A multicenter, randomized, placebo-controlled study to select the minimum effective dose of estetrol in postmenopausal participants (E4Relief): part 2—vaginal cytology, genitourinary syndrome of menopause, and health-related quality of life

Abstract

Objective 

A phase 2 study showed that 15 mg estetrol (E4) alleviates vasomotor symptoms (VMS). Here, we present the effects of E4 15 mg on vaginal cytology, genitourinary syndrome of menopause, and health-related quality of life.

Methods 

In a double-blind, placebo-controlled study, postmenopausal participants (n = 257, 40-65 y) were randomized to receive E4 2.5, 5, 10, or 15 mg or placebo once daily for 12 weeks. Outcomes were the vaginal maturation index and maturation value, genitourinary syndrome of menopause score, and the Menopause Rating Scale to assess health-related quality of life. We focused on E4 15 mg, the dose studied in ongoing phase 3 trials, and tested its effect versus placebo at 12 weeks using analysis of covariance.

Results 

Least square (LS) mean percentages of parabasal and intermediate cells decreased, whereas superficial cells increased across E4 doses; for E4 15 mg, the respective changes were −10.81% (P = 0.0017), −20.96% (P = 0.0037), and +34.17% (P < 0.0001). E4 15 mg decreased LS mean intensity score for vaginal dryness and dyspareunia (−0.40, P = 0.03, and −0.47, P = 0.0006, respectively); symptom reporting decreased by 41% and 50%, respectively, and shifted to milder intensity categories. The overall Menopause Rating Scale score decreased with E4 15 mg (LS mean, −3.1; P = 0.069) and across doses was associated with a decreasing frequency and severity of VMS (r = 0.34 and r = 0.31, P < 0.001).

Conclusions 

E4 demonstrated estrogenic effects in the vagina and decreased signs of atrophy. E4 15 mg is a promising treatment option also for important menopausal symptoms other than VMS.

In Brief

15 mg of E4 once daily, an effective dose for alleviating vasomotor symptoms of menopause, also has a benefi cial effect on vaginal cytology and maturation, vaginal dryness, vaginal pain with sexual activity, as well as quality of life, in particular on the somato-vegetative and psychological symptoms commonly observed with estrogen deprivation in postmenopausal women.

Vulvovaginal atrophy (VVA) progresses with menopausal age. It is a distressing condition that can occur during menopause because of estrogen depletion. Symptoms include vaginal dryness, dyspareunia, postcoital bleeding, vulvar itching, and burning and, in some cases, vulvar skin atrophy and development of fissures.^1,2 Women with VVA also experience dysuria, an increase in urinary frequency and urgency, and recurrent lower urinary tract infections.³ These vulvovaginal and urinary complaints caused by estrogen deficiency are described using the term genitourinary syndrome of menopause (GSM).⁴ GSM is underreported and not always considered by healthcare providers, leading to delayed management and undertreatment.⁵ Both large US and European REVIVE surveys (Real Participants' Views of Treatment Options for Menopausal Vaginal Changes) report a prevalence of more than 50% for GSM in untreated participants.^6,7 The European Vulvovaginal Epidemiological Survey (EVES) found that, in postmenopausal participants attending gynecology/menopause clinics, 90% were diagnosed with GSM, when symptom scores and a clinical examination were combined.⁸ In a recently updated North American Menopause Society (NAMS) position statement, GSM is recognized to be an important part of the spectrum of symptoms of menopause.⁹ There is also a large consensus that GSM symptoms may severely impact the quality of life (QoL) of women during menopause, especially sexual function, self-perception, and body image.¹⁰ Moreover, it is suggested that menopausal symptoms, particularly severe hot flashes, are predictive or associated with health problems (eg, cardiovascular disease, cognition impairment).^11,12 All these effects may impact health-related quality of life (HR-QoL).¹³

Local or systemic estrogen administration is the most effective therapy to alleviate GSM,¹⁴ as well as providing significant relief of other menopause specific symptoms and health status. Results from the EVES survey showed that postmenopausal Spanish and Italian participants (n = 918) who received systemic menopausal treatment experienced fewer and less severe VVA symptoms compared with participants on nonhormone or local therapy.⁸ In addition, the same participants scored better on the EuroQoL questionnaire (EQ5D3L), which measures the impact of VVA on QoL, the Day-to-Day Impact of Vaginal Aging scale, the Female Sexual Function Index, and the Female Sexual Distress Scale. They also reported better vaginal and vulvar health indices, with systemic estrogen treatment.^15-20

Estetrol (E4), produced by the human fetal liver, is a native estrogen with selective tissue activity.^21-25 The high oral availability of E4 and its long elimination half-life of between 24 and 32 hours are important prerequisites for administration of a once-daily oral drug.^26-28 A limited phase 1/2a study showed that E4 2 mg (n = 5) and 10 mg (n = 8) improved vasomotor symptoms (VMS) and had a positive trophic estrogenic effect on vaginal cytology.²⁹ Subsequently, a phase 2b study (E4Relief) that included both hysterectomized and nonhysterectomized participants was performed to select the minimum effective dose of E4 for alleviating VMS. We previously published data on the treatment effect of E4 2.5, 5, 10, or 15 mg on VMS and on endometrial and overall safety.³⁰ Here, we report the effects of E4 on the vaginal epithelial cell maturation index (VMI) and maturation value (MV), GSM, and HR-QoL scored by the Menopause Rating Scale (MRS), with special attention on the effect of E4 15 mg, which is an effective dose for the treatment of VMS as documented by the phase 2b study³⁰ and is the dose studied in two ongoing phase 3 trials (Clinicaltrials.gov NCT04209543, EudraCT 2019-001289-14).

METHODS

Study design and objectives

This was a multicenter, randomized, double-blind, placebo-controlled, phase 2 study in postmenopausal hysterectomized or nonhysterectomized women conducted in Europe (Clinicaltrials.gov NCT02834312, EudraCT 2015-004018-44). The study was approved by independent ethics committees of the participating centers and conducted in accordance with current ethical principles. All participants provided written informed consent.

