Number of vaginal lactobacilli in postmenopausal women with vaginal atrophy before and after treatment with erbium YAG laser, a randomized sham-controlled trial

doi:10.21203/rs.3.rs-2128372/v1

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Number of vaginal lactobacilli in postmenopausal women with vaginal atrophy before and after treatment with erbium YAG laser, a randomized sham-controlled trial

https://doi.org/10.21203/rs.3.rs-2128372/v1

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published 26 Sep, 2023

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Primary objective: To evaluate the effect of erbium YAG laser on the number of vaginal lactobacilli in postmenopausal women.

Secondary objectives: To evaluate the effects of erbium YAG laser on vaginal atrophy symptoms and vaginal pH in postmenopausal women.

Materials and Methods: A total of 44 postmenopausal women meeting inclusion criteria were randomized in laser group (n=22) and sham group (n=22). Vaginal lactobacilli grading, vaginal pH, vaginal atrophy score and vaginal atrophy symptoms (dryness, irritation, soreness, dyspareunia) wereassessed before and after treatment with erbium YAG laser for 2 consecutive times, 4 weeks interval; the results were compared with the effects of sham procedure. Any adverse events after the treatment were recorded. Statistical analysis was performed by using SPSS program and p < 0.05 was considered statistically significant.

Results: 44women were included and 5were lost to follow-up. Compared with sham procedure, there was an improvement of vaginal lactobacilli grading in laser group (5/20 in laser group and 1/19 in sham group). However, the improvement did not reach statistically significant (adjusted OR = 5.32, 95%CI = 0.5-56.21). Vaginal atrophy symptoms measured by VAS and vaginal pH were improved in both groups without statistically significant difference between groups. Vaginal “dryness” VAS and vaginal atrophy score after treatment were significantly lowered in laser group compared to sham group.

Conclusions: This study showed an improvement in vaginal lactobacilli grading after vaginal laser treatment. However, the difference of vaginal lactobacilli grading after treatment in both groups did not reach statistically significant.

Erbium YAG laser

Vaginal lactobacilli

Randomized sham-controlled trial

Menopause

Vaginal atrophy

Vaginal pH

Vaginal atrophy or genitourinary syndrome of menopause is commonly found in postmenopausal women. As many as 50% of postmenopausal women suffered from vaginal atrophy symptoms.¹ This condition is caused by low estrogen level after menopause.² The diagnosis of vaginal atrophy is clinical diagnosis which are vaginal dryness, irritation, pain, dyspareunia and urinary symptoms.³

Treatments of vaginal atrophy can be either local hormonal treatment or non-hormonal treatments. Currently, vaginal estrogen therapy is one of the standard treatments of vaginal atrophy. However, caution was suggested in hormone sensitive cancer survivors.⁴ Moreover, number of patients denied hormonal treatment. This leads to choices of non-hormonal treatments including vaginal erbium YAG laser.

Vaginal laser is a novel treatment for vaginal atrophy. There are two types of laser used for intravaginal therapy – CO₂ and erbium YAG laser.⁵ The advantage of erbium YAG laser is that it has 15 times more water absorption than CO₂ laser causing less penetration, faster tissue healing phase, fewer side effects and lesser pain compared with CO₂ laser.⁴

The effect of vaginal laser is thermal effect to vaginal epithelium resulting in an expression of heat-shock proteins which stimulate growth factors activities, neovascularization, neocollagenesis, extracellular matrix formation and increase vaginal thickness and elasticity. However, thermal effect of erbium YAG laser is less than that of CO₂ laser causing no tissue damage.⁴

Many prospective studies reported that vaginal erbium YAG laser can improve vaginal atrophy symptoms and its effect can be compared with vaginal estrogen therapy.^6–8 A systematic review and meta-analysis of laser therapy for genitourinary syndrome of menopause publish in 2017, included 14 studies of both CO₂ and erbium YAG laser, showed that vaginal laser improved all genitourinary syndrome of menopause (GSM) symptoms, female sexual function index (FSFI), vaginal health index score (VHIS) and vaginal maturation value (VMV). However, the quality of evidences was “low” or “very low” because there was no randomized sham-controlled study that can decrease the bias and placebo effect.⁵

