Design

The study was designed as a single-center, prospective, double-blind, randomized, placebo-controlled trial, which randomized participants into two groups in a 1:1 ratio. The study adhered to CONSORT guidelines and was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB No. 039/2561). The study was also reviewed by the Thai Clinical Trial Registry Committee and prospectively approved for registration since 2018-02-19 11:33:21 and Thai Clinical Trial Registry identification number TCTR20180219001. After approval, participants were included from August 2018 to March 2019. A thorough explanation of the study details was given to all enrolled women. Informed written consent was obtained prior to the start of the study.

Literate Thai women aged 4570years who experienced spontaneous menopause attending the General Gynecologic Clinic, Climacteric and Gender Health Clinic at King Chulalongkorn Memorial Hospital were recruited. Menopause was defined according to WHO criteria as the cessation of the period for at least 12 consecutive months [17].

These women were asked for their vaginal atrophy symptoms and other detailed histories. Participants were included if they reported at least one self-assessed vaginal atrophy symptom in moderate or severe intensity and reported engaging in penile-vaginal penetrative sexual intercourse at least once a month. Those with pathological or surgical causes of menopause, using menopausal hormone therapy (MHT) or non-hormonal treatment that might affect the vaginal epithelium, having abnormal vaginal bleeding/discharge without prior appropriate investigation and treatment, cervical or vaginal surgical history within the previous 3months, history of sex steroid hormone use 2months prior to study, history of psychiatric disorders or having partner with sexual dysfunction, contraindications for MHT including personal or family history of estrogen-related cancer, severe liver or kidney diseases and any suspected allergy to MHT, were excluded from the study. The recruited subjects were then asked to complete a 19-item Thai version of the Female Sexual Function Index questionnaire, and those that scored >26.55 points were then excluded as having no FSD. The Thai version of the Female Sexual Function Index questionnaire was validated in previous study among Thai postmenopausal women, aged 4060years, with high reliability coefficients and internal consistency (r=0.790.86, Cronbachs alpha value=0.82) [18]. After pelvic examination and test for vaginal pH, those with pH more than 5 were qualified for the study as they were likely to have poor estrogenic vaginal mucosa and might benefit from the treatment.

A computer generated block of four randomizations was used to randomize participants into two groups in a 1:1 ratio. The allocation and concealment of placebo and the drugs into identical opaque envelopes were done by a nurse who was not involved in contacting with patients or analyzing any data, thus blinding investigators, assessors, cytologist, microbiologist and all participants.

The sample size of this study was estimated based on our pilot study, conducted with 10 participants enrolled in both arms. The following formula was used for comparing continuous data in a randomized controlled trial [19].

$$ {displaystyle begin{array}{c}{n}_{trt}=frac{{left({z}_{1-frac{alpha }{2}}+{z}_{1-beta}right)}^2;left[sigma {}_{trt}{}^2 + frac{sigma {}_{con}{}^2}{r}right]}{varDelta^2}\ {}r=frac{n_{con}}{n_{trt}},varDelta ={mu}_{trt}-{mu}_{con}end{array}} $$

According to our pilot study, trt (mean value) and trt (standard deviation) of total FSFI at 12week follow up in treatment group were 26.8 and 5.7, respectively; con and con of total FSFI at 12week follow up in control group were 23.0 and 4.7, respectively. The ratio between groups (r) was set as 1 for 1:1. With using =0.05 and =0.2, the sample size needed for this study was 30 participants per group. Predicting a 10% drop out rate, the sample size was increased to 33 participants per group.

After completion of history taking and filling all the questionnaires, participants who met the inclusion criteria mentioned above were asked to undergo a pelvic examination, all conducted by single investigator (T.B.). A dry speculum was inserted without lubrication. A pH-indicator strip (pH range 014, Merck, Germany) was placed over the upper to the middle third of the lateral vaginal wall with contact time between the pH-indicator strip and the examined vaginal wall for 3s.

Two dry cotton buds were used to scrape contralateral vaginal wall. Each cotton bud was smeared onto each different glass slide. One slide was left to air dry and sent for evaluation of Normal Flora Index (NFI) with Gram staining by single microbiologist (T.C.), unaware of treatment allocation, participant symptoms and characteristics. NFI, representing vaginal microenvironment, consisted of 4 parameters, i.e., the number of lactobacilli, pathogenic microorganisms, leukocytes, and vaginal pH. Each parameter was graded on a 4-point scale (Table1). Pathogenic microorganisms included Gardnerella, Bacteroides, Mobiluncus and gram-variable bacilli [21, 22].

The other slide was fixed with 95% ethanol solution for 30min and sent for staining in accordance with Papanicolaou test. The slide was evaluated for Vaginal Maturation Value (VMV) by single cytologist (C.A.), unaware of treatment allocation, participant symptoms/characteristics. VMV, calculated from the formula: (% of intermediate cells 0.5)+(% of superficial cells 1), is considered as a surrogate of vaginal epithelium estrogen status [23, 24,25,26].

