In vivo volumetric visualization and quantification of cumulus expansion in mice with intravital optical coherence tomography

Abstract

Ovulation is preceded by a critical physiological process known as cumulus expansion, during which the cumulus cell layer surrounding the oocyte undergoes structural remodeling. Despite the recognized importance of this process for reproductive success, live quantitative imaging of cumulus expansion has not been previously achieved due to limitations of current imaging technologies for deeply located ovaries. In this study, we employed intravital optical coherence tomography for three-dimensional visualization of mouse follicles containing cumulus-oocyte complexes (COC) within the physiological context of the ovary, both ex vivo and in vivo. This method enabled time-lapse measurement of cumulus layer thickness and COC volume. Longitudinal imaging in live mice revealed the physiological spatiotemporal dynamics of cumulus matrix expansion preceding ovulation. These findings establish a novel in vivo platform for dynamic investigation of previously inaccessible preovulatory processes within a physiological context.

1. Introduction

The ovary contains numerous oocytes housed within follicles. In preovulatory follicles, the oocyte is enveloped by several layers of specialized somatic cells called cumulus cells, which results in the formation of the cumulus-oocyte complex (COC). The cumulus cells not only serve as a physical barrier, but also play a critical role in protecting the oocyte from proteolytic and oxidative stress [1]. Bidirectional communication between somatic cells and the oocyte is essential for oogenesis and directly influences the developmental competence of the resulting gamete [2]. It has been previously shown that cumulus cells regulate meiotic arrest and provide metabolic and nutritional support to the oocyte, thereby ensuring the maturation of a fertilization-ready oocyte capable of sustaining embryonic development [3,4].

A crucial event in the preovulatory phase is the expansion of the cumulus matrix. In response to the luteinizing hormone (LH) surge, the cumulus cell layer undergoes expansion, synthesizing a highly viscoelastic extracellular matrix, primarily composed of hyaluronan. The assembly and expansion of the cumulus matrix are essential for ovulation, oocyte transport, and fertilization [5,6]. Disruptions in cumulus expansion have been linked to subfertility or infertility in various mouse models [7–11], underscoring its clinical significance.

Despite its importance, investigations into cumulus matrix expansion have been largely limited to static, endpoint analyses. Traditionally, assessing cumulus matrix expansion relies on histological techniques such as hematoxylin-eosin staining and immunohistochemistry. Kitasaka et al. conducted hematoxylin-eosin staining of fixed ovaries and quantified the area of COCs in preovulatory follicles [12]. Fülöp et al. performed both hematoxylin staining and immunohistochemistry using hyaluronan-binding protein to visualize the cumulus matrix in the preovulatory follicles [7]. While these approaches offer high-resolution and cumulus-specific visualization, they require dissection and fixation of the tissue, limiting their ability to capture dynamic, spatiotemporal changes within the functional follicle. Development of in vitro culture protocols for isolated COCs enabled numerous studies of cumulus expansion mechanisms. For example, Babayev et al. isolated preovulatory COCs from mouse ovaries, and cultured them in vitro for temporal analysis of the cumulus expansion, including measurements of pre- and post-expansion COC area and cumulus layer thickness [13]. Dunning et al. demonstrated, using both in vivo and in vitro matured mouse COCs, that the expanded cumulus matrix functions as a molecular filter, establishing a unique extracellular environment that regulates metabolite diffusion and retains essential signaling molecules for the oocyte [14]. Carvalho et al. performed immunostaining analyses on in vitro cultured mouse COCs and showed that non-muscle myosin II-dependent cumulus cell migration facilitates cumulus layer expansion and is required for sperm to reach the oocyte [15]. Although these approaches have advanced our understanding of cumulus biology, they fall short of capturing the dynamic, spatiotemporal nature of cumulus expansion within the physiological ovarian environment. Given the physiological complexity of hormonal regulation of ovulation and biomechanical interplay between follicular structures, surrounding ovarian tissues, and other organs, there is a critical need for dynamic imaging technologies that preserve the native tissue environment and enable longitudinal studies. However, due to the inherent trade-off between the imaging depth and resolution in existing modalities, deeply embedded ovarian structures have remained inaccessible at the resolution required to visualize cumulus expansion in vivo, limiting our ability to study its real-time regulation within the native physiological context.

