Treatment of Genitourinary Syndrome of Menopause with Microablative CO₂ Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol

Treatment of Genitourinary Syndrome of Menopause with Microablative CO₂ Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Treatment of Genitourinary Syndrome of Menopause with Microablative CO₂ Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol Cassia Caroline Garcia Dalbem Teles, Catarina Maria Mesquita Garcia Dalbem, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6699446/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Genitourinary Syndrome of Menopause (GSM) affects a significant number of postmenopausal women, leading to symptoms such as vaginal atrophy, dryness, and discomfort. Hormonal therapies are not suitable for all patients, particularly those with contraindications to hormone replacement therapy. This study aimed to compare the efficacy and safety of two non-hormonal treatments—microablative CO₂ laser and fractional microablative radiofrequency—in the management of GSM. Methods: This is a double-blind, randomized, non-inferiority clinical trial involving 40 postmenopausal women diagnosed with GSM. Participants will be randomized to receive either microablative CO₂ laser or fractional microablative radiofrequency in three monthly sessions. The primary outcome is the Vaginal Health Index Score (VHIS), which assesses elasticity, pH, lubrication, epithelial integrity, and mucosal condition. Secondary outcomes include sexual function (Female Sexual Function Index - FSFI), GSM symptom severity, and urinary symptoms. Follow-up assessments will occur at 3, 6, and 12 months after the intervention. Histological analysis will be performed on vaginal biopsies from a subset of participants. The study was approved by the Research Ethics Committee of Universidade Brasil (approval no. 5.357.602), and all participants will provide informed consent prior to enrollment. Discussion: The findings from this study contribute to the comparative understanding of the effectiveness of CO₂ laser and fractional microablative radiofrequency as non-hormonal treatment options for GSM. The results have the potential to influence clinical practice by providing safe and effective therapeutic alternatives for women who are not candidates for hormonal therapy. Trial registration: This trial is registered in the Brazilian Clinical Trials Registry (ReBEC): RBR-2hgwvgy. Registered on July 2023. UTN: U1111-1282-9195. https://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy . This is a retrospectively registered trial. Obstetrics & Gynecology Genitourinary syndrome of menopause CO₂ laser fractional microablative radiofrequency vaginal atrophy non-hormonal therapy Vaginal Health Index Score clinical trial Figures Figure 1 Administrative Information Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/). Title {1} Treatment of Genitourinary Syndrome of Menopause with Microablative CO2 Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol Trial registration {2a and 2b}. This clinical trial was registered in the Brazilian Clinical Trials Registry (ReBEC): RBR-2hgwvgy https://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy UTN code: U1111-1282-9195 National registration on Plataforma Brasil (CAAE): 55003622.8.0000.5494 Research Ethics Committee approval: 5.357.602 (University Brasil Research Ethics Committee) The trial was retrospectively registered in July 2023. Although ethics approval was obtained on April 19, 2022, and screening began in September 2022, clinical interventions and data collection only started in March 2023. At the time, the research team was not fully aware of the need for prospective public registration as a publication requirement. The protocol was prospectively developed and strictly followed. The principal investigator remains blinded to group allocation, and no statistical analysis has been performed to date. This is the first formal submission of the full protocol. Protocol version {3} Version 1.0, Date: 22/04/2025 Funding {4} This study has not received external funding. The devices used in the interventions (laser and radiofrequency equipment) were provided by the principal investigator. Logistical support — including clinical infrastructure, human resources, and technical assistance for the procedures — was provided by the Regional Hospital de Cáceres Dr. Antônio Fontes. Author details {5a} Cássia Caroline Garcia Dalbem Teles, Universidade do Estado de Mato Grosso and Universidade Brasil. Physician. Principal investigator. Responsible for study conception, protocol development, and clinical assessments prior to the interventions. Nivaldo Antonio Parizotto, Universidade Brasil PhD. Academic supervisor. Contributed to the methodological design and generated the randomization sequence. Matheus Rodrigues de Souza, Universidade do Estado de Mato Grosso Physician. Member of the Data Monitoring Committee (DMC). Monitored clinical data integrity and safety throughout the study. Catarina Maria Mesquita Garcia Dalbem, Universidade do Estado de Mato Grosso and Universidade Brasil. Physician. Performed the clinical interventions using CO₂ laser and radiofrequency. Did not participate in data collection or outcome analysis. Marcos Araújo Chaves Júnior, Universidade do Estado de Mato Grosso Pathologist. Conducted histological analyses of vaginal biopsies collected during the study. Gabriela Fonseca Arenhart, Guilherme Bezerra, Leticia Andrioli da Cunha, Rayanni Freire Alves Pedroso, Viviane Ribeiro, Universidade do Estado de Mato Grosso Medical students. Provided technical research support, organized study data, and maintained participant contact during the trial. Name and contact information for the trial sponsor {5b} Primary Sponsor: Cássia Caroline Garcia Dalbem Teles Contact: [email protected] Role of sponsor {5c} The principal investigator, Cássia Caroline Garcia Dalbem Teles, is a permanent faculty member at Universidade do Estado de Mato Grosso (UNEMAT), where she conducts academic and clinical activities. She is also a doctoral student at Universidade Brasil, the academic institution responsible for the ethical oversight and doctoral supervision of this study. The principal investigator conceived and conducted the study and provided the equipment used in the interventions (microablative CO₂ laser and fractional microablative radiofrequency), which were her personal property. Hospital Regional de Cáceres Dr. Antônio Fontes provided logistical support, clinical infrastructure, and technical staff for the procedures. None of the institutions involved — Universidade do Estado de Mato Grosso (UNEMAT), Universidade Brasil, and Hospital Regional de Cáceres Dr. Antônio Fontes — participated in the study design, data collection, data analysis, interpretation of results, manuscript writing, or the decision to submit this manuscript for publication. All scientific and methodological decisions were the sole responsibility of the principal investigator and the study team. Introduction Background and rationale {6a} Genitourinary syndrome of menopause (GSM) is a common and debilitating condition affecting a significant proportion of postmenopausal women. It manifests through symptoms such as vaginal dryness, irritation, dyspareunia, and urinary complaints, negatively impacting quality of life and female sexual function (1). Although local estrogen therapy is considered the standard treatment, not all patients are willing or eligible to use it, especially those with contraindications to hormonal therapy. This has led to the search for non-hormonal alternatives. Among these alternatives, fractional CO₂ laser therapy is considered promising. Initial clinical studies report significant improvement in vulvovaginal atrophy symptoms following treatment, with a favorable safety profile (2–4). Similarly, microablative radiofrequency is being investigated as a non-hormonal intervention, and preliminary findings suggest relevant clinical benefits in alleviating genitourinary symptoms (5). Despite these findings, most available studies present important methodological limitations, such as lack of randomization, control groups, or long-term follow-up. Therefore, well-designed controlled clinical trials are considered necessary to strengthen the evidence base regarding the efficacy and safety of these therapies. Objectives {7} Primary objective: To evaluate the non-inferiority of fractional microablative radiofrequency compared to CO₂ laser in the treatment of Genitourinary Syndrome of Menopause (GSM), using the Vaginal Health Index Score (VHIS) as the primary outcome, as outlined in a double-blind, randomized, non-inferiority trial. Hypotheses: Null hypothesis (H₀): Fractional microablative radiofrequency is inferior to CO₂ laser in improving vaginal health (VHIS). Alternative hypothesis (H₁): Fractional microablative radiofrequency is not inferior to CO₂ laser in improving vaginal health (VHIS). Trial design {8} This study was a randomized, controlled, double-blind, non-inferiority clinical trial, conducted at a single center. A total of 36 postmenopausal women were included and were randomized to receive three monthly sessions of either microablative CO₂ laser or fractional microablative radiofrequency. Participants were allocated to the intervention groups in a 1:1 ratio. Methods: Participants, interventions and outcomes This protocol has been written in accordance with the SPIRIT 2013 guidelines. Study setting {9} The study was conducted at Hospital Regional de Cáceres Dr. Antônio Fontes, located in the city of Cáceres, Mato Grosso, Brazil. This is a public tertiary-level hospital affiliated with the Brazilian Unified Health System (SUS), providing specialized outpatient care and serving as a teaching and research center. Eligibility criteria {10} Inclusion criteria Women were included if they met all of the following criteria: being in physiological postmenopause (amenorrhea > 1 year), with FSH > 40 U/L and estradiol ≤ 25 pg/mL; aged between 50 and 69 years; presenting at least one GSM symptom, such as vaginal dryness, burning, itching, dyspareunia, and/or mild dysuria; and having a negative cervical cytology for neoplasia within the last 6 months (or according to screening protocols for women aged 65 years or older). Exclusion criteria Women were excluded if they refused to participate in the study; had received hormonal therapy (local or systemic) in the past 6 months; had used non-hormonal vaginal lubricants or moisturizers in the past month; had an active genital infection or suspected malignancy; had a history of genital herpes without adherence to Acyclovir prophylaxis; had severe comorbidities, including autoimmune diseases, HIV, hepatitis B or C, condylomatosis, neoplasms, thrombophilias, collagen disorders, or chronic use of corticosteroids or immunosuppressants; presented with grade II or III vaginal prolapse (POP-Q); had a history of skin photosensitivity; had previously undergone CO₂ laser or vaginal radiofrequency treatment; or had participated in another clinical trial within the past 12 months. Who will take informed consent? {26a} Informed consent was obtained prior to participant inclusion and randomization, through a printed document signed by both the participant and the principal investigator. The informed consent process took place during an outpatient clinical visit, in a private setting, where detailed information was provided regarding the study objectives, procedures, potential risks and benefits, and participants were reminded of their right to withdraw at any time. Participation decisions were made freely and after sufficient time for reflection. Participants were allowed to take the consent form home to consider participation, and later return to the clinic with the required exams for eligibility confirmation. At that time, they were able to review the document again and clarify any remaining questions before formal inclusion. Upon signing, a copy of the form was immediately provided to each participant. As part of the protocol, a reduced subgroup of participants was randomly selected to undergo vaginal biopsies before and after treatment, for histopathological evaluation. This possibility was clearly explained in advance, and all participants provided prior consent to this possibility before randomization. Refusal to undergo biopsy resulted in exclusion from the study. The biopsy procedure was performed under local anesthesia by a trained professional. The biological samples were used exclusively for the purposes of this study. Any additional use would require new ethical approvals if necessary. No intervention was initiated before formal consent was obtained. Additional consent provisions for collection and use of participant data and biological specimens {26b} Participants provided informed consent, through the Informed Consent Form, for the use of their clinical data and biological samples (vaginal biopsies) strictly for the purposes of this study, limited to histopathological analysis using hematoxylin-eosin (HE) and Masson's trichrome staining. The consent included authorization for temporary storage of the samples until completion of the analysis, including potential reprocessing or quality control prior to statistical evaluation. After this period, all samples will be discarded. No additional use is authorized. Any future use will require new ethics approval and specific participant consent. Interventions Explanation for the choice of comparators {6b} The choice of comparators, microablative CO2 laser and fractional microablative radiofrequency, was based on evidence demonstrating the efficacy of both therapies in treating Genitourinary Syndrome of Menopause (GSM). These non-hormonal treatments have shown promising results in reducing urogenital symptoms and are particularly recommended for women with contraindications to hormone therapy. However, there is still a gap in the scientific literature regarding a direct comparison of these approaches, which justifies conducting a non-inferiority clinical trial to determine if there are significant clinical differences between the techniques. Intervention description {11a} . CO₂ Laser Group: Treatment was performed using the MonaLisa Touch device (SmartXide2 V2LR, DEKA, Italy), with a 360° vaginal probe for intravaginal application. Three monthly sessions were conducted, with 30-day intervals between them. Application parameters included 40 watts of power, 1000 µs dwell time, 1000 µm spacing between points, using the Deka Pulse mode. Ablation depth (Stack 2 to 4) was adjusted based on the clinical severity of vulvovaginal atrophy: Stack 2 for severe, Stack 3 for moderate, and Stack 4 for mild atrophy. The vestibular region was treated using a vulvar probe with 20 watts of power, also in Deka Pulse mode. Radiofrequency Group: Treatment was performed using the Wavetronic 6000 Touch device with the Megapulse HF FRAXX system (Loktal Medical Electronics, Brazil), employing a disposable vaginal probe with 64 microneedles (200 µm in diameter and 1 mm in length). Three monthly sessions were administered. The first session used the Low Energy Soft Treatment mode (40 Hz), and the subsequent sessions used the Medium Energy Soft Treatment mode (60 Hz). Energy was delivered in a fractional and randomized manner across the microneedle columns to avoid thermal summation. The vaginal mucosa was treated in all sessions, and the vestibular area was addressed with the low-energy mode (40 Hz) in each application. Criteria for discontinuing or modifying allocated interventions {11b} The intervention could be discontinued or modified for individual participants in the following cases: explicit request by the participant to stop treatment for any reason; occurrence of a significant adverse event attributed to the intervention, such as intense pain, abnormal bleeding, local infection, or tissue injury; development of a clinical intercurrent condition not related to the intervention — such as systemic infection, hospitalization, acute cardiac condition, or need for emergency surgery — which, at the discretion of the team, would contraindicate continuation in the protocol; noncompliance with protocol criteria after the start of the intervention, such as the use of systemic or local hormonal therapy during follow-up; or recommendation by the external safety monitoring committee to interrupt the intervention due to unexpected adverse events or clinically relevant differences between groups, as described in item {21a}. No intervention was discontinued during the course of the study. Safety data have not yet been analyzed, as the final statistical analysis has not been initiated. Strategies to improve adherence to interventions {11c} Participants were contacted by phone calls or WhatsApp messages, according to their preference, to reinforce the dates of treatment sessions and follow-up visits. Communication was carried out in a personalized manner, using clear and supportive language to emphasize the importance of completing all treatment stages. The research team was available throughout the follow-up period to clarify questions and provide additional support when needed. Attendance at each session was recorded in a protected spreadsheet, and any absences were followed up with a new contact for rescheduling. This active monitoring strategy allowed the team to identify adherence difficulties and adapt the approach to the individual profile of each participant. Relevant concomitant care permitted or prohibited during the trial {11d} During the study, the use of any hormonal therapy, whether topical or systemic, as well as vaginal creams, lubricants, or other non-hormonal products that could affect the intervention outcomes, was prohibited. The use of systemic medications, such as antibiotics, was permitted only when clinically necessary, provided they did not interfere with primary and secondary outcomes. Any medication with potential impact on genitourinary health was evaluated by the clinical team, and in cases of significant interference, the participant could be withdrawn from the study. Provisions for post-trial care {30} Participants remained under follow-up for up to 12 months after the last intervention, with scheduled visits at months 6 and 12. The objective was to assess the maintenance of therapeutic effects and to identify any late adverse events. If any participant experienced harm related to the intervention, appropriate medical care was provided at no cost. After this period, no further follow-up was planned, given the safety profile of the technologies used. No financial compensation was offered, but clinical support was ensured for any adverse events directly related to the study. Outcomes {12} The primary outcome was improvement in vaginal health, measured by the Vaginal Health Index Score (VHIS) (2), which includes five parameters: mucosal elasticity, vaginal pH, fluid volume, epithelial integrity, and moisture. Secondary outcomes included female sexual function, assessed using the validated Female Sexual Function Index (FSFI) (6), developed by Rosen et al. and validated for Brazilian Portuguese by Borges and Medeiros (7); genitourinary symptoms (dryness, burning, itching, and dyspareunia), measured with a Visual Analog Scale (VAS); self-reported sexual satisfaction, also measured on a 0–10 cm VAS; urinary symptoms, assessed using the International Consultation on Incontinence Questionnaire – Urinary Incontinence Short Form (ICIQ-UI SF) (8); and histopathological evaluation of vaginal biopsies in a subgroup of participants (n = 4 per group), stained with hematoxylin-eosin (HE) and Masson's trichrome for tissue response analysis. All outcomes were assessed at T0 (baseline, before the first session); T1, T2, and T3 (30 days after the 1st, 2nd, and 3rd intervention sessions, respectively); and T4 and T5 (6 and 12 months after the first session). Statistical analysis of the outcomes has not yet been conducted and will be performed following acceptance of this protocol for publication. Participant timeline {13} Participants were followed over seven scheduled visits across a 12-month period, beginning with the screening visit (T0). The first intervention was performed on Day 1, preceded by the collection of a vaginal biopsy in a subgroup of participants (n = 4 per group). Subsequent interventions occurred on Days 30 and 60. Follow-up visits took place on Days 90, 180, and 365. The initial visit (T0) included eligibility assessment, signing of the Informed Consent Form (ICF), random allocation, and clinical data collection. The validated instruments — Vaginal Health Index Score (VHIS), Female Sexual Function Index (FSFI), Visual Analog Scale (VAS) for genitourinary symptoms, and ICIQ-UI Short Form for urinary symptoms — were applied immediately prior to each intervention (Days 1, 30, and 60) and during follow-up visits (Days 90, 180, and 365), enabling longitudinal analysis of treatment efficacy. Vaginal biopsies in the selected subgroup were performed on Days 1 (pre-intervention) and 180 (six months after the first session). Sample size {14} The sample size calculation was based on a non-inferiority clinical trial model, using the Vaginal Health Index Score (VHIS) as the primary outcome. The following statistical parameters were considered: type I error (α) of 0.025; type II error (β) of 0.10, corresponding to 90% power; a non-inferiority margin of 3 points; an estimated standard deviation of 2.5 points; and an expected mean difference of 0 between groups. The formula used followed the model described by Flight and Julious (9), with the data entered into the SampSize® application (https://app.sampsize.org.uk). Based on these parameters, a minimum of 32 valid participants (16 per group) was estimated. To account for potential losses, the study originally planned to recruit 40 participants. However, 4 women who signed the informed consent later declined to participate prior to the start of the interventions. Therefore, a total of 36 participants were included, which was considered sufficient to maintain the planned statistical power, as all exclusions occurred before randomization. Recruitment {15} Recruitment was carried out through consecutive screening at the gynecology outpatient clinic of the Hospital Regional de Cáceres Dr. Antônio Fontes, between July 2022 and February 2023. Eligible patients were approached in person by the research team during scheduled visits and received full information about the study, with the option to take the Informed Consent Form home for review. Initially, 40 women signed the consent form. However, four of them declined to participate at the time the interventions were scheduled to begin. Therefore, a total of 36 participants were included in the study. Recruitment ended once the planned sample size necessary to ensure statistical power had been reached. Some recruitment challenges were observed, including participants’ lack of prior knowledge about the proposed procedures, concerns about vaginal biopsy and pain, and transportation difficulties to the study site. Nevertheless, the overall adherence rate was considered satisfactory and enabled the trial to proceed according to the original design. Assignment of interventions: allocation Sequence generation {16a} Randomization was performed by a member of the research team who was not involved in data collection or clinical interventions, using a sequence electronically generated on the Sealed Envelope™ platform (https://www.sealedenvelope.com). Allocation was defined prior to the start of data collection and remained concealed from the clinical team and the monitoring committee until the end of the intervention phase. The sequence was used to allocate 36 participants (18 per group), according to the final sample included in the trial. Randomization followed a 1:1 allocation ratio between the two groups. Concealment mechanism {16b} The association between the participant enrollment number and the intervention group was performed prior to inclusion, by a researcher responsible for randomization who was not involved in data collection or intervention delivery. Allocation was determined using a previously generated electronic sequence and securely stored. The assigned group was revealed only at the time of intervention, through restricted access, ensuring allocation concealment and the integrity of the double-blind design. After the start of the first treatment session, no additional participants were included in the study. Implementation {16c} The allocation sequence was generated by an independent researcher who did not participate in data collection, clinical interventions, or assessments, and had no contact with participants. After recruitment and signing of the informed consent form by eligible women — a process conducted by the principal investigator — the participants’ names were forwarded to the person responsible for randomization, who assigned case numbers and carried out the allocation using the Sealed Envelope™ application, generating a random sequence. This sequence remained confidential and was accessed only at the time of intervention, ensuring blinding integrity. Only the intervention physician and assisting nurse had access to the allocation, exclusively for technical purposes. Neither was involved in data collection, clinical assessments, or statistical analysis. Assignment of interventions: Blinding Who will be blinded {17a} This was a randomized, controlled, double-blind clinical trial. Participants, the investigator responsible for clinical assessments, the pathologist who analyzed the biopsies, the data monitoring physician (DMC), and all other members of the clinical and support team remained blinded to group allocation. Only the intervention physician and the assisting nurse were aware of the allocation, due to technical necessity for the procedure; neither was involved in data collection, clinical assessments, or statistical analysis. Blinding was ensured through standardized strategies: interventions were performed in a dedicated room equipped with both devices (laser and radiofrequency); participants wore noise-canceling headphones; an opaque divider and surgical drape were used to block the participant’s view during the procedure; and the application team had no contact with the principal investigator or outcome assessors after the intervention. Any breach of blinding by the applicator would have led to the participant’s exclusion from the study. Procedure for unblinding if needed {17b} Unblinding was not planned as part of the standard protocol, as the study was designed to remain double-blinded until the completion of analyses. However, in exceptional situations involving serious adverse events or unexpected clinical reactions, group allocation could be disclosed for participant safety purposes. In such cases, only the researcher responsible for randomization would be authorized to access the information, upon justified request from the principal investigator or the data monitoring physician (DMC). Any unblinding event would be fully documented and reported to the Research Ethics Committee, including the reason, date, and individuals involved. Data collection and management Plans for assessment and collection of outcomes {18a} The primary outcome was assessed using the Vaginal Health Index Score (VHIS), composed of five clinical parameters: mucosal elasticity, vaginal pH, vaginal discharge volume, epithelial integrity, and moisture. The total score was used for between-group comparisons. Secondary outcomes were assessed as follows: sexual function using the validated Female Sexual Function Index (FSFI), which covers desire, arousal, lubrication, orgasm, satisfaction, and pain domains; genitourinary symptoms (dryness, burning, itching, and dyspareunia) using a 0–10 cm Visual Analog Scale (VAS); self-reported sexual satisfaction, also using a VAS, with 0 indicating no satisfaction and 10 maximum satisfaction; and urinary symptoms assessed by the ICIQ-UI Short Form, validated for urinary incontinence. Histopathological evaluation was performed in a subgroup of participants (n = 4 per group), using vaginal biopsies stained with hematoxylin-eosin (HE) and Masson’s trichrome to analyze epithelial alterations and tissue response. All assessments were conducted at the following time points: T0 (baseline, before the first session); T1, T2, and T3 (30 days after the 1st, 2nd, and 3rd intervention sessions, respectively); and