Effect of radiofrequency and pelvic floor muscle training in the treatment of women with vaginal laxity: A study protocol.

BACKGROUND: Vaginal laxity is an underreported condition that negatively affects women's sexual function and their relationships. Evidence-based studies are needed to better understand this complaint and to discuss its treatment options. Thus, we present a study protocol to compare the effect of radiofrequency and pelvic floor muscle training in the treatment of women with complaints of vaginal laxity. METHODS/
DESIGN: This is a prospective, parallel-group, two-arm, randomized clinical trial (Registry: RBR-2zdvfp-REBEC). Participants will be randomly assigned to one of the two groups of intervention (Radiofrequency or Pelvic Floor Muscle Training). The study will be performed in the Urogynecology outpatient clinic and in the physiotherapy outpatient clinic at the State University of Campinas-UNICAMP and will include women aged ≥ 18 years and with self-reported complaints of vaginal laxity. Participants will be assessed at baseline (pre-intervention period) and will be followed up in two periods: first follow-up (30 days after intervention) and second follow-up (six months after intervention). EXPECTED
RESULTS: The results of this randomized clinical trial will have a positive impact on the participants' quality of life, as well as add value to the development of treatment options for women with complaints of vaginal laxity. TRIAL REGISTRATION: Registry: RBR-2zdvfp-Registro Brasileiro de Ensaios Clínicos-REBEC (19/02/2020).

Introduction

Vaginal laxity (VL) is defined by the International Urogynecological Association (IUGA) and the International Continence Society (ICS) as a complaint of excessive vaginal flaccidity [1]. This condition is rarely discussed between patients and their doctors, possibly due to the lack of evidence-based treatments, embarrassment and lack of knowledge in the assessment of this condition [2]. According to urogynecologists, VL still presents itself as an underreported condition with reports of discomfort that can affect sexual function and relationships [3, 4]. The way women perceive their genitalia has a strong and positive impact on their sexual function [5].

The prevalence of VL is 24% and appears to be associated with younger age, vaginal births, pelvic organ prolapse (POP) symptoms or physical exam findings. Therefore, it is a somatic, not psychogenic, dysfunction [6].

It is speculated that pregnancy and childbirth play a role in VL [3]. Although there is no proven link between VL and childbirth, research shows that vaginal delivery can result in pelvic floor injury [6, 7]. Pelvic floor and vagina trauma during pregnancy and vaginal delivery can lead to the lengthening of the vaginal opening leading to permanent changes in sexual and physical sensitivity during sexual intercourse. These changes promote an important reduction in the quality of life of women and their partnership [8, 9].

Potential consequences associated with vaginal delivery that extend beyond the postpartum period are urinary incontinence (UI), POP, chronic pelvic pain (CPP), and sexual dysfunction [10-13]. Not all women adapt to the psychological and physical changes in the postpartum period, which can lead to changes in the emotional relationship with their partner [14]. Both vaginal delivery and levator ani muscle trauma are associated with an increase in the diameter of the genital hiatus [15]. The genital hiatus is limited by the puborectalis muscle, a component of the levator ani muscle, and appears to play an important role in defining the high vaginal pressure zone [16]. Avulsion of the levator ani muscle, especially if proven bilaterally, would have some effect on female sexual function [17].

The diagnosis of VL has been based on patients’ self-reporting. A comprehensive medical history, physical examination and psychosexual assessment are the initial steps to properly identify patients with VL [18].

The reduction in vaginal sensation during sexual intercourse may be related to anatomical damage to the perineal body, POP stage 1, laxity of the vaginal canal or introitus, damage to the nerves and connective tissue during pregnancy and childbirth or, potentially, a combination of these factors [19].

Surgical and non-surgical treatments for VL have been proposed. Surgical procedures for VL such as posterior colporrhaphy or perineorrhaphy are more commonly recommended. These procedures aim to reduce the size of the vaginal introitus, not necessarily treating the VL pathophysiology. Besides, 83% of the interviewed urogynecologists reported concerns with a potential risk for post-operative dyspareunia [3]. Post-surgical dyspareunia would further impair the quality of life of a woman who already complained of sexual dysfunction. Thus, it is necessary to develop non-surgical techniques that can assist in the treatment of other factors associated with VL, such as muscle hyperdistensibility and not just surgically reducing the size of the genital hiatus.

A non-surgical option for the treatment of VL includes pelvic floor muscle training (PFMT), which was initially recommended as a first-line treatment for UI [2, 20]. Pelvic floor muscle function appears to play an important role in female sexual function, and contraction of the levator ani muscle appears to increase the sexual response [21]. The contraction of the pelvic floor muscles also plays an important role in the female orgasmic response. Women with weak muscles who receive pelvic floor rehabilitation and strengthen the muscles in that region perceive a positive effect on their sex life [22]. Pelvic floor muscle training could have an effect on the hypertensile muscles of women complaining of VL.

Another non-surgical therapeutic possibility to treat VL is radiofrequency (RF). Despite the scarcity of controlled clinical trials to evaluate the therapeutic advantages, safety and efficacy of radiofrequency [23], the studies carried out to date have shown good tolerance, as well as, subjective improvement of vaginal narrowing, sexual function and decreased sexual discomfort [2] with effects maintained by 12 months and without any adverse events [8]. RF seems to improve vaginal vascularization and collagen fiber reorganization, which may also contribute to a decrease in the sensation of VL [24].