Treatments

Eligible participants were randomly allocated (1:1:1:1:1) to one of five treatment groups: E4 2.5, 5, 10, or 15 mg (SEQENS VLG CHEM, Villeneuve-la-Garenne, France) or placebo. Treatments were administered orally once daily for 12 consecutive weeks. E4 is a new chemical entity, and therefore, its effects on menopausal symptoms and the endometrium needed to be established as a monotherapy for a period of 12 weeks.²⁴ Therefore, not monthly, but after completing the E4 treatment period, nonhysterectomized participants received 10 mg dydrogesterone (Duphaston, first batch: Abbott Healthcare Products, Weesp, the Netherlands; second batch: BGP Products, Hoofddorp, the Netherlands) once daily for 14 days. The latter administration had a full progestogenic effect on the endometrium in all participants.³⁰

Participant selection

Included participants were healthy, aged 40 to 65 years (inclusive), had a body mass index between 18.0 and 35.0 kg/m² (inclusive), with at least 7 moderate to severe hot flashes per day or at least 50 moderate to severe hot flashes in the week preceding randomization. Participants with an intact uterus were eligible when transvaginal ultrasound showed a bilayer endometrial thickness of ≤5 mm. More detailed selection criteria have been described previously, as well as potential washout conditions.³⁰

Assessments

To calculate the VMI, cytological samples (vaginal Pap smears) were collected at baseline and at week 12 (W12) and assessed at a central laboratory. The VMI was defined as the percentage of parabasal cells (least mature type), intermediate cells (moderately mature), and superficial cells (most mature) in the total sampled number of squamous cells from the vaginal epithelium. The relative percentage of these cells change in women after menopause, with a shift to fewer intermediate and superficial cells and more parabasal cells. The corresponding MV, a clinical measure of vaginal estrogenization, was derived by multiplying these percentages by 0, 0.5, and 1, for parabasal, intermediate, and superficial cells, respectively, and summed up as a single score.^31,32 Vaginal pH was measured onsite by the study staff using a standardized vaginal pH paper test at baseline and at W12.

Genitourinary symptoms were self-scored in an e-diary at baseline and at W12. Vaginal dryness, vaginal and/or vulvar irritation/itching, dysuria, and vaginal pain associated with sexual activity (dyspareunia) were scored as 0 (none), 1 (mild), 2 (moderate), or 3 (severe). Vaginal bleeding associated with sexual activity was scored as 0 (absent) or 1 (present).

The burden of menopause specific symptoms can be assessed by standardized HR-QoL questionnaires, with several subscales encompassing different symptomatologic domains, allowing a psychometric approach.¹³ We used the MRS, a menopause-specific HR-QoL questionnaire that is short and easy to complete and frequently used in clinical studies. The MRS was developed in the 90s and has been validated in different countries and languages.^33,34 It is a self-administered questionnaire of 11 standardized items submitted to factor analysis, allowing the participants to record noticeable changes during menopausal hormone therapy (MHT), and for healthcare professionals to appreciate valuable changes.³⁵ The MRS has three symptom domains; psychological (four items), somato-vegetative (four items), and urogenital (three items).^33,34 The severity of each item is rated on a 5-point Likert scale as 0 (none), 1 (mild), 2 (moderate), 3 (severe), and 4 (very severe). The total score is the sum of the symptom scores and ranges from 0 to 44. A total score of 0 to 4 indicates “no/little complaints”; 5 to 8, “mild complaints”; 9 to 15, “moderate complaints”; and 16 to 44, “severe complaints.”³⁶ The MRS was completed by the participants at baseline, week 4 (W4), and W12.

Statistical analysis

All analyses were based on all randomized participants who received at least one study dose. Sample size for this study was based on the expected change from baseline in weekly frequency of moderate to severe VMS at W12, which was the primary endpoint. We used the last observation carried forward method to account for missing data. All efficacy variables except vaginal bleeding associated with sexual activity were analyzed as continuous variables. The effect of E4 at W4 and W12 was compared with placebo using analysis of covariance with treatment as the fixed effect and baseline as the covariate. Post hoc, a treatment by center interaction was included in case of a significant center effect. The analysis of covariance treatment least square (LS) means, that is, adjusted for the effect of the other covariates, were compared over all treatment groups, and pairwise differences versus placebo were conducted using Dunnett's adjustment for multiplicity. Post hoc, the change in the percentage of participants in each GSM severity category (none, mild, moderate, and severe) in each E4 group was compared with the change in the placebo group using logistic regression. All statistical tests and CI were two-sided, and α was set at 0.05. The parameter of vaginal bleeding associated with sexual activity was analyzed as a binary variable by using a logistic regression model with treatment group, study center, and baseline value as covariates.

Based on the data from all participants (ie, irrespective of treatment), the relationship between changes from baseline to W12 in the frequency and intensity of moderate to severe VMS and the changes in MRS overall and domain scores was post hoc examined using Spearman correlation.

To assess the overall improvement in individual MRS parameters in the E4 15 mg group, nonparametric Friedman tests of differences among repeated measures (baseline, W4, and W12) were conducted post hoc for the MRS total score, the 3 domains, and each of the 11 items individually.

RESULTS

Participant disposition and baseline characteristics

Of the 260 randomized participants, 3 did not receive study treatment.³⁰ Thirty-two of the 257 treated participants (12.5%) were hysterectomized. The mean (SD) age was 54.2 (4.4) years, and the mean (SD) body mass index was 26.1 (3.9) kg/m². Treatments groups did not differ for any of the baseline characteristics (Table 1).