The number of lactobacilli, pathogenic microorganisms and VMV was evaluated under the microscope with 1000x magnification (HPF). The number of leukocytes was evaluated under the microscope with 400x magnification.

Participants were then examined with transvaginal ultrasonography for baseline endometrial thickness [Samsung SONOACE R7, 2D imaging mode, grayscale 256 (8 bits), EVN49 probe 3.5MHz, single operator (T.B.)]. Blood samples were taken for baseline hematocrit, SGOT, SGPT, alkaline phosphatase, total cholesterol, LDL, HDL, triglycerides and estradiol level. The samples were analyzed immediately, or stored at 4 degrees Celsius until analysis no more than 24h later. All blood sample analyses were done with routine laboratory testing platform for research projects at the King Chulalongkorn Memorial Hospital, strictly adhering to the manufacturers protocol and in accordance with the laboratorys standard operating procedures for good laboratory practice.

After the recruitment process, participants were randomized into the conjugated estrogens or the placebo groups. The estrogens or placebo was given in numbered as identical opaque envelopes with instruction leaflet. The treatment arm was conjugated estrogens tablet (EstromonTM, 0.625mg). The placebo arm was lactose 90%, polyvinyl pyrrolidone K30 5%, magnesium stearate 3%, talcum 1% erythrosinelahe dye 1% and water which was evaporated completely during manufacturing. The placebo was made by the Faculty of Pharmaceutical Science, Chulalongkorn University and was visually identical to the conjugated estrogens tablet. No participants reported allergic reactions to lactose, talcum or any substances used in the study. Participants were required to insert each pill vaginally as deeply as possible every day for 3weeks. After 3weeks of participation, the investigator called to each participant to check on adherence, problems of drug administration or adverse reactions and willingness to continue the study, after which participants were advised to continue the study by inserting a pill vaginally twice weekly on every Monday and Friday night for the next 9weeks. The regimen was extrapolated from recommendation for treating dyspareunia with conjugated estrogen vaginal cream [11].

After the 12th week of study, each participant came back for reevaluation of symptoms, filling out questionnaires, undergoing pelvic examination for vaginal pH, smear for NFI and VMV, transvaginal ultrasonography and blood samples.

The primary outcome of this study was the changes of FSFI of the two treatment arms. FSFI was one of the validated standard questionnaires frequently used for assessing female sexual function and quality of life in clinical trials or epidemiological studies concerning sexual study [20, 27]. Since there were no validated measurement tools specifically designed for female sexual function and dysfunction in menopausal population, and FSFI was frequently used in researches concerning female sexual function and quality of life in menopausal population, the index was selected for this study. The questionnaires consisted of 19 self-reporting rating-scale items, assessing 6 domains of sexual function; desire, arousal, lubrication, orgasm, satisfaction and pain. Each item has a scaled response ranging from 0 to 5 or 1 to 5; with higher scores representing better sexual function (Table1). Each domain score is calculated by summation of scores from every item in the domain multiplied by the domain factor (i.e., 0.6 for desire, 0.3 for arousal and lubrication, 0.4 for orgasm, satisfaction and pain), thus the full score of each domain is 6. The possible full scores of total FSFI ranges from 2.0 to 36.0, with cut-off point of 26.55 or less considered FSD in premenopausal and postmenopausal women population [28]. However, there is no specific cut-off score for FSD in a population with GSM [29].

Secondary outcomes were changes in vaginal pH, VMV, NFI, and the Most Bothersome Symptoms (MBS). The MBS, also known as the vaginal atrophy symptoms consists of 4 symptoms; vaginal dryness, vaginal/vulvar irritation/itching, vaginal/vulvar soreness, and dyspareunia. Each symptom is self-graded by participants on a 4-point scale (0=no symptom, 1=mild, 2=moderate, 3=severe) [30]. Only those reported at least one self-assessed vaginal atrophy symptom in moderate or severe intensity were included. The safety parameters assessed were changes in hematocrit, SGOT, SGPT, alkaline phosphatase, total cholesterol, LDL, HDL, triglycerides, estradiol level and endometrial thickness.

IBM SPSS statistics version 18.0 for Windows was used for statistical analysis. The treatment effect was evaluated with intention-to-treat analysis, with missing data assumed by multiple imputation method. Baseline demographic characteristics were presented using descriptive statistics; mean and standard deviation (SD), median and interquartile range (IQR), or number and percentage as appropriate. Primary and secondary outcome comparisons between groups were evaluated with Mann-Whitney U test or analysis of covariance (ANCOVA), with treatment arm as a fixed effect in the model and the baseline value used as a covariate, according to data distribution characteristics. P-value of less than 0.05 is determined statistically significant.

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Effects of vaginal administration of conjugated estrogens tablet on sexual function in postmenopausal women with sexual dysfunction: a double-blind,...

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