Randomized, Double-blind, Placebo-controlled Clinical Trial for Evaluating the Efficacy of Fractional CO₂ Laser Compared With Topical Estriol in the Treatment of Vaginal Atrophy in Postmenopausal Women

Vera L. Cruz, MD; Marcelo L. Steiner, MD, PhD; Luciano M. Pompei, MD, PhD; Rodolfo Strufaldi, MD, PhD; Fernando L. Afonso Fonseca, PhD; Lucila H. Simardi Santiago, MD, PhD; Tali Wajsfeld, MD; Cesar E. Fernandes, MD, PhD

Disclosures

Menopause. 2018;25(1):21-28.

Methods

Study Design

Forty-five women were randomized in a double-blind, placebo-controlled clinical trial to compare the therapeutic responses to CO₂ laser, topical estriol, and the combination of both in the treatment of postmenopausal VVA. Participants were block-randomized into three groups of 15 participants consisting of CO₂ vaginal laser + placebo vaginal cream (laser, L), vaginal estriol cream + sham laser (estriol, E), or vaginal estriol cream + CO₂ vaginal laser (laser/estriol, LE) in an outpatient menopause clinic, located at Centro Atencao Integral Saude da Mulher (CAISM) in Sao Bernardo do Campo, Brazil, between January 2015 and May 2015. The study protocol was approved by ABC School of Medicine IRB and informed consent was obtained from all participants. Study was registered at the US National Institutes of Health (ClinicalTrials.gov) #NTC39495014.0.0000.0082.

Study Population

Study population included women between the ages of 45 and 70 who presented with amenorrhea for 24 months or longer and at least one moderate symptom of VVA (dyspareunia, dryness, or burning). Participants rated each of three VVA symptoms from 0 (no symptom) to 10 (very severe symptom) using the Visual Analog Scale (VAS) and symptoms were considered moderate if reported to be equal to or greater than 4 in VAS. Participants were excluded in the presence of BMI ≥35 kg/m²; chronic kidney or liver disease; drug-induced menopause; history of any form of cancer; previous vaginal radiotherapy; pap smear consistent with atypical squamous cells of undetermined significance, low-grade intraepithelial lesion or high-grade intraepithelial lesion in the previous 12 months; current use of vaginal lubricants or moisturizers; use of anabolic steroids, ospemifene or systemic estrogen therapy in the past 6 months or diagnosis of vulvovaginitis within 30 days prior to the study.

Study Interventions

Participants were randomized into one of three treatment arms: combined laser and estriol arm (LE), laser arm (L), and estriol arm (E). LE arm underwent two sessions of fractional CO₂ laser treatment at weeks 0 and 4 using the SmartXide2 system (Monalisa Touch, DEKA Laser, Florence, Italy) combined with 1 mg vaginal estriol therapy (Stele, Biolab, Brazil) 3 times a week for 20 consecutive weeks; L arm underwent two sessions of fractional CO₂ laser treatment at weeks 0 and 4 using the SmartXide2 system, combined with 1 mg estriol-substitute placebo 3 times a week for 20 consecutive weeks; and the E arm was assigned to two sessions of sham laser treatment at weeks 0 and 4 combined with 1 mg vaginal estriol (Stele, Biolab, Brazil) 3 times a week for 20 consecutive weeks.

Laser

The fractional microablative CO₂ laser SmartXide2 system (Monalisa Touch) was used in this study. A vaginal probe was gently inserted and manually rotated to provide a 360° treatment of the vaginal mucosa. Laser settings were based on previous studies and set to a power of 300 W, dwell time of 1,000 ms, dot spacing of 1,000 μm, and smart stack of 2.0.^[16] Participants were advised to avoid sexual activity for at least 3 days after each laser application as a mild inflammatory reaction previously reported could last up to 48 hours.^[17]

Placebo

Adequate placebo of fractional CO₂ laser was provided by adjusting power to 0.0 W while maintaining dwell time of 1,000 ms, point spacing of 1,000 μm, and smart stack of 2.0. This procedure is identical to real treatment; even the noise involved with firing the laser was maintained. The 1 mg estriol-substitute placebo topical cream was similar in appearance, odor, consistency, and packaging to the estriol cream.

Blinding

An unblinded nurse was responsible for programming laser parameters before the doctor and participant entered the room. Once programmed, a cover was placed over the screen to ensure proper blinding. The same nurse was responsible for the selection of the appropriate topical cream of either placebo or estriol for each participant. The same physician conducted all appointments and laser sessions.