TABLE 1 - Baseline demographics and characteristics of postmenopausal women randomized to E4 2.5, 5, 10, or 15 mg or placebo

	E4 2.5 mg n = 52	E4 5 mg n = 47	E4 10 mg n = 54	E4 15 mg n = 49	Placebo n = 55	Total N = 257	P
Age, mean (SD), y	54.0 (4.4)	53.8 (4.8)	54.3 (4.4)	55.2 (4.0)	53.7 (4.4)	54.2 (4.4)	0.45 a
BMI, mean (SD), kg/m2	25.4 (3.7)	26.1 (4.3)	26.0 (3.7)	26.2 (3.9)	26.6 (3.9)	26.10 (3.9)	0.64 a
FSH, mean (SD), IU/mL	86.1 (29.4)	88.0 (28.3)	92.2 (25.4)	93.8 (26.1)	91.0 (29.9)	90.2 (27.8)	0.63 a
No. pregnancies, median (range)	2 (0-5)	2 (0-5)	2 (0-5)	2 (0-8)	2 (0-6)	2 (0-8)	0.16 b
Time since menopause (range), y	3 (1-28)	3 (1-22)	3.5 (1-22)	3 (1-22)	4 (1-21)	3 (1-28)	>0.99 b
Hysterectomized, n (%)	8 (15.4)	4 (8.5)	5 (9.3)	9 (18.4)	6 (10.9)	32 (12.5)	0.51 c
Smoking, n (%)	6 (11.5)	4 (8.5)	11 (20.4)	2 (4.1)	8 (14.5)	31 (12.1)	0.12 c

P value: associated with the null hypothesis of homogeneous treatment group.

BMI, body mass index; E4, estetrol; FSH, follicle-stimulating hormone.

^aOne-way analysis of variance.

^bKruskal-Wallis test.

^cχ² Test.

Vaginal cytology: maturation index and MV

In all E4 groups, the arithmetic mean percentage of parabasal and intermediate cells decreased from baseline to W12, whereas the mean percentage of superficial cells increased. In the E4 15 mg group, a significant decrease of parabasal and intermediate cells and an increase of superficial cells were observed (LS means: −10.81% [95% CI, −18.29% to −3.32%], P = 0.0017; −20.96 [95% CI, −36.43% to −5.49%], P = 0.0037; +34.17% [95% CI, 19.80%-48.53%], P < 0.0001, respectively) (Table 2). In the placebo group, the changes in the percentages of each cell type from baseline were minimal (Table 2).

TABLE 2 - Vaginal maturation index: mean percentage of squamous parabasal cells, intermediate cells, and superficial cells (LOCF)

	Parabasal cells (%)					Intermediate cells (%)					Superficial cells (%)
	BL (SD)	W12 (SD)	CH	LS mean a (95% CI)	P	BL (SD)	W12 (SD)	CH	LS mean a (95% CI)	P	BL (SD)	W12 (SD)	CH	LS mean a (95% CI)	P
E4 2.5 mg (n = 52)	13.6 (24.7)	1.2 (2.6)	−12.3	−11.38 (−18.67 to −4.08)	0.0006	73.2 (27.9)	65.8 (26.6)	−7.6	−6.57 (−21.32 to 8.18)	0.6532	13.2 (21.9)	32.9 (26.8)	+19.9	17.45 (3.44-31.46)	0.0089
E4 5 mg (n = 44)	26.6 (35.8)	5.5 (15.9)	−20.7	−9.56 (−17.15 to −1.97)	0.0080	60.8 (33.8)	50.4 (32.0)	−11.9	−17.86 (−33.20 to −2.51)	0.0164	12.7 (19.5)	44.1 (34.0)	+32.6	28.68 (14.09-43.26)	<0.0001
E4 10 mg (n = 52)	17.2 (28.5)	1.6 (3.9)	−15.6	−11.78 (−19.07 to −4.49)	0.0004	69.8 (28.2)	47.2 (31.8)	−21.8	−22.95 (−38.01 to −7.90)	0.0009	13.0 (19.2)	51.2 (32.9)	+37.4	35.18 (21.15-49.21)	<0.0001
E4 15 mg (n = 47)	28.4 (38.7)	4.9 (19.0)	−23.4	−10.81 (−18.29 to −3.32)	0.0017	58.1 (35.5)	44.1 (31.5)	−13.4	−20.96 (−36.43 to −5.49)	0.0037	13.4 (22.0)	51.0 (33.0)	+36.8	34.17 (19.80-48.53)	<0.0001
Placebo (n = 54)	21.9 (33.8)	16.3 (27.8)	−7.1	—		69.2 (31.8)	68.8 (29.5)	0.8			8.9 (14.2)	14.9 (22.0)	+6.3

Sampling was not performed in 1, 3, 1, 2, and 1 participant in the E4 2.5, 5, 10, 15 mg and placebo groups, respectively.

BL, baseline; CH, mean change from baseline; E4, estetrol; LOCF, last observation carried forward; LS, least square; W12, week 12.

^aAnalysis of covariance on the mean change from baseline at W12 with treatment and center as fixed effects and baseline as covariate, including pairwise comparisons versus placebo (with adjustment for multiple comparisons according to Dunnett). For intermediate cells, the interaction treatment by site was included in the model. Values in bold are significant (P < 0.05).

The MV at baseline and at W12 is shown in Figure 1. At baseline, the mean (SD) MV was 43.5 (20.50) in the placebo group and ranged from 42.5 (26.01) to 49.8 (18.71) in the E4 treatment groups. At W12, the mean MV increased in all E4 groups. The largest absolute arithmetic mean change from baseline to W12 was observed with E4 15 mg (+30.6) (from 42.5 [SD, 26.01] to 73.1 [SD, 21.85]), which was significant (LS mean: +27.69 [95% CI, 22.30-33.07], P < 0.0001). Placebo had a negligible effect (mean change, 5.8; from 49.3 [SD, 20.28] to 43.5 [SD, 20.50]).