Vaginal lactobacilli are normal vaginal flora that can produce antimicrobial compounds such as hydrogen peroxide and lactic acid, and compete with pathogens for vaginal adherence.⁹ Number of vaginal lactobacilli increases after vaginal epithelial maturation because of an increase of glycogen storage in superficial vaginal epithelium.^10–11 Therefore, it can be used as an objective measurement of vaginal atrophy. A prospective study of vaginal CO₂ laser and vaginal flora reported a significant increase of vaginal lactobacilli and a significant decease of vaginal pH after 3 applications of CO₂ laser.⁹ However, there was no study to date about the effect of vaginal erbium YAG laser and number of vaginal lactobacilli.

This study aimed to measure the effect of vaginal erbium YAG laser on number of vaginal lactobacilli. Besides, we also evaluate vaginal pH, vaginal atrophy symptoms and adverse effects after vaginal erbium YAG laser treatment.

This randomized sham-controlled study was conducted at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand, between May 2019 and September 2020. The target population were postmenopausal women attending gynecology clinic or gender health clinic with vaginal atrophy symptoms (dryness, dyspareunia, irritation, pain). Inclusion criteria were postmenopausal women with at least one symptom of moderate to severe vaginal atrophy and age more than 40 years. (Moderate to severe vaginal atrophy was defined by the most bothersome symptom score at least “2” in at least one vaginal atrophy symptoms (0 = no symptom, 1 = mild, 2 = moderate, 3 = severe). Exclusion criteria were history of hormonal therapy, vaginal lubricants, vaginal moisturizers or spermicide in previous 3 months, active or recent genitourinary tract infection in previous 1-month, abnormal vaginal bleeding, genital organ prolapse stage at least II, history of vaginal surgery in previous 3 months and previous vaginal laser treatment.

For sample size calculation, two independent proportions formula was used. The sample size was calculated using data from our pilot study. The proportion in study group and control group were 0.4 and 0.9 respectively. (Outcome for sample size calculation was proportion of participants with grade 1 vaginal lactobacilli after vaginal erbium laser treatment and after sham operation) Using 5% type I error and 20% type II error, the calculated sample size was 17 per group. Adding 20% drop out, total of 42 participants were needed.

After completed written informed consent, all participants were interviewed to collect demographic data and randomized into laser group and sham group using block of four randomization with sealed enveloped. Participants in laser group were treated with erbium in yttrium aluminum-garnet crystal (Er:YAG) laser (Fotona Smooth™ XS, Fotona, Ljubljana, Slovenia), wavelength of 2940 nm, spot size 7 mm, frequency 1.6 Hz, laser energy 6.0 J/cm2 2 applications every 4 weeks. Participants in sham group were treated with 2 sham operations every 4 weeks. Sham operation was done while patient was in lithotomy position and placed an exactly the same equipment of vaginal erbium laser, but there was no laser energy from the equipment. A duration of sham operation was equal to that of vaginal erbium laser treatment or about 5 minutes. All participants were appointed for follow-up visit 4 weeks after the second laser or sham application. Vaginal atrophy symptoms were measured by visual analog scale (VAS) in every visit. Vaginal swabs were collected from posterior fornix and vaginal pH was measured using pH indicator strip applied to lateral vaginal wall. Vaginal atrophy score, as shown in Table 1, was assessed in every visit. The vaginal swabs were sent to a microbiologist to evaluate the vaginal lactobacilli grading. Vaginal lactobacilli were reported into 4 grades according to number of lactobacilli per high power field. (grade 1: < 6/HPF, grade 2: 6–20/HPF, grade 3: 21–50/HPF, grade 4: > 50/HPF) All participants were blinded during the study period. All vaginal swab slides were sent to a microbiologist with identification numbers that did not mention about study group. Therefore, a microbiologist who evaluated vaginal lactobacilli grading was also blinded.

