
0.62 0.50
Table 5. Prediction of preterm birth 532 weeks from premature rupture of membranes in twin pregnancies, based on serial fetal fibronectin and cervical length testing from 22 to 32 weeks.
fFN positive CL 20 mm
Sensitivity
Specificity
PPV
NPV
+LR
LR
37.5 25.0%
90.7 81.1%
5.7% 1.9%
99.0% 98.7%
4.0 1.4
0.69 0.92
with overall preterm birth 532 weeks (24.6% versus 4.6%, p50.001), preterm birth 532 weeks from preterm labor (17.0% versus 2.4%, p50.001), and preterm birth 532 weeks from PPROM (5.7% versus 1.0%, p ¼ 0.034). A positive fFN was not associated with indicated preterm birth 532 weeks (1.9% versus 1.2%, p ¼ 0.514). The testing characteristics of CL and fFN in predicting preterm birth 532 weeks from preterm labor and from PPROM are shown in Tables 4 and 5, respectively. A positive fFN had a higher positive likelihood ratio than did a short CL for both causes of preterm birth 532 weeks.
Discussion In this study, a positive fFN was associated with spontaneous preterm birth532 weeks arising from preterm labor as well as PPROM. Although a short CL was associated with preterm birth arising from preterm labor, a short CL was not
associated with preterm birth arising from PPROM. This suggests that the mechanisms leading to spontaneous preterm birth influence the accuracy of the particular screening test chosen. PPROM in twin pregnancies does not appear to be preceded by a short cervix, but is preceded by a positive fFN. Neither test is associated with indicated preterm birth. Spontaneous preterm birth may result from both inflammatory and non-inflammatory pathways [10]. The inflammatory cascade that leads to PPROM results in alterations to the normal membrane remodeling process that takes place throughout pregnancy. As membranes expand to accommodate a growing fetus, a balance is normally achieved between proliferative and degradative changes to the amnion and chorion. Extracellular membrane proteins such as collagens, laminins and fibronectins stabilize the fetal membranes and provide the required tensile strength to withstand increased intra and extra-amniotic pressures [14,15]. Fluctuations in the local concentrations of matrix metalloproteinases (MMPs) which breakdown the extracellular scaffold, and tissue inhibitors of metalloproteinases (TIMPs) promote a dynamic molecular milieu which adjusts to a changing environment. Premature increases in MMP activity or decreases in TIMPs may therefore lead to PPROM via membrane weakening and have been associated with the inflammatory response to abruption as well as to infection or colonization with different genital tract pathogens [14]. Specifically, inflammatory cytokines such as TNF-a and IL-1b as well as antioxidants produced as by-products of infection, may enhance production of MMPs leading to accelerated membrane weakening [16–19]. Genetic influences may also play a major role in this balance as there is evidence to suggest that certain racial groups may be predisposed to mount an exaggerated inflammatory response and thus experience increased rates of PPROM [20]. Fetal fibronectin is an extracellular matrix protein which is rarely found in cervicovaginal secretions beyond 22 weeks of gestation. Its presence in concentrations greater than 50 ng/dL implies an ongoing process of extracellular matrix protein degradation and separation of the decidual–fetal membrane interface which often precedes rupture [21]. It may therefore be reflective of the final pathway in an inflammatory process by which PPROM leads to preterm birth. This could explain our finding that a positive fFN in twin pregnancies was associated with spontaneous preterm birth arising from PPROM. Cervical shortening is associated with preterm birth in twin pregnancies and may occur via both inflammatory and non-inflammatory pathways. The physical effects of pathologic over-distension due to multi-gestation could lead to premature activation of the adaptive process of cervical softening, ripening, effacement and dilation. In response to amniochorion stretch, cervical collagen content may be altered via biochemical mechanisms involving regulatory hormones such as prostaglandins and progesterone, cytokines and nitric oxide [22–30]. Whether or not inflammation is the end result or the inciting event in cervical shortening is unclear. As a mechanical barrier to ascending infection, a shortened or dilated cervix may increase the risk of amnionitis [31–33]. Furthermore, cervical mucous has both innate and adaptive immune functions which may predispose to