FIG. 1: Vaginal MV at baseline and at W12 (LOCF). One participant was randomized to E4 15 mg but temporarily took E4 2.5 mg and was excluded from the analysis. One participant was randomized to E4 2.5 mg and temporarily took E4 10 mg but was included in the 2.5 mg group for the analysis. *P < 0.001 versus placebo, analysis of covariance with fixed factors treatment and pooled site and covariate baseline value, including pairwise comparisons versus placebo (with adjustment for multiple comparisons according to Dunnett). E4, estetrol; LOCF, last observation carried forward; W12, week 12; MV, maturation value.

Vaginal pH

At baseline, the mean vaginal pH was 4.6 in the placebo group and ranged from 4.6 to 4.8 in the E4 treatment groups. The pH decreased in all E4 groups and increased slightly in the placebo group (−0.08 [SD, 0.78] to −0.22 [SD, 1.17] for E4; +0.12 [SD, 0.72] for placebo). The changes versus baseline were not significantly different between any of the E4 treatment groups and the placebo group (Supplemental Table 1, https://links.lww.com/MENO/B84).

Self-reported genitourinary symptoms

Overall, at baseline, the percentage of participants reporting GSM was comparable between all treatment groups (Supplemental Table 2, https://links.lww.com/MENO/B85). The overall percentage of participants reporting any symptoms at baseline was 16.8% (43 of 256) for dysuria, 41.0% (105 of 256) for vaginal and/or vulvar irritation, 47.1% (120 of 255) for vaginal pain associated with sexual activity, and 66.8% (171 of 256) for vaginal dryness (Supplemental Table 2, https://links.lww.com/MENO/B85).

Compared with placebo, the mean intensity score at W12 significantly decreased for vaginal dryness in the E4 15 mg group (LS mean: −0.40 [95% CI, −0.76 to −0.03], P = 0.03) and for vaginal pain in the E4 5, 10, and 15 mg groups (LS mean E4 15 mg: −0.47 [95% CI, −0.77 to −0.17], P < 0.05), whereas there were no significant differences for irritation/itching and dysuria (Table 3).

TABLE 3 - GSM symptom scores at baseline and at W12 (LOCF)

		Vaginal dryness					Vaginal pain associated with sexual activity a					Vaginal and/or vulvar irritation/itching					Dysuria
		BL (SD)	W12 (SD)	LS mean (95% CI)	Pb		BL (SD)	W12 (SD)	LS mean (95% CI)	Pb		BL (SD)	W12 (SD)	LS mean (95% CI)	Pb		BL (SD)	W12	LS mean (95% CI)	Pb
E4 2.5 mg c (n = 53)		1.0 (0.9)	0.5 (0.8)	−0.23 (−0.58 to 0.13)	0.33		0.6 (0.8)	0.3 (0.7)	−0.27 (−0.56 to 0.02)	0.076		0.7 (1.0)	0.3 (0.6)	−0.23 (−0.53 to 0.06)	0.17		0.2 (0.6)	0.0 (0.2)	−0.12 (−0.31 to 0.06)	0.29
E4 5 mg (n = 47)		1.3 (0.9)	0.7 (0.9)	−0.30 (−0.66 to 0.05)	0.12		1.0 (1.1)	0.5 (0.8)	−0.33 (−0.62 to −0.03)	0.025		0.6 (0.9)	0.4 (0.7)	−0.06 (−0.37 to 0.24)	0.96		0.2 (0.6)	0.0 (0.2)	−0.12 (−0.31 to 0.07)	0.35
E4 10 mg (n = 53)		1.0 (0.9)	0.5 (0.8)	−0.32 (−0.67 to 0.03)	0.08		0.6 (0.8)	0.2 (0.4)	−0.47 (−0.76 to −0.18)	0.0004		0.7 (0.9)	0.3 (0.6)	−0.21 (−0.51 to 0.09)	0.25		0.2 (0.6)	0.1 (0.2)	−0.12 (−0.30 to 0.07)	0.34
E4 15 mg c (n = 48)		1.1 (1.0)	0.5 (0.7)	−0.40 (−0.76 to −0.03)	0.03		0.7 (0.9)	0.3 (0.5)	−0.47 (−0.77 to −0.17)	0.0006		0.5 (0.9)	0.4 (0.7)	−0.08 (−0.38 to 0.23)	0.93		0.3 (0.6)	0.3 (0.6)	0.08 (−0.10 to 0.27)	0.64
Placebo (n = 55)		1.3 (1.1)	0.9 (1.0)	—	—		1.0 (1.1)	0.7 (1.0)	—	—		0.8 (0.9)	0.5 (0.8)	—	—		0.3 (0.6)	0.2 (0.6)	—	—

BL, baseline; E4, estetrol; GSM, genitourinary syndrome of menopause; LOCF, last observation carried forward; LS, least square; W12, week 12.

^aOne participant did not complete the item for vaginal pain associated with sexual activity at baseline.

^bPairwise comparisons versus placebo (with Dunnett's adjustment for multiple comparisons) using an analysis of covariance with treatment and center as fixed effects and baseline as covariate. For vaginal pain and vaginal dryness, the interaction treatment by site was included in the model. Values in bold are significant (P < 0.05).

^cOne participant was randomized to E4 15 mg but temporarily took E4 2.5 mg and was excluded from the analysis. One participant was randomized to E4 2.5 mg and temporarily took E4 10 mg but was included in the 2.5 mg group for the analysis.

For vaginal dryness, vaginal pain, and irritation/itching, there was a greater percentage of participants shifting to a less intense category from baseline to W12 with E4 than in the placebo group. The percentage of participants who reported a change from baseline from having symptoms to having no symptoms increased in the E4 groups to a larger extent than in the placebo group, with a commensurate decrease in those reporting severe, moderate, and mild symptoms (Fig. 2 and Supplemental Table 2, https://links.lww.com/MENO/B85). For dysuria, the decrease in the percentage of participants reporting mild, moderate, or severe symptoms with E4 was comparable with the placebo group (Supplemental Table 2, https://links.lww.com/MENO/B85).

