**1. Introduction**

The most common reason for women to seek medical attention is dysuria, and it is believed that in 40% of cases urethritis and/or urethral syndrome are involved [1]. According to the US National Institutes of Health, one third of women with chronic pelvic pain (CPP) have urethral pain syndrome (UPS) [2,3]. The European Association of Urology defines UPS as the occurrence of chronic or recurrent episodic pain lasting for more than 6 months, and felt in the urethra, in the absence of proven infection or other obvious local pathology. It is often associated with negative cognitive, behavioral, sexual or emotional consequences [4], as well as with symptoms suggestive of lower urinary tract, sexual, intestinal, or gynecological dysfunction [5].

The problem of pain in the urethra with unchanged urinalysis, the absence of any other clinical manifestations, and the absence of somatically explainable causes, is complex and ultimately remains unresolved, since the exact pathogenetic mechanisms are not ye<sup>t</sup> fully understood [6–9]. Neither are there any clear recommendations for the prevention and treatment of UPS, as a result of which the only e ffective form of medical care today is symptomatic therapy—involving the continuing intake of strong pain medications, antidepressants, and anticonvulsants [4,9]. In the methodological recommendations on CPP, published under the auspices of the the Moscow Department of Health (dated 14 July 2016), it is noted that there is no specific accepted treatment for UPS [10]. The approach should be interdisciplinary and the treatment should be multimodal, with the general principles of chronic pain syndrome managemen<sup>t</sup> being applied [11–13].

The close embryological relationship between the urethra and the bladder makes it likely that there are causes similar to ones connected with the development of painful bladder syndrome [14]. According to the classification of the International Association for the Study of Pain (IASP, 2019) the mechanism of CPP and possible causes of its occurrence may include vascular lesions, persistent inflammatory processes, or violation of the innervation of organs due to mechanical compression in the pelvic region, but often the reason is not clear [15].

The connective tissue matrix of organs plays a key role in the occurrence and persistence of pain, as shown by the number of studies [16,17]. It is believed that connective tissue, as well as performing its supporting, protective and trophic functions, acts as a network-wide mechanosensitive signaling system—as a global unifying network [16,18].

Thus, it can be surmised that the above reasons for the development of CPP could be associated with factors that a ffect the state of the connective tissue matrix of the lower urinary tract. However, there are currently no methods for adequate, appropriate study of the structure of urethral tissues. According to the standards for examination of patients with CPP when using the UPOINT (Urinary, Psychosocial, Organ Specific, Infection, Neurologic/Systemic, Tenderness of Skeletal Muscles) classification [19] in the urology domain, the recommended list of examinations includes keeping a urination diary, cystoscopy, and the use of ultrasound (US) and uroflowmetry, while for complaints involving the urethra, urethroscopy is recommended. These methods allow only indirect assessment of the urethral tissues. Objective evaluation and accurate diagnosis of a disease that does not cause any visual changes, and results from a "diagnosis of exclusion" when using standard instrumental diagnostic methods, is important for understanding the pathogenetic aspects of the disease. In this work, we used traditional diagnostic methods, including US and uroflowmetry, and the non-traditional methods of ultrasound elastography (USE) and cross-polarization optical tomography (CP OCT) to study changes in the functioning of organs and their structure in UPS in comparison with the norm, and assessed the role of background diseases in the development of UPS.

The USE is a medical imaging modality that measures tissue mechanical properties by monitoring the response of tissue to acoustic energy [20,21]. In clinical settings, USE is emerging as a powerful tool for imaging and quantitatively monitoring cancer and fibrosis [22]. It provides a rapid visualization of the tissue elasticity using color-coding mode, even in organs deep within the body. Recently, USE is applied especially on the breast and liver, but the technique has been increasingly used for other tissues including the thyroid, prostate, lymph nodes, gastrointestinal tract, kidney, spleen, pancreas, and the musculoskeletal and vascular systems [22,23]. There are several USE techniques used in clinical practice, but USE with strain (compression) being the most common one allowing real-time visualization of the elastographic map on the screen [24]. With regard to the study of the urethra in UPS, the method can be useful for detecting fibrous changes in the urethral wall and adjacent tissues.

