Lisa Reusch

Nonlinear optical microscopy and ultrasound imaging of human cervical structure.

Abstract. The cervix softens and shortens as its collagen microstructure rearranges in preparation for birth, but premature change may lead to premature birth. The global preterm birth rate has not decreased despite decades of research, likely because cervical microstructure is poorly understood. Our group has developed a multilevel approach to evaluating the human cervix. We are developing quantitative ultrasound (QUS) techniques for noninvasive interrogation of cervical microstructure and corroborating those results with high-resolution images of microstructure from second harmonic generation imaging (SHG) microscopy. We obtain ultrasound measurements from hysterectomy specimens, prepare the tissue for SHG, and stitch together several hundred images to create a comprehensive view of large areas of cervix. The images are analyzed for collagen orientation and alignment with curvelet transform, and registered with QUS data, facilitating multiscale analysis in which the micron-scale SHG images and millimeter-scale ultrasound data interpretation inform each other. This novel combination of modalities allows comprehensive characterization of cervical microstructure in high resolution. Through a detailed comparative study, we demonstrate that SHG imaging both corroborates the quantitative ultrasound measurements and provides further insight. Ultimately, a comprehensive understanding of specific microstructural cervical change in pregnancy should lead to novel approaches to the prevention of preterm birth.

Detecting cervical microstructure via ultrasound and optical microscopy

A novel method for characterizing the microstructural integrity of the human cervix is under development. The cervix is composed of mostly aligned collagen and variations in acoustic backscattered power as a function of beam steering is an indicator of anisotropic scatterers. Results of experiments with phantoms and hysterectomy specimens suggest that this approach is a sensitive tool for describing the microstructure.

Comparison of ultrasonic measurements of nulliparous versus multiparous cervices

Premature remodeling of uterine cervical microstructure plays a large role in preterm birth. Invasive studies confirm that collagen, the major contributor to cervical strength, undergoes rearrangement long before changes associated with pre-delivery remodeling (shortening, softening, dilation) are grossly detectable. However, a lack of noninvasive technology sophisticated enough to interrogate the cervical microstructure makes quantitative assessment of the cervix challenging. Therefore, clinical cervical assessment is subjective. For instance, clinicians can feel a difference in softness between a nulliparous (never had babies) and multiparous (had babies) cervix. Further, they become concerned about preterm birth risk when the cervix feels “too soft” in midgestation. The inability to objectively describe these parameters impedes clinical management; in other words, currently there is no means to answer critical questions such as “how soft is too soft?”. Our preliminary results using quantitative ultrasound (QUS) techniques and shear wave sound speeds (SWS) to objectively describe cervical microstructure and softening suggest that these measurements are sensitive enough to detect differences between the nonpregnant nulliparous and multiparous cervix, and thus are promising for quantitative assessment of the cervix in pregnancy.

Registration of multiphoton optical images of cervical tissue to quantitative ultrasound data

Collagen remodeling, leading to cervical softening and ripening, is essential for normal vaginal delivery. If this process happens too early there is increased risk of preterm birth, which puts the child at risk for life long health complications and early death. Collagen content and organization in the cervix plays a large role in cervical function. The cervix is highly aligned and layered, and this organization breaks down during remodeling. Cervical collagen remodeling begins early in pregnancy, but there is currently no reliable, non-invasive, quantitative metric for determining whether a cervix is changing too rapidly, thus no effective measure of the risk of preterm birth. We have been developing quantitative ultrasound (QUS) techniques to measure the alignment, and corroborating those QUS measurements with optical microscopy and quantitative image analysis in the form of curvelet transforms (a multi-scale transform similar to wavelets). We show how different choices of scale for the curvelet transform characterize features at different spatial scales. In particular we are investigating which spatial scale, chosen for curvelet analysis, is most predictive of the ultrasound alignment measurements. The spatially-registered US echo power loss measurements are consistent with gross inspection of the optical image itself and with a curvelets based analysis done at the largest spatial scale.

Quantitative ultrasound for evaluating human cervical microstructure

This study uses quantitative ultrasound techniques to investigate the possibility of monitoring changes in cervical microstructure with acoustic backscatter. Standard “general imaging” ultrasound transducers and a novel endocavity transducer were used to detect changes in the power spectrum of the ultrasonic backscattered echo signal as a function of the angle between the acoustic beam and the dominant macrostructure of cervix specimens from hysterectomy. The echo signal power was assessed by integrating the echo signal power spectrum between consistent frequency limits. The echo signal power was found to decrease as a function of steering angle. Normalizing the echo signal power to that found at 0° (acoustic beams normal to the cervical canal) it was found that there was a monotonically increasing loss of power with increasing beam angle, and that power loss was symmetric about 0°. Those data were then compared to identically acquired and processed data from phantoms with spherical scatterers. Data from phantoms with spherical scatterers serve as a normalization for the expected backscatter signal power loss (with increasing steering angle) that would be expected due to the loss of effective aperture and directivity of the transducer. The power loss from the cervix consistently exceeded that from the phantoms, was statistically significant for all angles greater than 50°, and the excess power loss is likely due to the presence of aligned collagen fibers in the cervical microstructure.