FIG. 2: Mean shift at W12 (minus baseline values) of the percentage of participants reporting no, mild, moderate, or severe vaginal dryness (A) or vaginal pain with sexual activity (B) in the E4 2.5, 5, 10, and 15 mg and placebo groups and for all participants (total) (LOCF). One participant was randomized to E4 15 mg but temporarily took E4 2.5 mg and was excluded from the analysis. One participant was randomized to E4 2.5 mg and temporarily took E4 10 mg, but was included in the 2.5 mg group for the analysis. E4, estetrol; LOCF, last observation carried forward.

In the E4 15 mg group in particular, the percentage of participants reporting any vaginal dryness decreased from 60.4% to 35.4%. Reporting of mild dryness was stable (25.0%) and decreased from 25.0% to 10.4% for moderate and from 10.4% to 0.0% for severe dryness (Supplemental Table 2, https://links.lww.com/MENO/B85). Reporting of any vaginal pain decreased from 45.8% to 22.9%, from 25.0% to 18.8% for mild, from 16.7% to 4.2% for moderate, and from 4.2% to 0.0% for severe pain. Reporting of irritation/itching was low at baseline (27.1%) and stayed low at W12 (25.0%). Reporting of any dysuria decreased from 22.9% to 18.7%, from 16.7% to 10.4% for mild, increased slightly from 6.3% to 8.3% for moderate, and remained at 0.0% for severe dysuria (Supplemental Table 2, https://links.lww.com/MENO/B85).

Five participants reported having vaginal bleeding associated with sexual activity at baseline, that is, three in the E4 5 mg group and two in the placebo group: at the end of treatment, all three vaginal bleeding episodes had resolved in the E4 5 mg group, whereas the two bleeding episodes persisted in the placebo group. At W12, two other episodes of minimal bleeding at intercourse were recorded in the E4 15 mg group. Compared with placebo, the change in intensity score at W12 for E4 15 mg was not significant (LS proportion: 10.38 [95% CI, −223.76 to 244.51]).

Menopause Rating Scale

The MRS total and domain scores at baseline, W4, and W12 are presented in Table 4 and shown in Supplemental Figure 1, https://links.lww.com/MENO/B86. At baseline, the arithmetic mean MRS total scores varied from 16.4 (SD, 8.14) (E4 15 mg) to 18.2 (SD, 8.85) (placebo), with a mean value of 17.1 (SD, 7.83) for all groups; the somato-vegetative symptoms score was the highest score across all groups. Overall, scores improved (ie, decreased) in all treatment groups. The largest reductions in the MRS total score both at W4 and W12 were observed in the E4 15 mg group. This improvement in the MRS total score in the E4 15 mg group was partly driven by a reduction of the somato-vegetative domain score, which, in comparison with the change in the placebo group, was statistically significant at W4 (LS mean: −1.62 [95% CI, −2.72 to −0.51], P = 0.0014) but not significant at W12 (LS mean: −1.28 [95% CI, −2.59 to 0.03], P = 0.058). No statistically significant differences versus placebo were observed for the psychological and urogenital symptom scores. Numerically, among these latter scores, the change versus baseline was most pronounced for the effect of E4 15 mg on psychological symptoms.

TABLE 4 - MRS total and domain scores at baseline, W4, and W12 (LOCF)

	E4 2.5 mg	E4 5 mg	E4 10 mg	E4 15 mg	Placebo
	n = 53 a	n = 47	n = 53	n = 48 a	n = 55
MRS total score
Baseline, mean (SD)	16.5 (7.2)	16.5 (7.3)	17.6 (7.6)	16.4 (8.1)	18.2 (8.9)
W4
Absolute change from baseline, mean (SD)	−6.2 (5.7)	−6.0 (6.2)	−6.0 (6.4)	−7.8 (7.6)	−5.4 (6.5)
Change from baseline b , LS mean (95% CI)	−1.44 (3.98 to −1.09)	−1.41 (−4.02 to 1.19)	−0.71 (−3.23 to −1.81)	−3.15 (−5.74 to −0.56)	—
P value vs placebo c	0.44	0.48	0.90	0.011
W12
Absolute change from baseline, mean (SD)	−7.0 (6.3)	−5.5 (7.2)	−7.9 (8.3)	−8.3 (7.9)	−6.8 (8.7)
Change from baseline b , LS mean (95% CI)	−1.61 (−4.74 to 1.52)	−0.35 (−3.51 to 2.81)	−1.69 (−4.77 to 1.39)	−3.07 (−6.32 to 0.17)	—
P value vs placebo c	0.53	>0.99	0.47	0.069
Psychological symptoms score
Baseline, mean (SD)	5.3 (3.5)	5.6 (3.5)	6.0 (3.5)	5.9 (4.0)	6.3 (4.1)
W4
Absolute change from baseline, mean (SD)	−2.3 (3.1)	−2.6 (2.6)	−2.3 (3.1)	−3.0 (3.4)	−2.0 (2.8)
Change from baseline b , LS mean (95% CI)	−0.63 (−1.77 to 0.51)	−0.89 (−2.06 to 0.28)	−0.36 (−1.50 to 0.77)	−1.11 (−2.28 to 0.06)	—
P value vs placebo c	0.47	0.19	0.85	0.067
W12
Absolute change from baseline, mean (SD)	−2.6 (3.2)	−2.0 (3.8)	−2.8 (3.6)	−3.4 (4.0)	−2.5 (3.6)
Change from baseline b , LS mean (95% CI)	−0.71 (−2.15 to 0.72)	−0.16 (−1.61 to 1.29)	−0.32 (−1.74 to 1.09)	−1.21 (−2.69 to 0.27)	—
P value vs placebo c	0.56	>0.99	0.95	0.15
Somato-vegetative symptoms score
Baseline, mean (SD)	7.6 (2.8)	7.0 (2.9)	7.8 (2.5)	7.2 (2.6)	7.7 (3.3)
W4
Absolute change from baseline, mean (SD)	−2.4 (2.5)	−2.0 (2.4)	−2.2 (2.7)	−3.4 (2.9)	−2.1 (2.5)
Change from baseline b , LS mean (95% CI)	−0.32 (−1.39 to 0.76)	−0.25 (−1.36 to 0.86)	−0.03 (−1.11 to 1.05)	−1.62 (−2.72 to −0.51)	—
P value vs placebo c	0.89	0.95	>0.99	0.0014
W12
Absolute change from baseline, mean (SD)	−2.5 (2.8)	−2.1 (2.9)	−3.5 (3.1)	−3.7 (2.8)	−2.6 (3.7)
Change from baseline b , LS mean (95% CI)	0.05 (−1.22 to 1.33)	0.16 (−1.16 to 1.48)	−0.74 (−2.02 to 0.54)	−1.28 (−2.59 to 0.03)	—
P value vs placebo c	>0.99	>0.99	0.42	0.058
Urogenital symptoms score
Baseline, mean (SD)	3.5 (2.3)	4.0 (2.7)	3.8 (2.9)	3.3 (2.9)	4.2 (3.1)
W4
Absolute change from baseline, mean (SD)	−1.5 (1.7)	−1.4 (2.4)	−1.5 (2.3)	−1.3 (2.5)	−1.3 (2.4)
Change from baseline b , LS mean (95% CI)	−0.52 (−1.36 to 0.32)	−0.25 (−1.12 to 0.61)	−0.33 (−1.17 to 0.51)	−0.44 (−1.30 to 0.43)	—
P value vs placebo c	0.36	0.88	0.73	0.54
W12
Absolute change from baseline, mean (SD)	−1.8 (1.8)	−1.4 (2.8)	−1.7 (2.8)	−1.2 (2.6)	−1.7 (3.0)
Change from baseline b , LS mean (95% CI)	−0.62 (−1.54 to 0.30)	0.10 (−0.85 to 1.04)	−0.28 (−1.20 to 0.64)	−0.10 (−1.04 0.85)	—
P value vs placebo c	0.28	>0.99	0.87	>0.99

