R RheometryFigure 1. (A) MRI image of patient at 34 weeks gestation. Arrow points to cervical mucus plug. Scale bar: 1 cm. (B) Diagram of the cervical mucus plug in situ. doi:10.1371/journal.pone.0069528.gTo elucidate differences in extensional rheology, a Capillary Breakup Extensional Rheometer (CaBER) [28] was used. The CaBER draws a material apart into a filament at a fixed rate for rheological observation and the determination of spinnbarkeitCervical Mucus Properties and Preterm Birth RiskFigure 4. (A) Diagram of bead permeability assay. (B) Bead permeability assay results. High risk cervical mucus samples displayed significantly more permeability to the biotin labeled polystyrene beads compared to low risk controls (5.6 beads/field (+/ 22.6) vs. 2.2 beads/field (+/21.2), p = 0.006). doi:10.1371/journal.pone.0069528.gMeasurement of Mucus PermeabilityTo study permeability of the native cervical mucus, a bead translocation assay was performed with streptavidin-activated glass slides as reported [30]. Prior to the experiment the slides were preincubated for 30 minutes in 0.5 BSA to eliminate non-specific binding, and encased in an Arrayit 24-well multiplex microarray cassette. 25 mL of mucus sample, or 20 mM HEPES buffer without mucin, were spread in triplicate in individual wells. 5 uL biotinylated Fluospheres (0.2 mm, Invitrogen) at a concentration of 56106 particles/mL was added on top the mucus or buffer and allowed to diffuse for 2 hours at room temperature. The glass slides were washed of the mucus three times in washing buffer, with 0.1 Triton-X detergent added to the first washing step. Next, three images per well were acquired with a fluorescence microscope at 106 to quantify the number of streptavidin-bound biotin beads that had passed through the mucus to the underlying surface. The mean of the nine images per sample was taken to represent the number of beads that passed through each sample. The investigator responsible for quantifying the number of beads passing through each sample was blinded to the cervical mucus sample study group.Figure 3. (A) Diagram of shear rheology. inhibitor Rotational shear force applied to cervical mucus sample. (B) Example linear viscoelastic spectra of high risk and low risk cervical mucus samples. Storage modulus G9 and loss modulus G” of low risk mucus is an order of magnitude greater than that of high-risk mucus, indicating that highrisk mucus is more weakly cross-linked than low-risk mucus. doi:10.1371/journal.pone.0069528.g(capacity to form filaments) [29]. 2006100 mL of cervical mucus sample was Autophagy placed between two circular metal plates that were 6 mm in diameter, with an initial gap of 2 mm. The plates were then separated to a distance of 20 mm (maximum separation distance attainable with our CaBER device) at a constant rate of 3.6 mm/s and the separation distance at which the sample broke (“break point”) was recorded. For statistical calculations, a “break point” of 20 mm was used for those samples which remained intact. Shear rheometry was performed with a TA instruments ARG2 controlled stress rheometer. Approximately 75 mL of cervical mucus was placed in a 1.5 mm gap between an 8 mm diameter steel plate, and a Peltier plate whose temperature was controlled at 25uC. During the test, we apply a sinusoidally varying strain to the mucus sample, and measure the resulting stress response. The storage modulus G9 (storage modulus, quantifying the elastic, solid-like, recoverable property of a sub.R RheometryFigure 1. (A) MRI image of patient at 34 weeks gestation. Arrow points to cervical mucus plug. Scale bar: 1 cm. (B) Diagram of the cervical mucus plug in situ. doi:10.1371/journal.pone.0069528.gTo elucidate differences in extensional rheology, a Capillary Breakup Extensional Rheometer (CaBER) [28] was used. The CaBER draws a material apart into a filament at a fixed rate for rheological observation and the determination of spinnbarkeitCervical Mucus Properties and Preterm Birth RiskFigure 4. (A) Diagram of bead permeability assay. (B) Bead permeability assay results. High risk cervical mucus samples displayed significantly more permeability to the biotin labeled polystyrene beads compared to low risk controls (5.6 beads/field (+/ 22.6) vs. 2.2 beads/field (+/21.2), p = 0.006). doi:10.1371/journal.pone.0069528.gMeasurement of Mucus PermeabilityTo study permeability of the native cervical mucus, a bead translocation assay was performed with streptavidin-activated glass slides as reported [30]. Prior to the experiment the slides were preincubated for 30 minutes in 0.5 BSA to eliminate non-specific binding, and encased in an Arrayit 24-well multiplex microarray cassette. 25 mL of mucus sample, or 20 mM HEPES buffer without mucin, were spread in triplicate in individual wells. 5 uL biotinylated Fluospheres (0.2 mm, Invitrogen) at a concentration of 56106 particles/mL was added on top the mucus or buffer and allowed to diffuse for 2 hours at room temperature. The glass slides were washed of the mucus three times in washing buffer, with 0.1 Triton-X detergent added to the first washing step. Next, three images per well were acquired with a fluorescence microscope at 106 to quantify the number of streptavidin-bound biotin beads that had passed through the mucus to the underlying surface. The mean of the nine images per sample was taken to represent the number of beads that passed through each sample. The investigator responsible for quantifying the number of beads passing through each sample was blinded to the cervical mucus sample study group.Figure 3. (A) Diagram of shear rheology. Rotational shear force applied to cervical mucus sample. (B) Example linear viscoelastic spectra of high risk and low risk cervical mucus samples. Storage modulus G9 and loss modulus G” of low risk mucus is an order of magnitude greater than that of high-risk mucus, indicating that highrisk mucus is more weakly cross-linked than low-risk mucus. doi:10.1371/journal.pone.0069528.g(capacity to form filaments) [29]. 2006100 mL of cervical mucus sample was placed between two circular metal plates that were 6 mm in diameter, with an initial gap of 2 mm. The plates were then separated to a distance of 20 mm (maximum separation distance attainable with our CaBER device) at a constant rate of 3.6 mm/s and the separation distance at which the sample broke (“break point”) was recorded. For statistical calculations, a “break point” of 20 mm was used for those samples which remained intact. Shear rheometry was performed with a TA instruments ARG2 controlled stress rheometer. Approximately 75 mL of cervical mucus was placed in a 1.5 mm gap between an 8 mm diameter steel plate, and a Peltier plate whose temperature was controlled at 25uC. During the test, we apply a sinusoidally varying strain to the mucus sample, and measure the resulting stress response. The storage modulus G9 (storage modulus, quantifying the elastic, solid-like, recoverable property of a sub.