DOI: 10.3109/14767058.2014.926883
Cervical length and fetal fibronectin in twins with preterm labor and PPROM
transcervical infection when disrupted [34]. Proteomic studies for cervicovaginal biomarkers could help to better understand the breakdown of this barrier and subsequent activation of pathways involved in cervical shortening and preterm labor [34–38]. Our study suggests that in twin pregnancies, a short cervix most likely represents a non-inflammatory pathway to preterm birth, or an inflammatory pathway that would only lead to contractions, but not membrane breakdown and PPROM. This likely differs from singleton pregnancies, as twin pregnancies have significantly more uterine stretch than do singleton pregnancies. As far as we know, this is the first study to identify CL and fFN as specific predictors of different pathways of preterm birth in asymptomatic twin pregnancies. Previous studies have only commented upon the association between these biomarkers and preterm birth overall or spontaneous preterm birth without regard to the underlying cause. Strengths to our study include the large sample size undergoing routine fFN and CL testing. Since we cared for all of these patients, we were able to accurately record whether preterm birth occurred due to preterm labor, PPROM or indicated causes. Using population data and birth certificate registries would likely be flawed or may not address this specific detail. Weaknesses to our study include the fact that patients and obstetricians were not blinded to fFN and CL results. However, as there is no known way to prevent spontaneous preterm birth in twin pregnancies, the knowledge of fFN and CL results is unlikely to have affected our findings. Another weakness of the study is that each specific test has a low positive predictive value for the outcome measured. For example, although we found a significant association between a positive fFN and spontaneous preterm birth from PPROM, the actual positive predictive value of fFN is only 5.7% for PPROM. Therefore, we do not advocate using fFN to screen for PPROM at this time. Rather, our data may lend insight into the pathways of preterm birth for twin pregnancies allowing for the development of more therapeutic interventions. Whether patients with twin pregnancies should undergo routine fFN and CL screening at all is debatable. We have previously demonstrated that routine fFN and CL screening does not lead to a reduction in preterm birth [39]. However, routine fFN and CL screening in twin pregnancies appears to be associated with improved delivery and timing of antenatal corticosteroids [39]. Ultimately, only a randomized, prospective trial could determine the clinical and cost-effectiveness of routine fFN and CL screening. In conclusion, in twin pregnancies a positive fFN is associated with preterm birth532 weeks arising from preterm labor and PPROM. However, a short CL is only associated with preterm birth arising from preterm labor. Future research should focus on the specific pathways of preterm birth in twin pregnancies.
Declaration of interest The authors report no conflicts of interest.
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34. Buhimschi CS, Baumbusch MA, Campbell KH, et al. Insight into innate immunity of the uterine cervix as a host defense mechanism against infection and preterm birth. Expert Rev Obstet Gynecol 2009;4:9–15. 35. Gravett MG, Thomas A, Schneider KA, et al. Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection. J Proteome Res 2007;6: 89–96. 36. Buhimschi IA, Buhimschi CS, Weiner CP, et al. Proteomic but not enzyme-linked immunosorbent assay technology detects amniotic fluid monomeric calgranulins from their complexed calprotectin form. Clin Diagn Lab Immunol 2005;12: 837–44. 37. Havelock JC, Keller P, Muleba N, et al. Human myometrial gene expression before and during parturition. Biol Reprod 2005;72: 707–19. 38. Sakamoto Y, Moran P, Bulmer JN, et al. Macrophages and not granulocytes are involved in cervical ripening. J Reprod Immunol 2005;66:161–73. 39. Lifshitz SJ, Razavi A, Bibbo C, et al. Routine cervical length and fetal fibronectin screening in asymptomatic twin pregnancies: is there clinical benefit? J Matern Fetal Neonatal Med 2014; 27: 566–70.