Table 1

Assessment of vaginal atrophy score
	Not present (0)	Mild (1)	Moderate (2)	Severe (3)
Dryness	Normal lubrication	Slightly decreased	Minimal lubrication	Dry
Rugae	Normal number and depth	Reduced rugae	Rare rugae	Smooth vagina
Pallor	Normal pink	Light pink	Very pale	White/deep red
Petechiae	None	Bleeds on scraping	Bleeds on contact	Clearly seen
Mucosal elasticity	Normal	decreased	None	Stenosis

This study was funded by Ratchadapisek Sompot fund and was approved by Institutional Review Board of Faculty of Medicine, Chulalongkorn University. Statistical analysis was performed by SPSS version 22.0. For descriptive statistic, Number, percentage and mean ± standard deviation (SD) was used for continuous data while number and percent were used for categorical data. For analytical statistic, ANOVA repeated measures and Friedman test were used for continuous and categorial data, respectively. An overall statistical significance was considered when the p-value of < 0.05.

Total 44 participants were recruited and randomized to 22 in laser group and 22 in sham group. As 5 of them were lost to follow-up, total 39 participants were completed (the detail was shown by consort diagram in flow chart 1). Table 2 shows similar baseline characteristics of participants in laser group and sham group except for “age” that participants in laser group were younger than those in sham group (mean age in sham group was 60.4, mean age in laser group was 55.5). There was no significant difference in parity, menopausal age, year since menopause, baseline vaginal atrophy symptoms measured by VAS, baseline vaginal pH and baseline vaginal atrophy score between participants in both groups. There was no significant difference of BMI between two groups. There were similar baseline vaginal lactobacilli between laser group and sham group. The most presenting symptom of participants was dyspareunia (54.5% in both groups).

Table 2

Baseline characteristics of the subjects in laser group and sham group
	Laser group (mean ± SD)	Sham group (mean ± SD)	p value
Age (year)	55.5 ± 7.6	60.4 ± 6.6	0.03
Parity	1.5 ± 1.0	1.8 ± 1.1	0.47
Menopausal age (year)	47.3 ± 4.3	49.7 ± 5.3	0.10
Year since menopause (year)	9.0 ± 6.4	10.7 ± 7.1	0.41
Vaginal atrophy symptoms (VAS)	17.5 ± 8.6	16.3 ± 7.5	0.62
Vaginal pH	6.3 ± 1.4	6.6 ± 1.2	0.35
Vaginal atrophy score	6.5 ± 2.9	7.9 ± 2.9	0.13
	Laser group N(%)	Sham group N(%)	p value
BMI			1.00
< 18.5	1(4.5)	0(0)
18.5-24.99	12(54.6)	13(59.1)
25-29.99	7(31.8)	7(31.8)
≥ 30	2(9.1)	2(9.1)
Lactobacilli grade			0.55
1	17(77.3)	21(91)
2	2(9.1)	1(4.5)
3	1(4.5)	0(0)
4	2(9.1)	1(4.5)
Most symptoms			0.27
Dryness	8(36.5)	4(18.3)
Dyspareunia	12(54.5)	12(54.5)
Irritation	1(4.5)	5(22.7)
Pain	1(4.5)	1(4.5)

Table 3 shows vaginal lactobacilli grading in each visit of both groups. Visit 1 is pre-treatment. Visit 2 and 3 are after 1st and 2nd treatment respectively. In the laser group, grade of vaginal lactobacilli was higher significantly after 2nd treatment (p = 0.02). However, there was no statistically significant change of vaginal lactobacilli after 2 sham procedures. To compare between groups, using logistic regression for calculation of odd ratio (OR) of improvement after 2 laser treatment, adjusted OR for age was 5.32 (95%CI = 0.5–56.21, p = 0.17). Therefore, compared with sham group, the improvement of vaginal lactobacilli grading of participants in laser group was not reach statistically significant.