at 6 and 12 months after the first intervention. Plans to promote participant retention and complete follow-up {18b} Adherence to the protocol was promoted through close follow-up by the clinical support team, under the supervision of the principal investigator, who was responsible for clinical assessments and follow-up. Reminders were sent by phone and WhatsApp, and session scheduling was adjusted flexibly to accommodate each participant's availability. Participants had direct access to the principal investigator via mobile phone and could report any intercurrences at any time. As an active monitoring measure, the team contacted participants in a standardized manner five days after each procedure via WhatsApp to check on their well-being and identify potential adverse events. In the event of technical difficulties during the intervention, management was determined by the intervention physician, who documented the occurrence without communicating with the principal investigator, in order to maintain blinding. All adverse events were recorded using a specific form, with causality assessment performed by a member of the Data Monitoring Committee (DMC). Serious adverse events were immediately reported to the Research Ethics Committee, in accordance with regulatory requirements. All clinical and outcome assessments were maintained, even in cases of partial intervention discontinuation, following the principle of intention-to-treat analysis. Data management {19} Clinical and outcome data were collected through two distinct methods. Validated questionnaires (FSFI, ICIQ-UI Short Form, and VAS) were completed directly by participants, with on-site support from a trained member of the clinical team, who remained nearby solely for technical guidance, without influencing responses. Clinical evaluations (VHIS) were conducted exclusively by the principal investigator, using a gynecological exam with a vaginal speculum. All instruments were implemented using digital forms (Google Forms), accessed via electronic devices at the time of collection. The system was configured to prevent submission of incomplete forms, ensuring automatic data completeness. All data were stored in a secure digital database with restricted access throughout the collection phase, ensuring blinding. During follow-up, the physician responsible for the Data Monitoring Committee (DMC) had access to the forms for periodic review and safety monitoring. The principal investigator remains blinded to group allocation and has not yet accessed intervention-specific data, maintaining the double-blind condition of the protocol. Full access to the dataset is contingent upon acceptance of this protocol for publication. Vaginal biopsies were analyzed by a pathologist blinded to group allocation, using standard hematoxylin-eosin and Masson’s trichrome staining. All study personnel received prior training to standardize instrument administration and data recording in accordance with the study timeline. Confidentiality {27} All information obtained from participants was treated with strict confidentiality, in accordance with ethical principles for research involving human subjects. Each participant was identified solely by a code number assigned at the time of enrollment, and no identifying information was recorded in data collection forms or databases. Data were stored in a secure digital system (Google Forms), with restricted access during the study limited to operational information, such as contact lists and pre-visit clinical records. The principal investigator remains blinded to clinical outcomes and has not yet accessed the data collected through instruments, maintaining the double-blind condition of the protocol until this manuscript is accepted for publication. During the study, data were accessed only for safety monitoring purposes by the physician responsible for the Data Monitoring Committee (DMC), with no access to participant identities. Individual data will be used exclusively for scientific purposes, and no participant will be identified in any presentation or publication related to the study. After the trial is completed, data will be stored securely for five years, in accordance with regulatory requirements, and subsequently disposed of safely. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} Biological specimens were collected through vaginal mucosa biopsies from a randomly selected subgroup of participants, at baseline and again six months after the intervention. Samples are currently stored under controlled temperature at the partner laboratory of the Regional Hospital of Cáceres, and will be used exclusively for the histological evaluation defined in the protocol, using hematoxylin-eosin (HE) and Masson's trichrome staining. This temporary storage aims to ensure technical feasibility, allowing for reprocessing or quality control prior to the final statistical analysis. All participants were informed in advance about the biopsy procedures and the exclusive use of their samples for this study, with specific consent obtained before randomization, as detailed in the Informed Consent Form and its addendum. All specimens will be discarded upon completion of the analyses. No molecular or secondary use is authorized. Any further use will require new ethics committee approval and specific participant consent. Storage, analysis, and disposal of the samples will fully comply with the regulations of the Brazilian Health Regulatory Agency (ANVISA). Statistical methods Statistical methods for primary and secondary outcomes {20a} Statistical analysis will be conducted according to the intention-to-treat (ITT) principle, including all randomized participants regardless of protocol adherence. For the primary outcome (VHIS): Group comparison will be performed using a two-sided 95% confidence interval for the difference in means, adopting a non-inferiority margin of 3 points in the VHIS score. Statistical significance will be considered at α = 0.025. For secondary outcomes:Categorical variables will be analyzed using Pearson’s chi-square test or Fisher’s exact test, as appropriate. For numerical variables, Student’s t-test (with equal or unequal variances) will be used, or the Mann-Whitney test when normality is violated. Repeated measures comparisons over time will be conducted using ANOVA or the Friedman test for non-parametric data. Normality will be tested using the Shapiro-Wilk test, and variance homogeneity will be assessed using Levene’s test. Statistical significance will be considered at p < 0.05. All analyses will be performed using IBM SPSS software, version 27. Interim analyses {21b} No formal interim statistical analyses are planned for this trial. However, participant safety will be periodically monitored by the Data Monitoring Committee (DMC), with particular attention to the occurrence of serious adverse events or unforeseen risks. If significant safety concerns are identified, the DMC may recommend temporary suspension, protocol modifications, or study termination. Methods for additional analyses (e.g. subgroup analyses) {20b} Sensitivity analyses will be conducted to assess the impact of missing data and protocol deviations on the robustness of results. Although the study does not formally plan additional analyses, exploratory stratifications may be reported if relevant findings arise during the primary analysis. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} Missing data will be handled using multiple imputation, assuming, when plausible, that data are missing at random (MAR). However, in cases of follow-up interruption due to participant withdrawal or if other non-random missing data patterns are identified, sensitivity analyses will be conducted to assess the impact of these losses on the robustness of the primary results. Plans to give access to the full protocol, participant level-data and statistical code {31c} Following completion of the study, the full protocol, anonymized participant-level dataset, and statistical code may be made available to other researchers who submit a justified scientific request, are affiliated with recognized research institutions, and commit to ethical and confidential use of the data. Sharing of materials will be subject to prior approval by the Research Ethics Committee, when applicable, and will comply with all applicable confidentiality and research ethics standards. Oversight and monitoring Composition of the coordinating centre and trial steering committee {5d} The coordinating team was composed of the principal investigator, who was responsible for study design and clinical assessments, along with healthcare professionals affiliated with the participating center, including physicians, nurses, nursing technicians, and volunteer medical students. This team was responsible for implementing the interventions, collecting data, and clinically monitoring participants, ensuring the methodological integrity of the trial. Professor Dr. Nivaldo Antonio Parizotto, PhD, academic supervisor of the project, also generated the randomization sequence. Participant safety was monitored by a Data Monitoring Committee (DMC), composed of a single independent member who was blinded to allocation: Dr. Matheus Rodrigues de Souza, a physician and statistician not affiliated with the study team. The interventionist physician, who did not participate in clinical assessments, was responsible for reporting any adverse events directly to the DMC. The principal investigator did not participate in the analysis of these events, in order to maintain blinding. Monthly meetings were held by the coordinating team to review study progress, focusing on operational and logistical aspects, while maintaining blinding with respect to outcomes and adverse events. Composition of the data monitoring committee, its role and reporting structure {21a} The Data Monitoring Committee (DMC) was composed of a single independent member: Dr. Matheus Rodrigues de Souza, a physician and statistician, who was blinded to group allocation and had no affiliation with the study team. His role was to periodically review participant safety, focusing on the identification of serious adverse events. The DMC could recommend trial suspension or protocol modifications if relevant risks to participant safety were identified. The committee did not conduct interim efficacy analysis, focusing exclusively on safety monitoring and harm control. As no serious adverse events were identified during the study, no safety reports were issued to the Research Ethics Committee or the principal investigator. The DMC did not have authority to implement changes directly; any recommendations would be evaluated by the principal investigator and, when applicable, submitted for prior ethics approval, in accordance with regulatory standards. Adverse event reporting and harms {22} All adverse events (AEs) were systematically recorded using specific forms from the time of informed consent signing until the end of follow-up. As an active monitoring measure, participants were contacted via WhatsApp five days after each intervention for clinical evaluation and early identification of AEs. Information regarding severity, duration, and potential relationship to the intervention was collected. Causality assessment was performed by a member of the Data Monitoring Committee (DMC), based on medical chart review and clinical records. No serious adverse events (SAEs) were identified during the study, and therefore no reporting to the Research Ethics Committee was required. All study personnel were trained beforehand to identify and report adverse events in a standardized manner. Frequency and plans for auditing trial conduct {23} No formal independent audit was planned for this study. Internal monitoring was conducted by the trial team itself, with the goal of ensuring protocol compliance and participant safety. Safety was periodically monitored by the Data Monitoring Committee (DMC), with a focus on identifying serious adverse events. Reports to the Research Ethics Committee (REC) were planned for any relevant intercurrences, protocol modifications, or identified risks, in accordance with regulatory requirements. As the study proceeded as planned and no serious adverse events or protocol changes occurred, no formal reports to the REC were required. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} All substantial protocol amendments (including methodological changes, outcomes, sample size, inclusion/exclusion criteria, or ethical conduct) would be communicated to the Research Ethics Committee (REC) and registered in Plataforma Brasil and the ReBEC, as applicable. No changes would be implemented without prior ethical approval. If the Data Monitoring Committee (DMC) had recommended adjustments to the protocol for safety reasons, such recommendations would have been reviewed by the principal investigator and, if appropriate, submitted to the REC for prior approval. During the course of the study, no substantial amendments to the protocol were necessary. Dissemination plans {31a} The results of this clinical trial will be disseminated through publications in peer-reviewed scientific journals, presentations at relevant medical conferences, and registration in public platforms such as ClinicalTrials.gov and ReBEC. A summary of the results in lay language will be made available to participants upon request. There will be no restrictions on data publication, ensuring full transparency in communication with healthcare professionals, participants, and the scientific community. Full dissemination of the findings will occur only after this protocol is accepted for publication. Discussion This clinical trial faced relevant practical and operational challenges, particularly regarding participant retention throughout the 12-month follow-up period. One of the main obstacles was maintaining consistent communication with participants during follow-up visits, especially among those with logistical limitations or transportation difficulties. There was a tendency toward reduced adherence to long-term clinical assessments (at 6 and 12 months), especially after completion of the intervention sessions, possibly due to the perception that treatment had ended. To overcome this limitation, a more active follow-up approach was necessary, with personalized contact and reinforcement of the importance of clinical monitoring for participant safety and data quality. Detailed planning of the interventions and biological sample collection proved essential to avoid delays and ensure adherence to the timeline. To minimize losses and ensure protocol compliance, the research team carried out systematic follow-up, with an emphasis on organized scheduling and direct support to participants. Trial status This protocol corresponds to version 1.0, dated April 22, 2025. Ethics approval was granted on April 19, 2022. Participant screening began in September 2022. The trial was registered in the Brazilian Clinical Trials Registry – ReBEC (RBR-2hgwvgy) in July 2023, retrospectively. At the time, the research team was not fully aware that prospective registration in a public trial registry was a mandatory condition for publication in international journals, which explains the delay. Clinical interventions were initiated in March 2023, and the study was formally concluded in September 2025. At the time of this submission, the trial database has been finalized and technically prepared for analysis, including multiple imputation to handle missing data. The technical preparation was conducted by an independent data analyst under the supervision of the research team, while maintaining the blinding of the principal investigator. The principal investigator remains blinded to group allocation and has not accessed intervention-specific data. Final statistical analysis has not yet been initiated and will be performed only after acceptance of this protocol for publication. Abbreviations The following abbreviations are used throughout the manuscript: GSM: Genitourinary Syndrome of Menopause RF: Radiofrequency VHIS: Vaginal Health Index Score FSFI: Female Sexual Function Index Declarations Acknowledgements We thank the study participants, the clinical team at Hospital Regional de Cáceres Dr. Antônio Fontes, the nurses and nursing technicians from Clínica Dalbem who volunteered in carrying out the interventions, and the students from Universidade do Estado de Mato Grosso (UNEMAT) who contributed to data collection and participant follow-up throughout the study. Authors’ contributions {31b} CCGDT was the principal investigator. She conceived the study, developed the protocol, conducted the pre-intervention clinical assessments, and drafted the manuscript. CMMGD contributed to the protocol development and conducted the clinical interventions as outlined in the study design. MRS acted as the Data Monitoring Committee (DMC) member. He monitored the integrity of the clinical data and contributed to the technical review of the manuscript. NAP served as academic advisor. He contributed to the methodological design, generated the randomization sequence, and critically reviewed the manuscript. All authors read and approved the final version of the protocol. Funding {4} This study has not received external funding. The devices used in the interventions (laser and radiofrequency equipment) were provided by the principal investigator. Logistical support — including clinical infrastructure, human resources, and technical assistance for the procedures — was provided by the Regional Hospital de Cáceres Dr. Antônio Fontes. Availability of data and materials {29} The raw data generated and/or analyzed during this study may be made available upon reasonable request to the corresponding author, provided that ethical principles and participant confidentiality are upheld. Data sharing will be subject to approval by the Research Ethics Committee, when applicable. Ethics approval and consent to participate {24} This study was approved by the Research Ethics Committee (REC) of Universidade Brasil under approval number 5.357.602. Written informed consent was obtained from all participants prior to the initiation of any study procedures, in accordance with national and international ethical guidelines. The trial was also registered with ReBEC (Registro Brasileiro de Ensaios Clínicos), as required by current regulatory standards. Consent for publication {32} This study included the collection of clinical images for documentation purposes; however, no specific consent was obtained for their use in scientific publications. Therefore, no identifiable or non-identifiable images will be used in any reports, publications, or dissemination materials. The absence of this additional consent did not affect the ethical conduct of the study, and all other ethical guidelines were strictly followed. Competing interests {28} The authors declare that they have no financial or non-financial interests that could be interpreted as conflicts of interest related to this study. C.C.G.D.T., N.A.P., M.R.S., and C.M.M.G.D. confirm the absence of competing interests. Authors’ information (optional) Cássia Caroline Garcia Dalbem Teles is a gynecologist and obstetrician, holds a Master’s degree, and is a faculty member in the medical program at Universidade do Estado de Mato Grosso (UNEMAT) and also affiliated with Universidade Brasil, with specialization in lower genital tract pathology and colposcopy. Catarina Maria Mesquita Garcia Dalbem is a gynecologist, holds a Master’s degree, and is a professor at Universidade do Estado de Mato Grosso (UNEMAT), also affiliated with Universidade Brasil. Matheus Rodrigues de Souza is a physician specializing in statistical analysis and clinical trials, affiliated with Universidade do Estado de Mato Grosso (UNEMAT). Nivaldo Antonio Parizotto is a physiotherapist with a PhD in Electrical Engineering from the University of Campinas (UNICAMP) and postdoctoral training at the Wellman Center for Photomedicine, Harvard Medical School. He is currently a Senior Full Professor at the Federal University of São Carlos (UFSCar) and a faculty member of the Graduate Program in Biomedical Engineering at Universidade Brasil. References Nappi RE, Palacios S, Panay N, Particco M, Krychman ML. Vulvar and vaginal atrophy in four European countries: evidence from the European REVIVE Survey. Climacteric . 2016;19(2):188–97. doi:10.3109/13697137.2015.1107039. Salvatore S, Nappi RE, Zerbinati N, Calligaro A, Ferrero S, Origoni M, et al. A 12-week treatment with fractional CO₂ laser for vulvovaginal atrophy: a pilot study. Climacteric . 2014;17(4):363–9. doi:10.3109/13697137.2014.899347. Perino A, Calligaro A, Forlani F, Tiberio C, Cucinella G, Svelato A, et al. Vulvo-vaginal atrophy: a new treatment modality using thermo-ablative fractional CO₂ laser. Maturitas . 2015;80(3):296–301. doi:10.1016/j.maturitas.2014.12.006. Gambacciani M, Palacios S. Laser therapy for the restoration of vaginal function. Maturitas . 2017;99:10–5. doi:10.1016/j.maturitas.2017.01.012. Kamilos MF, Borrelli CL. New therapeutic option in genitourinary syndrome of menopause: pilot study using microablative fractional radiofrequency. Einstein (São Paulo) . 2017;15(4):445–51. doi:10.1590/S1679-45082017AO4051. Rosen R, Brown C, Heiman J, Leiblum S, Meston C, Shabsigh R, et al. The Female Sexual Function Index (FSFI): a multidimensional self-report instrument for the assessment of female sexual function. J Sex Marital Ther . 2000;26(2):191–208. doi:10.1080/009262300278597. Borges VL, Medeiros SF. Validação de questionário para avaliar a função sexual feminina após menopausa. Rev Bras Ginecol Obstet . 2009;31(6):293–9. doi:10.1590/s0100-72032009000600005. Avery K, Donovan J, Peters TJ, Shaw C, Gotoh M, Abrams P. ICIQ: a brief and robust measure for evaluating the symptoms and impact of urinary incontinence. Neurourol Urodyn . 2004;23(4):322–30. doi:10.1002/nau.20041. Flight L, Julious SA. Practical guide to sample size calculations: non-inferiority and equivalence trials. Pharm Stat. 2016 Jan-Feb;15(1):80-9. doi: 10.1002/pst.1716. Epub 2015 Nov 25. PMID: 26604186. Additional Declarations The authors declare no competing interests. Supplementary Files TelesSPIRITChecklistversion1.1May2025.docx SPIRIT 2013 Checklist for Clinical Trial Protocol Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6699446","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":458769829,"identity":"6d95f485-491a-4008-9dcc-ea258fa13f16","order_by":0,"name":"Cassia Caroline Garcia Dalbem Teles","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYLCCBAZmBj4JIOMDVICZKC1sQC2MM6AaCGthgGph5iFGC3/78ccfHtRYy7NJNx/dbNt2R063/fwB5sI9uLVInMkxk0g4lm7YJnMs7XZu2zNjszPJDMwznuHWYsCQw8aQwHaYsU0ixwyo5XDitgNALTwH8Gjhf/74Q8K/w/ZtEvnfblu2Ha7fdv4xAS0SCQYSiUDDgbaw3WZsO5xgdoOALRI33phJJPalJwP9Ynaz59xhw203HhscnoFHC39/+uOPP75Z2/ZLNz+78aPssLzZ+cSHjwvwaMEOSNYwCkbBKBgFowAVAABZsVXZo41arAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5191-6890","institution":"UNEMAT; Universidade Brasil","correspondingAuthor":true,"prefix":"","firstName":"Cassia","middleName":"Caroline Garcia Dalbem","lastName":"Teles","suffix":""},{"id":458770872,"identity":"a069866b-fd45-4140-9ef1-f1d55942f3bb","order_by":1,"name":"Catarina Maria Mesquita Garcia Dalbem","email":"","orcid":"https://orcid.org/0000-0001-8444-9216","institution":"UNEMAT; Universidade Brasil","correspondingAuthor":false,"prefix":"","firstName":"Catarina","middleName":"Maria Mesquita Garcia","lastName":"Dalbem","suffix":""},{"id":458770873,"identity":"ce253ada-ffde-4bd2-af63-1c47671d64c7","order_by":2,"name":"Matheus Rodrigues de Souza","email":"","orcid":"https://orcid.org/0000-0001-5517-0777","institution":"UNEMAT","correspondingAuthor":false,"prefix":"","firstName":"Matheus","middleName":"Rodrigues","lastName":"de Souza","suffix":""},{"id":458770874,"identity":"603b2628-ad78-4436-8734-b26fa43b18ea","order_by":3,"name":"Nivaldo Antonio Parizotto","email":"","orcid":"https://orcid.org/0000-0003-1774-9053","institution":"Universidade Brasil","correspondingAuthor":false,"prefix":"","firstName":"Nivaldo","middleName":"Antonio","lastName":"Parizotto","suffix":""}],"badges":[],"createdAt":"2025-05-19 13:35:24","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6699446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6699446/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83294410,"identity":"45727b22-45aa-4502-84ef-5ce885442864","added_by":"auto","created_at":"2025-05-22 13:32:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119922,"visible":true,"origin":"","legend":"\u003cp\u003eSPIRIT Figure: Schedule of enrolment, interventions, and assessments\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6699446/v1/c5ed47ddace2264d9c94f193.png"},{"id":83295344,"identity":"34b5a99f-f3a9-4673-b136-7ff333fd6954","added_by":"auto","created_at":"2025-05-22 13:40:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1618118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6699446/v1/7abff7eb-2012-4bb3-b694-e865044131b7.pdf"},{"id":83293771,"identity":"522830e3-ca60-44c7-b5b1-a872899b6da8","added_by":"auto","created_at":"2025-05-22 13:24:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":231977,"visible":true,"origin":"","legend":"\u003cp\u003eSPIRIT 2013 Checklist for Clinical Trial Protocol\u003c/p\u003e","description":"","filename":"TelesSPIRITChecklistversion1.1May2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6699446/v1/87788b5deafee695d655fb7c.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTreatment of Genitourinary Syndrome of Menopause with Microablative CO₂ Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Administrative Information","content":"\u003cp\u003eNote: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"639\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTitle {1}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eTreatment of Genitourinary Syndrome of Menopause with Microablative CO2 Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTrial registration {2a and 2b}.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eThis clinical trial was registered in the Brazilian Clinical Trials Registry (ReBEC): RBR-2hgwvgy\u003cbr\u003e \u003ca href=\"https://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy\"\u003e\u0026nbsp;\u003c/a\u003ehttps://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy\u003cbr\u003e\u0026nbsp; UTN code: U1111-1282-9195\u003cbr\u003e\u0026nbsp; National registration on Plataforma Brasil (CAAE): 55003622.8.0000.5494\u003cbr\u003e\u0026nbsp; Research Ethics Committee approval: 5.357.602 (University Brasil Research Ethics Committee)\u003c/p\u003e\n \u003cp\u003eThe trial was retrospectively registered in July 2023. Although ethics approval was obtained on April 19, 2022, and screening began in September 2022, clinical interventions and data collection only started in March 2023. At the time, the research team was not fully aware of the need for prospective public registration as a publication requirement. The protocol was prospectively developed and strictly followed. The principal investigator remains blinded to group allocation, and no statistical analysis has been performed to date. This is the first formal submission of the full protocol.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eProtocol version {3}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eVersion 1.0, Date: 22/04/2025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eFunding {4}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eThis study has not received external funding. The devices used in the interventions (laser and radiofrequency equipment) were provided by the principal investigator. Logistical support \u0026mdash; including clinical infrastructure, human resources, and technical assistance for the procedures \u0026mdash; was provided by the Regional Hospital de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eAuthor details {5a}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eC\u0026aacute;ssia Caroline Garcia Dalbem Teles, Universidade do Estado de Mato Grosso and Universidade Brasil. Physician. Principal investigator. Responsible for study conception, protocol development, and clinical assessments prior to the interventions.\u003c/p\u003e\n \u003cp\u003eNivaldo Antonio Parizotto, Universidade Brasil \u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePhD. Academic supervisor. Contributed to the methodological design and generated the randomization sequence.