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ORIGINAL ARTICLE
The association between maternal biomarkers and pathways to preterm birth in twin pregnancies Eric Bergh1, Andrei Rebarber1,2, Sandip Oppal2, Daniel H. Saltzman1,2, Chad K. Klauser1,2, Simi Gupta1,2, and Nathan S. Fox1,2 1
Department of Obstetrics, Gynecology, and Reproductive Science, Icahn School of Medicine at Mount Sinai, New York, NY, USA and Maternal Fetal Medicine Associates, PLLC, New York, NY, USA
2
Abstract
Keywords
Objective: We sought to estimate the association between cervical length (CL) and fetal fibronectin (fFN) and each pathway leading to preterm birth in twin pregnancies. Methods: Cohort study of 560 patients with twin pregnancies who underwent routine serial CL and fFN screening from 22 to 32 weeks in one maternal fetal medicine practice during 2005–2013. We calculated the association between a short CL (20 mm) or positive fFN with overall preterm birth 532 weeks, and then subdivided the analysis into preterm birth 532 weeks from preterm labor, preterm premature rupture of membranes (PPROM) and indicated causes. We excluded cases of monochorionic-monoamniotic placentation, vasa previa, twin–twin transfusion and patients with cerclage. Results: The overall rate of preterm birth 532 weeks was 6.9% (3.9% from preterm labor, 1.6% from PPROM and 1.4% indicated). A short cervix was associated with preterm birth 532 weeks arising from preterm labor (12.4% versus 2.0%, p50.001), but not PPROM (1.9% versus 1.3%, p ¼ 0.651). Positive fFN was associated with preterm birth 532 weeks both from preterm labor (17.0% versus 2.4%, p50.001) as well as from PPROM (5.7% versus 1.0%, p ¼ 0.034). Neither was significantly associated with preterm birth 532 weeks from indicated causes. Conclusions: The mechanism leading toward preterm influences the accuracy of screening tests chosen to assess risk in twin pregnancies. A shortened cervical length and positive fFN is associated with spontaneous preterm labor and birth 532 weeks. However, PPROM does not appear to be preceded by a short cervix, but is preceded by a positive fFN. Neither test is associated with an indicated preterm birth.
Cervical length, fetal fibronectin, PPROM, prematurity, preterm labor, twins
Introduction Preterm birth continues to be a major determinant of infant morbidity and mortality in twin pregnancies. In 2011, the twin birth rate was 33.1 per 1000 live births in the United States [1]. Among twin gestations, approximately 60% are born preterm with an average gestational age of approximately 35 weeks [2]. Despite advancements in neonatal intensive care, both short-term and long-term complications of prematurity remain significant causes of mortality in preterm infants. Of note, infant mortality rate in twins is fivefold higher than in singletons, likely due to complications of prematurity [3]. Cervical length (CL) and fetal fibronectin (fFN) have been studied extensively as independent markers of preterm delivery in multiple gestations. A large retrospective review
Address for correspondence: Nathan S. Fox, MD, Maternal Fetal Medicine Associates, PLLC, 70 East 90th Street, New York, NY 10128, USA. Tel: 212-722-7409. Fax: 212-722-7185. E-mail: [email protected]
History Received 18 February 2014 Revised 24 April 2014 Accepted 19 May 2014 Published online 19 June 2014
of CL measured in asymptomatic twin pregnancies identified both CL and gestational age at time of measurement as independent risk factors for preterm delivery [4]. These findings have been confirmed in large meta-analysis as well [5]. A positive fFN in twin pregnancies has also been shown to be significantly associated with preterm birth in a large meta-analysis [6]. Previously published data by our group has also identified fFN as a strong predictor of preterm birth 532 weeks in asymptomatic twin gestations with normal cervical length (425 mm) [7]. The combination of CL and fFN has also been studied in twin pregnancies. In the Preterm Prediction Study, both shortened cervical length (25 mm) and fFN were predictive of preterm delivery 532 weeks in twin pregnancies, the strongest association occurring when both tests resulted positive [8]. We have published similar findings as well. In our own experience following 155 asymptomatic women with twin pregnancies, a positive screen for both shortened cervical length (20 mm) and fFN had a 54.5% risk of spontaneous preterm birth532 weeks gestation in contrast to 4.2% risk among women who screened negative for both risk factors [9].
DOI: 10.3109/14767058.2014.926883
Cervical length and fetal fibronectin in twins with preterm labor and PPROM
Despite the known association between CL, fFN and preterm birth in twins, studies typically only examine the outcome of preterm birth overall or spontaneous preterm birth. However, preterm birth can arise through three primary pathways: preterm labor, preterm premature rupture of membranes (PPROM) or medically-indicated [10]. The objective of this study was to estimate the association between a short CL, positive fFN and preterm birth in twin pregnancies, separately examining each of the three pathways of preterm birth in twin pregnancies: preterm labor, PPROM and indicated preterm birth. This information may also add to our overall understanding of the pathways of preterm birth in twin pregnancies.