Table 3

Vaginal lactobacilli grading, vaginal atrophy symptoms (VAS), vaginal atrophy score, vaginal pH in each visit of participants in laser group and sham group
	Laser group				Sham group
Lactobacilli grade	Grade 1	Grade 2	Grade 3	Grade 4	Grade 1	Grade 2	Grade 3	Grade 4
Visit 1	15	2	1	2	17	1	0	1
Visit 2	16	0	1	3	16	2	0	1
Visit 3	13	1	1	5	17	1	1	0
p value	0.02				0.72
Vaginal atrophy symptoms (VAS)	Laser group (mean ± SD)				Sham group (mean ± SD)
Visit 1	17.5 ± 8.6				16.3 ± 7.5
Visit 2	10.5 ± 8.7				13.8 ± 8.9
Visit 3	6.8 ± 5.5				10.0 ± 8.2
p value	< 0.001				0.08
Vaginal atrophy score
Visit 1	6.5 ± 2.9				7.9 ± 2.9
Visit 2	4.3 ± 2.1				6.6 ± 2.3
Visit 3	3.9 ± 2.4				5.6 ± 1.9
p value	< 0.001				< 0.001
Vaginal pH
Visit 1	6.3 ± 1.4				6.6 ± 1.2
Visit 2	6.0 ± 1.3				6.6 ± 1.2
Visit 3	6.1 ± 1.2				6.2 ± 1.1
p value	0.20				0.15

Vaginal atrophy symptoms, which were measured by visual analog scale (VAS), were significantly decreased after treatment in laser group. However, there was not statistically difference of vaginal atrophy symptoms after sham operations. Table 3 also demonstrates that vaginal atrophy score after treatment were significantly lowered in both laser group and sham group (p < 0.001). There was no statistically significant difference of vaginal pH before and after treatment in both groups (p = 0.20 and 0.15 in laser group and sham group, respectively).

To determine secondary outcomes, mixed effect multiple linear regression was used and showed that there was significant change in vaginal dryness (mean change = -2.28, p = 0.01) and vaginal atrophy score (mean change = -1.49, p = 0.03) before and after treatment in laser group as compared to sham group. The changes of vaginal pH, total vaginal atrophy symptoms, pain, irritation and dyspareunia were not reach statistically significant. The secondary outcome results are shown in Table 4. For the adverse event, only one participant in sham group complained about pain during operation but it was improved after the operation.

Table 4

Secondary outcome adjusted with time and age due to imperfect randomization
Outcome	Mean change	p value	95% CI
VAS	-2.73	0.15	-6.44 to 0.98
Dryness	-2.28	0.01	-4.11 to -0.46
Irritation	-0.36	0.57	-1.64 to 0.91
Soreness	-1.18	0.07	-2.44 to 0.08
Dyspareunia	-1.50	0.09	-3.22 to 0.22
Vaginal atrophy score	-1.49	0.03	-2.82 to -0.16
pH	-0.0	0.86	-0.61 to 0.51

Vaginal erbium YAG laser is a novel treatment for vaginal atrophy or, newly termed, genitourinary syndrome of menopause (GSM). The literatures showed effectiveness and safety of intravaginal laser therapy while erbium YAG laser is considered as the safer choice as compared to fractional CO₂ laser because of its non-ablative effect.^4–8

Mechanism of action of erbium YAG laser is photothermal effect to vaginal epithelium causes superficial tissue shrinkage, neovascularization, neocollagenesis results in vaginal tissue thickening and increases vaginal elasticity.⁴

In the literature, there are few studies reporting an improvement of vaginal atrophy symptoms after vaginal erbium YAG laser treatment.^6–8 However, there was no study about vaginal erbium YAG laser and vaginal lactobacilli which can be used as an objective measurement. Vaginal lactobacilli are vaginal normal flora that dynamically changed with vaginal maturation and play a role in maintaining vaginal acidity and compete with pathogens.^9,10,12

