# Abstract

 

## Objective The aim of this study was to demonstrate the efficacy of an ultra-low-dose vaginal estriol 0.03 mg in combination with viable *Lactobacillus acidophilus* KS400 (Gynoflor^®^ vaginal tablets) in the short-term therapy and to investigate the long-term maintenance dose in the treatment of vaginal atrophy. 

## Methods This was a double-blind, randomized, placebo-controlled study (Controlled phase *--* initial therapy) followed by an open-label follow-up (Open phase -- test medication initial and maintenance therapy). Included were postmenopausal women with vaginal atrophy symptoms and Vaginal Maturation Index (VMI) of ≤ 40%. The method of treatment was initial therapy with test medication (or placebo in first phase), one vaginal tablet daily for 12 days, followed by maintenance therapy, one tablet on two consecutive days weekly for 12 weeks. 

## Results A total of 87 women completed the study. The Controlled phase results for a change in VMI demonstrated superiority of the 0.03 mg estriol--lactobacilli combination to placebo (*p* < 0.001). In the test group, the positive change in VMI was 35.2%, compared to 9.9% in the placebo group. In the Open phase after the initial therapy, the VMI was increased to 55.4% and, during maintenance therapy, it stayed at a comparable level (52.8--49.4%). The maturation of epithelium was followed by improvement of clinical symptoms and normalization of the vaginal ecosystem. 

## Conclusions The ultra-low-dose, vaginal 0.03 mg estriol--lactobacilli combination (Gynoflor^®^) was superior to placebo with respect to changes in VMI after the 12-day initial therapy, and the maintenance therapy of two tablets weekly was sufficient to prevent the relapse of vaginal atrophy. # Results An overview of subject disposition is given in [Figure 2](#). A total of 87 women completed both phases of the study as scheduled. Reasons for discontinuation were premature discontinuation at their own request or failure to show up at control visits. One woman in the placebo group withdrew her consent before visit C1 and was excluded from the ITT population due to no data. Another woman in the placebo group was excluded from the PPS population due to prior termination of the study after visit C1. During the Open phase, there was another major protocol violation as the woman did not comply with the treatment. All randomized subjects were part of the SAF population. <figure> <p><img src="" /></p> <figcaption>Disposition of subjects, Controlled phase. SAF, Safety population; ITT, intention-to-treat; PPS, per-protocol set</figcaption> </figure> Demographic and baseline characteristics were comparable between both treatment groups ([Table 1](#)). Most frequent concomitant diseases in both groups were dyslipidemia, hypertension, osteoporosis and osteoarthritis. Oral therapy with calcium carbonate was the most frequently concomitant medication. All women were Asian. ::::table-wrap

::: caption
Demographic and baseline characteristics
:::

```
                    *Estriol--lactobacilli*   *Placebo*                        
```

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*Mean* *SD* *Mean* *SD* *p Value*\
Age (years) 60.93 6.15 62.34 6.07 0.283
Menopause (years) 13.11 7.02 13.00 6.94 0.939
Height (cm) 154.86 5.45 153.43 5.88 0.239
Weight (kg) 56.16 8.10 57.82 8.24 0.343
Deliveries (number) 2.14 1.76 2.64 1.79 0.190

SD, standard deviation:::: All women included in the SAF (*n* = 89) took at least one dose of study drug. During the Controlled phase, 85 women (96%) used the study medication correctly. During the Open phase, 75 women (84%) adhered completely to the treatment schedule, 12 (13%) had minor deviation from the treatment schedule, but applied at least 20 out of the 24 scheduled tablets. Only one subject significantly deviated from the regimen. # Controlled phase The results of the confirmatory analysis (ITT) for the primary variable (change in VMI at C2) demonstrated superiority of the initial daily therapy with the 0.03 mg estriol--lactobacilli combination over placebo ([Table 2](#)). The PPS analysis yielded similar results. ::::table-wrap
::: caption
Change in Vaginal Maturation Index (VMI), the primary variable, in the
Controlled phase
:::

```
                               *Change in VMI at visit C2*               
```

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*n* *Mean* *SD* *p Value*\
*Intention-to-treat*\
Estriol--lactobacilli 44 35.2% 21.7% < 0.001
Placebo 44 9.9% 14.5%\
*Per-protocol set*\
Estriol--lactobacilli 44 35.2% 21.7% < 0.001
Placebo 43 10.2% 14.6%