Methods We reviewed the charts of all patients with twin pregnancies 422 weeks delivered by a single maternal-fetal medicine practice between June 2005 (when our electronic medical record was established) and June 2013. We excluded patients with monochorionic-monoamniotic placentation, vasa previa, twin–twin transfusion syndrome and patients with cerclage. We obtained baseline characteristics and pregnancy outcomes from our computerized medical record. Gestational age was determined by last menstrual period and confirmed by ultrasound in all patients. The pregnancy was redated if there was a 45 day discrepancy up to 14 weeks or a 47 day discrepancy after 14 weeks. If the pregnancy was the result of IVF, gestational age was determined from IVF dating. The Biomedical Research Alliance of New York Institutional Review Board approval was obtained before reviewing the charts. In our practice, patients with twin pregnancies are followed with serial CL and fFN testing every 2–4 weeks until 32 weeks [9]. fFN testing is done from 22 0/7 to 31 6/7 weeks, and CL screening is performed from 16 0/7 to 31 6/7 weeks; however, for the purpose of this study we only included CL results from 22 to 31 6/7 weeks as this was the window of fFN testing. All CL assessments and fFN testing were done in an outpatient setting on asymptomatic patients. All tests done on labor and delivery were excluded, as they were done on symptomatic patients as part of a preterm labor evaluation. Measurements of CL were performed using a 4- to 8-MHz transvaginal probe with an empty bladder according to criteria established by Iams et al. [11]. The shortest functional CL was used as this has been found to be the most reproducible measurement [12]. A short CL was defined a priori as a measurement 20 mm [9]. Fetal fibronectin testing was performed without the use of a speculum using a published protocol in singleton pregnancies [13] at least 24 h from the last reported intercourse or endovaginal ultrasound. Testing was not performed in the setting of vaginal bleeding. Swabs were sent for evaluation using a fetal fibronectin assay, and a concentration of 50 ng/mL or greater was considered positive. Physicians were not blinded to CL or fFN results. The primary outcome was preterm birth 532 weeks. We calculated the association between a short CL and a positive fFN with preterm birth 532 weeks. We then calculated the association between a short CL and positive fFN with each of
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the three pathways of preterm birth532 weeks: preterm labor, PPROM and indicated preterm birth. Preterm labor was defined as the onset of labor (contractions, cervical change) prior to rupture of membranes. PPROM was defined as ruptured membranes prior to the onset of labor. Indicated preterm birth included any indication for preterm birth due to fetal or maternal indications (e.g. preeclampsia or fetal growth restriction). If a patient had preterm labor or PPROM prior to 32 weeks, but delivered after 32 weeks, she was not considered as having a preterm birth 532 weeks (i.e. the outcome measured was preterm birth from each cause, and not the cause itself). Chi square and Fisher’s exact tests were used, as appropriate (SPSS for Windows 16.0, SPSS Inc., Chicago, IL). A p value of 0.05 was considered statistically significant.
