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Abstract
The uterine cervix plays a central role in the maintenance of pregnancy and in the process of parturition. Cervical remodeling involves dramatic changes in extracellular matrix composition and, in particular, of collagen and water content during cervical ripening (a term that describes the anatomical, biochemical, and physiologic changes in preparation for labor). Untimely cervical ripening in early gestation predisposes to preterm labor and delivery, the leading cause of infant death worldwide. Inadequate ripening of the cervix is associated with failure of induction or prolonged labor. The current approach to evaluate the state of the cervix relies on digital examination and sonographic examination. Herein, we present a novel imaging method that combines ultrasound (US) and photoacoustic (PA) techniques to evaluate cervical remodeling by assessing the relative collagen and water content of this organ. The method was tested in vitro in extracted collagen phantoms and ex vivo in murine cervical tissues that were collected in mid-pregnancy and at term. We report, for the first time, that our imaging approach provides information about the molecular changes in the cervix at different gestational ages. There was a strong correlation between the results of PA imaging and the histological assessment of the uterine cervix over the course of gestation. These findings suggest that PA imaging is a powerful method to assess the biochemical composition of the cervix and open avenues to non-invasively investigate the composition of this organ, which is essential for reproductive success.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Successful reproduction in placental mammals requires that the uterine cervix retains the conceptus throughout gestation and then allows its passage at the time of the onset of labor [1–3]. In early pregnancy, the cervix is stiff and long and subsequently softens and shortens as term approaches [4–7]. The term cervical ripening is used to refer to the anatomical, biochemical, biophysical, and clinical changes that precede the onset of labor in women [8]. Similar changes have been described in animal models [9–19]. The uterine cervix is fundamentally an extracellular matrix organ and the tensile strength is largely attributed to the presence of fibrillar collagen [20]. As the cervix ripens, there are dramatic changes in the composition of the cervix with a decrease in collagen content and in glycosaminoglycans as well as an increase in total water [15,21–29].
Untimely cervical ripening may lead to cervical insufficiency, which is responsible for recurrent spontaneous abortion and preterm birth [30–32]. A sonographic short cervix is a major risk factor for spontaneous preterm delivery (sPTD) [33], the leading cause of infant death below the age of five [34,35]. Failure of the cervix to ripen at term is thought to be the basis for a prolonged latent phase of labor and failure of induction of labor [36]. Collectively, these conditions are major causes of perinatal morbidity and mortality as well as labor disorders resulting in maternal morbidity.
Assessment of cervical status is performed with a digital examination to determine cervical length, dilatation, and consistency, which is called the Bishop’s score [37,38]. Ultrasound has gained a place in clinical practice by allowing the non-invasive assessment of cervical length [5,39,40]. Mothers with a short cervix are at high risk for preterm delivery, which can be reduced by the administration of vaginal progesterone [40–46]. In contrast, patients with a long cervix are at increased risk for requiring a cesarean delivery when they undergo labor at term [47,48]. However, the cervical length is a suboptimal means to assess cervical status because it does not provide information about cervical consistency. There is now compelling evidence that changes in the viscoelastic properties of the uterine cervix are key to its function [49,50]. Considerable efforts have been made to image the cervix with elastography to gain insights into tissue properties [51–55]. Hence, the direct estimation of cervical tissue composition for monitoring cervical ripening at the molecular level (fibrillar collagen remolding) had not been achieved.
Photoacoustic (PA) imaging has shown significant diagnostic potential by providing complementary functional and molecular information about a target tissue [56–58]. Its key advantage is the ability to provide high-resolution images of parameters (e.g. tissue vascularity, hemoglobin oxygen saturation or SO2 level, and tissue molecular composition) at clinically relevant depths [59]. Moreover, PA imaging complements conventional ultrasound (US) as both modalities share signal acquisition hardware and detection regimens [60–62]. Therefore, the combination of these techniques allows for the simultaneous acquisition of anatomical, functional, and molecular compositional information from the imaged tissue.
In this study, we report, for the first time, a combined US and PA imaging system and method that can assess the progress of collagen degradation in the murine cervical tissue using spectroscopic analysis of PA (sPA) images in the infrared range. The developed system and sPA method were first evaluated using in vitro control collagen phantom studies, and the robustness of the method was confirmed using ex vivo animal studies, including histological validation of the cervical ripening process.