SD, standard deviation:::: At C1, after half of the treatment period, the increase in VMI was 19.8% for the estriol--lactobacilli combination and 11.8% for the placebo. This difference was not significant (*p* = 0.061), indicating that a 6-day therapy with the 0.03 mg estriol--lactobacilli combination is not sufficient for full improvement. The development of VMI itself during the Controlled phase is shown in [Figure 3](#). <figure> <p><img src="" /></p> <figcaption>Dynamics of Vaginal Maturation Index (VMI), Controlled phase. E, entry visit; C1, follow-up 5–7 days after start of therapy; C2, follow-up 2–4 days after end of initial therapy</figcaption> </figure> The predominant vaginal symptoms ([Figure 4](#)) at the Entry visit were vaginal dryness and dyspareunia in both groups. The percentage of women with moderate or severe dryness decreased more in the estriol--lactobacilli combination group, resulting in a significant difference between the groups, both at C1 (*p* = 0.023) and at C2 (*p* < 0.001). The number of women who reported having intercourse was too low for a valid statistical analysis of dyspareunia (E: approximately 45%, C1 and C2: 11--21%). The percentage with moderate or severe dyspareunia at E was 84% in the estriol--lactobacilli combination group and 90% in the placebo group. The corresponding numbers at C1 were 40% for both groups, and at C2 -0% in the estriol--lactobacilli combination group and 22% in the placebo group. Individual results for dyspareunia, abnormal discharge, pruritus, and soreness therefore were not significantly different between the estriol--lactobacilli combination and placebo groups. <figure> <p><img src="" /></p> <figcaption>Dynamics of vaginal symptoms, Controlled phase. E, entry visit; C1, examination 5–7 days after start of therapy; C2, examination 2–4 days after end of initial, daily therapy</figcaption> </figure> Development of other secondary variables is shown in [Table 3](#). The total symptom score improved during estriol--lactobacilli combination therapy compared to placebo: at C1 without significant difference, and at C2 with significant difference. The percentages of the increase of superficial cells and the decrease of parabasal cells showed a similar dynamic: at C1 there were no significant differences between the groups; however, at C2 significant improvements in favor of the estriol--lactobacilli combination were observed. The number of lactobacilli and LBG also improved in both groups at C1, with no significant difference between the groups. But again, the corresponding results at C2 were significantly better in the test medication group. The mean vaginal pH decreased significantly more in the estriol--lactobacilli combination group as compared to placebo both at C1 and at C2. The global efficacy of the estriol--lactobacilli combination by investigator was significantly better than that of placebo, both at C1 and at C2. The rating by the patient was not significantly different at C1, but at C2 was significantly better for the test group. ::::table-wrap
::: caption
Secondary efficacy and safety variables in the Controlled phase. Data
are given as mean or %
:::

______________________________________________________________________

```
                              *Entry*       *C1*                       *C2*                                                   
```

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*Estriol--lactobacilli* *Placebo* *Estriol--lactobacilli* *Placebo* *Estriol--lactobacilli* *Placebo*\
(*n* = 44) (*n* = 44) (*n* = 44) (*n* = 44) (*n* = 44) (*n* = 43)

*Total symptoms score* 4.9 5.1 2.6 *2.7* 1.8 2.6

```
                              *p =* 0.767   *p =* 0.562                *p =* 0.022                                            
```

*Epithelial cells*

Parabasal 72.3% 64.6% 38.3% 45.2% 24.6% 45.0%

```
                              *p =* 0.2     *p =* 0.196                *p* < 0.001                                           
```

Intermediate 26.8% 33.9% 55.1% 47.9% 51.7% 52.0%

```
                              *p =* 0.229   *p =* 0.157                *p =* 0.955                                            
```

Superficial 0.9% 1.4% 6.6% 6.9% 23.6% 2.9%

```
                              *p =* 0.434   *p =* 0.9                  *p* < 0.001                                           
```

*Number of lactobacilli*

6--10 0.0% 2.3% 15.9% 18.2% 15.9% 14.0%

20--50 0.0% 4.5% 9.1% 6.8% 20.5% 9.3%

> 50 0.0% 0.0% 9.1% 2.3% 31.8% 2.3%

```
                              *p =* 0.212   *p =* 0.402                *p* < 0.001                                           
```

*Lactobacillary grade*

I 0.0% 2.3% 11.4% 4.5% 40.9% 4.7%

II 0.0% 2.3% 20.5% 20.5% 29.5% 30.2%

III 100% 95.5% 68.2% 75.0% 29.5% 65.1%

```
                              *p =* 0.359   *p =* 0.438                *p* < 0.001                                           
```

*pH value* 7.0 6.8 6.3 6.7 5.2 6.6

```
                              *p =* 0.114   *p =* 0.014                *p* < 0.001                                           
```

*Efficacy*

Investigator (very good/good) 47.7% 22.7% 61.4% 23.3%

```
                                                                       *p =* 0.006    *p* < 0.001                            
```

Patient (very good/good) 40.9% 34.1% 70.5% 46.5%

```
                                                                       *p =* 0.57     *p =* 0.031                             
```

*Tolerability*

Investigator (very good/good) 70.5% 61.4% 90.9% 79.1%

```
                                                                       *p =* 0.355    *p =* 0.01                              
```

Patient (very good/good) 50.0% 47.7% 65.9% 51.2%

```
                                                                       *p =* 0.809    *p =* 0.31                              
```

---------------------------------------------------------------------------------------------------------------------------------------:::: During the 12-day Controlled phase, no adverse event was reported. The majority of the investigators and the women judged the global tolerability of test medication as very good or good. 

