1. Introduction
Pelvic organ prolapse (POP) is a condition when organs inside the pelvis drop due to weakening of the supporting muscles and tissues of the vaginal wall. The prevalence of POP increases with age, affects the quality of life of the patients and can lead to social problems [
1]. The cause of prolapse is multifactorial but is primarily associated with pregnancy and vaginal delivery, which both lead to direct pelvic floor muscle and connective tissue/collagen injury [
2,
3,
4]. Epidemiological data also point to a genetic predisposition (polymorphisms of collagen-related and extracellular matrix-remodeling genes) and, therefore, the need for early detection of POP and timely treatment [
5].
Normal pelvic organ support is provided by the interaction between the levator ani muscles and the connective tissues that attach the uterus and vagina to the pelvic side walls [
6]. In the case of dysfunction of the supporting apparatus, the vaginal wall is subjected to pressure from the other pelvic organs, which leads to prolapse of the anterior, posterior or apical segment of the vagina. While pelvic floor muscle insufficiency is usually associated with vaginal birth and injury to the levator ani muscles [
6,
7], connective tissue insufficiency is explained by changes in the content, structure, biomechanics and catabolism of collagen [
8,
9]. It is collagen, organized into fibers and bundles, that normally provides tensile strength to tissues. It has previously been demonstrated that vaginal wall thickness decreases in women with prolapse, and this may be due to changes in the collagen bundles [
10]. Histological changes in pelvic floor tissue showed that collagen fibrils lost their native well-organized, tightly packed morphology and acquired loose, disorderly and discontinuous structures [
11]. However, research into changes in collagen bundles in the tissue of the vaginal wall is limited and controversial [
12]. We suggest that studies using new high-resolution optical techniques to assess collagen bundle changes in the vaginal wall would be useful.
Further issues requiring discussion are the treatment options for POP. The vaginal wall is the component of the pelvic support system that is targeted for treatment in the early stages of POP. Traditional treatments include observation, physical therapy for the pelvic floor, vaginal pessaries and surgery, but these approaches often do not provide entirely satisfactory results and can even cause urological problems [
13,
14,
15,
16]. Currently, the alternative to such traditional treatments for patients with early-stage POP is provided by various laser technologies (ablative and non-ablative laser exposure), these being highly effective and minimally invasive during treatment [
17,
18,
19,
20,
21]. However, laser treatment does not provide complete recovery, but it enables strengthening (tissue compaction) of the vaginal wall by remodeling the collagen framework to improve the results of further surgical treatment.
Intravaginal neodymium laser (Nd:YAG) with a wavelength of 1064 nm provides innovative, minimally-invasive non-ablative treatment of early-stage vaginal wall prolapse. The advantage of this approach compared to other laser technologies is that the treatment uses a photothermal effect. Laser radiation is absorbed predominantly by the deoxyhemoglobin and oxyhemoglobin of the erythrocytes in the microvasculature, causing a controlled increase in the temperature of the connective tissue of the vaginal wall and activating overexpression of heat shock proteins [
22,
23,
24]. The result of such thermal exposure is the remodeling of collagen bundles in addition to activation of angiogenesis and collagenogenesis [
25], which leads to an increase in the density of the connective tissue and, accordingly, the supporting function of the vaginal wall [
26]. In addition, due to the low absorption by the main components of the tissue (water and collagen), the effect is carried out over the entire depth of the mucosa (several millimeters), therefore including the lamina propria, whereas classical ablation lasers create micro-damage in the surface layer to the depth of only a few microns [
27].
A number of studies have demonstrated the initial results of the clinical efficacy of Nd:YAG lasers in gynecology [
28,
29,
30]. The advantages of this type of laser are the non-invasive exposure, the absence of complications and the reduction of the rehabilitation period to 1 week from 1 month following ablative laser exposure. At the same time, however, the lack of an objective method for monitoring the efficiency of Nd:YAG laser treatment has so far prevented its wide clinical application. It is necessary to use new high-resolution and non-invasive methods for visualization of the microstructure of the vaginal wall in order to obtain objective assessments of pathological changes in the lamina propria connective tissue before and after laser treatment without the need to take biopsies.
