Cervix sample collection and preparation

The cervical samples were divided into two groups, with eight samples per group, collected after hysterectomy and during cesarean delivery procedures, respectively. For the hysterectomy group, the cervical samples were taken immediately after the removal of the uterus in the operating room. For each individual, a five-cubic-millimeter (mm³) cervical sample was excised from the 12 o’clock position of the cervix. The excised sample was rinsed with phosphate-buffered saline (PBS, Corning^®, 1X without calcium and magnesium, PH 7.4 ± 0.1) to remove blood from the tissue surface before placement into Ambion^® RNALater^™ (Invitrogen Inc.) storage solution at 4°C. During imaging and transportation, samples were kept in RNALater at 4°C. After imaging, samples were embedded into the O.C.T. compound (Tissue-Tec, Sakura Finetek Inc.) and frozen with liquid nitrogen. Several (>2) ten-micrometer-thick sections were sliced using a cryostat and separated into two groups. Samples from one group were stained with hematoxylin and eosin (H&E), and samples from the other group were stained with Sirius Red for further histological analyses.

Cervical samples from women who underwent cesarean delivery were taken from the 12 o’clock position of the cervix after the procedure. Under aseptic conditions, a sterile speculum was placed, and Kevorkian biopsy forceps were used to obtain the cervical sample. The sample was rinsed with PBS to remove blood on the tissue surface before preservation in RNALater. Imaging, transportation, and histology procedures followed the same protocol as the cervix-hysterectomy samples.

Principles of photoacoustic imaging to measure collagen-to-water ratio (CWR)

The proposed bimodal imaging system includes a TVUS probe coupled to a fiber-optic light delivery system surrounding the TVUS transducer for spectroscopic photoacoustic (sPA) imaging. This custom-built light delivery system consists of 19 fibers (NA = 0.39, Thorlabs, Inc., USA). Of the 19 fibers, 18 delivered light around the TVUS probe, and one fiber was used to monitor real-time energy. The fiber-holding sheath is designed to hold the fibers in place. The total diameter of this combined probe is 29 mm, which fits well with clinical requirements. The fibers were bent close to the probe’s tip to enhance the illumination patterns. A Monte-Carlo model simulation to optimize the alignment and bending angles of the fibers was performed to achieve the optimal light delivery for PA imaging [59]. The proximal end of the fiber bundle was coupled to a self-cooled nanosecond, tunable pulsed laser (Phocus Core, OPOTek^® Inc., USA) across a range of wavelengths between 680 nm to 2500 nm at a pulse repetition of 10 Hz. A programmable digital US acquisition system (Vantage 128, Verasonics^®, Inc., USA) acquired US and PA images. A custom-built, high-speed, field-programmable gate array synchronized laser excitation and US acquisition and controlled the interleaved acquisition of US and PA frames. Interleaved US and PA images were reconstructed by using an adaptive beamforming algorithm [60]. Real-time TVUS and PA imaging sequences were developed in which three US frames were acquired between two consecutive PA frames, all synchronization signals were calibrated, and co-registered TVUS/PA images were reconstructed. Additionally, the imaging of a calibration phantom characterized the axial resolution of 260 μm and lateral resolution (at 25 mm depth) of 450 μm [44].

Spectroscopic photoacoustic evaluation of CWR

Spectroscopic PA (sPA) is a widely used PA imaging technology that utilizes laser illumination at different wavelengths to the tissue of interest and probes the object’s optical properties as broadband acoustic pressure waves. To estimate the CWR in cervical tissue, we developed a two-step sPA-CWR measurement algorithm. First, amplification of the PA signal acquired between 1150 and 1250 nm (window 1: collagen domination, where collagen has its peak absorption) detected the relatively small variations [61]. At wavelength (λ) of 1150 to 1250 nm, water absorption remained constant, but collagen absorption had a clear peak [62,63]. Since collagen is still relatively weak compared to water (Fig 1) [61], we used an algorithm (Eq 1) [64] to amplify collagen’s PA.

Fig 1

Optical absorptions of oxyhemoglobin, lipid, collagen, and water.