To our knowledge, this study is the first randomized sham-controlled study to assess the effect of vaginal erbium YAG laser on vaginal lactobacilli, vaginal pH and vaginal atrophy symptoms. This study reports the objective and subjective measurements of vaginal atrophy after vaginal erbium YAG laser treatment. The results show an increase of vaginal lactobacilli grading after two laser applications. The increase of vaginal lactobacilli grading can be described by the effect of vaginal laser on vaginal epithelial maturation that superficial cells of vaginal epithelium are increased after treatment with vaginal laser. The superficial cells are glycogen-rich cells and lactobacilli use glycogen as their nutrients and produce lactic acid. Moreover, vaginal atrophy symptoms assessed by visual analog scale (VAS) and vaginal atrophy score were improved after laser treatments. The reduction in VAS score in this study is similar to the results of previous studies.^{5–8,13−15} However, unlike several reports,^9,16 this study does not revealed statistically difference of vaginal pH before and after vaginal laser applications. This can be resulted from the majority of participants with grade 1 vaginal lactobacilli after vaginal laser treatment. Their vaginal epithelium contains only few lactobacilli that cannot produce enough lactic acid to lower vaginal pH.

The safety of intravaginal laser therapy is one of major concerns. The result form previous studies showed only few minor adverse events after vaginal laser applications.^5,8,16,17 In our study, there was only one patient in sham group complained about pain after treatment and it was improved during the follow-up period. While there was no reported side effect after treatment in laser group. Therefore, the safety result form our study is concordant with previous studies^5,8 and can confirm the short-term safety of vaginal erbium YAG laser treatment. However, long term follow-up is needed to evaluate the long-term safety of this treatment.

There are several strengths in this study. First, this study is a randomized sham-controlled study which can diminish the bias and placebo effect. Second, our study evaluates both subjective outcome (VAS score) and objective outcomes (vaginal lactobacilli, vaginal pH, vaginal atrophy score) of vaginal atrophy after vaginal laser treatment.

The limitation of this study is different baseline characteristic between laser group and sham group (participants in laser group were younger that participants in sham group) which is resulted from imperfect randomization.

There was significant amount of participants loss to follow-up during study period because of COVID-19 situation and personal illness that did not related to the procedure. However, the amount of participants loss to follow-up were similar in both laser group and sham group (2/22 and 3/22, respectively). Therefore, an effect of loss to follow-up might not interfere with the outcome of this study.

For further research, increases sample size or vaginal laser applications might show statistically improvement of both subjective and objective measurements of vaginal atrophy after vaginal erbium laser.

There was no statistically significant difference of vaginal lactobacilli grading before and after treatment with erbium YAG laser as compared to sham procedure. However, grade of lactobacilli tends to increase after two applications of vaginal laser. Vaginal “dryness” symptom was improved significantly after treatment with erbium YAG laser. There was no reported serious adverse event.

Ethics approval and consent to participate: this study had been performed in accordance with the Declaration of Helsinki and was approved by Institutional Review Board of Faculty of Medicine, Chulalongkorn University
Consent for publication: Not applicable
Availability of data and materials: all data and materials are described in the manuscript and tables
Competing interests: the authors declare that they have no competing interests
Funding: the study was funded by the Ratchadapisek Sompoch Endowment Fund Chulalongkorn University Fund.
Authors' contributions: N.P. wrote the main manuscript text, prepared all figures and tables, K.P and A.S. reviewed the manuscript
Acknowledgements: we would like to thank the Gynecologic Outpatient Clinic and Gender Health Clinic at King Chulalongkorn Memorial Hospital for their support in collecting the data. Special thanks to Dr.Tanittha Chatsuwan, Department of Microbiology, Faculty of Medicine, Chulalongkorn University and her colleagues who evaluated the grade of vaginal lactobacilli. Of course, we are grateful to all subjects in this study.

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No competing interests reported.

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Number of vaginal lactobacilli in postmenopausal women with vaginal atrophy before and after treatment with erbium YAG laser, a randomized sham-controlled trial

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