Optical coherence tomography (OCT) is a high-resolution (5–15 μm) optical imaging technique that can be clinically used for noninvasive assessment of the cervical and vaginal mucosa to an image depth of 1.5 mm—approaching the biopsy depth for histological examination and the corresponding depth of neodymium laser exposure. In earlier studies, conventional OCT has been successfully used to quickly identify vaginal epithelial damage [
31], for monitoring its treatment with nonoxynol-9 vaginal gel [
32] and for analysis of human ovarian tissue [
33,
34]. The first encouraging results of using OCT in vivo for tracking vaginal tissue changes after treatment with fractional-pixel CO
2 laser therapy for genitourinary syndrome of post-menopausal women have also been obtained [
35,
36]. The emerging OCT modalities, such as polarization-sensitive (PS) OCT, OCT-angiography and OCT-elastography, as well as endoscopic and handheld scanning probes, significantly extend the prospects and possibilities of the application of OCT techniques to various types of tissues and organs [
37,
38,
39]. More recently, it has been shown that compression optical coherence elastography (C-OCE) can provide a robust method for the detection of the early stages of vaginal wall prolapse based on significant decreases in tissue stiffness of the vaginal wall and can be used for assessing the increase in stiffness of the wall after laser treatment [
30].
PS OCT mode allows qualitative and quantitative assessment of the collagen fibers’ state (optically anisotropic structures) in different types of biological tissue using birefringence properties. Birefringence is a phase retardation change with depth which characterizes orientation and spatial collagen fiber organization [
37]. Cross-polarization OCT (CP OCT) is a variant of PS OCT that allows imaging of collagen fibers state not only due to its birefringence but also cross-scattering properties. CP OCT devices have two channels to register the backscattered light: co-channel for waves which retained the initial polarization state and cross-channel for waves with a changed polarization state to the orthogonal one. It has been shown that OCT signal intensity in cross-polarization channels depends on the thickness, orientation and density arrangement of collagen fibers [
40]. The attenuation coefficient of the OCT signal is commonly used to quantify the CP OCT data. It helps to improve the assessment of the collagen state, for example, in non-tumorous and tumorous tissues [
41], in the identification of an early stage of vulvar lichen sclerosus [
42], and in the detection of areas of inflammation vs. norm and necrosis [
43]. More recently, it became possible to build depth-resolved attenuation maps in the B-scan projection for the earlier detection of ovarian and fallopian tube malignancy [
44]. It has been shown that depth-resolved attenuation map** of the structural B-scan helps better visualization of the borders between the epithelium and the lamina propria.
The purpose of the current study was to assess connective tissue changes in the vaginal wall under prolapse and after treatment with a neodymium (Nd:YAG) laser, using CP OCT with depth-resolved attenuation map**. To the best of our knowledge, this study is the first to demonstrate B-scan attenuation maps of human vaginal wall tissue in various conditions in the co- and cross-polarization channels. In future, this technique might also be useful for in vivo detection of early stages of vaginal wall prolapse and assessment of the efficacy of laser treatments.
4. Discussion
In this paper, for the first time, we calculated the attenuation coefficient from CP OCT images of human vaginal wall tissue in different states: age norm, stage I–II prolapse and stage I–II prolapse after Nd:YAG laser treatment. Attenuation coefficient calculation from OCT data allowed us to achieve improved biological structure visualization and objectively differentiate between tissues of different morphology [
41,
42,
43,
44]. In the present study, the depth-resolved method for attenuation coefficient was used for the first time to evaluate connective tissue changes in the vaginal wall. As has been shown in [
49], the depth-resolved method for attenuation coefficient calculations can identify additional structural features. Unlike more commonly used methods based on the linear fit of the logarithm of the signal, the method utilized in the study avoids axial resolution deterioration from the selection of the fitting range, decreases the variance of the attenuation coefficient estimation of the optically uniform specimen and avoids biases from the poor choice of fitting range in the case of the layered object. For the first time, cross-sectional (B-scan) color-coded maps to represent attenuation coefficient value distributions have been built for two polarization channels for the vaginal wall. The construction of B-scan attenuation maps is most applicable for the visualization of layered biological tissues, including the vaginal wall. In our study, this allowed us to clearly distinguish the epithelium from the underlying lamina propria and assess changes in collagen bundles in the latter layer. Previously, attenuation map** has predominantly been used in the en face plane, which is preferable for non-layered structures, such as, for example, tumors or inflammations [
41,
43].
