Creased compared with women at low-risk for preterm delivery (2.860.8 cm vs. 0.0060.0 cm, p,.01). The demographics of the high-risk and low-risk patients were similar, but we observed more African-American patients in the low-risk cohort. Less cervical mucus was collected from low-risk patients, likely due to the limited access of the closed cervix and the thickened consistency of the mucus. In evaluation of the clinical outcome, the high-risk patients delivered earlier compared to lowrisk controls (34.464.3 weeks versus 37.162.1 weeks, p,.05). While there was a significant 10457188 difference in delivery gestational age between the two groups, we noted that some low-risk patients also delivered at or before 37 weeks of gestation. The reason for this is that several of the `low-risk’ patients who were recruited from the inpatient antepartum service had risk factors for indicated preterm delivery (i.e. preeclampsia). Hence, the frequency of indicated preterm birth was increased in the `low-risk’ cohort, which resulted in a lower than expected mean delivery gestational age for this cohort (37 weeks).Shear rheometry reveals a higher elasticity of high-risk mucus than low risk mucusSince rheological properties are key determinants of hydrogel barrier functions [31], we Title Loaded From File performed a more detailed rheological characterization of cervical mucus from high-risk and low-risk patients. Title Loaded From File Specifically, we investigated the viscoelasticity of the mucus samples of three high-risk and three gestational age matched controls using a rotational shear rheometer, measuring G9 (storage modulus) and G99 (loss modulus). Across all pairs, both high-risk and low-risk mucus had a higher storage modulus than loss modulus, indicating that all cervical mucus samples are more solid-like than liquid-like. In addition, both the storage and loss moduli of cervical mucus samples from patients at high-risk of preterm delivery were found to be an order of magnitude lower than that of cervical mucus samples from gestational age matched low-risk controls (Figure 3). This result suggests that the gelforming mucins (and potentially other molecules) within high-risk mucus may be less effectively cross-linked, thereby generating a weaker gel, possibly with larger pores.Table 1. Patient Characteristics.Characteristic Age (Years) Gravidity Parity Race ( ) White Black Hispanic Other Gestational Age (wks) Dilation (cm) Prior PTB ( ) Positive GBS carrier ( ) Mucus collected (ml) Gestational Age at Delivery (wks)High Risk (n = 18) 27.4 (+/26.6) 2.7 (+/21.6) 1.0 (+/21.1)Low Risk (n = 18) 29.6 (+/25.8) 3.0 (+/21.9) 0.7 (+/20.8)P alue 0.36 0.71 0.50 ,0.22 0 50 28 30.8 (+/23.4) 2.8 (+/20.8) 22 17 264.0 (+/2117.0) 34.4 (+/24.3)50 11 28 11 30.4 (+/23.3) 0.00 (+/20.0) 16 17 192.0 (+/261.5) 37.1 (+/22.8) 0.74 ,0.001 0.64 1.0 0.018 0.Expressed as Mean (+/2 SD) or percentage; GBS: Group B Streptococcus. doi:10.1371/journal.pone.0069528.tCervical Mucus Properties and Preterm Birth RiskScanning Electron Microscopy reveals heterogeneity within mucus samplesIn an attempt to directly visualize the mucin cross-linking, Scanning Electron Microscopy (SEM) was performed on two highrisk and two low-risk gestational age matched controls (n = 4). The sample preparation resulted in dehydrated, brittle samples, which were fractured prior to imaging. We noted a high degree of heterogeneity within each sample, but we imaged regions of filamentous networks in matched locations (subsurface regions along fractures) for compa.Creased compared with women at low-risk for preterm delivery (2.860.8 cm vs. 0.0060.0 cm, p,.01). The demographics of the high-risk and low-risk patients were similar, but we observed more African-American patients in the low-risk cohort. Less cervical mucus was collected from low-risk patients, likely due to the limited access of the closed cervix and the thickened consistency of the mucus. In evaluation of the clinical outcome, the high-risk patients delivered earlier compared to lowrisk controls (34.464.3 weeks versus 37.162.1 weeks, p,.05). While there was a significant 10457188 difference in delivery gestational age between the two groups, we noted that some low-risk patients also delivered at or before 37 weeks of gestation. The reason for this is that several of the `low-risk’ patients who were recruited from the inpatient antepartum service had risk factors for indicated preterm delivery (i.e. preeclampsia). Hence, the frequency of indicated preterm birth was increased in the `low-risk’ cohort, which resulted in a lower than expected mean delivery gestational age for this cohort (37 weeks).Shear rheometry reveals a higher elasticity of high-risk mucus than low risk mucusSince rheological properties are key determinants of hydrogel barrier functions [31], we performed a more detailed rheological characterization of cervical mucus from high-risk and low-risk patients. Specifically, we investigated the viscoelasticity of the mucus samples of three high-risk and three gestational age matched controls using a rotational shear rheometer, measuring G9 (storage modulus) and G99 (loss modulus). Across all pairs, both high-risk and low-risk mucus had a higher storage modulus than loss modulus, indicating that all cervical mucus samples are more solid-like than liquid-like. In addition, both the storage and loss moduli of cervical mucus samples from patients at high-risk of preterm delivery were found to be an order of magnitude lower than that of cervical mucus samples from gestational age matched low-risk controls (Figure 3). This result suggests that the gelforming mucins (and potentially other molecules) within high-risk mucus may be less effectively cross-linked, thereby generating a weaker gel, possibly with larger pores.Table 1. Patient Characteristics.Characteristic Age (Years) Gravidity Parity Race ( ) White Black Hispanic Other Gestational Age (wks) Dilation (cm) Prior PTB ( ) Positive GBS carrier ( ) Mucus collected (ml) Gestational Age at Delivery (wks)High Risk (n = 18) 27.4 (+/26.6) 2.7 (+/21.6) 1.0 (+/21.1)Low Risk (n = 18) 29.6 (+/25.8) 3.0 (+/21.9) 0.7 (+/20.8)P alue 0.36 0.71 0.50 ,0.22 0 50 28 30.8 (+/23.4) 2.8 (+/20.8) 22 17 264.0 (+/2117.0) 34.4 (+/24.3)50 11 28 11 30.4 (+/23.3) 0.00 (+/20.0) 16 17 192.0 (+/261.5) 37.1 (+/22.8) 0.74 ,0.001 0.64 1.0 0.018 0.Expressed as Mean (+/2 SD) or percentage; GBS: Group B Streptococcus. doi:10.1371/journal.pone.0069528.tCervical Mucus Properties and Preterm Birth RiskScanning Electron Microscopy reveals heterogeneity within mucus samplesIn an attempt to directly visualize the mucin cross-linking, Scanning Electron Microscopy (SEM) was performed on two highrisk and two low-risk gestational age matched controls (n = 4). The sample preparation resulted in dehydrated, brittle samples, which were fractured prior to imaging. We noted a high degree of heterogeneity within each sample, but we imaged regions of filamentous networks in matched locations (subsurface regions along fractures) for compa.