Study Outcomes

The study consisted of five clinical visits (participant selection, weeks 0, 4, 8, and 20). Primary outcomes included improvement in Vaginal Health Index (VHI) and in VVA symptoms using the VAS. Both VHI and VVA were assessed at weeks 0, 8 and 20.

VHI analyzes the following five components on a scale of 1 to 5: elasticity, fluid volume, pH, epithelial integrity, and moisture. A minimal total score of 5 points indicates severe VVA, and a maximal total score of 25 points indicates no clinical signs of VVA.

Participants reported intensity of VVA symptoms using a 10-cm VAS. The scale's left extremity indicates the complete absence of symptoms (0) and the right extremity indicates the worst possible symptom (10). Participants rated VVA symptoms (dyspareunia, dryness, or burning) from 0 to 10.

Secondary outcome measures were assessed at weeks 0, 8, and 20 and consisted of the analysis of vaginal smear samples and the assessment of quality of sexual function using the validated Portuguese version of the Female Sexual Function Index (FSFI).^[21]

Vaginal smear samples were collected in a single scraping of the middle third of the lateral vaginal wall with an Ayre spatula; 100 cells were analyzed per specimen by the Pathology Department. Parabasal (P), intermediary (I), and superficial (S) cell counts were performed and multiplied by 0.0, 0.5, and 1.0, respectively. The sum of all three values comprises the maturation value (MV) of Meisels, and an increased percentage of P cells and I cells suggests a decrease in estrogen levels.^[22] MV values ranging from 0 to 49 indicate low estrogen effect, 50 to 64 indicate moderate estrogen effect, and 65 to 100 indicate high estrogen effect on the vaginal epithelium.^[23]

The FSFI questionnaire addresses six different domains (desire, arousal, lubrication, orgasm, satisfaction, and pain/discomfort) ranging from 0 (no sexual activity in the past 4 wk) or 1 (very dissatisfied) to 5 (very satisfied). The sum of questions related to each individual domain was multiplied by its unique predetermined factor and the sum of the 6 domain final scores was reported as the full-scale score. Full scale scores ranging from 2.0 (severe dysfunction) to 36.0 (absence of dysfunction) were used to evaluate sexual function throughout the study, with increased FSFI scores correlating to an improvement of symptoms.^[19] An optimal cut score of 26, reported by Wiegel et al,^[24] is currently used to differentiate between women with and without sexual dysfunction.

Sample Size

A standard deviation of 2.5 was adopted from an observational study on CO₂ laser therapy for VVA symptoms.^[16] To detect a three-point difference in the VHI with a P < 0.05 and a power ≥0.8, 12 women per arm were required. Taking into consideration a potential loss of 20% of participants throughout the study, 15 women were included in each treatment arm for a total study enrollment of 45 women.

Randomization

An independent nurse included women in either the combined, laser, or estriol treatment group according to a computer-generated randomization list. Randomization was carried out in blocks (block randomization) of 15 individuals with the help of a computer program.

Statistical Analysis

Average and standard deviation were performed in quantitative continuous data analysis, medians, and interquartile range in quantitative discrete data and qualitative variables were summarized using absolute numbers and percentages. Intention-to-treat (ITT) analysis and per-protocol analysis were used for analysis of primary outcomes. ANOVA was used in the comparison among study arms and multiple significant comparisons among arms were obtained using the least significant difference test. Comparison of treatment groups was performed using the Kruskal–Wallis test when data presented nonnormal distribution. A repeated measures ANOVA was used for data analysis in multiple time points and the Friedman test was used when data presented nonnormal distribution. Paired t test and Wilcoxon signed-rank test was used when data presented nonnormal distribution for comparison of baseline and week 20 within groups. Comparison of categorical data was performed using the χ ² test. Cohen d test was used to calculate effect sizes for primary outcomes and effect sizes were considered small if 0.2, medium if 0.5, and large if 0.8. Multivariate analysis was used for analysis of outcomes when baseline characteristics were statistically different among groups. Differences were considered significant with a P value of 0.05. Data were analyzed using the statistical analysis tool WinStat for Microsoft Excel version 2009.