## Open phase For the Open phase, all women (*n* = 87) in the two groups in the Controlled phase were combined (see Methods), and results were analyzed at Follow-up 1 (after daily initial therapy), Follow-up 2 and Follow-up 3 (weekly maintenance therapy). After the daily initial therapy with the estriol--lactobacilli combination, VMI was clearly increased ([Figure 5](#)). During the weekly maintenance therapy, the mean VMI stayed at a comparable level at Follow-up visits 2 and 3, showing moderate estrogenic stimulation. Thus, the 0.03 mg estriol--lactobacilli combination at a regimen of two vaginal tablets per week was on average sufficient to maintain the improved VMI after the initial 12-day therapy. <figure> <p><img src="" /></p> <figcaption>Dynamics of Vaginal Maturation Index (VMI), Open phase. C2, examination 2–4 days after the end of initial therapy; C3, follow-up visit 2 weeks after start of maintenance therapy; C4, follow-up visit at end of therapy (12 weeks)</figcaption> </figure> The percentage of women with moderate or severe vaginal dryness clearly decreased from 77% at E to 13.7% at Follow-up 1 (after initial therapy), and further improved during maintenance therapy to 2.3% at Follow-ups 2 and 3. In women having intercourse, dyspareunia after initial therapy clearly decreased from 64.2% to 21.4% at Follow-up 1, to 14.3% at Follow-up 2, and further to 3.2% at Follow-up 3. All other secondary variables are shown in [Table 4](#). The total symptom score improved strongly during initial therapy and further improved slightly during maintenance therapy. The percentage of epithelial cells clearly improved during initial therapy and remained at similar levels at Follow-up visits 2 and 3. The percentage of women with more than 50 lactobacilli per field of view clearly increased not only during initial therapy, but also during maintenance therapy, reaching the highest level at the end of the study. A similar development was observed for LBG: the percentage of women with a normal flora (LBG I) increased also during the maintenance therapy, resulting in 67% of all women having a normal flora and only 6% having an abnormal flora at the end of the study. The global assessments of efficacy (good or very good) by investigator and patient were 89% and 92%, respectively, and highest at the end of the Open phase. ::::table-wrap
::: caption
Secondary efficacy and safety variables in the Open phase. Data are
given as mean or %
:::

______________________________________________________________________

```
                              *Initial therapy*     *Maintenance therapy*                     
```

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*Entry* *Follow-up 1* *Follow-up 2* *Follow-up 3*\
*E/C2* (*n* = 87) *C2/C2A* (*n* = 87) *C3* (*n* = 87) *C4* (*n* = 87)

*Total symptom score* 3.8 1.2 0.8 0.6

*Epithelial cells*

Parabasal 58.8% 19.0% 15.5% 19.4%

Intermediate 39.3% 51.6% 62.0% 62.2%

Superficial 1.9% 29.6% 21.7% 28.4%

*Number of lactobacilli*

6--10 6.9% 14.9% 9.2% 10.3%

20--50 4.6% 23.0% 19.5% 13.8%

> 50 1.1% 39.1% 47.1% 52.9%

*Lactobacillary grade*

I 2.3% 51.7% 56.3% 66.7%

II 14.9% 28.7% 29.9% 27.6%

III 82.8% 19.5% 13.8% 5.7%

*pH value* 6.8 5.0 5.0 4.9

*Efficacy*

Investigator (very good/good) 77.0% 83.9% 88.5%

Patient (very good/good) 71.3% 86.2% 91.9%

Tolerability

Investigator (very good/good) 94.2% 98.9% 95.4%

Patient (very good/good) 93.6% 87.4% 88.5%
-------------------------------------------------------------------------------------------------------:::: During the 14-week Open phase with the 0.03 mg estriol--lactobacilli combination, a total of four adverse events were reported by four of the 89 women. All four adverse events were judged to be not related to study treatment. The global assessment of tolerability at all follow-up visits evaluated by the investigator was rated as very good/good and constantly over 90%, while the assessment of this parameter by the patient was rated on average 80% and increased over time ([Table 4](#)).


Extracted trial result:
Outcome: Baseline characteristics: menopause (continuous)
Intervention: Vaginal estriol plus viable Lactobacillus acidophilus KS400
Comparator: Placebo
Intervention group size: 44
Intervention mean: 13.11
Intervention standard deviation: 7.02
Comparator group size: 44
Comparator mean: 13
Comparator standard deviation: 6.94