In our previous paper [
30] describing quantification of changes in the state of collagen bundles localized in the laminae propria of the same studied groups, C-OCE methods and quantitative morphological analysis of the collagen bundles (local thickness, uniformity and orientation) were used. In the case of vaginal wall prolapse, we observed a statistically significant decrease in tissue stiffness and its increase after Nd:YAG laser treatment. The results of quantitative morphological analysis also showed a decrease in the local thickness of collagen bundles and the uniformity of their arrangement during stage I–II prolapse. However, after Nd:YAG laser treatment, an increase in the local thickness of the collagen bundles, a change in their orientation and an increase in the uniformity of their arrangement were demonstrated. In the current study, we did not carry out morphometry; however, we visually observed similar processes in the connective tissue: a reduced density and thinning of collagen bundles during prolapse, an increase in the thickness of the collagen bundles and an increase in their density after laser treatment. It should be noted that CP OCT, in comparison with C-OCE, has greater potential to be used for further, more rapid derivation of 2D or 3D images of the vaginal wall tissue in vivo with higher resolution (~10–15 µm) without additional compression of the tissue and using a reference layer. In addition, compared to our previous C-OCE study [
30], the number of study samples was increased, which confirmed our results on the nature of the changes in the collagen bundles in vaginal wall prolapse without and after laser treatment. Our study is also consistent with studies using electron microscopy, in which it was observed that collagen fibrils lost their normal parallel structures in the affected vaginal tissue and also had large gaps between them [
52,
53].
In this study, we have demonstrated, for the first time, specific changes in the collagen bundles of the vaginal wall connective tissue are associated with the decrease in its scattering and polarization properties. The level and nature of cross-polarization backscattering in the cross-channel on OCT images and, accordingly, the attenuation coefficient of the OCT signal in the cross-channel mainly depended on the number, orientation and density of collagen bundles in the lamina propria of the vaginal wall. This is an important factor in the development of the early stages of vaginal wall prolapse and in assessing the restoration of the mechanical properties of the vaginal wall after laser treatment. Using CP OCT, it is possible to acquire a clear distinction between areas with loosely arranged collagen bundles and densely arranged ones based on the ability of collagen bundles to cross-polarization backscatter. Two parameters within the lamina propria were targeted and calculated: the median value and the percentages of high (>4 mm
−1) and low (<4 mm
−1) attenuation coefficient values. In this case, all tissue structures that do not generate an OCT signal (mainly lymphatic vessels or spaces between collagen bundles) or generate a weak signal (thin collagen bundles) fell into the category of structures with an attenuation coefficient value of <4 mm
−1. The median values of the Att(co) and Att(cross) coefficients in the lamina propria were significantly (
p < 0.0001) lower in the stage I–II prolapse compared with those in the age norm (
Figure 3). This was associated with thinner collagen bundles and large slit-like spaces between them in the case of prolapse of the vaginal wall. After the laser treatment, significantly higher median values of the Att(co) and Att(cross) coefficients were noted, compared to the vaginal wall prolapse without treatment (
p < 0.0001). This was associated with an increase in the local thickness of the collagen bundles, changes in their orientation and an increase in the uniformity of their arrangement, leading to more noticeable polarization effects. The difference in the spatial localization of the high and low regions in the attenuation maps in the cross-channel for the age norm and the stage I–II prolapse, both without and after laser treatment, can additionally be useful for differentiation between these three conditions. It was shown that when calculating the percentage of high (≥4 mm
−1) and low (<4 mm
−1) attenuation coefficient values (
Figure 4), the Att(cross) coefficient better reflects the presence of the densely arranged and thickened collagen bundles and distinguishes them from areas occupied by loosely arranged collagen bundles or dilated lymphatic vessels. Significantly lower values were found for stage I–II prolapse compared to the age norm (24% vs. 55%), with a return to normal values after Nd:YAG laser treatment (58%). This new knowledge—the significant difference between stage I–II prolapse and the age norm and stage I–II prolapse after Nd:YAG laser treatment—holds promise for using CP OCT both to diagnose early stages of vaginal wall prolapse and to enable quantitative monitoring of the changes in the lamina propria of the vaginal wall after Nd:YAG laser treatment.
These results are consistent with the results of earlier studies involving calculations of the attenuation coefficients of OCT signal in the tissues of the pelvic organs. In particular, a decrease in the attenuation coefficient for cancerous ovaries when compared with normal or benign ovaries has been shown [
44] that could have similarly originated from the remodeling of collagen fibers during the progression of such ovarian cancers [
54]. Using CP OCT technology, the quantitative parameters of the attenuation coefficients for dermal lesions in vulvar lichen sclerosus have also been established, which makes it possible to detect the disease in the case of an early lesion based on a decrease in the values of the attenuation coefficients due to decreases in the thickness of collagen bundles as well as the presence of dermal edema [
42]. Previously, our group has also demonstrated the use of CP OCT to visualize the contours of lymphatic vessels more clearly using depth-resolved attenuation map** in vulvar lichen sclerosus [
49]. In this study, while clear contours of the lymphatic vessels were also well visualized in the attenuation maps of the vaginal wall, we found no significant difference in the percentage areas involved between stage I–II prolapse and the age norm and stage I–II prolapse after Nd:YAG laser treatment.