In general, OCT is similar to the ultrasonic technique, except for using light instead of sound and is centered on interferometry in the near-infrared range of wavelength (700–1300 nm) [25,26]. It measures the time delay and amplitude of backscattered light. The aim of the OCT technology is to perform a real-time, in vivo, optic biopsy, with direct label-free visualization of the histological structure of the human tissues at the level of the general architectonics to a depth of 1.5 mm [27]. High spatial resolution (5–15 μm) and performance simplicity with minimal expertise are the main advantages of OCT in contrast to US. The endoscopic nature of OCT probes not only enhances patient comfort and safety but also makes it especially suitable for assessing narrow tubular organs as well as for using standard guidewires for examining deeply located objects in the body [28].

CP OCT is a functional extension of OCT that enables the detection of changes in the state of polarization of light caused by birefringence and coupling between two polarization states due to scattering in the random media (cross-scattering) [29]. As a result, two types of images are obtained simultaneously: in the initial (co-) polarization and orthogonal (cross-) polarization, which allow assessing isotropic (cells) and anisotropic (collagen and elastic fibers of connective tissue) structures separately [30,31]. This is important in cases when precise observation of only connective tissue structures is needed.

The goal of the study was to assess the condition of the tissue in the female urethra in UPS, by using non-traditional methods for this pathology—compression US and CP OCT.

#### **2. Materials and Methods**

## *2.1. Patients*

In total, 69 female patients were enrolled in this study: 55 with established UPS ("UPS" group, aged from 21 to 66 years) and 14 with a healthy urethra as a control group ("Norm" group, aged from 24 to 62 years). Patients with UPS received treatment in the urology department of the N.A. Semashko Nizhny Novgorod Regional Clinical Hospital between 2014 and 2019.

Inclusion criteria for the UPS group consisted of: age 18 years and older; presence of recurrent episodic pain localized in the urethra lasting more than 6 months; absence of infectious lesion or obvious organ pathology [32]. Exclusion criteria for UPS group were: age under 18; the presence of inflammatory processes in the lower urinary tract; the presence of tumors of the pelvic organs; radiation damage to the pelvic organs; pregnancy; lactation. The control group included women whose age was 18 years and older, with no detected pathology and complaints from the lower urinary tract. Otherwise, people were excluded from the study.

This study was approved by the review board of the Privolzhsky Research Medical University (Protocol #6 from 28 April 2020). The research was carried out within the framework of the RFBR project #19-07-00395, agreements 1236/19 from 11 April 2019 and #1365/20 from 30 March 2020. Informed consent to participate in the study was obtained from the participants. All conducted studies and the number of patients included are presented in Table 1.




**Table 1.** *Cont.*

\* carried out to exclude the presence of interstitial cystitis; 0 patients with stones of the upper urinary tract but without pyelonephritis who have been assigned cystoscopy; # performed in conjunction with cystoscopy.

## *2.2. Transvaginal US*

Transvaginal US (TVUS) was performed using a Philips Epiq5 system (Philips Ultrasound., Inc., 22100 Bothell-Everett Highway, Bothell, Washington, 98021-8431, USA). The sensor was inserted directly into the vagina, allowing visualization of the state of the bladder neck and urethra (assessment of their structure, the condition of their walls, and the width of the internal lumen) and detecting abnormalities in the structure of the urethra compared with the norm. This was also the first study in which patients with UPS underwent compression elastography of the adjacent urethral tissues. Compression elastography is a technique that displays the relative deformation of tissues in the form of their color mapping in real time [33]. When the tissue is subjected to an external force (deformation), the harder/denser areas of the tissue exhibit relatively less compression than the softer areas [24]. In our study, on the USE images, the adjustment scale was set to display the harder areas in blue, with the softer areas appearing in red [34].