E4, estetrol; LOCF, last observation carried forward; MRS, Menopause Rating Scale; W4, week 4; W12, week 12.

^aOne participant was randomized to E4 15 mg but temporarily took E4 2.5 mg and was excluded from the analysis. One participant was randomized to E4 2.5 mg and temporarily took E4 10 mg but was included in the 2.5 mg group for the analysis.

^bRelative to the change versus baseline in the placebo group.

^cAnalysis of covariance with treatment and center as fixed factors and baseline value as a covariate. For the total score and the psychological symptom score at visit 4, the interaction treatment by site was included in the model. Values in bold are significant (P < 0.05).

The observed changes in the E4 15 mg group from baseline to W12 were highly significant for all domain items except for bladder problems (Table 5).

TABLE 5 - Improvement in individual MRS domain items in the 15 mg E4 group (n = 48^a), based on scoring (mean [SD]) at baseline, W4, and W12 (LOCF)

MRS parameters	Score at baseline	Score at W4	Score at W12	Friedman test Pb
Psychological symptoms	5.9 ± 4.0	2.9 ± 2.6	2.6 ± 2.4	<0.0001
Depressive mood	1.5 ± 1.2	0.6 ± 0.8	0.6 ± 0.7	<0.0001
Anxiety	1.2 ± 1.1	0.5 ± 0.7	0.4 ± 0.8	<0.0001
Irritability	1.5 ± 1.1	0.9 ± 0.9	0.8 ± 0.9	0.0001
Exhaustion	1.7 ± 1.2	0.9 ± 0.9	0.7 ± 0.9	<0.0001
Somato-vegetative symptoms	7.2 ± 2.6	3.7 ± 2.3	3.5 ± 2.4	<0.0001
Hot flashes and sweating	2.8 ± 0.7	1.4 ± 1.0	1.0 ± 0.8	<0.0001
Heart discomfort	0.8 ± 0.9	0.4 ± 0.6	0.4 ± 0.7	0.0016
Sleep problems	2.1 ± 1.1	1.0 ± 0.9	1.0 ± 0.9	<0.0001
Joint and muscular discomfort	1.5 ± 1.2	0.9 ± 1.0	1.1 ± 1.2	0.0424
Urogenital symptoms	3.3 ± 2.9	2.0 ± 2.3	2.1 ± 2.1	0.0151
Sexual problems	1.3 ± 1.2	0.7 ± 0.9	0.7 ± 1.0	0.0004
Bladder problems	0.8 ± 1.1	0.7 ± 1.0	0.9 ± 1.1	0.7739
Vaginal dryness	1.1 ± 1.3	0.6 ± 0.9	0.6 ± 0.7	0.0024
MRS total	16.4 ± 8.1	8.7 ± 6.2	8.1 ± 5.8	<0.0001

E4, estetrol; LOCF, last observation carried forward; MRS, Menopause Rating Scale; W4, week 4; W12, week 12.

^aOne participant was randomized to E4 15 mg but temporarily took E4 2.5 mg and was excluded from the analysis.

^bW12 versus baseline after Friedman's test for repeated measures.

The correlation between the changes from baseline of VMS frequency/severity and the MRS overall scores at W12, irrespective of treatment, was moderate though statistically significant (r = 0.34 and r = 0.31, P < 0.001). For the psychological symptoms score and the somato-vegetative symptoms score, the correlations were moderate, and for urogenital symptoms score, the correlations were low (all significant, except for VMS frequency and urogenital symptoms) (Table 6).