\u003c/p\u003e\n \u003cp\u003eMatheus Rodrigues de Souza, Universidade do Estado de Mato Grosso \u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePhysician. Member of the Data Monitoring Committee (DMC). Monitored clinical data integrity and safety throughout the study.\u003c/p\u003e\n \u003cp\u003eCatarina Maria Mesquita Garcia Dalbem, Universidade do Estado de Mato Grosso and Universidade Brasil. Physician. Performed the clinical interventions using CO₂ laser and radiofrequency. Did not participate in data collection or outcome analysis.\u003c/p\u003e\n \u003cp\u003eMarcos Ara\u0026uacute;jo Chaves J\u0026uacute;nior, Universidade do Estado de Mato Grosso \u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePathologist. Conducted histological analyses of vaginal biopsies collected during the study.\u003c/p\u003e\n \u003cp\u003eGabriela Fonseca Arenhart, Guilherme Bezerra, Leticia Andrioli da Cunha, Rayanni Freire Alves Pedroso, Viviane Ribeiro, Universidade do Estado de Mato Grosso \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMedical students. Provided technical research support, organized study data, and maintained participant contact during the trial.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eName and contact information for the trial sponsor {5b}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003ePrimary Sponsor: C\u0026aacute;ssia Caroline Garcia Dalbem Teles\u003c/p\u003e\n \u003cp\u003eContact: [email protected]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eRole of sponsor {5c}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eThe principal investigator, C\u0026aacute;ssia Caroline Garcia Dalbem Teles, is a permanent faculty member at Universidade do Estado de Mato Grosso (UNEMAT), where she conducts academic and clinical activities. She is also a doctoral student at Universidade Brasil, the academic institution responsible for the ethical oversight and doctoral supervision of this study.\u003c/p\u003e\n \u003cp\u003eThe principal investigator conceived and conducted the study and provided the equipment used in the interventions (microablative CO₂ laser and fractional microablative radiofrequency), which were her personal property. Hospital Regional de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes provided logistical support, clinical infrastructure, and technical staff for the procedures.\u003c/p\u003e\n \u003cp\u003eNone of the institutions involved \u0026mdash; Universidade do Estado de Mato Grosso (UNEMAT), Universidade Brasil, and Hospital Regional de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes \u0026mdash; participated in the study design, data collection, data analysis, interpretation of results, manuscript writing, or the decision to submit this manuscript for publication. All scientific and methodological decisions were the sole responsibility of the principal investigator and the study team.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eBackground and rationale {6a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenitourinary syndrome of menopause (GSM) is a common and debilitating condition affecting a significant proportion of postmenopausal women. It manifests through symptoms such as vaginal dryness, irritation, dyspareunia, and urinary complaints, negatively impacting quality of life and female sexual function (1). Although local estrogen therapy is considered the standard treatment, not all patients are willing or eligible to use it, especially those with contraindications to hormonal therapy. This has led to the search for non-hormonal alternatives.\u003c/p\u003e\n\u003cp\u003eAmong these alternatives, fractional CO₂ laser therapy is considered promising. Initial clinical studies report significant improvement in vulvovaginal atrophy symptoms following treatment, with a favorable safety profile (2\u0026ndash;4). Similarly, microablative radiofrequency is being investigated as a non-hormonal intervention, and preliminary findings suggest relevant clinical benefits in alleviating genitourinary symptoms (5).\u003c/p\u003e\n\u003cp\u003eDespite these findings, most available studies present important methodological limitations, such as lack of randomization, control groups, or long-term follow-up. Therefore, well-designed controlled clinical trials are considered necessary to strengthen the evidence base regarding the efficacy and safety of these therapies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives {7}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary objective: To evaluate the non-inferiority of fractional microablative radiofrequency compared to CO₂ laser in the treatment of Genitourinary Syndrome of Menopause (GSM), using the Vaginal Health Index Score (VHIS) as the primary outcome, as outlined in a double-blind, randomized, non-inferiority trial.\u003c/p\u003e\n\u003cp\u003eHypotheses:\u003c/p\u003e\n\u003cp\u003eNull hypothesis (H₀): Fractional microablative radiofrequency is inferior to CO₂ laser in improving vaginal health (VHIS).\u003c/p\u003e\n\u003cp\u003eAlternative hypothesis (H₁): Fractional microablative radiofrequency is not inferior to CO₂ laser in improving vaginal health (VHIS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design {8}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was a randomized, controlled, double-blind, non-inferiority clinical trial, conducted at a single center. A total of 36 postmenopausal women were included and were randomized to receive three monthly sessions of either microablative CO₂ laser or fractional microablative radiofrequency. Participants were allocated to the intervention groups in a 1:1 ratio.\u003c/p\u003e"},{"header":"Methods: Participants, interventions and outcomes","content":"\u003cp\u003eThis protocol has been written in accordance with the SPIRIT 2013 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy setting {9}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted at Hospital Regional de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes, located in the city of C\u0026aacute;ceres, Mato Grosso, Brazil. This is a public tertiary-level hospital affiliated with the Brazilian Unified Health System (SUS), providing specialized outpatient care and serving as a teaching and research center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility criteria {10}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen were included if they met all of the following criteria: being in physiological postmenopause (amenorrhea \u0026gt; 1 year), with FSH \u0026gt; 40 U/L and estradiol \u0026le; 25 pg/mL; aged between 50 and 69 years; presenting at least one GSM symptom, such as vaginal dryness, burning, itching, dyspareunia, and/or mild dysuria; and having a negative cervical cytology for neoplasia within the last 6 months (or according to screening protocols for women aged 65 years or older).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen were excluded if they refused to participate in the study; had received hormonal therapy (local or systemic) in the past 6 months; had used non-hormonal vaginal lubricants or moisturizers in the past month; had an active genital infection or suspected malignancy; had a history of genital herpes without adherence to Acyclovir prophylaxis; had severe comorbidities, including autoimmune diseases, HIV, hepatitis B or C, condylomatosis, neoplasms, thrombophilias, collagen disorders, or chronic use of corticosteroids or immunosuppressants; presented with grade II or III vaginal prolapse (POP-Q); had a history of skin photosensitivity; had previously undergone CO₂ laser or vaginal radiofrequency treatment; or had participated in another clinical trial within the past 12 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will take informed consent? {26a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained prior to participant inclusion and randomization, through a printed document signed by both the participant and the principal investigator. The informed consent process took place during an outpatient clinical visit, in a private setting, where detailed information was provided regarding the study objectives, procedures, potential risks and benefits, and participants were reminded of their right to withdraw at any time.\u003c/p\u003e\n\u003cp\u003eParticipation decisions were made freely and after sufficient time for reflection. Participants were allowed to take the consent form home to consider participation, and later return to the clinic with the required exams for eligibility confirmation. At that time, they were able to review the document again and clarify any remaining questions before formal inclusion. Upon signing, a copy of the form was immediately provided to each participant.\u003c/p\u003e\n\u003cp\u003eAs part of the protocol, a reduced subgroup of participants was randomly selected to undergo vaginal biopsies before and after treatment, for histopathological evaluation. This possibility was clearly explained in advance, and all participants provided prior consent to this possibility before randomization. Refusal to undergo biopsy resulted in exclusion from the study.\u003c/p\u003e\n\u003cp\u003eThe biopsy procedure was performed under local anesthesia by a trained professional. The biological samples were used exclusively for the purposes of this study. Any additional use would require new ethical approvals if necessary.\u003c/p\u003e\n\u003cp\u003eNo intervention was initiated before formal consent was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional consent provisions for collection and use of participant data and biological specimens {26b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants provided informed consent, through the Informed Consent Form, for the use of their clinical data and biological samples (vaginal biopsies) strictly for the purposes of this study, limited to histopathological analysis using hematoxylin-eosin (HE) and Masson\u0026apos;s trichrome staining. The consent included authorization for temporary storage of the samples until completion of the analysis, including potential reprocessing or quality control prior to statistical evaluation. After this period, all samples will be discarded. No additional use is authorized. Any future use will require new ethics approval and specific participant consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExplanation for the choice of comparators {6b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe choice of comparators, microablative CO2 laser and fractional microablative radiofrequency, was based on evidence demonstrating the efficacy of both therapies in treating Genitourinary Syndrome of Menopause (GSM). These non-hormonal treatments have shown promising results in reducing urogenital symptoms and are particularly recommended for women with contraindications to hormone therapy. However, there is still a gap in the scientific literature regarding a direct comparison of these approaches, which justifies conducting a non-inferiority clinical trial to determine if there are significant clinical differences between the techniques.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention description {11a}\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCO₂ Laser Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment was performed using the MonaLisa Touch device (SmartXide2 V2LR, DEKA, Italy), with a 360\u0026deg; vaginal probe for intravaginal application. Three monthly sessions were conducted, with 30-day intervals between them. Application parameters included 40 watts of power, 1000 \u0026micro;s dwell time, 1000 \u0026micro;m spacing between points, using the Deka Pulse mode. Ablation depth (Stack 2 to 4) was adjusted based on the clinical severity of vulvovaginal atrophy: Stack 2 for severe, Stack 3 for moderate, and Stack 4 for mild atrophy. The vestibular region was treated using a vulvar probe with 20 watts of power, also in Deka Pulse mode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiofrequency Group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment was performed using the Wavetronic 6000 Touch device with the Megapulse HF FRAXX system (Loktal Medical Electronics, Brazil), employing a disposable vaginal probe with 64 microneedles (200 \u0026micro;m in diameter and 1 mm in length). Three monthly sessions were administered. The first session used the Low Energy Soft Treatment mode (40 Hz), and the subsequent sessions used the Medium Energy Soft Treatment mode (60 Hz). Energy was delivered in a fractional and randomized manner across the microneedle columns to avoid thermal summation. The vaginal mucosa was treated in all sessions, and the vestibular area was addressed with the low-energy mode (40 Hz) in each application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCriteria for discontinuing or modifying allocated interventions {11b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intervention could be discontinued or modified for individual participants in the following cases: explicit request by the participant to stop treatment for any reason; occurrence of a significant adverse event attributed to the intervention, such as intense pain, abnormal bleeding, local infection, or tissue injury; development of a clinical intercurrent condition not related to the intervention \u0026mdash; such as systemic infection, hospitalization, acute cardiac condition, or need for emergency surgery \u0026mdash; which, at the discretion of the team, would contraindicate continuation in the protocol; noncompliance with protocol criteria after the start of the intervention, such as the use of systemic or local hormonal therapy during follow-up; or recommendation by the external safety monitoring committee to interrupt the intervention due to unexpected adverse events or clinically relevant differences between groups, as described in item {21a}.