To our knowledge, to date, no clinical trial has been developed to assess the role of pelvic floor muscle and radiofrequency training in VL. Thus, the general objective will be to compare the effect of RF and PFMT in women with VL symptoms. The specific objectives are related to the assessment of the sexual function, vaginal symptoms, and sexual distress, as well as, to assess the impact of UI on patients’ quality of life. The POP staging, contractility, and pelvic floor muscle function will be also evaluated. Finally, we will assess the impression of improvement in VL complaints after the interventions.

Our hypothesis is that RF will be different from PFMT in treating women with VL symptoms.

Materials and methods

Trial design

This is a prospective, parallel-group, two-arm, randomized clinical trial. It involves three assessments in which primary and secondary outcomes will be evaluated: one pre-intervention visit, one 30-day post-intervention visit, and a six-month consultation after the intervention. Participants will be randomly assigned to one of the two groups of intervention (RF or PFMT). The study will follow the CONSORT recommendations [25] and the SPIRIT Statement (Standard Protocol Items: Recommendations for Interventional Trials) [26]. Fig 1 shows the detailed study steps.

Fig 1

Description of the study steps.

F1: Follow-up (30 days after intervention); F2: Follow-up (6-months after intervention); w: week; PFMT: Pelvic Floor Muscle Training; BMI: Body Mass Index; ICIQ-VS: International Consultation on Incontinence Questionnaire—Vaginal Symptoms; ICIQ-SF: International Consultation on Incontinence Questionnaire Short-Form.

The term VL was recently defined and little is known about this complaint. There is still no gold standard treatment for VL and further studies are needed to understand its pathophysiology. Although its pathophysiology is not completely known, there is a consensus on the association of VL with pregnancy and childbirth [2, 10, 27]. Some proposed mechanisms involve overstretching of the vaginal walls and introitus during vaginal birth and an increase in levator ani hiatal dimensions resulting from macro and microtrauma of the levator ani muscle [7, 17]. Although supervised PFMT is recommended as a first-line treatment for stress or mixed UI in women by most of the guidelines [28-30], more studies are needed to demonstrate the effect of PFMT on female sexual function. A randomized controlled trial concluded that women reporting improvement in sexual function demonstrated greatest increase in PFM strength and endurance [31].

Regarding RF, a recent randomized, multicenter, sham-controlled clinical trial found a statistically significant and clinically important improvement of VL with RF when compared with Sham treatment [32]. In our study, RF will be applied once every 4 weeks (a total of three applications) and will probably be less likely to face problems related to treatment adherence when compared to PFMT. Although the RF procedure has been shown to be well tolerated, adverse effects may occur [32, 33]. PFMT is generally free of adverse effects.

Study setting

Patient recruitment and assessment/treatment will be carried out in the Urogynecology outpatient clinic at the School of Medical Sciences and in the Physiotherapy outpatient clinic at the Centro de Atenção Integral à Saúde da Mulher (CAISM)—Hospital da Mulher Professor Dr. José Aristodemo Pinotti, both units affiliated to the State University of Campinas—UNICAMP.

Study population

Women with self-reported complaints of VL. There is no objective and standardized diagnostic evaluation for VL and its pathophysiological mechanism is not yet known [34].

Sample size

The sample calculation was based on the study by Krychman et al. [32], who demonstrated that RF therapy was associated with significant clinical and statistically significant improvement in sexual function in women with VL, when data analysis was performed in a group containing 73 patients. To calculate the sample of the present study, we used values of sexual function assessed using the FSFI questionnaire. There was an increase of 7 points in the FSFI score in the group treated with radiofrequency and an increase of 3 points in the control group. When considering a study power of 80%, an alpha of 0.05 with two-tailed test, it was found that the minimum number of participants required in each group will be added to a percentage of 30% loss in the sample, totaling 68 women, 34 in each group (isolated RF and isolated PFMT).

Eligibility criteria

We will include women aged ≥ 18 and ≤ 60 years, with VL complaints assessed by direct question (yes / no) and by the VLQ [2] (very loose, moderately loose, slightly loose), and willing to attend treatments on the scheduled date and places.

Participants who present the following conditions will be excluded from the study: use of a pacemaker; decompensated heart disease; cognitive deficit; peripheral or central neurological disorders; the presence of any type of cancer; the presence of cervical dysplasia; history of active urinary or vaginal infection; decompensated metabolic diseases; patients undergoing physical therapy for pelvic floor disorders; patients using vaginal estrogen in the last 6 months; patients already undergoing surgery for prolapse or urinary or anal incontinence; patients with stage 2 POP onwards; force of contraction of the pelvic floor muscles equal to zero according to the modified Oxford scale [35].

Recruitment

Women will be recruited from the Urogynecology outpatient clinic (CAISM / UNICAMP) and by social media advertisements, publicity posters, and printed ads from this study. All patients will be contacted by telephone by the researcher who applies the eligibility criteria and sends the VLQ via email or virtual communication platforms. The recruited participants will have their names, contact numbers, and VLQ responses recorded in a spreadsheet. A contact telephone with a virtual communication platform specific to this study is available for participants to contact the researchers whenever necessary. Participants will later be called by phone for the initial assessment. Participants interested in the study who did not pass the eligibility criteria are referred to the Urogynecology outpatient clinic for follow-up.

Allocation

The randomization sequence will be carried out through a computer program, in a 1:1 allocation ratio, in two blocks. The numbers corresponding to the study groups (1. Radiofrequency and 2. Pelvic Floor Muscle Training) will be placed in opaque sealed envelopes that will be opened by the study participants after signing the consent form and undergoing initial assessment in the first clinical visit.