2. Materials and methods
2.1 Principles of spectroscopic photoacoustic (sPA) evaluation of water and collagen
Spectroscopic PA (sPA) imaging is a widely used method to evaluate functional and molecular information by utilizing variant wavelengths of light into the tissue and probing the acoustic signal amplitude, which represents the optical properties in the imaged object. As previously reported, water and collagen have different optical properties [63,64], shown in Fig. 1. There are two localized maxima in the absorption spectrum of water and collagen: (a) 1100 to 1300 nm; and (b) 1400 nm to 1600 nm. In the first spectral range, water has a near-constant absorption and at the same time collagen peaks at 1200 nm. Therefore, one can relate the variations in sPA signal in the first spectral range to the changes in concentration of collagen. Lipid absorption has a significant overlap with collagen. However, since the lipid content of cervical tissue is not significant, its absorption can be disregarded in sPA calculations [65]. In the second spectral range, water and collagen have a local peak at approximately 1450 and 1470 nm respectively; however, water is a dominant absorber (by the factor of ∼6) within the range of these wavelengths (Fig. 1). Therefore, the change in sPA signal within this second spectral window is dominated by the variations in the water content of the tissue. By tuning laser excitation wavelengths within these two optical windows, there is a possibility that sPA imaging can distinguish between water and collagen within tissue using these optical signatures.
Fig. 1. (a) Optical absorption spectra of water, collagen, oxyhemoglobin, and lipid in the infrared range. In Window 1, collagen absorption varies while water is constant. In Window 2, water is dominant. (b) Collagen absorption and Scattering. (a) and (b) are adopted from Ref. [64].
2.2 Initial evaluation of sPA imaging for collagen and water in tissue-mimicking phantoms
We used collagen extracted from rat tail to evaluate the sensitivity of PA imaging for detecting collagen absorption. The collagen (type I) was isolated from rat tail and reconstituted to the desired concentrations based on a previously established protocol [66]. Briefly, the collagen solution was freeze-dried under sterile conditions and the dried powder was resuspended in sterile 1mM HCl solution. This process yielded a high-purity and uniform collagen solution in which the concentration was adjusted to 65 mg/ml. After the collagen solution was prepared, it was transferred to an imaging phantom and kept at low temperature (4 °C). The collagen solution was then scanned spectroscopically at a range of 1070-1650 nm to evaluate the collagen absorption signatures. As a control sample, pure distilled water was scanned in the same imaging phantom at 4 °C.
2.3 Photoacoustic imaging experimental setup
We developed a combined US/PA imaging system that integrated a high energy, tunable Nd: YAG laser (Phocus Core, 680 nm to 2400 nm wavelength range, 10 Hz repetition rate, 8 ns pulse duration, OPOTEK Inc.) with a commercial linear array ultrasound transducer (L11-4v, Philips) using a custom 19-fiber bundle (NA 0.39, FT1000EMT, Thorlabs Inc.). The combined system is connected to a research ultrasound platform (Vantage 128, Verasonics Inc.) to acquire real-time US and PA signals. The transmit center frequency was set to 9 MHz, giving an axial resolution of 166 µm and lateral resolution 175 µm (at the depth of 2 cm) on our US and PA images. The fiber bundle forms a 4 mm in diameter circular illumination pattern, which is able to accurately target the murine cervical tissue samples and to reduce the illumination of the surrounding environment, and thus reduces the generation of PA signal arising from coupling media. During our experiments, the fluence was kept below the ANSI maximum permissible exposure (MPE); the maximum fluence during our experiments was 24 mJ/cm2 at 680 nm [67]. In addition, the output beam from one of the 19 fibers was constantly monitored with a power meter (PE50-DIF-C, Ophir Inc.) to measure the laser energy variations across different wavelengths and compensate the PA signals with respect to these energy variations. The coupling medium used in our experiments was made using a polyvinyl alcohol (PVA, 10% wt/wt with Propylene Glycol/ water 40:60)-based phantom, which was 10 × 10 × 10 cm3 with a cubical hole of 1 cm3 at the center (Fig. 2(b)).
Fig. 2. (a) Collecting of murine cervical samples at different gestational age. Two sets of samples were collected at 13.5- and 19.5-days post coitus (dpc). (b) sPA imaging for murine cervices. The sample placed inside a PVA holder and imaging were performed on the top(c) Histologic analysis for the imaged murine cervices. Samples were frozen inside OCT compound and after slicing, they analyzed histologically.