\u003c/p\u003e\n\u003cp\u003eNo intervention was discontinued during the course of the study. Safety data have not yet been analyzed, as the final statistical analysis has not been initiated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrategies to improve adherence to interventions {11c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were contacted by phone calls or WhatsApp messages, according to their preference, to reinforce the dates of treatment sessions and follow-up visits. Communication was carried out in a personalized manner, using clear and supportive language to emphasize the importance of completing all treatment stages. The research team was available throughout the follow-up period to clarify questions and provide additional support when needed. Attendance at each session was recorded in a protected spreadsheet, and any absences were followed up with a new contact for rescheduling. This active monitoring strategy allowed the team to identify adherence difficulties and adapt the approach to the individual profile of each participant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelevant concomitant care permitted or prohibited during the trial {11d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the study, the use of any hormonal therapy, whether topical or systemic, as well as vaginal creams, lubricants, or other non-hormonal products that could affect the intervention outcomes, was prohibited. The use of systemic medications, such as antibiotics, was permitted only when clinically necessary, provided they did not interfere with primary and secondary outcomes. Any medication with potential impact on genitourinary health was evaluated by the clinical team, and in cases of significant interference, the participant could be withdrawn from the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProvisions for post-trial care {30}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants remained under follow-up for up to 12 months after the last intervention, with scheduled visits at months 6 and 12. The objective was to assess the maintenance of therapeutic effects and to identify any late adverse events. If any participant experienced harm related to the intervention, appropriate medical care was provided at no cost. After this period, no further follow-up was planned, given the safety profile of the technologies used. No financial compensation was offered, but clinical support was ensured for any adverse events directly related to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes {12}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary outcome was improvement in vaginal health, measured by the Vaginal Health Index Score (VHIS) (2), which includes five parameters: mucosal elasticity, vaginal pH, fluid volume, epithelial integrity, and moisture.\u003c/p\u003e\n\u003cp\u003eSecondary outcomes included female sexual function, assessed using the validated Female Sexual Function Index (FSFI) (6), developed by Rosen et al. and validated for Brazilian Portuguese by Borges and Medeiros (7); genitourinary symptoms (dryness, burning, itching, and dyspareunia), measured with a Visual Analog Scale (VAS); self-reported sexual satisfaction, also measured on a 0\u0026ndash;10 cm VAS; urinary symptoms, assessed using the International Consultation on Incontinence Questionnaire \u0026ndash; Urinary Incontinence Short Form (ICIQ-UI SF) (8); and histopathological evaluation of vaginal biopsies in a subgroup of participants (n = 4 per group), stained with hematoxylin-eosin (HE) and Masson\u0026apos;s trichrome for tissue response analysis.\u003c/p\u003e\n\u003cp\u003eAll outcomes were assessed at T0 (baseline, before the first session); T1, T2, and T3 (30 days after the 1st, 2nd, and 3rd intervention sessions, respectively); and T4 and T5 (6 and 12 months after the first session).\u003c/p\u003e\n\u003cp\u003eStatistical analysis of the outcomes has not yet been conducted and will be performed following acceptance of this protocol for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipant timeline {13}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were followed over seven scheduled visits across a 12-month period, beginning with the screening visit (T0). The first intervention was performed on Day 1, preceded by the collection of a vaginal biopsy in a subgroup of participants (n = 4 per group). Subsequent interventions occurred on Days 30 and 60. Follow-up visits took place on Days 90, 180, and 365.\u003c/p\u003e\n\u003cp\u003eThe initial visit (T0) included eligibility assessment, signing of the Informed Consent Form (ICF), random allocation, and clinical data collection. The validated instruments \u0026mdash; Vaginal Health Index Score (VHIS), Female Sexual Function Index (FSFI), Visual Analog Scale (VAS) for genitourinary symptoms, and ICIQ-UI Short Form for urinary symptoms \u0026mdash; were applied immediately prior to each intervention (Days 1, 30, and 60) and during follow-up visits (Days 90, 180, and 365), enabling longitudinal analysis of treatment efficacy. Vaginal biopsies in the selected subgroup were performed on Days 1 (pre-intervention) and 180 (six months after the first session).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size {14}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample size calculation was based on a non-inferiority clinical trial model, using the Vaginal Health Index Score (VHIS) as the primary outcome. The following statistical parameters were considered: type I error (\u0026alpha;) of 0.025; type II error (\u0026beta;) of 0.10, corresponding to 90% power; a non-inferiority margin of 3 points; an estimated standard deviation of 2.5 points; and an expected mean difference of 0 between groups. The formula used followed the model described by Flight and Julious (9), with the data entered into the SampSize\u0026reg; application (https://app.sampsize.org.uk).\u003c/p\u003e\n\u003cp\u003eBased on these parameters, a minimum of 32 valid participants (16 per group) was estimated. To account for potential losses, the study originally planned to recruit 40 participants. However, 4 women who signed the informed consent later declined to participate prior to the start of the interventions. Therefore, a total of 36 participants were included, which was considered sufficient to maintain the planned statistical power, as all exclusions occurred before randomization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment {15}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecruitment was carried out through consecutive screening at the gynecology outpatient clinic of the Hospital Regional de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes, between July 2022 and February 2023. Eligible patients were approached in person by the research team during scheduled visits and received full information about the study, with the option to take the Informed Consent Form home for review.\u003c/p\u003e\n\u003cp\u003eInitially, 40 women signed the consent form. However, four of them declined to participate at the time the interventions were scheduled to begin. Therefore, a total of 36 participants were included in the study. Recruitment ended once the planned sample size necessary to ensure statistical power had been reached.\u003c/p\u003e\n\u003cp\u003eSome recruitment challenges were observed, including participants\u0026rsquo; lack of prior knowledge about the proposed procedures, concerns about vaginal biopsy and pain, and transportation difficulties to the study site. Nevertheless, the overall adherence rate was considered satisfactory and enabled the trial to proceed according to the original design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: allocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence generation {16a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRandomization was performed by a member of the research team who was not involved in data collection or clinical interventions, using a sequence electronically generated on the Sealed Envelope\u0026trade; platform (https://www.sealedenvelope.com). Allocation was defined prior to the start of data collection and remained concealed from the clinical team and the monitoring committee until the end of the intervention phase.\u003c/p\u003e\n\u003cp\u003eThe sequence was used to allocate 36 participants (18 per group), according to the final sample included in the trial. Randomization followed a 1:1 allocation ratio between the two groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcealment mechanism {16b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe association between the participant enrollment number and the intervention group was performed prior to inclusion, by a researcher responsible for randomization who was not involved in data collection or intervention delivery. Allocation was determined using a previously generated electronic sequence and securely stored. The assigned group was revealed only at the time of intervention, through restricted access, ensuring allocation concealment and the integrity of the double-blind design.\u003c/p\u003e\n\u003cp\u003eAfter the start of the first treatment session, no additional participants were included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplementation {16c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe allocation sequence was generated by an independent researcher who did not participate in data collection, clinical interventions, or assessments, and had no contact with participants. After recruitment and signing of the informed consent form by eligible women \u0026mdash; a process conducted by the principal investigator \u0026mdash; the participants\u0026rsquo; names were forwarded to the person responsible for randomization, who assigned case numbers and carried out the allocation using the Sealed Envelope\u0026trade; application, generating a random sequence.\u003c/p\u003e\n\u003cp\u003eThis sequence remained confidential and was accessed only at the time of intervention, ensuring blinding integrity. Only the intervention physician and assisting nurse had access to the allocation, exclusively for technical purposes. Neither was involved in data collection, clinical assessments, or statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: Blinding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will be blinded {17a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a randomized, controlled, double-blind clinical trial. Participants, the investigator responsible for clinical assessments, the pathologist who analyzed the biopsies, the data monitoring physician (DMC), and all other members of the clinical and support team remained blinded to group allocation. Only the intervention physician and the assisting nurse were aware of the allocation, due to technical necessity for the procedure; neither was involved in data collection, clinical assessments, or statistical analysis.\u003c/p\u003e\n\u003cp\u003eBlinding was ensured through standardized strategies: interventions were performed in a dedicated room equipped with both devices (laser and radiofrequency); participants wore noise-canceling headphones; an opaque divider and surgical drape were used to block the participant\u0026rsquo;s view during the procedure; and the application team had no contact with the principal investigator or outcome assessors after the intervention. Any breach of blinding by the applicator would have led to the participant\u0026rsquo;s exclusion from the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcedure for unblinding if needed {17b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnblinding was not planned as part of the standard protocol, as the study was designed to remain double-blinded until the completion of analyses. However, in exceptional situations involving serious adverse events or unexpected clinical reactions, group allocation could be disclosed for participant safety purposes.\u003c/p\u003e\n\u003cp\u003eIn such cases, only the researcher responsible for randomization would be authorized to access the information, upon justified request from the principal investigator or the data monitoring physician (DMC). Any unblinding event would be fully documented and reported to the Research Ethics Committee, including the reason, date, and individuals involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for assessment and collection of outcomes {18a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary outcome was assessed using the Vaginal Health Index Score (VHIS), composed of five clinical parameters: mucosal elasticity, vaginal pH, vaginal discharge volume, epithelial integrity, and moisture. The total score was used for between-group comparisons.\u003c/p\u003e\n\u003cp\u003eSecondary outcomes were assessed as follows: sexual function using the validated Female Sexual Function Index (FSFI), which covers desire, arousal, lubrication, orgasm, satisfaction, and pain domains; genitourinary symptoms (dryness, burning, itching, and dyspareunia) using a 0\u0026ndash;10 cm Visual Analog Scale (VAS); self-reported sexual satisfaction, also using a VAS, with 0 indicating no satisfaction and 10 maximum satisfaction; and urinary symptoms assessed by the ICIQ-UI Short Form, validated for urinary incontinence.\u003c/p\u003e\n\u003cp\u003eHistopathological evaluation was performed in a subgroup of participants (n = 4 per group), using vaginal biopsies stained with hematoxylin-eosin (HE) and Masson\u0026rsquo;s trichrome to analyze epithelial alterations and tissue response.\u003c/p\u003e\n\u003cp\u003eAll assessments were conducted at the following time points: T0 (baseline, before the first session); T1, T2, and T3 (30 days after the 1st, 2nd, and 3rd intervention sessions, respectively); and at 6 and 12 months after the first intervention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to promote participant retention and complete follow-up {18b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdherence to the protocol was promoted through close follow-up by the clinical support team, under the supervision of the principal investigator, who was responsible for clinical assessments and follow-up. Reminders were sent by phone and WhatsApp, and session scheduling was adjusted flexibly to accommodate each participant\u0026apos;s availability. Participants had direct access to the principal investigator via mobile phone and could report any intercurrences at any time.