The researchers who will assist in completing the questionnaires and physical examination, the researchers responsible for ultrasound and the data analysts will be blinded for the treatment group to which the participants were randomized to.

Initial assessment (first clinical visit)

Patients registered on the recruitment spreadsheet will be contacted by phone and the initial evaluation will be scheduled. In the initial evaluation, the participants will go through a lecture given by the researcher (G.M.V.P) in order to present the study, the assessments, the interventions, and the follow-up periods. At this point, the participants will be able to have all questions answered about the study. Participants who agree to participate in the study will receive a consent form for reading and signing. Participants will have their personal details protected and a number will substitute their identities.

Interventions

The intervention period for both groups will be 12 weeks.

1- Radiofrequency

The RF group will receive three radio frequency applications at 4-week intervals, (an initial application, a second application after four weeks, and a third application also after four weeks) comprising 12 weeks of intervention. The four-week period between applications will allow adequate healing of the vaginal tissues submitted to the application of radiofrequency. The procedure will be performed by a trained researcher (G.M.V.P) with a supervision of an experienced urogynecologist (C.R.T.J/L.G.O.B).

The Wavetronic 6000 Touch device with the Megapulse HF FRAXX system (Loktal Medical Electronics, São Paulo, Brazil) will be used, equipped with an electronic energy fractionation circuit, connected to a vaginal electrode with 64 microneedles 200μ in diameter and 1mm in length, and divided into an array of eight columns, with eight needles each. When pressing the trigger pedal, these 64 needles are not energized simultaneously and the energy release is randomized in columns of eight needles in a predefined sequence, which does not allow two adjacent columns to fire in sequence, preventing the thermal sum of the columns (control fractional firing system (Smart Shoot). This allows for cooling between the points and the preservation of tissues adjacent to the vaporized points, so that neocolagenesis and neoelastogenesis can occur, through fibroblastic stimulation [24].

1.1-Procedure. Topical anesthesia in the posterior vestibule and vaginal opening (mucosa) with 2% lidocaine gel, 2 to 3 minutes before the procedure (Table 1). A patient in a lithotomy position, with the lower limbs flexed and supported, will be introduced a disposable (high-impact polystyrene) vaginal speculum. A careful vaginal examination will be performed for any changes in the vaginal wall. Whiff test will be performed using a swab to collect vaginal discharge. A drop of 10% potassium hydroxide will be added over the vaginal secretion [36]. If a characteristic fishy odor is felt, the patient will not undergo RF and will be referred for evaluation of possible vaginosis. If the vaginal wall is intact and the whiff test is negative, the procedure will be continued, starting with topical anesthesia of the vaginal walls with 10% lidocaine spray. After 2 minutes, vaginal antisepsis with 2% aqueous chlorhexidine and cleaning with sterile 0.9% saline will be performed. After cleaning, the entire liquid content of the serum will be wiped with sterile gauze before starting to apply the RF.

Table 1

Pelvic floor muscle training and radiofrequency sessions according to the treatment duration.

Period	Interventions
	Radiofrequency	Pelvic Floor Muscle Training
1 to 4 weeks	1st application	1st phase	2nd phase	3rd phase
	• 2% Lidocaine Gel• Vaginal Examination with speculum• Whiff Test• 10% lidocaine spray• Cleaning: 2% aqueous chlorhexidine and sterile 0.9% saline• RF: 45Watts, Low Energy program (40 milliseconds)• Post-procedure orientation: 10-day sexual abstinence	• PFM maximum contraction (6 r / sustained 6 s/ 6 s rest: 1 time). Supine position.• PFM maximal contraction + transverse contraction (6 r / sustained 6 s/ 6 s rest: 1 time). Supine position.• PFM maximal contraction + hip elevation (6 r / sustained 6 s/ 6 s rest: 1 time). Supine position.	• PFM maximum contraction (1 cough / 3 r). Supine position.• PFM maximal contraction with the lower limbs extended and abducted (6 r / sustained 6 s/ 6 s rest: 2 times). Supine position.• PFM contraction in three stages—mild, moderate, maximum (6 r: 2 times). Sitting position.• PFM maximal contraction (6 r / sustained 6 s/ 6 s rest: 2 times). Standing position.	• Pelvic mobilization (anterior and posterior tilts, lateral tilts and rotation of the pelvis). Standing position. No PFM contraction in this phase. 10 repetitions each pelvic movement.
5 to 8 weeks	2nd application	• PFM maximum contraction (6 r / sustained 8 s/ 8 s rest: 1 time). Supine position.• PFM maximal contraction + transverse contraction (6 r / sustained 8 s/ 8 s rest: 1 time). Supine position.• PFM maximal contraction + hip elevation (6 r / sustained 8 s/ 8 s rest: 1 time). Supine position.	• PFM maximum contraction (2 cough / 3 r). Supine position.• Fast PFM maximal (8 r: 2 time). Supine position.• PFM contraction in six stages—mild, moderate, maximum–maximum, moderate, mild (8 r: 2 times). Sitting position.• PFM maximal contraction (8 r /sustained 8 s/ 8 s rest: 2 times). Standing position.• PFM maximal contraction (8 r /sustained 8 s/ 8 s rest: 2 times). Four supports (hands and knees).	• Same Intervention (above)
	Same procedure (above)
9 to 12 weeks	3rd application	• PFM maximum contraction (6 r / sustained 10 s: 1 time). Supine position.• PFM maximal contraction + transverse contraction (6 r / sustained 10 s/ 10 s rest: 1 time). Supine position.• PFM maximal contraction + hip elevation (6 r / sustained 10 s/10 s rest: 1 time). Supine position.	• PFM maximum contraction (3 cough / 3 r). Supine position.• Fast PFM maximal (10 r: 2 time). Sitting position.• PFM contraction in six stages—mild, moderate, maximum–maximum, moderate, mild (10 r: 2 times). Sitting position.• PFM maximal contraction (10 r /sustained 10 s/10 s rest: 2 times). Standing position.• PFM maximal contraction (10 r /sustained 10 s/10 s rest: 2 times). Four to two supports (right hand and left knee/ left hand and right knee).	• Same Intervention (above)
	Same procedure (above)