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Table 1. Demographic and clinical characteristics of study participants at baseline
Characteristic	Laser (n = 15)	Estriol (n = 15)	Laser + estriol (n = 15)	P
Demographic
Age	55.9 ± 5.2	56.9 ± 6.0	55.7 ± 4.4	0.83
BMI	26.2 ± 3.7	27.8 ± 2.7	24.9 ± 3.9	0.1
Pregnancies	2.9 ± 1.6	2.5 ± 1.1	2.5 ± 0.9	0.62
Years since menopause	8.2 ± 7.0	8.7 ± 6.4	8.3 ± 6.7	0.98
VHI	11.7 ± 2.9	12.6 ± 4.2	12.6 ± 4.2	0.76^a
FSFI	19.60 ± 7.55	21.80 ± 8.95	16.19 ± 8.78	0.21
VAS
Dyspareunia	4.2 ± 3.8	3.2 ± 3.4	6.5 ± 3.9	0.065
Dryness	6.9 ± 3.7	5.6 ± 2.9	7.9 ± 3.0	0.153
Burning	3.4 ± 4.4	0.9 ± 1.6	4.9 ± 3.8	0.01 ^b
No. (%)
Previous HT	1 (7.7)	5 (35.7)	5 (33.3)	0.18^c
Smoking	2 (15.5)	2 (14.3)	2 (13.3)	0.98^c
Hot flushes	7 (53.8)	6 (42.9)	10 (66.7)	0.43^c
Orgasm	7 (53.8)	9 (64.3)	9 (60.0)	0.85^c
Libido	7 (53.8)	11 (78.6)	11 (73.3)	0.34^c

Items listed as means ± SD. P values of 0.05 were considered statistically significant.
BMI, body Mass Index; FSFI, Female Sexual Function Index; HT, hormone therapy; VAS, Visual Analog Scale (0-10, where 0 = no symptom and 10 = severe symptom); VHI, Vaginal Health Index.
^aKruskal-Wallis test.
^bLeast significant difference analysis showed group E vs LE and E vs L: P < 0.05.
^cChi-square test.
All others: ANOVA.

Table 2. Visual Analog Scale scores at 0, 8, and 20 weeks by treatment group
	Laser (n = 15)	Estriol (n = 15)	Laser + estriol (n = 15)	P ^a
Dyspareunia
Baseline	4.9 ± 3.7	3.2 ± 3.4	6.5 ± 3.9	0.09
Week 8	2.9 ± 2.9	0.6 ± 1.7	2.5 ± 3.8	0.16
Week 20	0.7 ± 1.5	0.2 ± 0.6	0.9 ± 1.8	0.95
P ^b	0.01	0.058	0.009
Dryness
Baseline	8.0 ± 2.6	5.6 ± 2.9	7.9 ± 3.0	0.07
Week 8	3.6 ± 2.6	2.4 ± 2.0	3.3 ± 2.9	0.57
Week 20	1.4 ± 2.0	0.5 ± 1.4	0.3 ± 0.7	0.35
P ^b	<0.001	<0.001	<0.001
Burning
Baseline	3.9 ± 4.5	0.9 ± 1.6	4.9 ± 3.8	0.017 ^c
Week 8	1.0 ± 2.0	0.1 ± 0.5	1.2 ± 2.7	0.33
Week 20	0.5 ± 1.5	0.1 ± 0.3	0.4 ± 1.1	0.95
P ^a	0.02	0.51	0.002

Intention-to-treat analysis. Items listed as mean ± SD. P values of 0.05 were considered statistically significant.
^aANOVA.
^bFriedman test.
^cLeast significant difference analysis showed group E vs LE and E vs L: P<0.05.
All others: Visual Analog Scale (0-10, where 0¼no symptom and 10 = severe symptom).