2.4 Murine cervical imaging
C57BL/6 mice (Jackson Laboratories) were housed under a circadian cycle (12 hours light:12 hours dark) in the animal care facility at the C. S. Mott Center for Human Growth and Development, Wayne State University (Detroit, MI). Pregnant mice were euthanized on 13.5 or 19.5 days post coitum (dpc; n = 10 samples per gestational age group) and the cervices were collected and stored in RNAlater solution, following the manufacturer’s instructions. Figure 2 shows the sequence of sample collection, transportation for sPA imaging, and histological analysis. Murine cervical samples were imaged within one hour after extraction. During transportation and the imaging procedure, the samples were kept in RNAlater solution. Wide-spectrum range sPA (1150-1650 nm) data acquisition was performed and the laser energies at different wavelengths were recorded to normalize the acquired PA signals with respect to energy variations. The selected wavelengths covered the absorption peaks for both collagen and water. In addition, the RNAlater solution was spectroscopically imaged in the same wavelength range to assure no background interference from the solution. Upon completion of imaging, the tissue was washed with PBS, embedded in optimal cutting temperature (OCT) compound, and snap-frozen using liquid nitrogen, followed by storage at -80°C prior to sectioning and histological analyses. Using a cryostat instrument, 10-µm-thick sections of the frozen tissues were stained using the Picrosirius Red Stain Kit (Polysciences Inc.), according to the manufacturer’s instructions, and imaged under a light microscope to examine the stained collagen as previously described [68]. To semi-quantitatively determine the amount of collagen and non-collagen proteins in each tissue, Sirius Red/Fast Green collagen staining was used, following the manufacturer’s instructions, as previously described [69]. A set of hematoxylin and eosin (H&E)-stained and Picrosirius red-stained (PSR) tissue section images of murine cervices showing the cervical canal or lumen were obtained as references to visualize the cervical collagen network disorganization. The quantitative determination of collagen was performed by washing cervical tissues with 1X PBS (Fisher Scientific), followed by incubation of tissues in 0.2 - 0.3 ml of dye solution at room temperature for 30 minutes. The dye solution was aspirated, and the tissue slide was repeatedly rinsed with 0.5 mL of distilled water. Dye Extraction Buffer (1 mL) was applied to the tissue and gently mixed by pipetting until the color was eluted from the tissue section. The eluted dye solution was collected in a cuvette and read with a spectrophotometer at OD values of 540 nm and 605 nm to quantitatively calculate the total collagen amount in each sample.
3. Results and discussion
3.1 sPA imaging for collagen and water samples
The evaluation of PA imaging sensitivity for detecting collagen and water absorption is shown in Fig. 3. Both samples were spectroscopically imaged within the wavelength range of 1050 to 1650 nm. The acquired PA signals were averaged over the same size region of interest (ROI) in both collagen and control distilled water samples. The mean and standard deviation of the PA signals are plotted in Fig. 3. A relatively constant absorption of water sample within the first spectral window is demonstrated. The PA signal amplitude varied between 0 and 351 (a.u.) for each point inside the ROI. The PA signal of the collagen phantom shows a stronger PA signal due to the presence of 65 mg/ml of collagen. Both control and collagen samples show a relatively high PA signal within the second spectral window. These results are predictable since the concentration of collagen in our sample is only 6.5% (65 mg of collagen vs. 935 mg of water /ml). During these controlled studies, experimental variations such as laser energies at each wavelength, illumination pattern, and probe receiving parameters were kept the same. Therefore, these results truly represent the sPA signature of collagen and water samples. The concentration of collagen in cervical tissue is reported to be more than 100 mg/g at term [21], which is significantly larger than what could be achieved in making phantoms. Therefore, we anticipate an even more significant distinction between spectral features of collagen and water is detectable in tissue samples. These results indicate a clear peak of the collagen sample at 1200 nm within the collagen-dominant range. For the water-dominant range, we can also observe that pure water has an absorption peak around 1450 nm and collagen peaks at about 1470 nm, which is correlated with the collagen-water absorption reported in Fig. 1. To summarize, the results of collagen and water phantoms indicate the capability of PA imaging to track collagen and water absorptions that can enable the distinction between these two components by using their optical properties and through sPA signal acquisition at their peak absorption wavelengths (i.e. 1200 nm, 1450 nm, 1470 nm).
Fig. 3. Spectroscopic PA measurements of collagen (65 mg/mL) and blank (water) samples. sPA indicates the collagen has absorption peaks at 1200 and 1470 nm, while water remains near-constant around 1200 nm and has an absorption peak at 1450 nm.