In the collagen dominating (window #1), water has nearly constant absorption and, by contrast, collagen peaks around 1200 nm. In the water dominating (window #2), water peaks about 1470 nm, and collagen peaks around 1520 and 1540 nm (figure adopted from [61]).

Later, the amplified images were passed through a standard sPA wavelength unmixing method (Eq 2) [41] to calculate the final CWR. This spectroscopic process, performed for each pixel of the image, was guided by the B-mode US to identify the true location of the cervical tissue. The sPA wavelength unmixing method took the average of two pairs of PA data, which were 1150 with 1200 and 1200 with 1250. The higher wavelength features (window 2: water dominating) were not applied in the unmixing method given that water has a strong absorption peak at λ = ~1450 nm. The CWR calculation method may be further enhanced by including sPA data acquired between 1400 and 1600 nm.

Collagen phantoms

Before applying the sPA-CWR method in human samples, we validated the method in collagen phantoms with known concentrations (1.0, 0.75, and 0.5 mg/ml) and blanks (containing only PBS). The collagen phantoms (Fig 2a) were prepared with collagen solutions and then freeze-dried under sterile conditions. The dried powder was resuspended in sterile 1mM HCl solution to form a high-purity, uniform collagen solution. After its preparation, the collagen solution was transferred to an imaging phantom and kept at low temperature (4°C). The collagen solution was then scanned using sPA imaging technology (wavelengths of 1070 to 1650 nm, 10 nm step size) to evaluate absorption behavior. The maximum fluence is 24 mJ/cm² at 1070 nm, which is about 25% of the maximum permissible exposure (MPE) [65]. The CWRs were then calculated by the sPA unmixing method, and the range of detected CWRs was normalized to the highest concentration of the prepared collagen samples (1 mg/ml). The detected results are shown in Fig 2b, which indicated that the proposed sPA wavelength unmixing method was sensitive in its detection of the CWR in controlled collagen samples. Additionally, the concentration of collagen gels used in this study was below the collagen concentration in cervical tissue [15,66–68]. Therefore, the sPA measurement of collagen variations was anticipated to be even more sensitive in in-vivo human studies.

Fig 2

Collagen-to water-ratio in collagen phantoms.

(a) Photograph of collagen phantoms at different concentrations ranging from 0 (PBS) to 1 mg/mL. (b) Normalized sPA-CWR to estimate the collagen concentration for the collagen phantoms at different concentrations. The linear curve fit showed a high correlation between the detected CWR to the collagen concentration, with the coefficient of determination (R2) of 99.66%.

CWR in tissue samples of the human cervix

We performed another study in human cervical samples to indicate the capability of the sPA method to detect the CWR. Wide-spectrum range sPA (from 1150 nm to 1650 nm) imaging was acquired, and the laser energies at different wavelengths were recorded to normalize the sPA signals with respect to energy variations, with the maximum fluence of 20 mJ/cm² at 1150 nm. Although water is generally diffuse in soft tissue, the light beam acts locally. Hence, it produces the gradient required for generating PA signals. RNALater solution was also spectroscopically imaged in the same wavelength range to assure no interference from the background solution. Upon completion of the imaging studies, the tissue samples were washed with PBS, embedded into optimal cutting temperature compound, and underwent snap-freezing using liquid nitrogen, followed by storage at −80°C prior to histologic analyses. There were two types of stains applied to cervix samples: 1) Haemotoxylin and eosin (H&E) to depict edema (increased water content), in which water was visualized as void (uncolored) space [69], and 2) Sirius Red to semi-quantitatively determine the amount of collagen and non-collagen proteins [70]. By utilizing polarized light microscopic imaging, the organized concentric network of collagen can be visualized as a red/yellow rod-like structure.

Cervix samples from the hysterectomy groups were separated in those from women still in reproductive age (<41 years of age), and women at the end of the reproductive age (≥41 years old). We aimed to evaluate if changes in collagen and water content were related only to the effect of pregnancy and not by hormonal changes in the perimenopausal period.

