Genitourinary syndrome of menopause (GSM) is a condition affecting appro- ximately 50% of postmenopausal women, and to have significant impacts in the sexual sphere [1] [2] [3] . GSM develops as a direct result of reduced ovarian function and circulating hormone levels [4] . The syndrome is characterised by an involution of the genitourinary mucosa and adjacent vulvo-vaginal tissues and by histologically and clinically apparent changes in type I versus type III collagen ratios and in a reduction in both the quantity of elastic fibres and in vascularisation of the genitourinary tissues [5] . Consequently, the diameter of the vagina is reduced and the vaginal epithelium becomes pale and tends to develop infections. Clinically, these changes present as dryness, burning sensation, severe dyspareunia, dysuria and stress urinary incontinence (SUI) [6] [7] . Considering the progressive rise in the age of the general population, many women face this problem for more than one third of their adult life [1] .

A variety of therapeutic options, including topical and systemic hormonal therapies and non-hormonal lubrication provide relief of GSM symptoms [8] . Topical hormonal therapy is the gold standard treatment approach, however, patient compliance remains poor and is contraindicated in patients with a history of breast cancer [9] [10] .

In recent years, a novel therapeutic fractionated CO₂ laser-elicited vaginal mucosa-regenerating treatment option has been introduced, and has demon- strated positive effects on collage nogenesis-driven tissue remodelling. The treat- ment exploits a unique combination of minimal superficial ablation and deeper thermal deposition of the fractionated CO₂ laser energy, delivered via a probe designed specifically for the vaginal anatomy. In response, collagen and elastic fibre remodelling, glycogen synthesis and transudate production are induced, yielding a state typical of the reproduction age. In parallel, the microablative action of the fractionated CO₂ laser stimulates the interaction of heat shock proteins, which activate fibroblasts to produce other components of the extra- cellular matrix, such as proteoglycans, glycosaminoglycans [11] . A fundamental role is played by transforming growth factor-beta (TGF-β), and epidermal growth factor (FGF and EGF), that stimulate angiogenic activity [12] [13] [14] . Overall, the resulting light-induced eutrophication process involves early thermal damage (within 48 - 72 h of treatment), which is followed by a proliferation phase, with production of collagen and extracellular matrix (in the subsequent 30 days). Lastly, the remodelling phase (after 40 days) involves the apposition of mature collagen fibres and new elastic fibres [15] .

The purpose of this study was to evaluate the efficacy of CO₂ laser therapy in treating vulvo-vaginal atrophy (VVA) and mild-to-moderate SUI symptoms in postmenopausal women.

The 33 participating patients presented symptoms of vaginal atrophy only (48.5%, n = 16) or both vaginal atrophy and SUI (51.5%, n = 17). Patients were of a mean age of 52.3 ± 9.9 years, and after an average 7.0 ± 5.8 years from menopause onset. Fifteen of the 33 patients (45.4%) had a history of cancer, with a distribution of 20% ovarian, 27% cervical, 7% breast and 46% endometrial cancer. Patient baseline characteristics are summarized in Table 1.

Progressive improvements in SUI symptoms were noted between the first and last treatment sessions, as manifested by a mean 2.6-point reduction in SUI scores (p-value < 0.05, Table 2), and an increase in VHI scores (p-value < 0.05, Table 2). Improved moisture and objective characteristics of the vaginal mucosa, as expressed by a mean 6-point rise in VHI-S, were recorded as well (Table 2). In parallel, dyspareunia VAS scores significantly declined over the course of treatment and stood at 4.3 at the 3-month follow-up visit (p-value < 0.05 Table 2). Slightly greater improvements in subjective assessments and clinical signs were observed among patients with surgery-induced menopause, as compared to those who underwent physiological menopause (Figures 1-3 and Table 2). No adverse events were reported during the study period. All the patients were satisfied with the procedure, and reported a significant improvement in their quality of life (VAS scale).

Before treatment, the stratified squamous epithelium appeared atrophic and thin, with no evidence of the crests of the Malpighian layer and epithelial papillae, and with a flattened sub-epithelial junction (Figure 4(a), Figure 5(a), Figure 6(a)). Post-treatment histology samples showed restoration of the epithelial and subepithelial structures, characteristic of reproductive-age vaginal mucosa

*Difference between treatment sessions P < 0.05.