The device will be calibrated in FRAXX mode, 45 Watts, Low (initial application) and Medium (second and third application) Energy program (40 and 60 milliseconds, respectively), and the fractional vaginal electrode will be used. Applications will be under direct view. The electrode will be lightly pressed against the mucosa, without pressing, so that all microneedles are in uniform contact with the tissue. The application will be carried out sequentially on the lateral vaginal walls, in rows, avoiding overlapping, starting from the proximal third of the vagina to the distal third in the vestibule exposed by the opening of the speculum. The speculum will be gently rotated to the anteroposterior position for the application of the anterior and posterior vaginal walls. At the end of the procedure, the speculum will be gently removed. Patients will be advised on post-treatment care and the use of 5% dexpanthenol cream in the vaginal opening is recommended two to three times daily if there is any discomfort in the region for 2 to 3 days and interrupt sexual intercourse for 10 days. Patients will be instructed to contact the researchers if they experience any discomfort or notice any changes in vaginal discharge. These patients will be evaluated by an experienced gynecologist. There will be three vaginal applications at 4-week intervals, totaling 12 weeks of treatment.

2- Pelvic floor muscle training

The patients in the PFMT group will be assisted during the sessions by an experienced physiotherapist (G.M.V.P). The participants of this group will have 12 individual sessions of supervised PFMT, lasting 40–60 minutes, once a week, and totaling 12 weeks of treatment and continue their treatment with home PFMT program. To follow the treatment at home, patients will receive a printed diary containing the complete PFMT program, with figures illustrating the positions and orientations for each exercise for the pelvic floor muscles. Patients will have the possibility to contact the physiotherapist for questions regarding home treatment by video call, audio call or messages through a telephone number made available exclusively for the study. Patients will be instructed not to perform other exercises for the pelvic floor, different from the exercises proposed by the intervention program of the present study. Our intervention program for PFMT is based on the studies of Bo et al. [37] and Dumoulin et al. [38].

2.1- Procedure. The first PFMT session is longer and focused on the careful evaluation of the pelvic floor muscles in order to identify any muscle condition that interferes with the progress of the intervention; guidance on the correct performance of the pelvic floor muscle contraction with the aid of vaginal palpation and educational material; presentation of the PFMT program; and finally, the first sequence of exercises.

Patients will be instructed to perform three phases of exercise in each session, with at least two sessions of the PFMT program per day (Table 1). The exercises will undergo progression every 4 weeks of intervention, totaling three progressions (6 repetitions, sustained for 6 seconds, 6 seconds for rest, 8 repetitions, sustained for 8 seconds, 8 seconds for rest, and 10 repetitions, sustained for 10 seconds, 10 seconds for rest). The first phase comprises three exercises of maximum contraction of the pelvic floor muscles in the supine position, with the lower limbs semiflected and the feet supported, associated with contraction transverse exercise, and hip elevation. The second phase comprises pre-contraction of the pelvic floor muscles associated with cough; maximum sustained contraction in the supine position with the lower limbs extended and abducted; fast contractions in both supine and sitting positions, contraction in three stages (mild, moderate, maximum) in a sitting posture; maximum sustained contraction in standing posture and four supports (hands and knees). The third phase comprises the relaxation period with breathing exercises associated with pelvic mobilization in a standing posture. No pelvic floor muscles contraction in this phase. Patients will be instructed on the importance of adhering to the PFMT. In case of two absences, the patients will be contacted and their permanence in the study will be reassessed.

Primary outcomes

The primary outcome will be the indication of improvement in the VL symptoms after the proposed interventions assessed through a single question with seven possible answers. The Global Response Assessment (GRA) [2] is a 7-level scale with response to the question: “How are you now (levels of vaginal laxity/tightness and sexual satisfaction) compared to before treatment” (markedly improved, moderately improved, slightly improved, no change, slightly worse, moderately worse, markedly worse?). This scale item has been already used to evaluate improvement in vaginal laxity symptoms after treatment [2].

Secondary outcomes

The description of secondary outcomes is described below together with their measurement instruments.

Female Sexual Function Index (FSFI) [39]: a brief and multidimensional instrument to assess sexual function in women. The questionnaire was developed and validated by Rosen et al. and consists of 19 items that investigate sexual response over the past four weeks and performance in six domains: sexual desire, arousal, lubrication, orgasm, satisfaction, and pain [39]. The validation in Portuguese took place in 2008 by Thiel et al. [40]. The answers are scored according to the sum of the items that make up each domain (simple score) and multiplied by the domain factor generating the weighted score [40]. The maximum score is 36 points, adding up the total of each domain. Wiegel et al. proposed a cut-off score to differentiate women with or without sexual dysfunction in the amount of 26.55 [41].