Here, we implemented intravital optical coherence tomography (OCT) to address this limitation. OCT is a noninvasive, label-free, and depth-resolved imaging modality that provides microscale spatial resolution with an imaging depth of 1 to 2 mm [16]. Unlike fluorescence-based methods, OCT relies on intrinsic tissue contrast and does not require exogenous contrast agents, making it well-suited for longitudinal studies in live animals as well as clinical applications. While OCT has been widely adopted in clinical ophthalmology, cardiology, and oncology, its application in reproductive biology is only beginning to gain momentum. We previously established a set of structural and functional OCT-based methods for live volumetric imaging of reproductive processes in female mice. These include intravital imaging of oocyte and embryo transport, as well as sperm migration within the female reproductive tract [17,18]. Such intravital imaging approaches have also enabled us to perform in vivo mapping of ciliary beat frequency and cilia coordination in the mouse fallopian tube [19,20], demonstrating its utility not only for structural imaging but also for dynamic functional analysis in mouse reproductive research [21].

In this study, we advance intravital OCT imaging by presenting an ex vivo and in vivo volumetric imaging approach, that enables high-resolution, label-free visualization and quantitative analysis of cumulus matrix expansion. By integrating semi-automated segmentation and computational image analysis, we performed volumetric measurements of the COC parameters within the ovary. This study establishes a platform for a variety of future studies investigating normal and abnormal follicular or cumulus dynamics, contributing to a more comprehensive understanding and improved management of female infertility, particularly in the context of ovulatory disorders.

2. Materials and methods

2.1. OCT system and imaging

A lab-built spectral domain OCT system utilized in this study was previously described [22]. The system employs the supercontinuum laser (NKT Photonics) using a central wavelength of ∼800 nm and a bandwidth of ∼100 nm. A fiber-based Michaelson interferometer was employed, directing the interference of light from the reference and sample arms to a spectrometer based on a 250 kHz e2 V OctoPlus camera (Teledyne Technologies Inc). Fast Fourier transform was used to obtain the OCT intensity A-line from the equally k-spaced interference fringes. The system provides an A-line rate of up to 250 kHz and has an axial and transverse resolutions of approximately 4 µm. Three-dimensional transverse scanning was performed using a set of galvanometer mirrors (GVS012, Thorlabs Inc), offering high flexibility in adjusting the number of pixels and scanning distance. Various settings were applied to accommodate the size and structural differences in reproductive organ regions.

2.2. Mouse manipulations

The wild-type ICR female mice aged 6 to 21 weeks were used in this study. All animal procedures have been approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine, and all experiments were conducted following the approved guidelines and protocols. A conventional superovulation treatment was used to induce cumulus expansion and maximize the yield of COCs. 3.75 IU equine chorionic gonadotropin with inhibin antiserum (CARD HyperOva, Cosmo Bio) was injected intraperitoneally (i.p.), followed 48 hours later by an i.p. injection of 7.5 IU human chorionic gonadotropin (hCG, MilliporeSigma). The animals were euthanized at 0 or 12 hours after hCG injection. For ex vivo imaging, we dissected the female reproductive tract including ovary, oviduct, and part of the uterus. The freshly extracted reproductive organs were transferred to a 35-mm petri dish containing pre-heated 37°C phosphate buffered saline (PBS)(Sigma-Aldrich). All samples were imaged within 1 hour after the dissection on a pre-heated stage maintained at 37°C. Ex vivo volumetric OCT imaging was performed on the freshly extracted ovary to visualize and quantify the COCs in the preovulatory follicle.

2.3. COCs collection and in vitro brightfield imaging

The animals were euthanized at 0 or 12 hours after hCG injection. COCs were collected from the freshly extracted ovary by puncturing preovulatory follicles with a 26-gauge needle. The isolated COCs were transferred to a 35-mm petri dish containing pre-heated 37°C PBS. In vitro brightfield imaging was conducted using a stereo microscope (Stemi 508, Carl Zeiss) equipped with a high-speed camera (Axiocam 705, Carl Zeiss). All samples were imaged within 1 hour after dissection. The obtained data were exported using a ZEN 2 Blue Edition software (Carl Zeiss).

