This randomized controlled, unblended, clinical trial was conducted between February 2017 and February 2018 in the Department of Gynecology at Universidade Federal de Sao Paulo. It was approved by the institutional committee of the Federal University of Sao Paulo (protocol No. 101162/2017) and entered into the Brazilian Clinical Trials Registry (ReBEC) under ID RBR-228FSY. All participants who met the inclusion criteria and were asked to participate in the study gave their consent for participation before the start of the study. The investigator obtained hard copies of informed consent from all the women who participated in the study.
First, we determined that the sample size would correspond to the number of participants who met the inclusion criteria over a 1-year period at the Gynecological Disease Prevention Nucleus (NUPREV), Department of Gynecology. Twenty-six participants were initially selected for the study: one patient was excluded because she did not meet the inclusion criteria and hence, 25 women were randomized into two groups: 13 were allocated to receive LT and 12 were to receive TE. Randomization of participants was carried out through the Random. org website, which generates random numbers (https://www.random.org/).
The study included women aged 50 to 65 years with amenorrhea for at least 12 months, their serum FSH levels (>40 IU/L) were compatible with the postmenopausal stage, and they had a current clinical complaint of at least one of the following symptoms: dyspareunia or vaginal dryness. Exclusion criteria included women who had hormone therapy contraindications, used hormonal medication (systemic or topical) in the past 12 months before the study, or had cervical changes, autoimmune diseases, coagulopathies, immunosuppression, pelvic organ prolapse, and/or previous pelvic radiotherapy.
Participants from both groups underwent vaginal hormonal cytology, which was performed using a plastic Ayres spatula, and the smear was collected from the vaginal walls and posterior vaginal fornix. Further, colposcopy of the cervix (when they did not undergo total hysterectomy) and the vagina, and biopsy of the proximal third of the right lateral vaginal wall was performed approximately 6.0 cm from the introitus. All procedures were performed by the same clinician. Transvaginal ultrasound was performed at the start of both treatments, and women with an endometrial echo <5 mm were included in the study. However, ultrasound control was performed since low-dose vaginal estrogen preparations were generally considered safe for vulvovaginal atrophy treatment. Each woman was subjected to treatment and follow-up for 4 months.
The LT group was subjected to three sessions of fractional CO2 laser application, 30 days apart, using the SmartXide2 system (Monalisa Touch, Deka Laser, Florence, Italy).[13,14,23,26] Manual rotation of the vaginal probe was performed along the length of the vaginal wall (360°). The parameters used were determined based on previous studies, with power of 30 W, dwell time of 1,000, dot spacing of 1,000, and smart stack 2.[12,22] Participants were informed about the possibility of minor bleeding and were recommended to avoid coital sexual activity and the use of tampon for at least 3 days.[31,32] There was no requirement of analgesia or anesthesia during any of the clinical procedures. The participants underwent a new biopsy and hormonal cytology 30 days after their last session.
The procedure in the case of treatment in the TE group included daily vaginal application of 1 mg of estriol cream for a period of 30 days, followed by a treatment regimen of twice a week application, for 2 months. Hormonal cytology and vaginal biopsy of each participant was performed 30 days after the last application of estriol. The study investigator (P.F.S.P.D.) at the obstetrics and gynecology clinic performed the procedures in both the groups, and cytological and histological evaluations were performed by two pathologists who were blinded to the treatment groups and stage of the study at which they were involved.
The Frost index represents the relative proportion of parabasal, intermediate, and superficial cells of the VE,[13,33,34] which is also calculated from the cell maturation index (Frost), and the number of cells in each category was multiplied by the point value (deep × 0 + intermediate × 0.5 + superficial × 1). A value of 0 to 49 indicated a low estrogen effect, a value of 50 to 64 indicated a moderate estrogen effect, and a value of 65 to 100 indicated a high estrogen effect.[13,35,36]
Epithelium biopsies were evaluated before the first treatment and at the fourth month follow-up after the third treatment, using a common optical microscope (Nikon Eclipse E200, Nikon Instruments Inc, NY), and the Breslow thickness of the mucosa was measured in millimeters (mm). A 4 mm biopsy was performed from the proximal third of the right vaginal wall. The conventional histopathological method was performed as described by our group previously. Hormonal and histological cytologies were measured by pathologists who were blinded to the treatment groups or time points of treatment.
Participants from both groups responded to the Brazilian SQ-F (female sexual quotient) questionnaire at baseline and at the end of their treatment. The SQ-F is a Brazilian validated instrument composed of 10 questions, each of which must be answered on a scale ranging from 0 to 5. Higher scores indicated better sexual performance and satisfaction. The sum of the 10 answers was multiplied by 2, resulting in a total index ranging from 0 to 100. The seventh question required a different calculation method; that was, the value of the answer given (from 0 to 5) had to be subtracted from 5 to achieve the final score for the question.[35,39–41]
Statistical analysis was initially performed using the mean, median, minimum, and maximum values, standard deviation, and absolute and relative frequencies (percentages). Inferential analyses employed to confirm or refute the evidence found in the descriptive analysis were performed using the Shapiro-Wilk test to verify the assumption of normality of numerical information. Student's t test was used for independent samples when comparing groups (TE and LT) for numerical information with normal distribution (current age, body mass index, and number of deliveries). Chi-square or Fisher's exact tests were used to compare the groups (TE and LT) according to marital status and education. We used the Mann–Whitney U test to compare groups (TE and LT) according to age at the beginning of menopause (numerical information with skewed distribution). Analysis of variance with repeated measures was performed for the comparison of epithelium thickness (SQ-F), number of deep, intermediate, and superficial cells (Frost), and maturation index (Meisel), between groups (TE and LT) and times (initial and final) in addition to multiple Bonferroni comparisons whenever needed. An alpha significance level of 5% was used for the inferential analyses. Data were entered in Excel 2010 for Windows, and statistical analyses were performed using SPSS Statistics version 24 (IBM Corp., Armonk, NY) and R version 3.6.3 (R Core Team, 2016).
Menopause. 2021;28(7):756-763. © 2021 The North American Menopause Society