CWR in collagen phantoms

Fig 2 shows the sPA-CWR detection for the collagen phantoms. The collagen concentrations in the collagen phantoms ranged from 0% (PBS) to 1 mg/mL. The detection results were normalized to 1 mg/ml. The linear-fitted curve shows a high correlation (R² = 0.9966) along with the collagen concentration increment. This result demonstrated that the sPA-CWR method is capable of detecting the collagen concentration in the collagen phantoms with high accuracy.

CWR in samples of the human cervix

The age range for pregnant women was 21 to 36 years of age (median, 30 years), whereas it varied from 29 to 58 years (median, 42 years) for the hysterectomy group. In the hysterectomy group, there were four patients under 41 years of age and four patients over the age of 43 years. The demographic characteristics of all patients are detailed in Table 1. Fig 3 shows the averaged sPA amplitude changes across eight cervical tissue samples in each group along wavelengths, histological H&E staining, and Sirius Red-stained images of non-pregnant and pregnant women. From the detected PA signal amplitudes (Fig 3a and 3b), the non-pregnant cervical tissues had a clear peak from collagen absorptions in the collagen-dominating range (1150 to 1250 nm). By contrast, in the water-dominating range (1300 to 1650 nm), the cervical tissues from pregnant women had higher PA amplitude. H&E microscopic images (Fig 3c) of cervix samples taken from non-pregnant humans showed compact collagen bundles with minimal or no intervening edema. When examined with Sirius Red stain, it was evident that the collagen fibers form bundles with distinct fasciculation (Fig 3e). By contrast, the cervical samples from pregnant women showed loose collagen fibers with no discrete bundles and the presence of water (edema) separating the collagen fibers (Fig 3d). The absence of discrete collagen bundles, as well as the loss of the distinct fasciculation seen in samples from non-pregnant women, was evident (Fig 3f). We further quantified the CWR with the sPA wavelength unmixing method for pregnant (18.7%, SD 7.5%) and non-pregnant (55.0%, SD 20.3%) women and demonstrated a significant higher CWR in the non-pregnant group (two samples one-tailed t-test with equal variance, p = 0.00016). Subgroup analysis in pregnant vs. non-pregnant women <41 years of age also demonstrated a significant difference in the CWR between these two groups (pregnant 18.7%, SD 7.5% and non-pregnant ≤ 41 years, 46.3%, SD 23.1%; p = 0.0049) (Fig 4). The detected CWRs for each sample are shown in Table 1.

Fig 3

Averaged PA signal amplitudes and histological images of non-pregnant and pregnant women.

(a, b) Spectroscopic photoacoustic (sPA) imaging signals of cervix samples obtained from pregnant and non-pregnant women (n = 8 for each group). The cervix samples from non-pregnant women showed higher PA signal amplitudes in the collagen absorption peak (around 1200 nm). By contrast, the cervix samples from pregnant women presented higher PA signal amplitudes in water absorption peak (around 1470 nm). (c, d) H&E-stained images indicated the loss of tissue composition (uncolored space) in non-pregnant cervix samples compare to pregnant cervix samples. (e, f) Sirius Red-stained images of non-pregnant and pregnant tissue visualized by polarized light microscope. The latter revealed less packed collagen fibers (red/yellow rod-like structure).

Fig 4

Spectroscopic photoacoustic (sPA) imaging estimated CWR in pregnant and non-pregnant ≤ 41 year-old and in non-pregnant > 41 year-old cervix samples.

The analysis of all cervix samples from non-pregnant and pregnant women showed a significantly lower CWR in the pregnant cervix (at-term). A subgroup analysis comparing pregnant women and non-pregnant women age ≤ 41 years also indicated a significantly lower CWR in the cervix samples from pregnant women.

Table 1

Patient information and collagen-to-water ratio (CWR) [%] estimated by spectroscopic photoacoustic (sPA) imaging.