2.4. Implantation of an imaging window and in vivo imaging

The imaging window was implanted on the right dorsal side of the female mouse for in vivo imaging, as previously described [18,23]. Briefly, female mice were anesthetized with isoflurane and placed on a 37°C heating platform. Ophthalmic ointment (AACE Pharmaceuticals) was applied to prevent eye dehydration during surgery and imaging. The fur on the right dorsal side was shaved, and Nair hair removal cream was used to remove the hair. The depilated skin was swabbed with iodine solution and 70% ethanol. A circular part of skin tissue was removed, and the window was sutured to the edge of the skin through 14 eyelets along the rim using 4–0 nylon suture (Ethicon Inc). A ∼2 mm incision was made in the muscle layer underneath the window to expose the reproductive organs. The ovary, the oviduct, and part of the uterus were gently withdrawn through the muscle incision and stabilized by securing the surrounding fat pad associated with the ovary onto the window tissue holders using a tiny droplet of surgical glue (Covetrus). A 37°C sterile saline solution was added to the exposed tissues to prevent dehydration before closure. The window was then closed with a 12 mm diameter circular cover glass and secured with an O-ring. The window was stabilized with two clamps and slightly lifted to minimize movement artifacts due to animal breathing. In vivo volumetric OCT imaging was conducted at 0, 6, and 12 hours post hCG injection on the mouse under isoflurane anesthesia placed on a heating platform.

2.5. Quantification of cumulus expansion

Volumetric rendering and dynamic visualizations were conducted using Imaris software (Bitplane). The Clipping plane function was used to present the cross-sectional views through the volume. The Measurement Points function was used to measure the oocyte diameter and the thickness of cumulus cell layer. The cumulus thickness was measured at 3, 6, 9, and 12 o'clock positions and averaged as previously described [13].

The coarse 3D volume often includes extraneous structures that are unambiguously not part of the COC. Additionally, the complex often appears in contact with the follicular wall such that there often is no clear delineation of some portion of the complex-wall interface, which confounds thresholding and edge detection. Therefore, a semi-automated segmentation of the complex was tuned to each experiment. The Surfaces function of Imaris was applied to manually segment the entire follicular structure containing the COC. The volume of interest was projected onto 2D, cross-sectional slices spaced 0.95 µm apart along the z-axis of the follicle; these masks define a roughly elliptical region of interest (ROI) in each cross-sectional slice with a stack of several hundred images. The single median gray value of all ROIs in the stack was computed. The bounding box of each ROI was extracted. Each gray value in a box is divided by the stack median and squared; this has the effect of suppressing noise and expanding the grayscale range. The boxes were smoothed via a Gaussian filter (radius = 4) and the grayscale values within clustered via k-means (k = 3); this gives roughly dark (background), intermediate (else), and bright (complex) segmentation, however, some extra-complex structures and imaging artifacts can be included. Standard morphological operations were performed to fill-in and smooth each candidate structure. The area, centroid, mean, and standard deviation of the original images within each candidate structure was computed. Visual inspection of relatively few images (say 5-10%) was found to be sufficient to tag structures as extraneous. Using these labels, a random forest classifier was used to predict whether a candidate structure is more likely complex or extraneous. The outer boundary of each remaining complex mask was saved as a set of points. The result is a stack of boundaries, one per image. A final smoothing operation was performed on each boundary by rejecting points corresponding to large changes in the derivative of the convex hull of the boundary points. As this is a user-tuned parameter, each stack of boundaries was inspected visually and minor, rare errors manually corrected in ImageJ (National Institutes of Health). Once done, the volume of the COC is straightforwardly computed as the total number of bounded voxels times the known voxel volume (2.9 µm³).

2.6. Statistical analysis

Quantitative data are presented as mean ± standard error of the mean. Data analysis was conducted using GraphPad Prism 9. Unpaired Student’s t-test was used to analyze statistical differences between two groups. One-way ANOVA with Tukey’s multiple comparison test was used to assess the significance of differences between more than three groups. Any p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Volumetric visualization of mouse preovulatory follicles