In this paper, it is also worth noting the positive dynamics of changes in the condition of the vaginal mucosa after neodymium non-ablative laser treatment, which may, in the near future, compete with treatments traditionally used in the clinic today, as well as with microablative laser therapy approaches [
17,
18,
19] for treating gynecological and urological problems. At the same time, CP OCT with depth-resolved attenuation map** may provide an objective method for monitoring the efficiency of Nd:YAG laser treatment of the early stages of vaginal wall prolapse since the depth of the OCT imaging (~2 mm) corresponds to the depth of the laser exposure. It has been shown that the nature of the remodeling of the vaginal wall tissues after Nd:YAG laser exposure can be determined by an increase in the quantitative parameters of the attenuation coefficients in the area of the lamina propria, thus indicating an intensification of the regenerative reactions of the tissue. Certainly, further accumulation of CP OCT data and rigorous statistical analysis are required to verify the diagnostic value of the encouraging results presented above, based on our examination of a set of 26 samples comprising three conditions of the vaginal wall. However, even these data demonstrate that, in addition to the conventionally discussed mean attenuation values, analysis of the CP OCT-based spatial localization of high and low values of the attenuation coefficient regions opens very promising prospects for distinguishing between effective and ineffective laser treatment.
In the future, we anticipate that the CP OCT can be a robust method for detecting early stages of vaginal wall prolapse in vivo based on reducing the attenuation coefficient values in the cross-channel. In addition, the use of CP OCT will allow us to conduct dynamic monitoring of the effectiveness of Nd:YAG laser treatment of prolapse and to identify patients who can undergo repeated laser therapy courses to prevent a relapse of POP. However, it should be taken into account that during CP OCT examination of the vaginal wall in vivo, the values of attenuation coefficients may differ slightly (be lower) from the values obtained during ex vivo examination. We believe that the main reason for this difference is the fullness of blood and lymphatic vessels in living tissue. The presence of large dilated lymphatic vessels in the lamina propria, which have a signal at the noise level when calculating the attenuation coefficient within this layer, leads to a decrease in its average value in in vivo calculations compared to ex vivo tissue, where the contribution of low values from collapsed lymphatic vessels to the overall signal is not that significant. As for blood vessels, in vivo, they are characterized by a high level of co-channel backscatter, comparable to surrounding tissue (therefore, they are indistinguishable from other structures in structural OCT images), and the absence of cross-channel scattering, which reduces the average attenuation coefficient values in the cross-channel. In ex vivo tissue imaging, blood vessels have narrowed lumens, which virtually excludes these areas from the calculations and leads to an increase in the attenuation coefficient in the cross-channel, with almost no effect on the signal in the co-channel. Unlike vessels, collagen fibers do not significantly change their scattering properties before or after tissue excision. Therefore, we demonstrate the ability of the CP OCT method to assess the number and organization of collagen bundles in this pathology and the ability to monitor changes in their number and location after laser treatment. In the future, we plan to compare CP OCT data of the vaginal wall in in vivo and ex vivo studies.
One limitation of this study is that some of the early stage prolapse of the vaginal wall are focal; thus, the selected OCT B-scans in these cases may represent a mixture of the diseased and normal tissues. Moreover, given the likelihood of heterogeneous areas of connective tissue remodeling in the early stages after restoration of the anisotropic properties, at later dates, such differences will probably not be so noticeable. Therefore, in the early stages of monitoring the effectiveness of treatment in the future, it will be more appropriate to use an intravaginal OCT probe [
36], which could enable examination of several different sections of the vaginal wall tissue and accordingly exclude the need for invasive (punch or excision biopsy) manipulations in such patients. Current efforts are underway for the construction and implementation of a circumferential-scanning intravaginal CP OCT probe for in vivo imaging of the vaginal wall.
In summary, the present study provides a new diagnostic approach both to non-invasive early detection of prolapse and to monitoring the efficiency of Nd:YAG laser treatment based on the assessment of the attenuation coefficients calculated from real-time CP OCT data. The results obtained in this study have shown several advantages of using attenuation coefficient maps in the cross-channel over conventional log-scale OCT images and attenuation coefficient maps in the co-channel. Firstly, they better reflect the true state of the lamina propria connective tissue. Secondly, the contours of the abundant low-attenuating slit-like structures (lymphatic vessels) located in the lamina propria are more clearly represented. Thirdly, the values of the Att(cross) coefficient in the tissues can themselves be used to provide an objective diagnostic parameter for the condition of the connective tissue.