Results During the study period, 611 patient with twin pregnancies 422 weeks were delivered in our practice. 51 were excluded from analysis (25 with cerclage, 10 monochorionicmonoamniotic, nine late transfers with no CL/fFN testing, four with twin–twin transfusion, and three with vasa previa) leaving 560 patients for analysis. The characteristics of the population are listed in Table 1. The incidence of preterm birth 532 weeks overall was 6.9%. Subdivided by cause of preterm birth 532 weeks, the incidence of preterm birth 532 weeks from preterm labor was 3.9%, from PPROM 1.6%, and from an indicated cause 1.4%. Three patients (0.5%) had PPROM less than 32 weeks and delivered after 32 weeks. 552 patients had CL measurements, 105 (19.0%) of whom had a short CL 20 mm prior to 32 weeks. 544 patients underwent fFN testing, 53 (9.7%) of whom had a positive fFN prior to 32 weeks. The association between a short CL and preterm birth 532 weeks is shown in Table 2. A short CL was associated with overall preterm birth 532 weeks (16.2% versus 4.4%, p50.001) and was associated with preterm birth 532 weeks from preterm labor (12.4% versus 2.0%, p50.001). However, a short CL was not associated with preterm birth 532 weeks from PPROM (1.9% versus 1.3%, p ¼ 0.651) or indicated preterm birth 532 weeks (1.9% versus 1.1%, p ¼ 0.623). The association between a positive fFN and preterm birth 532 weeks is shown in Table 3. A positive fFN was associated Table 1. Characteristics of the study population. Number of pregnancies Total cervical length measurements Total fetal fibronectin tests Maternal age (years) Conception Spontaneous Ovulation induction In-vitro fertilization Multifetal pregnancy reduction Caucasian Prepregnancy BMI (kg/m2) Prior term birth Prior preterm birth Prior LEEP or cone biopsy Chorionicity Monochorionic Dichorionic
560 2247 2140 34.2 ± 7.5 years 23.0% 11.6% 65.2% 7.3% 87.4% 23.5 ± 4.5 33.9% 6.2% 2.8% 12.9% 87.1%
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Table 2. Likelihood of preterm birth 532 weeks from all causes in twin pregnancies, based on serial cervical length testing from 22 to 32 weeks. CL 20 mm CL420 mm 22–32 weeks 22–32 weeks N ¼ 447 N ¼ 105 Any preterm birth 532weeks Preterm birth 532 weeks from: Preterm labor PROM Indicated
p
16.2%
4.4%
50.001
12.4% 1.9% 1.9%
2.0% 1.3% 1.1%
50.001 0.651 0.623
CL, cervical length; PROM, premature rupture of membranes. Table 3. Likelihood of preterm birth 532 weeks from all causes in twin pregnancies, based on serial fetal fibronectin testing from 22 to 32 weeks. fFN positive fFN negative 22–32 weeks 22–32 weeks N ¼ 491 N¼53 Any preterm birth 532weeks Preterm birth 532 weeks from: Preterm labor PROM Indicated
p
24.6%
4.6%
50.001
17.0% 5.7% 1.9%
2.4% 1.0% 1.2%
50.001 0.034 0.514
fFN, fetal fibronectin; PROM, premature rupture of membranes. Table 4. Prediction of preterm birth 532 weeks from preterm labor in twin pregnancies, based on serial fetal fibronectin and cervical length testing from 22 to 32 weeks.
fFN positive CL 20 mm
Sensitivity
Specificity
PPV
NPV
+LR
LR
42.9% 59.1%
91.6% 82.6%
17.0% 12.4%
97.6% 98.0%
5.1 3.4
0.62 0.50
Table 5. Prediction of preterm birth 532 weeks from premature rupture of membranes in twin pregnancies, based on serial fetal fibronectin and cervical length testing from 22 to 32 weeks.
fFN positive CL 20 mm
Sensitivity
Specificity
PPV
NPV
+LR
LR
37.5 25.0%
90.7 81.1%
5.7% 1.9%
99.0% 98.7%
4.0 1.4
0.69 0.92
with overall preterm birth 532 weeks (24.6% versus 4.6%, p50.001), preterm birth 532 weeks from preterm labor (17.0% versus 2.4%, p50.001), and preterm birth 532 weeks from PPROM (5.7% versus 1.0%, p ¼ 0.034). A positive fFN was not associated with indicated preterm birth 532 weeks (1.9% versus 1.2%, p ¼ 0.514). The testing characteristics of CL and fFN in predicting preterm birth 532 weeks from preterm labor and from PPROM are shown in Tables 4 and 5, respectively. A positive fFN had a higher positive likelihood ratio than did a short CL for both causes of preterm birth 532 weeks.