To test the potential of OCT imaging for volumetric, time-lapse investigations of cumulus matrix expansion, we performed ex vivo OCT imaging of the extracted mouse female reproductive tract, including the ovary, oviduct, and part of the uterus, at 0 or 12 hours post hCG injection. A representative 3D OCT reconstruction of the mouse ovary and oviduct is shown in Fig. 1. The OCT field of view covers a large portion of the ovarian tissues (Fig. 1(a) and (f)). Figure 1(b) and (g) shows the top-view cross-sectional images of several preovulatory follicles, each containing a single COC, at 0 and 12 hours post hCG. With the millimeter-scale imaging depth, the COC within the preovulatory follicles was also visualized in the depth-resolved cross-sections at both time points (Fig. 1(c) and (h)). Ex vivo OCT imaging captured the structural features within the follicle, including granulosa cell layers, antrum, cumulus cell layer, and oocyte surrounded by the zona pellucida (Fig. 1(d)-(e) and i-j). Notably, the unexpanded and expanded cumulus matrices were identified at 0 and 12 hours, respectively. These findings demonstrate the capability of the OCT-based approach for studying follicular dynamics within the mouse ovary.

Fig. 1.

Volumetric visualization of mouse preovulatory follicles. (a) Three-dimensional OCT image of extracted mouse ovary and oviduct at 0 hours post hCG injection. Dashed line represents the location for the cross-sectional image in panel (c). (b) Top-cropped OCT image of mouse ovary at 0 hours post hCG. Arrowheads indicate the follicles. (c) Corresponding cross-section of mouse ovary and oviduct. Dashed rectangle represents the location for the enlarged image shown in panel (d). Arrowheads indicate the follicle with a COC. (d-e) Corresponding OCT image showing the follicular structure. Granulosa cells (yellow), antrum (black), cumulus cells (magenta), and oocyte (blue) are manually highlighted. (f) Three-dimensional OCT image of extracted mouse ovary and oviduct at 12 hours post hCG injection. Dashed line represents the location for the cross-sectional image in panel (h). (g) Top-cropped OCT image of mouse ovary at 12 hours post hCG. Arrowheads indicate the follicles. (h) Corresponding cross-section of mouse ovary and oviduct. Dashed rectangle represents the location for the enlarged image shown in panel (i). Arrowheads indicate the follicle with a COC. (i-j) Corresponding OCT image showing the follicular structure. Granulosa cells (yellow), antrum (black), cumulus cells (magenta), and oocyte (blue) are manually highlighted. Scale bars in (a-c) and (f-h) correspond to 500 μm, and scale bars in (d-e) and (i-j) correspond to 200 μm.

3.2. Quantification of cumulus matrix thickness changes

To evaluate the expansion of the cumulus matrix, we performed ex vivo OCT imaging at 0 and 12 hours post hCG injection, and quantified the thickness of the cumulus matrix as well as the oocyte diameter (Fig. 2). The cumulus matrix was dramatically expanded within the follicle at 12 hours post hCG compared to 0 hours post hCG (Fig. 2(a)-(b)). Consistent with this observation, the thickness of the cumulus matrix was significantly greater at 12 hours than 0 hours post hCG, while the oocyte diameter remained unchanged between these two time points (Fig. 2(c)-(d)). Both the thickness of the cumulus matrix and oocyte diameter measured using ex vivo OCT imaging were consistent with those obtained using in vitro brightfield imaging of isolated COCs at each time point, thereby validating our OCT imaging approach (Fig. 2(c)-(f)).

Fig. 2.

Quantification and validation of the thickness of cumulus matrix. (a-b) Ex vivo 3D OCT images of preovulatory follicles at 0 (a) and 12 (b) hours post hCG injection. Magenta and blue lines represent the diameter of the oocyte and the thickness of the cumulus matrix, respectively. Scale bars correspond to 100 μm. (c) Comparison of the oocyte diameter between 0 and 12 hours post hCG and between the two different imaging methods. (d) Comparison of the thickness of the cumulus matrix between 0 and 12 hours post hCG and between the two different imaging methods. The cumulus thickness was measured at 3, 6, 9, and 12 o'clock positions and averaged for comparison. Data are shown as the mean ± standard error with individual data points. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test indicated significant differences (* P < 0.01). n.s.: non-significant. (e-f) In vitro brightfield images of the isolated COCs at 0 (e) and 12 (f) hours post hCG. Magenta and blue lines represent the diameter of the oocyte and the thickness of the cumulus matrix, respectively. Scale bars correspond to 100 μm.