(Figure 4(b), Figure 5(b), Figure 6(b)). Immunostaining for collagen III and IV demonstrated a visible post-treatment enhancement. Microscopic examination of the biopsy sections revealed a striking increase in the thickness of epitelium, with resurfacing of the Malpighian layer and epithelial papillae. The median improvement from baseline in epithelium thickness was 97% (range: 9% - 203%).

The first use of CO₂ laser in gynaecologic surgery was reported in 1973 [17] . The technique involves transformation of light energy into heat, which then causes

evaporation of water from within the target cells. The fractionated/pixel CO₂ laser treatment used in this study triggers superficial thermo-ablation, and deep thermal cell activation to stimulate tissue remodelling. The process involves a small family of proteins, known as heat shock proteins (HSP), which is activated, [18] [19] leading to a change in cell metabolism and selective induction of ex- pression of certain receptors on the surface of the cells of the treated tissue [20] [21] . Among these, sub-types 43, 47 and 70 are overexpressed, and act as chaperones to collagen, and also play a predominant role in the induction of growth factors, such as TGF-β, a key cytokine in the inflammatory and fibrogenetic processes that produce collagen and extracellular matrix [22] [23] . This cascade of events stimulates restoration of metabolic trophism of the vaginal mucosa, which improves its elasticity and moisture, and provides relief from discomfort in menopausal women. The present findings demonstrated that fractional/pixel CO₂ laser treatment stimulated tissue remodelling, neocollageno genesis and reconstruction of the trabecular architecture typical of collagen. The thickened epithelial papillae, with enhanced production of glycogen, contributed to improved moisture and objective characteristics of the vaginal mucosa, as expressed in improved VHI-S and histologically evident qualitative and quantitative changes

in vaginal tissue. Overall, the treatment regimen provided effective relief from the predominant GSM symptoms, improved vaginal health and improved control of urination. Future urodynamic studies will be necessary to better quantify these improvements.

The laser therapy applied here induced neo-collagenogenesis, up regulated collagen type 3 expression, and increased epithelial papillae thickness, but due to limitations of the technique the degree of up regulation was not quantifiable. In addition. In addition, a significant enhancement of type III collagen production was observed in the lamina propria following treatment, likely to underlie its improved function, as well as the re-establishment of the physiological vaginal pH, probably due to the microbial lactobacillus flora (Figure 7 Figure 8). No side effects were observed and the procedure was well tolerated and did not require any local or systemic pharmacological or behavioural treatments. Of note, the ideal safety profile reported here can most likely be attributed to the stringent application of the study’s inclusion and exclusion criteria.

Women with surgically-induced menopause benefited from higher treatment efficacy, as compared to those with physiological menopause. The average patient age in the two subcohorts was significantly different, where patients with surgically-induced menopause were a mean 5 years younger than those with physiologic menopause, and the time from menopause was almost 4 years shor- ter compared to those who had experienced physiological menopause (Table 2). This interesting finding suggests that the rejuvenating prospects of fractional CO₂ laser therapy on the vaginal wall increase with proximity to onset of meno- pause.

These preliminary results warrant further assessments in a larger sample size and over a longer follow-up period, later to be followed by validation in randomised studies. These studies must consider the longevity of both subjective and objective improvements, as well as the need for maintenance treatments.

The fractional/pixel CO₂ laser is a minimally invasive precision tool, which provides for a novel, outpatient approach to treat GSM symptoms. The approach is bound to pave the way toward a new, safe and efficacious therapeutic modality for rejuvenation of the vaginal mucosa and improved urinary control. This approach provides particular hope for oncology patients with treatment-induced menopause and subsequent genitourinary atrophy and SUI, for whom hormonal therapy is contraindicated, as well as for women who are mutation carriers (e.g., BRCA1-2 mutations) who suffer from GSM or SUI, for whom hormonal therapy is contraindicated. Further scientific data will be necessary to validate the therapeutic efficacy of the presented laser therapy for genitourinary atrophy, SUI and sexual-dysfunction-related symptoms. Moreover, longer follow-up periods will establish the duration of the treatment effect.

Pagano, I., Gieri, S., Nocera, F., Scibilia, G., Fraggetta, F., Galia, A., Gioè, M., Pecorino, B. and Scollo, P. (2017) Evaluation of the CO₂ Laser Therapy on Vulvo-Vaginal Atrophy (VVA) in Oncological Patients: Preliminary Results. Journal of Cancer Therapy, 8, 452- 463. https://doi.org/10.4236/jct.2017.85039