Table 3. FSFI scores (individual domains and total) at 0, 8, and 20 weeks by treatment group
	Laser (n = 13)	Estriol (n = 14)	Laser + estriol (n = 15)	P
Desire
Baseline	2.4 [1.5; 3.6]	2.4 [2.1; 3.6]	1.8 [1.2; 3.0]	0.19
Week	2.4 [1.8; 3.6]	2.4 [2.2; 3.6]	3.0 [1.2; 3.6]	0.99
Week 20	2.4 [1.8; 3.6]	3.0 [2.4; 3.6]	3.6 [1.8; 3.6]	0.76
P (baseline vs week 20)^a	0.39	0.63	0.005
Arousal
Baseline	2.4 [1.4; 4.0]	3.6 [2.1; 4.8]	2.7 [1.5; 4.5]	0.66
Week	2.4 [1.4; 3.4]	3.1 [2.0; 4.9]	3.6 [1.8; 4.2]	0.35
Week 20	3.0 [1.5; 3.6]	4.0 [2.2; 4.6]	3.9 [1.8; 4.5]	0.68
P (baseline vs week 20)^a	1.00	1.00	0.17
Lubrication
Baseline	4.2 [2.7; 5.0]	4.2 [2.8; 5.6]	2.7 [1.2; 4.2]	0.15
Week	2.7 [1.0; 5.1]	4.5 [2.0; 5,1]	4.2 [2.7; 5.4]	0.49
Week 20	3.0 [0.8; 4.5]	3.9 [2.9; 5,2]	3.6 [2.7; 4.8]	0.29
P (baseline vs week 20)^a	0.24	0.58	0.02
Orgasm
Baseline	4.0 [2.0; 4.8]	4.2 [2.9; 4.7]	3.6 [1.2; 4.8]	0.68
Week	2.8 [0.6; 4.4]	4.0 [0.0; 6.0]	4.0 [1.6; 5.6]	0.60
Week 20	2.4 [0.0; 4,6]	4.2 [2.3; 6.0]	4.4 [2.8; 5.6]	0.14
P (baseline vs week 20)^a	0.26	0.95	0.11
Satisfaction
Baseline	3.2 [1.6; 4.8]	4.8 [2.1; 5.7]	3.6 [1.2; 4.8]	0.46
Week	4.0 [1.4; 4.8]	4.8 [2.3; 5.7]	4.8 [2.8; 5.2]	0.40
Week 20	3.6 [1.4; 4.0]	4.6 [3.0; 5.7]	4.8 [2.8; 4.8]	0.13
P (baseline vs week 20)^a	0.72	0.60	0.09
Pain
Baseline	4.4 [1.6; 5.6]	4.8 [2.2; 5.7]	2.4 [1.2; 3.6]	0.04 ^b
Week	2.0 [0.0; 4.4]	5.2 [0.0; 6.0]	3.6 [1.2; 4.8]	0.39
Week 20	2.0 [0.0; 3.6]	6.0 [3.9; 6.0]	2.8 [1.6; 5.6]	0.006 ^c
P (baseline vs week 20)^a	0.04	0.16	0.02
Total
Baseline	18.6 [16.4; 24.6]	23.6 [17.5; 29.8]	18.7 [7.2; 22.6]	0.21
Week	18.0 [11.4; 20.7]	22.9 [8.4; 29.7]	22.6 [11.3; 26.3]	0.39
Week 20	14.4 [7.8; 22.4]	25.4 [16.8; 29.3]	23.6 [14.9; 28.6]	0.10
P (baseline vs week 20)^a	0.26	0.56	0.02

Items listed as median [interquartile range]. P values of 0.05 were considered statistically significant.
FSFI, Female Sexual Function Index.
^aPaired student t test.
^bLeast significant difference analysis: E vs LE, P <0.05.
^cLeast significant difference analysis: E vs L, P <0.05.
All others: ANOVA.

Table 4. Parabasal cells (percentage per HPF) and Meisels (maturation value) at 0, 8, and 20 weeks by treatment group
	Laser	Estriol	Laser + estriol	P ^a
Baseline
n	12	11	10
P cells	35.4 ± 31.5	45.3 ± 38.1	26.2 ± 36.3	0.47
Meisels	42.4 ± 24.0	36.9 ± 29.7	48.4 ± 25.3	0.61
Week 8
n	12	14	13
P cells	14.3 ± 24.4	2.4 ± 2.6	5.2 ± 8.2	0.11
Meisels	64.5 ± 23.1	65.6 ± 6.5	65.0 ± 10.5	0.98
Baseline vs week 8^b
P cells	0.03	<0.01	0.09
Meisels	0.01	0.01	0.07
Week 20
n	11	9	11
P cells	16.7 ± 29.2	6.1 ± 11.7	3.9 ± 3.7	0.24
Meisels	58.5 ± 23.7	58.2 ± 8.5	60.4 ± 8.6	0.93
Baseline vs week 20^b
P cells	0.02	0.11	0.07
Meisels	0.01	0.46	0.07

Items listed as mean ± SD. P values of 0.05 were considered statistically significant.
HPF, higher power field; P cells, parabasal cells.
^aANOVA.
^bWilcoxon test.
Maturation value of Meisels: repeated measures ANOVA between groups (P = 0.12) and within groups (P < 0.001).