3.3. Volume measurement of the cumulus-oocyte complex ex vivo

To volumetrically investigate the expansion of the cumulus matrix during the preovulatory process, we performed ex vivo OCT imaging at 0 and 12 hours post hCG injection and quantified the volume of the COC within the follicle using semi-automated segmentation. The volume was calculated by combining Gaussian smoothing, k-means clustering, and random forest classification with manual correction to isolate the complex. The final volume was obtained as the sum of segmented voxels, and the segmented structure was rendered as a 3D reconstruction using Imaris software. While the oocyte surrounded by a thin layer of the cumulus cells was observed at 0 hours post hCG (Fig. 3(a)-(b)), the oocyte with an expanded cumulus matrix was observed within the follicle at 12 hours (Fig. 3(c)-(d)). Consistent with this observation, the volume of the COC was significantly greater at 12 hours than 0 hours (Fig. 3(e)). These findings demonstrate that the OCT-based approach is an effective and powerful tool for volumetric quantitative analysis of cumulus expansion within intact ovaries.

Fig. 3.

Volume measurement of the cumulus-oocyte complex ex vivo. (a) Cross-sectional OCT image of the follicle with a COC at 0 hours post hCG injection. (b) Segmented structure of the COC. Oocyte and cumulus matrix are highlighted in magenta and green, respectively. (c) Cross-sectional OCT image of the follicle with a COC at 12 hours post hCG injection. (d) Segmented structure of the COC. Oocyte and cumulus matrix are highlighted in magenta and green, respectively. Scale bars in (a-d) correspond to 200 μm. (e) Comparison of the volume of the COC between 0 and 12 hours post hCG. Data are shown as the mean ± standard error with individual data points. Statistical analysis by unpaired Student’s t-test indicated a significant difference (* P < 0.05).

3.4. Longitudinal quantification of the cumulus expansion in live mice

To visualize and evaluate the expansion of the cumulus matrix in vivo, we conducted survival surgery on female mice to implant an imaging window and performed intravital 3D OCT imaging longitudinally at 0, 6, and 12 hours post hCG injection. In between the imaging sessions, the animal was awake and was anesthetized repeatedly for the imaging. The position of the same follicle under investigation was easily located within the ovary based on overall structural features, including the size and spatial orientation of the target follicle and the relative distribution of neighboring follicles. The oocyte surrounded by cumulus cells, as well as the follicular structures, were clearly visualized in vivo at all stages (Fig. 4(a)-(c)). Longitudinal OCT imaging of the same follicle over time enabled the temporal tracking of structural and volumetric dynamics, highlighting the progressive expansion of the cumulus matrix during the preovulatory period. Temporal quantification demonstrated that the thickness of the cumulus matrix and the volume of the COC increased in a time-dependent manner, while the oocyte diameter remained consistent throughout the preovulatory period (Fig. 4(d)-(f)). These findings demonstrate the feasibility of the presented OCT-based approach for temporal, volumetric, microscale visualization and quantification of cumulus expansion in vivo, which is not achievable with any currently available methods.

Fig. 4.

Visualization and quantification of the cumulus expansion in live mice. (a) In vivo imaging setup with an implanted window on the mouse. (b) Image of the ovary through the intravital window. (c-e) In vivo time-lapse cross-sectional OCT images of the COC within the preovulatory follicle at 0 (c), 6 (d), and 12 (e) hours post hCG injections. Oocyte and cumulus matrix are manually highlighted in blue and magenta, respectively. Scale bars correspond to 200 μm. (f) Temporal change in the oocyte diameter. (g) Temporal change in the thickness of the cumulus matrix. (h) Temporal change in the volume of the COC.

4. Discussion and conclusion

Here, we present an OCT-based imaging approach that enables label-free, depth-resolved, three-dimensional microscale visualization of the oocyte surrounded by the cumulus matrix, as well as the follicular structures. Using this approach, we captured and quantified the expansion of the cumulus matrix within the intact follicle both ex vivo and in vivo, preserving the physiological context of the ovary throughout the imaging process. These findings demonstrate that the spatial resolution of the OCT system (approximately 4 μm in tissue in this study) is sufficient to visualize detailed structural features of the mouse preovulatory follicle, including the multi-layered cumulus cells and oocyte surrounded by the zona pellucida (distinguishable as dark circle separating the oocyte from the cumulus cell mass). The ability to visualize these structural features in three dimensions without the need for exogenous labeling represents a significant advancement over conventional histological methods. To the best of our knowledge, this study presents the first volumetric, quantitative measurement of cumulus matrix expansion in live animals.