TABLE 6 - Spearman correlation between changes versus baseline at W12 in the MRS overall and domain scores, and changes in moderate-to-severe VMS frequency and severity, irrespective of treatment

	Change in VMS frequency		Change in VMS severity
Change in MRS (N = 256)	r	P	r	P
Overall score	0.336	<0.0001	0.310	<0.0001
Psychological symptoms score	0.256	<0.0001	0.248	<0.0001
Somato-vegetative symptoms score	0.417	<0.0001	0.381	<0.0001
Urogenital symptoms score	0.092	NS	0.129	<0.05

MRS, Menopause Rating Scale; NS, not significant; r, Spearman correlation coefficient; VMS, vasomotor symptoms; W12, week 12.

DISCUSSION

A daily dose of 15 mg E4 is effective for alleviating VMS as shown in the E4Relief phase 2b dose finding study.³⁰ Here, we present the effect of E4 2.5, 5, 10, and 15 mg on vaginal cytology, GSM, and HR-QoL, with a focus on E4 15 mg. Overall, E4 15 mg had a beneficial effect on most of the reported endpoints and therefore is a promising treatment option for all the symptoms noted previously, which are a major source of distress for many postmenopausal women.

Vaginal cytology

At baseline, all participants had high follicle-stimulating hormone levels (mean [SD], 90.2 (27.8) IU/L across groups), low E2 levels (<20 pg/mL), and amenorrhea for more than 1 year.³⁰ The percentage of parabasal cells was elevated (13.6%-28.4%) and that of superficial cells was low (8.9%-13.2%) in accordance with the expected range for untreated postmenopausal participants (superficial cells <15%).² Coelingh Bennink et al²⁹ demonstrated that oral E4 2 mg to 40 mg per day decreases vaginal atrophy within 4 weeks. E4 doses >10 mg restored superficial cells to a level comparable with the one observed in premenopausal ovulatory participants and were more effective than oral E2 valerate 2 mg per day. In this study, we found a strong effect of oral E4 on vaginal cytology, occurring even at a dose of 2.5 mg per day, with further significant increases in the percentage of superficial cells with E4 5, 10, and 15 mg versus placebo. The changes induced by E4 15 mg are comparable with those observed with oral conjugated estrogens 1.25 mg per day.^37,38 According to some authors, systemic estrogen improves GSM symptoms more powerfully than local estrogen treatment.^2,14,18,39 However, this opinion is not universal and not a recommendation of NAMS.⁹

In our study, the mean VMI increase in superficial cells at 12 weeks with E4 15 mg (+36.8%) and is higher than those reported with local treatment with E2 vaginal soft gel capsules (IMVEXXY) 10 μg (+17.9%) or E2 vaginal tablets (Vagifem) (+15%).⁴⁰

An increase in the percentage of intermediate cells from baseline of roughly 20% is observed in vaginal or oral E2 users.^40,41 In contrast, the percentages of intermediate cell types decreased slightly with 10 and 20 mg of oral E4 in the study by Coelingh Bennink et al.²⁹ In our study, the mean intermediate cells percentages ranged between 58.1% and 73.2% across treatment groups at baseline, and they decreased by 7.6% to 21.8% at W12 with increasing E4 doses. This divergence from E2 treatments is the consequence of a higher conversion to superficial cells, indicating a higher vaginal estrogenic effect with E4 10 and 15 mg.

The MV is based on weighted proportions from the VMI three cell types combined in a single score for estimating local estrogenization.³² In a cohort of 868 postmenopausal participants in the United States with adequate vaginal cytology specimens from the National Social Life, Health and Aging Project, the MV weighted mean was 44.4 in women not using hormones in the last 12 months.³² In our study, comparable baseline MV were recorded (42.5-49.8, Fig. 1), despite age differences between the two studies. MV increased by nearly 12.4% among US National Social Life, Health and Aging Project study participants who used any type of MHT in the prior 12 months. In our study, E4 use at W12 increased the MV significantly versus baseline and placebo in all treatment groups. The mean percentage change from baseline with E4 15 mg was 72%. This reflects a marked increase in estrogenization, in line with the increased VMI.

Vaginal pH

Postmenopausal vaginal atrophy is associated with scarce superficial cells and therefore decreased lactic acid production by vaginal lactobacilli hydrolysis of cellular glycogen, as well as a vaginal pH of 5.5 to 6.0, whereas, in premenopausal women with a large amount of superficial cells, the pH value is <4.5.^2,42 In our study, a rather acidic pH level (4.6-4.8) was recorded at baseline, although the participants showed a typical atrophic vaginal morphology with a high percentage of parabasal cells. In several publications, a decrease in vaginal pH correlated with an estrogen-induced increase in the percentage of vaginal superficial cells. It was therefore postulated that pH may be a simple and effective surrogate method to assess vaginal cytology.^42,43 However, as underlined by Donders et al,⁴² pH alone is not a reliable diagnostic marker for atrophy, because of several variables such as interfering local treatments, infections, and age since menopause. In our study, starting from a rather low pH, despite typical vaginal atrophy, the small additional decrease due to E4 treatment was not significant versus placebo.

Genitourinary syndrome of menopause

The mean intensity GSM scores at baseline were lower in our study than in studies of different estrogens conducted in large populations of “selected” postmenopausal women who had severe symptoms (as quantified by an elevated mean intensity score) accompanied by proven vaginal atrophy.⁴¹ Unlike these studies, we included postmenopausal women with the primary aim to assess the effect of different E4 doses on the reduction of VMS and not to focus selectively on GSM symptoms. The baseline data are therefore more in line with large “nonselected” cross-sectional surveys conducted in postmenopausal women such as the EVES and the AN evaluation of GEnitourinary symptoms in perimenopausaL women (ANGEL) studies.^18,44 Despite the low mean intensity score of GSM symptoms, the frequency of GSM symptoms was elevated: 66.8% at baseline for vaginal dryness, 47.1% for vaginal pain associated with sexual activity, 41.0% for vaginal irritation/itching, and 16.8% for dysuria. These frequencies are identical to those reported in other large studies.^7,45