\u003c/p\u003e\n\u003cp\u003eAs an active monitoring measure, the team contacted participants in a standardized manner five days after each procedure via WhatsApp to check on their well-being and identify potential adverse events. In the event of technical difficulties during the intervention, management was determined by the intervention physician, who documented the occurrence without communicating with the principal investigator, in order to maintain blinding.\u003c/p\u003e\n\u003cp\u003eAll adverse events were recorded using a specific form, with causality assessment performed by a member of the Data Monitoring Committee (DMC). Serious adverse events were immediately reported to the Research Ethics Committee, in accordance with regulatory requirements. All clinical and outcome assessments were maintained, even in cases of partial intervention discontinuation, following the principle of intention-to-treat analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management {19}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical and outcome data were collected through two distinct methods. Validated questionnaires (FSFI, ICIQ-UI Short Form, and VAS) were completed directly by participants, with on-site support from a trained member of the clinical team, who remained nearby solely for technical guidance, without influencing responses. Clinical evaluations (VHIS) were conducted exclusively by the principal investigator, using a gynecological exam with a vaginal speculum.\u003c/p\u003e\n\u003cp\u003eAll instruments were implemented using digital forms (Google Forms), accessed via electronic devices at the time of collection. The system was configured to prevent submission of incomplete forms, ensuring automatic data completeness. All data were stored in a secure digital database with restricted access throughout the collection phase, ensuring blinding.\u003c/p\u003e\n\u003cp\u003eDuring follow-up, the physician responsible for the Data Monitoring Committee (DMC) had access to the forms for periodic review and safety monitoring. The principal investigator remains blinded to group allocation and has not yet accessed intervention-specific data, maintaining the double-blind condition of the protocol. Full access to the dataset is contingent upon acceptance of this protocol for publication. Vaginal biopsies were analyzed by a pathologist blinded to group allocation, using standard hematoxylin-eosin and Masson\u0026rsquo;s trichrome staining.\u003c/p\u003e\n\u003cp\u003eAll study personnel received prior training to standardize instrument administration and data recording in accordance with the study timeline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfidentiality {27}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll information obtained from participants was treated with strict confidentiality, in accordance with ethical principles for research involving human subjects. Each participant was identified solely by a code number assigned at the time of enrollment, and no identifying information was recorded in data collection forms or databases. Data were stored in a secure digital system (Google Forms), with restricted access during the study limited to operational information, such as contact lists and pre-visit clinical records.\u003c/p\u003e\n\u003cp\u003eThe principal investigator remains blinded to clinical outcomes and has not yet accessed the data collected through instruments, maintaining the double-blind condition of the protocol until this manuscript is accepted for publication.\u003c/p\u003e\n\u003cp\u003eDuring the study, data were accessed only for safety monitoring purposes by the physician responsible for the Data Monitoring Committee (DMC), with no access to participant identities. Individual data will be used exclusively for scientific purposes, and no participant will be identified in any presentation or publication related to the study. After the trial is completed, data will be stored securely for five years, in accordance with regulatory requirements, and subsequently disposed of safely.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiological specimens were collected through vaginal mucosa biopsies from a randomly selected subgroup of participants, at baseline and again six months after the intervention. Samples are currently stored under controlled temperature at the partner laboratory of the Regional Hospital of C\u0026aacute;ceres, and will be used exclusively for the histological evaluation defined in the protocol, using hematoxylin-eosin (HE) and Masson\u0026apos;s trichrome staining. This temporary storage aims to ensure technical feasibility, allowing for reprocessing or quality control prior to the final statistical analysis.\u003c/p\u003e\n\u003cp\u003eAll participants were informed in advance about the biopsy procedures and the exclusive use of their samples for this study, with specific consent obtained before randomization, as detailed in the Informed Consent Form and its addendum.\u003c/p\u003e\n\u003cp\u003eAll specimens will be discarded upon completion of the analyses. No molecular or secondary use is authorized. Any further use will require new ethics committee approval and specific participant consent. Storage, analysis, and disposal of the samples will fully comply with the regulations of the Brazilian Health Regulatory Agency (ANVISA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods for primary and secondary outcomes {20a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis will be conducted according to the intention-to-treat (ITT) principle, including all randomized participants regardless of protocol adherence.\u003c/p\u003e\n\u003cp\u003eFor the primary outcome (VHIS): Group comparison will be performed using a two-sided 95% confidence interval for the difference in means, adopting a non-inferiority margin of 3 points in the VHIS score. Statistical significance will be considered at \u0026alpha; = 0.025.\u003c/p\u003e\n\u003cp\u003eFor secondary outcomes:Categorical variables will be analyzed using Pearson\u0026rsquo;s chi-square test or Fisher\u0026rsquo;s exact test, as appropriate. For numerical variables, Student\u0026rsquo;s t-test (with equal or unequal variances) will be used, or the Mann-Whitney test when normality is violated. Repeated measures comparisons over time will be conducted using ANOVA or the Friedman test for non-parametric data.\u003c/p\u003e\n\u003cp\u003eNormality will be tested using the Shapiro-Wilk test, and variance homogeneity will be assessed using Levene\u0026rsquo;s test. Statistical significance will be considered at p \u0026lt; 0.05. All analyses will be performed using IBM SPSS software, version 27.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterim analyses {21b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo formal interim statistical analyses are planned for this trial. However, participant safety will be periodically monitored by the Data Monitoring Committee (DMC), with particular attention to the occurrence of serious adverse events or unforeseen risks. If significant safety concerns are identified, the DMC may recommend temporary suspension, protocol modifications, or study termination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods for additional analyses (e.g. subgroup analyses) {20b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSensitivity analyses will be conducted to assess the impact of missing data and protocol deviations on the robustness of results. Although the study does not formally plan additional analyses, exploratory stratifications may be reported if relevant findings arise during the primary analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMissing data will be handled using multiple imputation, assuming, when plausible, that data are missing at random (MAR). However, in cases of follow-up interruption due to participant withdrawal or if other non-random missing data patterns are identified, sensitivity analyses will be conducted to assess the impact of these losses on the robustness of the primary results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to give access to the full protocol, participant level-data and statistical code {31c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing completion of the study, the full protocol, anonymized participant-level dataset, and statistical code may be made available to other researchers who submit a justified scientific request, are affiliated with recognized research institutions, and commit to ethical and confidential use of the data. Sharing of materials will be subject to prior approval by the Research Ethics Committee, when applicable, and will comply with all applicable confidentiality and research ethics standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOversight and monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the coordinating centre and trial steering committee {5d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coordinating team was composed of the principal investigator, who was responsible for study design and clinical assessments, along with healthcare professionals affiliated with the participating center, including physicians, nurses, nursing technicians, and volunteer medical students. This team was responsible for implementing the interventions, collecting data, and clinically monitoring participants, ensuring the methodological integrity of the trial. Professor Dr. Nivaldo Antonio Parizotto, PhD, academic supervisor of the project, also generated the randomization sequence.\u003c/p\u003e\n\u003cp\u003eParticipant safety was monitored by a Data Monitoring Committee (DMC), composed of a single independent member who was blinded to allocation: Dr. Matheus Rodrigues de Souza, a physician and statistician not affiliated with the study team. The interventionist physician, who did not participate in clinical assessments, was responsible for reporting any adverse events directly to the DMC. The principal investigator did not participate in the analysis of these events, in order to maintain blinding. Monthly meetings were held by the coordinating team to review study progress, focusing on operational and logistical aspects, while maintaining blinding with respect to outcomes and adverse events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the data monitoring committee, its role and reporting structure {21a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Data Monitoring Committee (DMC) was composed of a single independent member: Dr. Matheus Rodrigues de Souza, a physician and statistician, who was blinded to group allocation and had no affiliation with the study team. His role was to periodically review participant safety, focusing on the identification of serious adverse events. The DMC could recommend trial suspension or protocol modifications if relevant risks to participant safety were identified.\u003c/p\u003e\n\u003cp\u003eThe committee did not conduct interim efficacy analysis, focusing exclusively on safety monitoring and harm control. As no serious adverse events were identified during the study, no safety reports were issued to the Research Ethics Committee or the principal investigator. The DMC did not have authority to implement changes directly; any recommendations would be evaluated by the principal investigator and, when applicable, submitted for prior ethics approval, in accordance with regulatory standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse event reporting and harms {22}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll adverse events (AEs) were systematically recorded using specific forms from the time of informed consent signing until the end of follow-up. As an active monitoring measure, participants were contacted via WhatsApp five days after each intervention for clinical evaluation and early identification of AEs.\u003c/p\u003e\n\u003cp\u003eInformation regarding severity, duration, and potential relationship to the intervention was collected. Causality assessment was performed by a member of the Data Monitoring Committee (DMC), based on medical chart review and clinical records. No serious adverse events (SAEs) were identified during the study, and therefore no reporting to the Research Ethics Committee was required. All study personnel were trained beforehand to identify and report adverse events in a standardized manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency and plans for auditing trial conduct {23}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo formal independent audit was planned for this study. Internal monitoring was conducted by the trial team itself, with the goal of ensuring protocol compliance and participant safety. Safety was periodically monitored by the Data Monitoring Committee (DMC), with a focus on identifying serious adverse events.\u003c/p\u003e\n\u003cp\u003eReports to the Research Ethics Committee (REC) were planned for any relevant intercurrences, protocol modifications, or identified risks, in accordance with regulatory requirements. As the study proceeded as planned and no serious adverse events or protocol changes occurred, no formal reports to the REC were required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll substantial protocol amendments (including methodological changes, outcomes, sample size, inclusion/exclusion criteria, or ethical conduct) would be communicated to the Research Ethics Committee (REC) and registered in Plataforma Brasil and the ReBEC, as applicable. No changes would be implemented without prior ethical approval.\u003c/p\u003e\n\u003cp\u003eIf the Data Monitoring Committee (DMC) had recommended adjustments to the protocol for safety reasons, such recommendations would have been reviewed by the principal investigator and, if appropriate, submitted to the REC for prior approval.\u003c/p\u003e\n\u003cp\u003eDuring the course of the study, no substantial amendments to the protocol were necessary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissemination plans {31a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this clinical trial will be disseminated through publications in peer-reviewed scientific journals, presentations at relevant medical conferences, and registration in public platforms such as ClinicalTrials.gov and ReBEC. A summary of the results in lay language will be made available to participants upon request.