3.2 sPA Imaging of Pregnant Murine Cervical Tissue Samples
Upon examining the sPA imaging in water and collagen samples, we evaluated the developed sPA imaging method using excised pregnant murine cervices. A set of energy-normalized co-registered US/PA images of a murine cervix at 13.5 dpc, acquired at 3 different wavelengths (1150, 1200, 1250 nm), are shown in Fig. 4. The PA images indicate the PA signal amplitude change across the selected wavelength range. The PA signal at 1200 nm shows the largest amplitude, which correlated with the absorption peak of collagen [64]. The co-registered US and PA images allow for the evaluation of localized collagen and water in different location of cervical tissue.
Fig. 4. (a) US image for murine cervix sample (13.5 dpc); (b-d) Energy normalized PA images overlapped with the US at 1150, 1200 and 1250 nm. The PA amplitude intensity peaks at ∼1200 nm (collagen peak absorption), indicating the highest collagen concentration.
To further investigate the contribution of collagen and water to sPA signals, a wider range of sPA data (1100-1650 nm) was collected and processed for two groups of murine cervical tissues. The sPA results for the murine cervical samples from 13.5 and 19.5 dpc are divided into two separate bandwidths: the collagen-dominant range (1100-1300 nm), where collagen absorption peaks at around 1200 nm and water absorption is nearly constant [63,64], and the water-dominant range (1300-1650 nm), where water has a significantly larger absorption compared to other endogenous tissue chromophores. Since the lipid content of cervical tissue is not significant, its contribution to the PA signal can be disregarded in the evaluation of collagen and water content of the tissue. Figure 5(a) shows the decrease in collagen-associated signal in cervical tissue from 19.5 dpc compared to those from 13.5 dpc, whereas in Fig. 5(b), the signal from the water is significantly increased in the ripened cervix (19.5 dpc). Furthermore, in order to perform a quantitative analysis of the sPA measurements, we defined an arbitrary metric that represents the collagen-to-water ratio (C/W) based on the measured PA spectra:
where $AU{S_{Collagen}}$ and $AU{S_{Water}}$ represent the area under the spectral in the collagen-dominating range (1100-1300 nm) and the water-dominating (1300-1650 nm) range, respectively. The C/W values for 13.5 and 19.5 dpc were calculated as 60.45% $\pm$ 8.95% and 41.04% $\pm $ 5.33%, respectively. To demonstrate the differences between the two groups, an unpaired T-test analysis was done, which led to a p-value of 0.00014 (t = 5.8923, df = 18, standard error of difference = 3.294), indicating that the measured C/W metric is significantly different between the two groups. We also compared our results to H&E staining of the murine cervices at 13.5 dpc and 19.5 dpc, shown in Fig. 5(c–d). These images depict the presence of edema (increased water content shown as white parts of the histological images) in the murine cervices at 19.5 dpc. At this gestational age, overall tissue organization is lost and the endocervical mucosa is no longer present. Fibrous components disappear, and the presence of an amorphous material is observed between muscular cells. A transversal slide of the murine cervix at 13.5 dpc, stained with Picrosirius Red and microscopically imaged under polarized light, is shown in Fig. 5(e). A dense, organized concentric network of collagen fibers has birefringence and appears as red/yellow rod-like structures. By contrast, at 19.5 dpc, the degradation of the collagen network results in no clear organization of collagen fibers, and the tissue had significantly lower overall collagen fiber content (Fig. 5(f)). An evaluation algorithm was created and applied in the Picrosirius red-stained (PSR) images. A threshold binary mask (20% histogram level was set as intensity threshold), selected to remove the background, and then evaluates the size of the masked off areas by which the binary mask indicated each individual collagen fiber. The collagen fiber shapes are detected in pixel values and the mass center of the shape was also calculated. The quantification of the average collagen fiber size takes the average of the integral binary mask size in which the individual collagen fiber was indicated. Figure 5(g–h) shows the pattern analysis of PSR tissues using image quantification algorithms to extract radiomic features such as intensity measurement and size of collagen regions. Furthermore, we analyzed the fluorescent intensity for these PSR images [24]. The 13.5 dpc murine cervical images have a mean of 78.11 (a.u.) and 19.5 dpc samples have a mean of 12.76 (a.u.). The unpaired t-test results indicated the two-tailed p-value equals 0.018 (<0.05), which demonstrated the statistically significant difference between the two groups of cervices at 13.5 and 19.5 dpc in PSR images.Fig. 5. (a, b): sPA signal amplitudes of murine cervices at two gestational ages (13.5 and 19.5 dpc, n = 10 for each); (c, d): H&E staining; (e, f): Sirius Red staining and polarized light microscopy images of transversal slide of murine cervices. Position of the cervical canal or lumen is indicated for reference; (g, h): Quantification of histological images to identify the collagen surface-area of collagen.