Discussion In this study, a positive fFN was associated with spontaneous preterm birth532 weeks arising from preterm labor as well as PPROM. Although a short CL was associated with preterm birth arising from preterm labor, a short CL was not
associated with preterm birth arising from PPROM. This suggests that the mechanisms leading to spontaneous preterm birth influence the accuracy of the particular screening test chosen. PPROM in twin pregnancies does not appear to be preceded by a short cervix, but is preceded by a positive fFN. Neither test is associated with indicated preterm birth. Spontaneous preterm birth may result from both inflammatory and non-inflammatory pathways [10]. The inflammatory cascade that leads to PPROM results in alterations to the normal membrane remodeling process that takes place throughout pregnancy. As membranes expand to accommodate a growing fetus, a balance is normally achieved between proliferative and degradative changes to the amnion and chorion. Extracellular membrane proteins such as collagens, laminins and fibronectins stabilize the fetal membranes and provide the required tensile strength to withstand increased intra and extra-amniotic pressures [14,15]. Fluctuations in the local concentrations of matrix metalloproteinases (MMPs) which breakdown the extracellular scaffold, and tissue inhibitors of metalloproteinases (TIMPs) promote a dynamic molecular milieu which adjusts to a changing environment. Premature increases in MMP activity or decreases in TIMPs may therefore lead to PPROM via membrane weakening and have been associated with the inflammatory response to abruption as well as to infection or colonization with different genital tract pathogens [14]. Specifically, inflammatory cytokines such as TNF-a and IL-1b as well as antioxidants produced as by-products of infection, may enhance production of MMPs leading to accelerated membrane weakening [16–19]. Genetic influences may also play a major role in this balance as there is evidence to suggest that certain racial groups may be predisposed to mount an exaggerated inflammatory response and thus experience increased rates of PPROM [20]. Fetal fibronectin is an extracellular matrix protein which is rarely found in cervicovaginal secretions beyond 22 weeks of gestation. Its presence in concentrations greater than 50 ng/dL implies an ongoing process of extracellular matrix protein degradation and separation of the decidual–fetal membrane interface which often precedes rupture [21]. It may therefore be reflective of the final pathway in an inflammatory process by which PPROM leads to preterm birth. This could explain our finding that a positive fFN in twin pregnancies was associated with spontaneous preterm birth arising from PPROM. Cervical shortening is associated with preterm birth in twin pregnancies and may occur via both inflammatory and non-inflammatory pathways. The physical effects of pathologic over-distension due to multi-gestation could lead to premature activation of the adaptive process of cervical softening, ripening, effacement and dilation. In response to amniochorion stretch, cervical collagen content may be altered via biochemical mechanisms involving regulatory hormones such as prostaglandins and progesterone, cytokines and nitric oxide [22–30]. Whether or not inflammation is the end result or the inciting event in cervical shortening is unclear. As a mechanical barrier to ascending infection, a shortened or dilated cervix may increase the risk of amnionitis [31–33]. Furthermore, cervical mucous has both innate and adaptive immune functions which may predispose to
DOI: 10.3109/14767058.2014.926883
Cervical length and fetal fibronectin in twins with preterm labor and PPROM
transcervical infection when disrupted [34]. Proteomic studies for cervicovaginal biomarkers could help to better understand the breakdown of this barrier and subsequent activation of pathways involved in cervical shortening and preterm labor [34–38]. Our study suggests that in twin pregnancies, a short cervix most likely represents a non-inflammatory pathway to preterm birth, or an inflammatory pathway that would only lead to contractions, but not membrane breakdown and PPROM. This likely differs from singleton pregnancies, as twin pregnancies have significantly more uterine stretch than do singleton pregnancies. As far as we know, this is the first study to identify CL and fFN as specific predictors of different pathways of preterm birth in asymptomatic twin pregnancies. Previous studies have only commented upon the association between these biomarkers and preterm birth overall or spontaneous preterm birth without regard to the underlying cause. Strengths to our study include the large sample size undergoing routine fFN and CL testing. Since we cared for all of these patients, we were able to accurately record whether preterm birth occurred due to preterm labor, PPROM or indicated causes. Using population data and birth certificate registries would likely be flawed or may not address this specific detail. Weaknesses to our study include the fact that patients and obstetricians were not blinded to fFN and CL results. However, as there is no known way to prevent spontaneous preterm birth in twin pregnancies, the knowledge of fFN and CL results is unlikely to have affected our findings. Another weakness of the study is that each specific test has a low positive predictive value for the outcome measured. For example, although we found a significant association between a positive fFN and spontaneous preterm birth from PPROM, the actual positive predictive value of fFN is only 5.7% for PPROM. Therefore, we do not advocate using fFN to screen for PPROM at this time. Rather, our data may lend insight into the pathways of preterm birth for twin pregnancies allowing for the development of more therapeutic interventions. Whether patients with twin pregnancies should undergo routine fFN and CL screening at all is debatable. We have previously demonstrated that routine fFN and CL screening does not lead to a reduction in preterm birth [39]. However, routine fFN and CL screening in twin pregnancies appears to be associated with improved delivery and timing of antenatal corticosteroids [39]. Ultimately, only a randomized, prospective trial could determine the clinical and cost-effectiveness of routine fFN and CL screening. In conclusion, in twin pregnancies a positive fFN is associated with preterm birth532 weeks arising from preterm labor and PPROM. However, a short CL is only associated with preterm birth arising from preterm labor. Future research should focus on the specific pathways of preterm birth in twin pregnancies.
Declaration of interest The authors report no conflicts of interest.
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