\u003c/p\u003e\n\u003cp\u003eThere will be no restrictions on data publication, ensuring full transparency in communication with healthcare professionals, participants, and the scientific community. Full dissemination of the findings will occur only after this protocol is accepted for publication.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis clinical trial faced relevant practical and operational challenges, particularly regarding participant retention throughout the 12-month follow-up period. One of the main obstacles was maintaining consistent communication with participants during follow-up visits, especially among those with logistical limitations or transportation difficulties.\u003c/p\u003e\n\u003cp\u003eThere was a tendency toward reduced adherence to long-term clinical assessments (at 6 and 12 months), especially after completion of the intervention sessions, possibly due to the perception that treatment had ended. To overcome this limitation, a more active follow-up approach was necessary, with personalized contact and reinforcement of the importance of clinical monitoring for participant safety and data quality.\u003c/p\u003e\n\u003cp\u003eDetailed planning of the interventions and biological sample collection proved essential to avoid delays and ensure adherence to the timeline. To minimize losses and ensure protocol compliance, the research team carried out systematic follow-up, with an emphasis on organized scheduling and direct support to participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis protocol corresponds to version 1.0, dated April 22, 2025.\u003c/p\u003e\n\u003cp\u003eEthics approval was granted on April 19, 2022.\u003c/p\u003e\n\u003cp\u003eParticipant screening began in September 2022.\u003c/p\u003e\n\u003cp\u003eThe trial was registered in the Brazilian Clinical Trials Registry \u0026ndash; ReBEC (RBR-2hgwvgy) in July 2023, retrospectively. At the time, the research team was not fully aware that prospective registration in a public trial registry was a mandatory condition for publication in international journals, which explains the delay.\u003c/p\u003e\n\u003cp\u003eClinical interventions were initiated in March 2023, and the study was formally concluded in September 2025.\u003c/p\u003e\n\u003cp\u003eAt the time of this submission, the trial database has been finalized and technically prepared for analysis, including multiple imputation to handle missing data. The technical preparation was conducted by an independent data analyst under the supervision of the research team, while maintaining the blinding of the principal investigator.\u003c/p\u003e\n\u003cp\u003eThe principal investigator remains blinded to group allocation and has not accessed intervention-specific data. Final statistical analysis has not yet been initiated and will be performed only after acceptance of this protocol for publication.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used throughout the manuscript:\u003c/p\u003e\n\u003cp\u003eGSM: Genitourinary Syndrome of Menopause\u003c/p\u003e\n\u003cp\u003eRF: Radiofrequency\u003c/p\u003e\n\u003cp\u003eVHIS: Vaginal Health Index Score\u003c/p\u003e\n\u003cp\u003eFSFI: Female Sexual Function Index\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the study participants, the clinical team at Hospital Regional de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes, the nurses and nursing technicians from Cl\u0026iacute;nica Dalbem who volunteered in carrying out the interventions, and the students from Universidade do Estado de Mato Grosso (UNEMAT) who contributed to data collection and participant follow-up throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions {31b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCCGDT was the principal investigator. She conceived the study, developed the protocol, conducted the pre-intervention clinical assessments, and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eCMMGD contributed to the protocol development and conducted the clinical interventions as outlined in the study design.\u003c/p\u003e\n\u003cp\u003eMRS acted as the Data Monitoring Committee (DMC) member. He monitored the integrity of the clinical data and contributed to the technical review of the manuscript.\u003c/p\u003e\n\u003cp\u003eNAP served as academic advisor. He contributed to the methodological design, generated the randomization sequence, and critically reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding {4}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has not received external funding. The devices used in the interventions (laser and radiofrequency equipment) were provided by the principal investigator. Logistical support \u0026mdash; including clinical infrastructure, human resources, and technical assistance for the procedures \u0026mdash; was provided by the Regional Hospital de C\u0026aacute;ceres Dr. Ant\u0026ocirc;nio Fontes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials {29}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data generated and/or analyzed during this study may be made available upon reasonable request to the corresponding author, provided that ethical principles and participant confidentiality are upheld. Data sharing will be subject to approval by the Research Ethics Committee, when applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate {24}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Research Ethics Committee (REC) of Universidade Brasil under approval number 5.357.602. Written informed consent was obtained from all participants prior to the initiation of any study procedures, in accordance with national and international ethical guidelines. The trial was also registered with ReBEC (Registro Brasileiro de Ensaios Cl\u0026iacute;nicos), as required by current regulatory standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication {32}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included the collection of clinical images for documentation purposes; however, no specific consent was obtained for their use in scientific publications. Therefore, no identifiable or non-identifiable images will be used in any reports, publications, or dissemination materials. The absence of this additional consent did not affect the ethical conduct of the study, and all other ethical guidelines were strictly followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests {28}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or non-financial interests that could be interpreted as conflicts of interest related to this study. C.C.G.D.T., N.A.P., M.R.S., and C.M.M.G.D. confirm the absence of competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information (optional)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC\u0026aacute;ssia Caroline Garcia Dalbem Teles is a gynecologist and obstetrician, holds a Master\u0026rsquo;s degree, and is a faculty member in the medical program at Universidade do Estado de Mato Grosso (UNEMAT) and also affiliated with Universidade Brasil, with specialization in lower genital tract pathology and colposcopy.\u003c/p\u003e\n\u003cp\u003eCatarina Maria Mesquita Garcia Dalbem is a gynecologist, holds a Master\u0026rsquo;s degree, and is a professor at Universidade do Estado de Mato Grosso (UNEMAT), also affiliated with Universidade Brasil.\u003c/p\u003e\n\u003cp\u003eMatheus Rodrigues de Souza is a physician specializing in statistical analysis and clinical trials, affiliated with Universidade do Estado de Mato Grosso (UNEMAT).\u003c/p\u003e\n\u003cp\u003eNivaldo Antonio Parizotto is a physiotherapist with a PhD in Electrical Engineering from the University of Campinas (UNICAMP) and postdoctoral training at the Wellman Center for Photomedicine, Harvard Medical School. He is currently a Senior Full Professor at the Federal University of S\u0026atilde;o Carlos (UFSCar) and a faculty member of the Graduate Program in Biomedical Engineering at Universidade Brasil.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNappi RE, Palacios S, Panay N, Particco M, Krychman ML. Vulvar and vaginal atrophy in four European countries: evidence from the European REVIVE Survey. \u003cem\u003eClimacteric\u003c/em\u003e. 2016;19(2):188\u0026ndash;97. doi:10.3109/13697137.2015.1107039.\u003c/li\u003e\n \u003cli\u003eSalvatore S, Nappi RE, Zerbinati N, Calligaro A, Ferrero S, Origoni M, et al. A 12-week treatment with fractional CO₂ laser for vulvovaginal atrophy: a pilot study.\u0026nbsp;\u003cem\u003eClimacteric\u003c/em\u003e. 2014;17(4):363\u0026ndash;9. doi:10.3109/13697137.2014.899347.\u003c/li\u003e\n \u003cli\u003ePerino A, Calligaro A, Forlani F, Tiberio C, Cucinella G, Svelato A, et al. Vulvo-vaginal atrophy: a new treatment modality using thermo-ablative fractional CO₂ laser.\u0026nbsp;\u003cem\u003eMaturitas\u003c/em\u003e. 2015;80(3):296\u0026ndash;301. doi:10.1016/j.maturitas.2014.12.006.\u003c/li\u003e\n \u003cli\u003eGambacciani M, Palacios S. Laser therapy for the restoration of vaginal function.\u0026nbsp;\u003cem\u003eMaturitas\u003c/em\u003e. 2017;99:10\u0026ndash;5. doi:10.1016/j.maturitas.2017.01.012.\u003c/li\u003e\n \u003cli\u003eKamilos MF, Borrelli CL. New therapeutic option in genitourinary syndrome of menopause: pilot study using microablative fractional radiofrequency.\u0026nbsp;\u003cem\u003eEinstein (S\u0026atilde;o Paulo)\u003c/em\u003e. 2017;15(4):445\u0026ndash;51. doi:10.1590/S1679-45082017AO4051.\u003c/li\u003e\n \u003cli\u003eRosen R, Brown C, Heiman J, Leiblum S, Meston C, Shabsigh R, et al. The Female Sexual Function Index (FSFI): a multidimensional self-report instrument for the assessment of female sexual function.\u0026nbsp;\u003cem\u003eJ Sex Marital Ther\u003c/em\u003e. 2000;26(2):191\u0026ndash;208. doi:10.1080/009262300278597.\u003c/li\u003e\n \u003cli\u003eBorges VL, Medeiros SF. Valida\u0026ccedil;\u0026atilde;o de question\u0026aacute;rio para avaliar a fun\u0026ccedil;\u0026atilde;o sexual feminina ap\u0026oacute;s menopausa.\u0026nbsp;\u003cem\u003eRev Bras Ginecol Obstet\u003c/em\u003e. 2009;31(6):293\u0026ndash;9. doi:10.1590/s0100-72032009000600005.\u003c/li\u003e\n \u003cli\u003eAvery K, Donovan J, Peters TJ, Shaw C, Gotoh M, Abrams P. ICIQ: a brief and robust measure for evaluating the symptoms and impact of urinary incontinence. \u003cem\u003eNeurourol Urodyn\u003c/em\u003e. 2004;23(4):322\u0026ndash;30. doi:10.1002/nau.20041.\u003c/li\u003e\n \u003cli\u003eFlight L, Julious SA. Practical guide to sample size calculations: non-inferiority and equivalence trials. Pharm Stat. 2016 Jan-Feb;15(1):80-9. doi: 10.1002/pst.1716. Epub 2015 Nov 25. PMID: 26604186.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidade do Estado de Mato Grosso (UNEMAT)","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Genitourinary syndrome of menopause, CO₂ laser, fractional microablative radiofrequency, vaginal atrophy, non-hormonal therapy, Vaginal Health Index Score, clinical trial","lastPublishedDoi":"10.21203/rs.3.rs-6699446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6699446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eGenitourinary Syndrome of Menopause (GSM) affects a significant number of postmenopausal women, leading to symptoms such as vaginal atrophy, dryness, and discomfort. Hormonal therapies are not suitable for all patients, particularly those with contraindications to hormone replacement therapy. This study aimed to compare the efficacy and safety of two non-hormonal treatments\u0026mdash;microablative CO₂ laser and fractional microablative radiofrequency\u0026mdash;in the management of GSM.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eThis is a double-blind, randomized, non-inferiority clinical trial involving 40 postmenopausal women diagnosed with GSM. Participants will be randomized to receive either microablative CO₂ laser or fractional microablative radiofrequency in three monthly sessions. The primary outcome is the Vaginal Health Index Score (VHIS), which assesses elasticity, pH, lubrication, epithelial integrity, and mucosal condition. Secondary outcomes include sexual function (Female Sexual Function Index - FSFI), GSM symptom severity, and urinary symptoms. Follow-up assessments will occur at 3, 6, and 12 months after the intervention. Histological analysis will be performed on vaginal biopsies from a subset of participants. The study was approved by the Research Ethics Committee of Universidade Brasil (approval no. 5.357.602), and all participants will provide informed consent prior to enrollment.\u003c/p\u003e\u003ch2\u003eDiscussion:\u003c/h2\u003e \u003cp\u003eThe findings from this study contribute to the comparative understanding of the effectiveness of CO₂ laser and fractional microablative radiofrequency as non-hormonal treatment options for GSM. The results have the potential to influence clinical practice by providing safe and effective therapeutic alternatives for women who are not candidates for hormonal therapy.\u003c/p\u003e\u003ch2\u003eTrial registration:\u003c/h2\u003e \u003cp\u003eThis trial is registered in the Brazilian Clinical Trials Registry (ReBEC): RBR-2hgwvgy. Registered on July 2023. UTN: U1111-1282-9195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy\u003c/span\u003e\u003cspan address=\"https://ensaiosclinicos.gov.br/rg/RBR-2hgwvgy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. This is a retrospectively registered trial.\u003c/p\u003e","manuscriptTitle":"Treatment of Genitourinary Syndrome of Menopause with Microablative CO₂ Laser versus Fractional Microablative Radiofrequency: A Double-Blind, Randomized, Non-Inferiority Clinical Trial Protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 13:24:54","doi":"10.21203/rs.3.rs-6699446/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b954a1e3-d6fe-4c20-bed3-2980453a5547","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48741292,"name":"Obstetrics \u0026 Gynecology"}],"tags":[],"updatedAt":"2025-05-22T13:24:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 13:24:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6699446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6699446","identity":"rs-6699446","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}