Female Sexual Distress Scale-Revised (FSDS-R): a self-report questionnaire with 13 questions scored on a 5-point Likert scale from 0 (never) to 4 (always) to assess the sexual distress. Sexual distress is characterized by a set of feelings (for example, unhappiness, guilt, frustration, stress, worry) and emotions that individuals have about their sexuality. It differs from sexual dysfunction related to symptoms of sexual function, such as arousal, orgasm and pain, separate from emotions [42]. The Portuguese translation was developed by Berenguer et al. [43].

International Consultation on Incontinence Questionnaire—Vaginal Symptoms (ICIQ-VS): is a 14-item questionnaire validated for the Portuguese language by Tamanini et al. that assesses the presence and the impact of vaginal symptoms, as well as their relationship with quality of life [44, 45].

International Consultation on Incontinence Questionnaire Short-Form (ICIQ-SF): validated in Portuguese by Tamanini et al., is a simple, brief, and self-administered questionnaire, capable of quickly and effectively assessing the impact of urinary incontinence on patients’ quality of life. It consists of four questions that assess the frequency, severity, and impact of urinary incontinence. Your score can vary from 0 to 21, the greater the commitment, the higher the total value [46, 47].

Pelvic Organ Prolapse Quantification (POP-Q): the ICS recommends POP description and staging using this instrument [48, 49]. The staging classification is defined as [50] Stage 0: there is no demonstrated prolapse; Stage I: the most distal part of the prolapse is more than 1 cm above the level of the hymen; Stage II: the most distal portion of the prolapse is between 1 cm above the hymen and 1 cm below the hymen; Stage III: the most distal portion of the prolapse is more than 1 cm beyond the plane of the hymen, but everted at least 2 cm less than the total vaginal length; Stage IV: complete eversion or eversion of up to 2 cm from the total length of the lower genital tract. The hymen is the reference point used to describe the quantitative prolapse and represents the zero point. Six anatomical points will be evaluated with the aid of a disposable graduated ruler. Two on the anterior vaginal wall (Aa and Ba). Two on the posterior vaginal wall (Ap and Bp) and two points on the upper vagina (C and D). The genital hiatus, the total vaginal length and the perineal body will also be measured. All points will be measured in Valsalva maximum, except the total vaginal length [50]. The ICS clinically defined significant POP in stage II or higher [49, 50].

Pelvic Floor Muscle Strength (PFMS): the strength of the pelvic floor muscles will be graduated according to the modified Oxford scale (5-point) by means of bi-digital vaginal palpation with the patients in the supine position with the lower limbs supported [35].

Pelvic Floor Muscle Morphometry (PFMM) and Vaginal Thickness (VT): the morphometry of the pelvic floor muscles will be assessed during rest, during contraction of the pelvic floor muscles, and during the Valsalva maneuver [51]. The vaginal thickness will be assessed in its proximal, middle, and distal third using two approaches—transabdominal and transvaginal [52, 53]. The equipment used for transabdominal, transvaginal and transperineal ultrasound measurements will be the GE Voluson 730 Expert® (GE Medical System Kretz-Technik GmbH and Co OHG, Zipf, Austria). The 2 to 6 MHz convex RAB4-8L 3D / 4D probe will be used to record the morphometry of the pelvic floor muscles. Measurements will be performed at rest, Valsalva maneuver, and pelvic floor muscle contraction with the patient in the supine position [51] for the angle of the levator plate, the anorectal angle, the thickness of the levator ani muscle, and the area of the levator hiatus in cm2. For vaginal thickness, probes 4C-D 2 at 5 MHz transabdominal and 5 to 9 MHz transvaginal will be used to measure the vagina in its proximal, middle, and distal thirds [52, 53].

Baseline assessment

After signing the consent form, patients will be submitted to an anamnesis that includes questions related to the date of birth, marital status, ethnicity, education, body mass index, physical activity, surgeries, prior diseases, and medication. Questions related to sexual life such as sexual activity (yes / no), type of sexual behavior (homosexual, heterosexual, bisexual), type of sexual intercourse (vaginal, anal or both), origin of VL complaint (participant, partner or both), time of VL symptoms (months). In addition, patients will be asked about their perception of VL symptoms. Questions related to obstetric history and urinary / intestinal habits will also be included.

Subsequently, patients who sign the consent form will be referred for a physical examination consisting of the assessment of the strength of the pelvic floor muscles and POP quantification. The questionnaires will be self-completed by the participants and collected at the end of the baseline assessment. Patients will then be referred for ultrasound exams. These procedures will be performed in the initial assessment (first visit).

Follow-up period assessment

Patients will be followed up in two periods after the interventions: first follow-up (30 days after intervention) and second follow-up (six months after intervention). The assessment procedures in the follow-up period will be the same as those used in the baseline physical examination, questionnaires, and ultrasound exams. We will add the Global Response Assessment.

Data collection and management

Researchers with over 20 years of research experience will coordinate data collection. An assistant researcher will be responsible for checking all signatures of the consent form and the answers to each questionnaire to ensure that there is no blank answer. A researcher will perform a physical examination of all patients in the study. An experienced physiotherapist (over 10 years) and specialist in women’s health, under the supervision of the main researchers, will carry out both the interventions and different researcher with experience and expertise in ultrasound will perform the ultrasound exams. Patients will be contacted and monitored by telephone.