At the end of study treatment (W12), the mean intensity symptom score had decreased for each symptom compared with baseline values for all treatment doses and significantly for vaginal dryness and pain with E4 15 mg. Of note, the decrease in mean intensity score at W12 versus baseline was 55% and 57% for vaginal dryness and pain, respectively. Decreases in intensity scores in treated groups versus placebo were also statistically significant for vaginal dryness and dyspareunia and nonsignificant for vaginal irritation and dysuria. These results are similar to those reported in other large “nonselected” trials using systemic estrogen treatment and in selected studies with systemic or local vaginal estrogen.^18,46,47

Vaginal pain with sexual activity and vaginal dryness are the most prominent symptoms leading to impairment of sexual functioning and related problems and are the most commonly reported symptoms in postmenopausal women.^18,32 In our study, 43% of the participants reported both dryness and pain, much more than the association with other symptoms of GSM. Interestingly, vaginal dryness itself may cause dyspareunia, although individuals may not be aware of the association between the two symptoms.^8,10,48 At W12, reporting of vaginal dryness and pain decreased by 41% and 50%, respectively. Changes for vaginal irritation and dysuria were less pronounced. These symptoms were less frequently reported at baseline, and for dysuria, the mean intensity score was low in all treatment groups (0.2-0.3). Changes were therefore small and not significant. Dysuria is also reported to be less frequent⁴⁶ and less sensitive to hormone treatment or even to worsen, although this was not a finding in our study.^49,50 For both symptoms, the already low percentage of participants with severe symptoms at baseline (2.1% and 0%, respectively) decreased to/remained at 0% at W12. Altogether, E4 15 mg restores vaginal physiology at a level encountered in cycling premenopausal women^29,51 and, in doing so, improves GSM symptoms. Administration of systemic estrogen treatment, in the absence of specific contraindications, is well indicated when VMS, GSM, and other symptoms are present, whereas local vaginal treatment will only improve GSM symptoms.^2,8,14 Moreover, previous studies showed that systemic hormone therapy results in improved sexual functioning (libido, arousal) and relief of VVA/GSM. Oral MHT may be more effective than local estrogen action because of central and other effects.^52,53 In addition, in the large EVES study, women on systemic treatment had fewer and milder VVA symptoms than women on local treatments.¹⁸

Menopause Rating Scale

The baseline mean value of the MRS score in the five treatment groups was 17.1, which corresponds to severe postmenopausal symptoms.^36,54

In all E4 treatment groups, the total MRS score decreased (ie, showing improvement) substantially by W4, followed by a further decrease at W12. This improvement was seen in all 3 domains (somato-vegetative [ie, essentially VMS], psychological, and urogenital symptoms scores) on all 11 items, except bladder problems. It was maximal with E4 15 mg with a significant difference versus placebo at W4 (P = 0.011) that almost reached significance at W12 (P = 0.069). This is the consequence of the low number of participants and of the moderate decrease by W12 of the MRS score in the placebo group, perhaps in relation to a consistent decrease in VMS in this group. In the E4 15 mg treatment group, the MRS total score decreased by 53.7% at W12. Fait et al⁵⁵ showed that an estradiol metered transdermal dose (1.56 mg/spray) caused a decrease of the total MRS score from 16.5 at baseline to 9.2 at W12, that is, a 44% decrease.

In the E4 15 mg group, a large improvement in the somatovegetative domain score was observed versus placebo at W4 (P = 0.0014) and W12 (P = 0.058). This improvement in the somato-vegetative domain corresponds to the marked improvement in QoL and well-being occurring with the MHT treatment of VMS as observed in numerous studies and is also the main component of the observed MRS overall score improvement.^13,56

The significant correlation between the MRS overall score and the domain scores, as well as the frequency and severity of VMS (Table 6), is an additional way of assessing a potential relationship of the reduction of hot flashes and the amelioration of HR-QoL. This correlation was also reported by Simon et al⁵⁶ using the menopause-specific QoL score.

Improvement in psychological symptoms and urogenital symptoms is not always consistent with hormone treatment. However, when analyzing isolated items of the MRS, it is interesting that sexual problems and vaginal dryness scores ameliorated rapidly versus baseline (1.3 [baseline] to 0.7 [W12] [P = 0.0004] and 1.1 to 0.6 [P = 0.0024], respectively) and concurred with improved QoL.^13,56

A severe MRS score at baseline (≥16-17) is more improved by hormone treatment than a mild or moderate one.⁵⁵ In that respect, it is noteworthy that older postmenopausal women with a low level of climacteric symptoms assessed in the Women's Health Initiative study did not have an improvement in QoL with hormone treatment (conjugated estrogens + medroxyprogesterone acetate).⁵⁷ These observations demonstrate that severe climacteric symptoms considerably affect QoL in all its domains, as reflected by impaired MRS, menopause-specific QoL score, and other scores and MHT in symptomatic participants improves HR-QoL.^18,34,54

Limitations of this study are the absence of a direct comparison with other oral or vaginal estrogens in the global population of postmenopausal women and in selected participants with GSM symptoms. It would also be worth performing head-to-head studies comparing oral and vaginal routes of administration using the same estrogen.

CONCLUSIONS

The 2022 update of the NAMS Hormone Therapy Position Statement confirmed that hormone therapy remains the most effective treatment for VMS and GSM.⁹ E4 15 mg once daily, an effective dose for alleviating VMS,³⁰ also has a beneficial effect on vaginal cytology and maturation, on vaginal dryness, vaginal pain with sexual activity, and QoL, in particular on the somato-vegetative and psychological symptoms commonly observed with estrogen deprivation in postmenopausal women.

Acknowledgments: We thank the personnel in the participating centers, for conducting the E4Relief study. Medical writing support was provided by Jan Egberts and Arjen van Willigenburg at T4C Communications, Hilversum, the Netherlands. We also thank the members of the Donesta Scientific Advisory Boards, for their valuable advice. Statistical support was provided by M. Filatenkova, M. Hennion, and F. Nollevaux (Pharmalex Belgium).