OC09.01: Correlation of human cervical collagen microstructure with quantitative ultrasound

Objectives: Left ventricular (LV) ejection causes a forward flow in the fetal aortic isthmus (AoI) while right ventricle (RV) has a retrograde influence. This study proposes reference values for an ISI reflecting the changing balance between fetal RV and LV performances. Methods: Doppler recordings of 104 normal fetuses from 17 to 38 weeks were reviewed. The ISI was calculated as follows: Nadir of end-systolic velocity (Ns) ÷ Peak systolic velocity (Ps). Right (QP) and LV outputs (QS) were also calculated. Results: Up to 27 weeks of gestation, the ISI is stable at +0.2. From about 28 weeks, a brief end-systolic retrograde flow (ESRF) is observed, increasing steadily with gestation and causing a fall of ISI whose mean reference value reached −0.3 at 38 weeks. (Figure). Simultaneous Doppler recordings of isthmus and ductus arteriosus demonstrate that the essential contributor to the ESRF is flow emanating from the RV. Significant association is found between Ps and QS (QS: P < 0.001; QP: P = 0.2) and between Ns and QP (QP: P < 0.0001; Qs: P = 0.4). Conclusions: The ISI expresses well the physiologic increase in fetal RV preponderance. ISI is a simple non-invasive mean of evaluating the interactive performance of the fetal ventricles.

OP08.01: Complex shear wave propagation in cervix is consistent with collagen microstructure

Objectives: Current health policy encourages the use of rapid trisomy testing rather than full karyotype analysis. We compared the results of QF-PCR with full karyotyping in the west of Scotland population. Methods: A retrospective audit of results was performed between 01/11/09 and 31/10/11. Results: From prenatal diagnostic procedures, 1726 CVS or amniocentesis samples were received in the regional genetics laboratory which serves a population of 3 million. Main referral reason for a diagnostic procedure was due to abnormal scan (52%), followed by increased risk at second trimester screening (31%). Family history of chromosomal anomalies (7%) and maternal age (5%) were less common indications. Three cases had inadequate sample for culture, 1 failed culture and 1 was not set up. Abnormal karyotype was seen in 181 samples: 71% of abnormalities were correctly identified at the time of QF-PCR. Approximately 50% had trisomy 21, 25% had trisomy 18 and the remainder had Turner’s syndrome, 69 XXX, or trisomy 13. QF-PCR did not detect the abnormality seen at full karyotyping in 51 cases. Of these, 16 cases had balanced translocation or inversion and 15 had normal ultrasound examinations. Thirteen had unbalanced changes and 10 of these also had abnormal scans. Due to referral reasons (e.g. maternal age or biochemical screening), sex chromosomes were not always examined at QF-PCR. Fourteen cases of XO/XXX/XXY were missed. There were 4 samples with mosaicism but only 1 case also had an abnormal scan. Conclusions: Full karyotyping can give valuable additional information. However, in those with severe chromosomal abnormalities, structural anomalies are often seen on ultrasound examination.

OP10.02: Quantitative ultrasound for detection of collagen fiber alignment in the layered microstructure of the human cervix

Objectives: Previous studies reported that oligohydramnios is associated with adverse perinatal outcome in preterm premature rupture of membranes (PPROM). However, patients with initially normal amniotic fluid volume (AFV) can maintain adequate AFV or progress to have oligohydramnios, and the clinical outcome of patients who maintain normal AFV has not been well examined. This study was conducted to address this issue. Methods: The AF index (AFI) was serially measured in patients with PPROM (< 34 weeks) with an interval of several days. Patients who delivered within 2 days of admission (n = 66) or in whom AFI was not measured serially were excluded from analysis. Oligohydramnios was defined as AFI < 5 and cases were divided into 3 groups according to the change of AFI during 1st and 2nd ultrasonography (USG): Group 1, maintaining normal AFI; Group 2, normal AFI at 1st USG but oligohydramnios at 2nd USG (developing oligohydramnios); Group 3, oligohydramnios at 1st USG. Results: The AF index (AFI) was serially measured in 188 patients with PPROM with a median interval of 3 days. The rate of oligohydramnios at 1st USG was 35% (66/188). Among 122 patients who had normal AF at 1st USG, 84 of patients (69%) maintained normal AFV and 38 patients (31%) developed oligohydramnios in 2nd USG. Cases in groups 2 and 3 had shorter mean interval-todelivery than those in group 1 (group 1, 4.8 ± 5.3 weeks; group 2, 1.9 ± 2.1 weeks; group 3, 1.6 ± 1.6 weeks; P < 0.001) and these differences remained significant after adjustment for gestational age. In 141 patients, amniocentesis was performed within 1 week of 1st USG measure and AF matrix metalloproteinase-8 was measured after delivery. Cases in groups 2 and 3 had higher rate of intra-amniotic infection and/or inflammation than those in group 1 (group 1, 28% [21/75]; group 2, 55% [16/29]; group 3, 70% [26/37]; P < 0.001). Conclusions: Cases with persistent normal AFI had better outcome than those with developing oligohydramnios. Serial measurement of AFI is needed in cases with PPROM.