The analysis of the collected data will be preceded by the elaboration of a computerized database where the variables will be coded in a data dictionary and validated. This database will often be filled in by an assistant researcher and supervised by the main researcher.

Harms

The suspension of the intervention will occur through the verification of significant levels of discomfort during the application of vaginal RF (Visual Analogue Scale), as well as the significant occurrence of events such as urinary tract infection, vulvovaginitis, irritation and vaginal injury after application of vaginal RF (telephone contact). In these cases, an appropriate medical treatment will be offered. Women will be discontinued if they miss any radiofrequency sessions or if their presence in physiotherapy sessions does not reach 80%.

Data analysis

Initially, a descriptive analysis of the data will be performed to characterize the research participants, in the form of values ​​of absolute frequency and percentage (relative) for categorical variables and values of mean and standard deviation for numerical variables.

Statistical analysis of comparison and correlation of the obtained data will be performed. The Kolmogorov-Smirnov test will be performed to analyze the sample’s normality. Depending on the results obtained in the normality test, the Analysis of Variance (data with normal distribution) or Wilcoxon and Mann-Whitney Test (non-parametric data) will be used for comparative analyzes between the groups. Likewise, Pearson or Spearman tests will be used for correlational analyzes. Categorical variables will be analyzed using the chi-square test or Fisher’s exact test. Statistical analyzes will be performed using the statistical program SAS System for Windows (Statistical Analysis System), version 9.4, adopting a significance level of 5% (p <0.05). Study endpoints will be analyzed primarily for the per protocol population, and repeated, for sensitivity reasons, for intention-to-treat population [54].

Ethical considerations

The present study has been analyzed and approved by the Research Ethics Committee of the State University of Campinas–UNICAMP–CAAE—12919119.9.0000.5404 (08/08/2019)–and CEP 3.495.558 (08/0/8/2019). This study is also registered in the Registro Brasileiro de Ensaios Clínicos–REBEC—RBR-2zdvfp as a clinical trial (19/02/2020).

All participants who agree to participate in the study will receive a consent form for reading and signing. In addition, participants will have their personal data protected and a number will replace their identities within the study.

Trial status

This trial is currently recruiting participants for the study. The initial assessments have also been started. This study was initiated in 2019 and is planned to finish in 2023.

Discussion

Vaginal laxity is a complaint that is still little discussed among patients and their physicians and the lack of evidence-based treatment negatively impacts the management of this condition. There is a need to evaluate non-surgical options that offer minimal adverse effects at lower costs for women with VL complaints.

The present study aims to investigate two types of non-surgical treatment for VL and to compare the effect of both therapies. If therapies prove to be equally effective for VL complaints, our study will open a path for non-surgical options for VL management. In addition, while we await evidence on the VL pathophysiology, our study will contribute to developing knowledge of treatment options for this condition that negatively affects women’s sexual lives and relationships.

Dissemination of study findings

The present study is a part of a Ph.D. thesis and its results will be presented to the scientific board of the State University of Campinas–UNICAMP and to national and international scientific conferences.

Study amendments

Any protocol amendments that are necessary will be effectively communicated and modified in the relevant parties (trial registry, Research Ethics Committee, funding agency, and journal). Any inquiries regarding the study will be properly answered by the researchers in the initial assessment period and during the period of the study.

SPIRIT 2013 checklist study protocol.

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24 Jun 2021

PONE-D-21-07963

EFFECT OF RADIOFREQUENCY AND PELVIC FLOOR MUSCLE TRAINING IN THE TREATMENT OF WOMEN WITH VAGINAL LAXITY: A STUDY PROTOCOL

PLOS ONE

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Kind regards,

Jose María Blasco, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent will be informed consent

3. Please include additional information regarding the surveys or questionnaires used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed the survey or questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. If the questionnaire is published, please provide a citation to the (1) questionnaire and/or (2) original publication associated with the questionnaire.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQv

5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 52. Abstract � It is not recommended to use acronyms directly without having explained them beforehand and this makes the abstract difficult to read. In this case, the action that each of the two groups is going to carry out is not clear.

Line 52. Introduction � The data presented in this section are correct, however, it is necessary to restructure this section because the thread of the reading is lost, not making clear until the end what one wants to study. It is also necessary to delve a little into the benefits of the different non-surgical actions in this pathology, in order to correctly guide the introduction.

Line 119. Introduction � The secondary objectives are somewhat confusing, they seem like a simple list, please rewrite this point to make it clearer.

Line 131. Trial design � Why is an assessment not carried out immediately after the intervention? It would be interesting to see if there is any kind of change in that phase.

Line 171. Study population � Why not put a roof or old cut? Are the same results expected in the elderly as in the adult, for example?

Line 192. Eligibility criteria � Data already exposed are repeated with the study population section, please review it.

Line 301. Procedure � Is rest time allowed between exercise and exercise? If yes, how long is this break?

Line 326. Primary outcomes� Is the proposed scale validated? Some more explanation of the main variable of the study is missing, since it is not clear.

Line 326 to 402. Outcomes (primary and secondary) � It would be interesting to provide the interrater and test-retest reliability data for all the variables.

Line 419. Follow-up Period Assessment � indicate that the measurements are from the end of the intervention.

Line 430 � It would be convenient to name the years of experience that the researchers who carry out both the intervention and the data collection have.