While the histological analysis of murine cervical tissue showed decreased amounts of collagen disorganization in late gestation, the direct quantitation of collagen revealed no significant difference in collagen/total protein (w/w) between cervical tissue at 13.5 dpc and 19.5 dpc. The calculated average collagen amount (absolute value) of 5 samples for 13.5 dpc is 52.97 µg collagen per mg protein (std: 8.13) and for 5 samples from 19.5 dpc, the average collagen is 45.48 µg collagen per mg protein (std: 12.89). These results indicate that the collagen amount remains relatively unchanged inside the cervical tissue. However, the collagen fiber network undergoes disorganization (as shown in Fig. 5e–f). The collagen disorganization along with the increased water content of the ripened mouse cervix leads to the lower local concentration of collagen and thus the variation in the collagen signal signature as was detected in Fig. 5(a).
sPA signature of collagen and water depends on both absorption and scattering of the tissue. The variation in tissue scattering can affect the distribution of the light [70] and thus varies the sPA signals. The process of collagen degradation affects the sPA signal through two mechanisms: (a) it reduces the collagen concentration that affects the effective absorption of the collagen, and (b) it affects the scattering properties of the tissue. Thus, the degradation of PA signals in the imaged samples may indicate that, while the tissue scattering due to the collagen network degradation varies with gestational age, the overall amount of collagen contained in the cervical tissue may remain constant but in a depolymerized and/or denatured state. Therefore, we can conclude that while the collagen content (absolute mass amount) remained relatively constant in non-ripe and ripe cervical tissue samples, the increment in water content of the tissue has effectively reduced the “relative concentration” of collagen. This could contribute to the reduction of the signal arising from the collagen in our measurements. Further studies to identify the sensitivity of sPA to the structural change in collagen is a subject of our future planned study. Also, in order to have a comprehensive understanding about the mechanism of sPA signal changes in non-ripe and ripe pregnant murine cervices, further studies will be performed to separate the absorption and scattering contributions obtained in our findings.
The designed sPA method is capable of acquiring co-registered US and PA images in order to localize information about collagen and water content in the target tissue. Therefore, a globalized collagen water estimation matrix can be introduced for further analysis, especially since the collagen:water ratio is one essential factor for evaluating cervical ripening [29]. The combination of two spectral ranges shown in Fig. 1, water dominant and collagen dominant, potentially increase the accuracy of calculating a more robust collagen water matrix and in the water dominant.
The proposed PA imaging can be integrated into a clinical transvaginal probe, enabling seamless integration into clinical use and allowing for real-time, robust, non-invasive estimation of cervical remodeling during pregnancy [71–75]. Given that transvaginal ultrasound (TVUS) is widely used for measuring cervical length, the additional sPA imaging will enable acquiring relevant information about cervical remodeling in a single TVUS exam. Using fast wavelength switching lasers [76], the sPA data can be acquired in a short time, allowing for easy adoption of the method into clinical practice. The low power laser pulses required for imaging shallow tissue (since the transvaginal probe can be placed in close proximity to the cervix and the stroma is only several mm deep from the cervical tissue surface) eliminates the safety concerns associated with using the laser in pregnant women. Further research and development of our technology could include a more advanced analysis of sPA spectra and other mathematical tools to estimate the concentration changes of collagen throughout gestation.
4. Conclusion
We have developed and demonstrated the abilities of a combined US and sPA imaging system and method that measures the cervical tissue optical properties in the near-infrared range and is capable of detecting variations in collagen organization in the murine uterine cervix. With this new technique, we imaged and compared the optical absorption of collagen phantom and water, acquired in the form of sPA signals, and showed distinct spectroscopic PA patterns between collagen and water. In addition, we observed detectable spectral changes that reflect cervical collagen network organization. Moreover, we compared the sPA results with histological tissue analysis of the murine cervix and confirmed the capability of the system to accurately evaluate the process of cervical remodeling. The developed imaging method introduces new possibilities for studying cervical tissue reorganization during pregnancy.
Funding
National Institutes of Health (HHSN275201300006C).
Acknowledgments
This research was supported, in part, by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS); and, in part, with Federal funds from NICHD/NIH/DHHS under Contract No. HHSN275201300006C. N.G-L was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. Dr. Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government. The authors would like to acknowledge George Schwenkel, Jiayin Dong, and Adeel Siddiqui for assisting in animal studies, Yang Jiang and Po-Jen Chiang for assistance in histology, Dr. Valeria Garcia-Flores for assisting in preparing some of the figures, and Derek Miller for proofreading the manuscript.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
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