This was a randomized controlled trial that was conducted on 96 postmenopausal women who suffered from vaginal atrophy in two health centers in Ahvaz, Iran. The inception date of this study was in April 2018 and the completion date was September 2018. The protocol of this study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ref No: IR.AJUMS.REC.1396.720). This study was registered in the Iranian Registry for Clinical Trials (Ref No: IRCT20160602028220N2). This study adheres to CONSORT guidelines for a randomized controlled trial (CONSORT checklist, supplementary material). The inclusion criteria were as follow: literate women, age 40-60, at least one year passed from their last menstrual period or the level of FSH>40IU, monogamous women with a sexual relationship. Women with following criteria were excluded from the study: vaginal infection, women who used hormone replacement therapy, any undiagnosed genitalia diseases, smoker’s women, the body mass index more than 30kg/m2, vaginal bleeding or spotting, any breast diseases with unknown cause, using vaginal lubricant at least 15 days before the intervention. All women provided written informed consent before data collection. Menopause was confirmed by results of FSH in some participants, but in most participants, the menopause were confirmed if the menses was ceased for one year.
Sample size calculation
We used the following formula with considering Al-Saqi et al’s study, [17] β=0.9, α=0.05, S12= S22= 1.795, d=1.0035, and considering 25% attrition the total sample size for each group was calculated to be 48. (see Formula 1 in the Supplementary Files)
Drug preparation
Oxytocin powder was purchased from Caspian Company at 0.08 % and stored at 2 to 8 ° C, away from light. For the vaginal gel preparation, at first, the 2% sodium carboxymethyl cellulose was added to the 20% propinyl glycol gradually when it was stirring. Then the boiled water containing 0.2% methylparaben was added and stirred until it was cool. Then 2gr oxytocin powder in the distilled water residue (39%) was added and stirred to form the oxytocin gel. The same steps were taken to make a placebo, except the oxytocin powder was not added. Oxytocin gel and placebo were placed in similar tubes and the pharmacist assigned codes to these tubes. The researcher and the participants were not aware of the content of each tube.
Recruitment
Eligible women were placed in the lithotomy position and the vagina was assessed regarding infection and any abnormal discharge. Women who did not have any of these symptoms were considered for intervention. Then women were evaluated regarding vaginal signs and symptoms of vaginal atrophy such as dryness, pallor, dyspareunia, redness, inflammation, and vulvovaginal erosion. The subjective symptoms of vaginal atrophy were assessed using the self- reported scale of burning, itching, feeling of dryness and dyspareunia. The severity of each symptom was determined by the patient and then an appropriate score was applied as follows: the absence of any symptoms received zero, mild symptoms received 1, moderate and intense symptoms received 2 and 3 respectively. The sum of scores was calculated for each woman. If the total score was <5, we considered the patient as vaginal atrophy.
For measuring vaginal PH, a disposable speculum (Bekr brand) was inserted into the vagina and a sample from the posterior fornix was taken using a cotton swab (Neeva) and placed in the lamella and fixed with fixator (Patofix) and sent to a reference laboratory for vaginal maturation index evaluation (VMI). The vaginal PH was measured using a PH gauge paper (Merck, Germany). The paper tape was contacted to the vaginal wall and kept for one minute and the corresponding strip was compared to the PH bar and the number recorded in the checklist. If the vaginal atrophy (signs and subjective symptoms) was confirmed, women were recruited for the intervention.
Randomization
Eligible women were randomly assigned in two groups of vaginal gel of oxytocin or placebo using block randomization with a block size of 4 and the ratio of 1:1. For allocation concealment, each woman received a code and all codes were kept in an opaque envelope until the time of intervention. The researcher and the participant were not aware of the groups and the intervention that they received. The oxytocin and placebo were placed in similar tubes and coded by the pharmacist. None of the researchers or participants was aware of real treatment.
Measurement
The following forms were used for data collection. A demographic questionnaire was used for recording characteristics such as age, education level, and economic situation. A checklist was used for recording signs and symptoms of vaginal atrophy and PH. Also, a checklist was used for recording the subjective symptoms of vaginal atrophy. Except for the demographic questionnaire that completed at baseline, other checklists were completed at baseline, two and eight weeks after intervention. The validity and reliability of all questionnaire and checklist were assessed using content validity. The body mass index was calculated by weight (kg)/height (m2) and self-reported by participants. The maturation index and vaginal PH were measured at baseline and eight weeks after the intervention, while the subjective symptoms of vaginal atrophy were measured at baseline, two and eight weeks after intervention.
Intervention
Women in the intervention group requested to use oxytocin gel (400IU) and women in the placebo group used placebo vaginal gel for eight weeks. An applicator contains 4 gr and participants requested to filled-up one- quarter of each applicator per night and continue to use for eight consecutive weeks.
Statistics
All data entered SPSS version 22. The normal distribution of data was assessed using the Shapiro–Wilk test. The continuous data were analyzed using independent t-test, while the categorical data was analyzed using the chi-square test. For variables with more than two measurements, the Generalized Estimating Equations (GEE) was used. P<0.05 was considered significant.