OCT is gaining popularity across various biological fields, including reproductive and developmental biology [21,24–26]. However, its application to the female reproductive system, particularly ovarian function, remains limited. This gap underscores the need for OCT-based applications that can address current technical limitations and offer deeper insights into ovarian physiology. The intravital OCT-based approach presented here addresses these challenges by providing several key advantages over traditional techniques: real-time in vivo imaging capability, greater imaging depth, a wide field of view, and not relying on exogenous contrast agents. These features enable the investigation of follicular dynamics with minimal alteration to native physiological environment.

Importantly, OCT uses low-intensity near-infrared light, which is considered to be safe by the U.S Food and Drug Administration and is routinely used in clinical settings, such as ophthalmology and cardiology [27]. Fluks et al. demonstrated optical coherence microscopy scanning to be safe for cultured immature mouse oocytes [28]. While further in-depth studies are needed to investigate the specific effect which this imaging procedure might have on follicular development, the presented data suggest a great promise for prolonged or longitudinal in vivo imaging studies in animal models.

Cumulus cells play multiple roles in oogenesis, ovulation, oocyte transport, and fertilization [2]. Prior to ovulation, the cumulus cell layer must undergo expansion within the follicle, forming a viscoelastic extracellular matrix. However, no existing technology enables noninvasive, real-time visualization and quantification of the resultant volumetric expansion of the cumulus cell layer in vivo. Thus, the spatiotemporal dynamics of cumulus matrix expansion within the follicle remain poorly understood. This study presents the first application of OCT for volumetric, time-lapse visualization of the cumulus matrix surrounding the oocyte within the functional follicle. Using this approach, we tracked structural and volumetric changes in the COC both in extracted ovaries and in live mice, revealing physiological cumulus dynamics preceding ovulation. Notably, our method does not require tissue dissection, fixation, or isolation of COCs, which can compromise structural integrity and disrupt cellular interactions within the follicle. By preserving the native tissue environment, our findings provide not only physiologically relevant insights that were previously inaccessible with existing techniques, but also unique perspectives on the spatial and longitudinal regulation of cumulus matrix expansion.

While OCT offers significant advantages for dynamic imaging of physiological processes, including reproductive processes [21], it is associated with certain limitations. Its label-free imaging capability is beneficial; however, it lacks molecular specificity, which can be achieved through imaging techniques such as fluorescence microscopy, confocal microscopy, or light-sheet microscopy. Integrating OCT with light-sheet microscopy [29] or confocal/multiphoton microscopy could allow for molecular labeling and provide a powerful tool for spatiotemporal tracking of folliculogenesis and oocyte maturation at both cellular and molecular levels. Moreover, combining OCT with fluorescence-based modalities could facilitate functional imaging of live tissues, allowing one to correlate structural and physiological changes with molecular events in real time. Such multimodal approaches would synergistically combine the advantages of each technology, offering comprehensive insights into reproductive physiology from both structural and functional perspectives.

In summary, we employed a volumetric OCT imaging approach, both ex vivo and in vivo, to investigate cumulus matrix expansion in real time at cellular-level resolution with millimeter-scale imaging depth. By integrating this imaging modality with semi-automated segmentation and computational image analysis, we revealed the physiological and spatiotemporal dynamics within preovulatory follicles, which are critical for successful ovulation. Since dysfunctions or disorders related to ovulation are a primary cause of female infertility [30], the OCT-based approach has significant potential for studying the etiology of ovulatory disorders and reproductive failure. Considering that numerous fertility-related molecules have been identified through genetic manipulation in mice [31,32], dynamic imaging under functionally disrupted conditions may help address previously unexplored questions in mammalian reproduction. The combination of advanced imaging techniques and genetic, molecular, or pharmacological approaches could provide valuable insights and inform improved strategies for the treatment of human infertility and other reproductive pathologies.

Funding

National Institutes of Health 10.13039/100000002 ( P41EB031772, R01HD116768, R01HD112102).

Disclosures

The authors declare no conflicts of interest.

Data availability

Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.