#### *2.3. CP OCT Study and Image Analysis*

Time-domain device "Polarization-sensitive optical coherence tomograph OCT-1300U" (BioMedTech LLC, Nizhny Novgorod, Russia) (Figure 1a), that provides two image acquisition in co- and cross-polarizations was used in the study [29,35]. The device is approved for clinical use (product license №FCP 2012/13479 of 30 May 2012) and is equipped with replaceable endoscopic probe (Figure 1b,c). It has the following characteristics: the radiation source is a superluminescent diode, of operating wavelength 1310 nm, spectrum width 100 nm, axial resolution 15 μm, lateral resolution 25 μm, and radiation power at the object 3 mW. OCT image size in each polarization is 1.8 × 1.3 mm (width × height), image acquisition time is 2 s. Due to the presence of a flexible endoscopic probe with an outer diameter of 2.7 mm, the examination of the urethral tissue could be carried out simultaneously with cystoscopy through a standard endoscope. Our group's application of the CP OCT method to the study of the female urethral wall in patients with UPS, is a global "first" [36].

From 4 to 13 images were obtained from each patient: of the bladder neck and three regions of the urethra (Figure 1d) at the 6 oclock position corresponding to a conventional clockface, and, if possible, with other additional images of the urethra in the three directions (9, 12, and 3 h of the clockface).

In the "UPS"/"N" groups, 169/58 CP OCT images were obtained, which included 43/16 CP OCT images of the bladder neck, as the section closest to the urethra and therefore potentially involved in processes occurring in the proximal urethra and 126/42 CP OCT images of the urethra (its proximal 41/14, middle 40/12, and distal 45/16 regions) (Table 2).

**Figure 1.** Cross-polarization optical tomography (CP OCT) device and areas under study shown on a diagram of the female urethra. (**a**) CP OCT device; (**b**) Flexible endoscopic forward-looking CP OCT probe; (**c**) Enlarged tip of the probe from (**b**). (**d**) Drawing of the urethra where it transitions to the bladder. Here, the circles indicate the locations from which CP OCT images were obtained in the proximal (blue), middle (green), and distal parts of the urethra (yellow) [37]. 1—urethra, 2—neck of urinary bladder, 3—triangle of urinary bladder, 4—lacunae and openings of urethral ducts, 5—openings of paraurethral Skene's ducts.


A visual assessment of the CP OCT images of the bladder neck and urethra was performed by two readers. The objects of interest were the epithelium and the state of the connective tissue structures of the urethra in patients with UPS, relative to the normal state of these structures. In the epithelium, the thickness was assessed as: normal, thickening (hyperplasia), or thinning (atrophy); in the connective tissue stroma, attention was paid to the presence of any element in the images corresponding to an inflammatory process or fibrosis. The CP OCT features of inflammation were: (1) lack of clarity of the border between the first (epithelial) and the second (connective tissue) layers, (2) the absence of horizontal ordering of the structures that are representative of the norm, and (3) the presence of any indistinctness in their images, which would correspond to cellular tissue infiltration. Significant thickening of the connective tissue layer, up to the lower border of the image with maintaining a high signal level was considered a sign of fibrosis [36,38]. Before qualitative evaluation of CP OCT images readers were trained by training test. After an independent blind visual assessment of the CP OCT images, the «UPS» group was divided into 2 age subgroups: patients under 50 and those over 50.

#### *2.4. Statistical Analysis*

The statistical analysis was performed using IBM SPSS Statistics software, V20 (IBM Corporation, Somers, NY, USA). The inter-reader reliability was calculated using the Fleiss' kappa (κ) coefficient: κ > 0.8—perfect agreement; 0.7 ≤ κ < 0.8—substantial agreement; κ < 0.7—poor agreement.