Line 461 � Indicate version of the SPSS program

Line 449. Data analysis � This is considered a non-inferiority work, please indicate it and explain in this section how it will be verified.

Line 542. References � Please keep the same line spacing as the rest of the article.

Reviewer #2: A study protocol entitled „EFFECT OF RADIOFREQUENCY AND PELVIC FLOOR MUSCLE TRAINING IN THE TREATMENT OF WOMEN WITH VAGINAL LAXITY: A STUDY PROTOCOL” has interesting topic and currently scoping.

Some methodological inacurracies should be addressed before the paper can be considered for publication.

Major

1. Describe the rationale for using a non-inferiority design of the study.

2. What are study objectives? What kind of effects of radiofrequency and pelvic floor muscle training are expected by the authors?

3. Please justify the established efficiency of PFMT in Vaginal Laxity

4. Where is the first visit taking place? Is the assessor going to be blindfolded?

5. Line 210-222

Authors wrote: „The numbers corresponding to the study groups (1. Radiofrequency and 2. Pelvic Floor Muscle Training) will be placed in opaque sealed envelopes that will be opened by the study participants after signing the consent form and undergoing initial assessment or first clinical visit.”

Will the participants know the group they are allocated to after signing the consent form and undergoing initial assessment or first clinical visit ?

6. Line 223-225

Is the person performing physical examination of PFMbe going to be blindfolded or is it going to be the same person conducting Pelvic Floor Muscle Training ? Which centre are PFM trainings going to be conducted in?

7. Line 374

Will Pelvic Organ Prolapse Quantification (POP-Q) be the procedure to compare the efficacy of two treatment methods? Will Pelvic Organ Prolapse Quantification (POP-Q) be used as criteria of exclusion? In lines 196 - 202 the authors state: Participants who present the following conditions will be excluded from the study: ……patients with stage 2 POP onwards;…”

8. Please describe in detail how the vaginal wall thickness measurements will be defined and how they will be performed by transvaginal and transabdominal USI.

9. Line 393

Which method will be used for morphometry of PFM? Which ultrasound parameters will be assessed?

10. Are all USI examinations going to be performed by the same examiner ?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Aug 2021

July 18th 2021

Dear Editors-in-Chief

Dr. Emily Chenette

PlosOne

I would like to thank you for the opportunity to have our study reviewed by the Plos One Editorial Board.

Dear Academic Editor

Dr. Jose María Blasco, Ph.D.

PlosOne

Thank you for considering our study for revision in this prestigious Journal. We believe that the comments will enrich enormously our work.

Our responses will be stated with track changes (highlighted in yellow) in the manuscript as recommended and in red in the present document.

Yours sincerely,

Luiz Gustavo Oliveira Brito MD/PhD

Corresponding Author (on behalf of the authors)

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: The style requirements were added as requested. Thank you.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether consent will be informed consent

Response: We removed the information from Study Amendments and added the information in lines 485-487. Thank you.

3. Please include additional information regarding the surveys or questionnaires used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed the survey or questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. If the questionnaire is published, please provide a citation to the (1) questionnaire and/or (2) original publication associated with the questionnaire.

Response: The description of the questionnaires are found in lines 339-344 and 350-379. Thank you.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQv

Response: The ORCID for the corresponding author has been provided. Thank you.

5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

Responde: OK. Thank you.

6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Response: The tables have been included as required. Thank you.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Partly

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Partly

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: No

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find

helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 52. Abstract � It is not recommended to use acronyms directly without having explained them beforehand and this makes the abstract difficult to read. In this case, the action that each of the two groups is going to carry out is not clear.

Response: Thank you for your suggestion. Please find the correction in lines 50-51.

Line 52. Introduction � The data presented in this section are correct, however, it is necessary to restructure this section because the thread of the reading is lost, not making clear until the end what one wants to study. It is also necessary to delve a little into the benefits of the different non-surgical actions in this pathology, in order to correctly guide the introduction.

Response: The Introduction has been changed as suggested. Thank you. Please find the text in lines 95-97; 102-111; 118-120, and 126-127.

Line 119. Introduction � The secondary objectives are somewhat confusing, they seem like a simple list, please rewrite this point to make it clearer.

Response: The secondary objectives have been corrected as required. Thank you. Please find the correction in lines: 130-134.

Line 131. Trial design � Why is an assessment not carried out immediately after the intervention? It would be interesting to see if there is any kind of change in that phase.

Response: We understand your question and appreciate your recommendations; however, it will not be possible to carry out the assessments immediately after the interventions due to the 10-day radiofrequency healing period. Therefore, due to healing and tissue repair, we decided to standardize the assessment after 30 days in both groups.

Line 171. Study population � Why not put a roof or old cut? Are the same results expected in the elderly as in the adult, for example?

Response: We thank you for this recommendation. Even though we are not unaware of the profile of the patient with vaginal laxity, we decided not to exceed 60 years of age. Please find this information in line 198.

Line 192. Eligibility criteria � Data already exposed are repeated with the study population section, please review it.

Response: We corrected the study population and inclusion criteria as suggested. Thank you. Lines 181-183, and 198.

Line 301. Procedure � Is rest time allowed between exercise and exercise? If yes, how long is this break?

Response: We included the rest time as required in the text and in table 1. Thank you. Lines: 316-317.

Line 326. Primary outcomes� Is the proposed scale validated? Some more explanation of the main variable of the study is missing, since it is not clear.

Response: We added more information regarding the primary outcome as required. Thank you. Lines: 339-344.