Sample No	Age (Years)	Ethnicity	Cervical Length [mm]	Gestational Age [Weeks]	BMI	sPA-CWR [%]
Pregnant Cervical Samples
1	29	AA	38.5	39.4	19.6	18.5
2	28	AA	42.0	39.3	12.6	12.3
3	38	AA	28.0	39.0	16.5	15.8
4	28	AA	22.5	40.3	18.6	17.7
5	24	AA	23.7	39.6	39.0	35.8
6	36	AA	23.6	38.0	22.7	21.3
7	32	AA	29.0	39.7	15.9	15.2
8	21	AA	28.6	39.1	13.3	12.9
Non-Pregnant Cervical Samples > 41 years of age
1	45	AA			22.2	55.3
2	50	AA			31.6	74.8
3	58	AA			27.7	77.9
4	43	AA			21.4	47.0
Non-pregnant Cervical Samples ≤ 41 years of age
1	41	Other			30.0	18.6
2	29	AA			37.3	72.9
3	36	Other			32.9	38.9
4	37	AA			27.2	54.7

AA, African American; BMI, Body mass index; Other, Caucasian/Oriental/Hispanic.

Potential clinical application

This study demonstrates, for the first time, the use of PA imaging to evaluate the characteristics of the cervix at term by using human cervical tissue. This emerging modality may serve as a promising ancillary tool to provide real-time, high-resolution molecular images by using non-ionizing radiation and the added advantage of compatibility with an ultrasound imaging system [77]. The acquisition of valuable information of cervical changes occurring at the molecular level during pregnancy by means of a non-invasive approach may be used to increase the predictive accuracy of conventional transvaginal sonography if combined with the PA imaging modality. Photoacoustic imaging could also be used to assess patient response to therapy, which can help to individualize the clinical management with progesterone, cerclage, or pessary in women at risk for spontaneous preterm birth [78–87]. This bimodal imaging method can potentially acquire data for tissue elasticity, blood perfusion, and tissue oxygenation. The single device is capable of measuring anatomical, biomechanical, molecular, and functional characteristics of the cervix and has the potential of optimizing the time for induction of labor.

Strengths and weaknesses

Among its strengths, the proposed imaging system is more advanced than standard TVUS and sonographic evaluation for cervical length; it can supplement the existing, widely used TVUS; and it has a high safety standard, making it reliable to use for pregnant women [88]. We anticipate that the sPA measurements can be performed in 3–5 minutes. The additional requirement of sPA imaging can be easily combined with the current ultrasound system, thus providing a point-of-care diagnostic tool. The whole system, including the laser, control, and ultrasound unit, can be easily fitted in a portable cart of similar size to a clinical ultrasound machine. The device holds the potential for a clinically acceptable routine examination capable of identifying cervical changes during pregnancy and throughout labor induction in a labor and delivery unit. Fig 5 shows the design and physical dimension of a developed prototype with current TVUS transducers.

Fig 5

Transvaginal ultrasound and photoacoustic imaging system for future in-vivo studies.

(a) Computer-assisted 3D design of the fiber-optical delivery system around a clinical transvaginal ultrasound transducer. (b) A photograph of the assembled combined photoacoustic (PA) and transvaginal ultrasound imaging transducer. This prototype can be a point-of-care device for the future clinical diagnoses by characterizing cervix status. The diameter of this prototype is 29 mm, which satisfies clinical requirements.

We noted certain limitations of the study and the PA imaging. The statistical results obtained in this study were limited given the small sample size. Although the sPA-CWRs were estimated in-vitro, it is still feasible to detect cervical changes leading to spontaneous preterm birth at a larger scale in the in-vivo study. The lack of Bishop scores associated with the pregnant cervices may lead to the loss of valuable information. The estimation of sPA-CWR of the whole cervix in PA imaging cannot be evaluated at one scanning. Data should be obtained from different cervical areas to create a global PA cervical score. A higher frequency linear US probe was used to image samples of about five cubic millimeters. By contrast, a TVUS probe set at lower frequency and bandwidth may offer a different sensitivity. The sensitivity may also vary during in-vivo studies. Future studies will focus on patients presenting a torn or insufficient cervix who undergo labor; however, the cervices of cesarean deliveries may not be ripened at the time of the study. Future studies are also required to determine the cost-effectiveness and long-term effects of this modality.