Line 326 to 402. Outcomes (primary and secondary) � It would be interesting to provide the interrater and test-retest reliability data for all the variables.

Response: As we only have one investigator performing the interventions, we did not consider the interrater and test-retest reliability data..

Line 419. Follow-up Period Assessment � indicate that the measurements are from the end of the intervention.

Response: We have indicated the required information in line 428. Thank you.

Line 430 � It would be convenient to name the years of experience that the researchers who carry out both the intervention and the data collection have.

Response: We added the required information in lines 435-439. Thank you.

Line 461 � Indicate version of the SPSS program

Response: We updated the required information in lines 473-475.Thank you.

Line 449. Data analysis � This is considered a non-inferiority work, please indicate it and explain in this section how it will be verified.

Response: We thank you for raising this question, which made us reflect on the study design. Since our sample size calculation has not added the range of an inferiority margin, we decided to review the study design. We decided to carry out a comparison between the interventions. We made the modifications in the following lines: 48; 135-136; 140, and 145 (reference 25).

Line 542. References � Please keep the same line spacing as the rest of the article.

Response: We corrected the reference spacing as required.

Reviewer #2: A study protocol entitled „EFFECT OF RADIOFREQUENCY AND PELVIC FLOOR MUSCLE TRAINING IN THE TREATMENT OF WOMEN WITH VAGINAL LAXITY: A STUDY PROTOCOL” has interesting topic and currently scoping.

Some methodological inacurracies should be addressed before the paper can be considered for publication.

Major

1. Describe the rationale for using a non-inferiority design of the study.

Response: We thank you for raising this question, which made us reflect on the study design. Since our sample size calculation did not include an inferiority margin, we decided to review the study design. We decided to carry out a comparison between the interventions. We made the modifications in the following lines: 48; 135-136; 140, and 145 (reference 25).

2. What are study objectives? What kind of effects of radiofrequency and pelvic floor muscle training are expected by the authors?

Response: Please find this information in lines 118-120, 126-127, and 129-136.

3. Please justify the established efficiency of PFMT in Vaginal Laxity

Response: As we said in the lines 112-120 and 495-498, there are no stablished, gold-standard treatments for vaginal laxity yet. Thus, we decided to compare non-surgical treatment options for vaginal laxity.

4. Where is the first visit taking place? Is the assessor going to be blindfolded?

Response: We added information as required. Lines: 227-229; 232, and 428-429. The assessors will not know which group the participants will belong.

5. Line 210-222

Authors wrote: „The numbers corresponding to the study groups (1. Radiofrequency and 2. Pelvic Floor Muscle Training) will be placed in opaque sealed envelopes that will be opened by the study participants after signing the consent form and undergoing initial assessment or first clinical visit.”

Will the participants know the group they are allocated to after signing the consent form and undergoing initial assessment or first clinical visit ?

Response: Study participants will only know which intervention group they will be allocated after they have signed the consent form and have undergone baseline assessment.

6. Line 223-225

Is the person performing physical examination of PFMbe going to be blindfolded or is it going to be the same person conducting Pelvic Floor Muscle Training ? Which centre are PFM trainings going to be conducted in?

Response: Thank you for raising this question. The person who will carry out the intervention will not be the same person who will carry out the assessment. Please find the correct information in lines 443-444.The training of the pelvic floor muscles will take place in the Urogynecology outpatient clinic at the School of Medical Sciences and in the Physiotherapy outpatient clinic at the Centro de Atenção Integral à Saúde da Mulher (CAISM) - Hospital da Mulher Professor Dr. José Aristodemo Pinotti.

7. Line 374

Will Pelvic Organ Prolapse Quantification (POP-Q) be the procedure to compare the efficacy of two treatment methods? Will Pelvic Organ Prolapse Quantification (POP-Q) be used as criteria of exclusion? In lines 196 - 202 the authors state: Participants who present the following conditions will be excluded from the study: ……patients with stage 2 POP onwards;…”

Response: Although we have excluded POP cases, the POP-Q classification is important because we would like to check if there will be changes in the measurements of genital hiatus, perineal body or other external measurement.

8. Please describe in detail how the vaginal wall thickness measurements will be defined and how they will be performed by transvaginal and transabdominal USI.

Response: Please find the information required in lines 411-413

9. Line 393

Which method will be used for morphometry of PFM? Which ultrasound parameters will be assessed?

Response: Please find the information required in lines 408-411.

10. Are all USI examinations going to be performed by the same examiner?

Response: Yes and the examiner will not be aware of the study groups.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Oct 2021

EFFECT OF RADIOFREQUENCY AND PELVIC FLOOR MUSCLE TRAINING IN THE TREATMENT OF WOMEN WITH VAGINAL LAXITY: A STUDY PROTOCOL

PONE-D-21-07963R1

Dear Dr. Brito,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jose María Blasco, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors,

After reviewing the corrections, it has been observed that all suggested changes have been noted to have been corrected or a rational response to the proposed suggestion justified.

Both the design and the language of the article are considered to be more than correct.

In addition, the article is considered to meet the minimum standards of the journal for publication.

finally indicate that for my part it is not necessary to make any additional changes.

Sincerely

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

29 Oct 2021

PONE-D-21-07963R1

Effect of radiofrequency and pelvic floor muscle training in the treatment of women with vaginal laxity: a study protocol

Dear Dr. Brito:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jose María Blasco

Academic Editor

PLOS ONE