**4. Discussion**

The purpose of this study was to test a new, objective measurement modality for vaginal moisture/dryness and to evaluate its validity. To achieve this, we used a calibrated filter-paper test strip, similar to the one used for the ophthalmic Schirmer test, in a standardized manner comparing symptomatic women suffering from GSM-associated vaginal dryness to women without dryness. The comparison yielded a statistically significant difference between the groups, suggesting that this test is correlated with symptoms and is useful in distinguishing symptomatic vaginal dryness from normal vaginal moisture.

Furthermore, the "modified Schirmer test" measurements showed strong and statistically significant correlations to the currently used indices of vaginal atrophy, i.e., pH, VHI, and VAS scores. These correlations imply a non-inferiority of the test compared to currently accepted measures.

It is important to note that the two study groups were significantly diverse in most characteristics (age, hormonal status, contraception, and lubricant use), as they represent essentially different phases of women's lives in terms of hormonal status.

We found no statistically significant distinction in the "modified Schirmer test" measurements between women who reported using hormonal contraceptives and those who reported using non-hormonal contraceptives; neither did we find differences between women with diverse hormonal statuses in the control group (i.e., normal menstruation, amenorrhea associated with hormonal IUD, and perimenopause). This could be a resultant of the small sample size, as these were subgroups of the entire control group. Although larger studies are needed to confirm our findings, the finding that this test is not altered by hormonal status or contraceptive modalities may indicate yet another advantage of its use, as it provides a direct and hormonal-independent indication of the vaginal moisture level.

Clinical trials evaluate treatment efficacy for GSM mostly using measures that represent vaginal changes occurring in response to the presence or absence of estrogen. Most frequently, these include pH level, the VHI, and the VMI [13]. Vaginal pH level is mainly determined by the presence or absence of lactobacilli. These bacteria produce lactic acid as they catabolize glycogen, which in turn decreases the vaginal pH level to 3.5–4.5. As glycogen is found in mature epithelial cells yet absent in para-basal epithelial cells (which represent atrophy), a vaginal pH level >5 in the absence of other causes (such as infection or presence of semen, cervical mucous, or blood) is indicative of glycogen absence, thus, of decreased estrogen and resultant atrophy [3,8]. The VHI, first described by Bachmann in 1992 [20], is widely used even though it has not been validated (Gloria Bachmann, personal communication) and uses mostly subjective parameters. The VHI is comprised of five parameters, observed during examination per speculum, graded from 1 to 5 each. Four parameters are not well defined and are subjective to inter and intra observer differences: vaginal elasticity, fluid volume (measured by fluid pooling in the fornix), epithelial integrity, and epithelial moisture, whereas only one measurement is objective—the vaginal pH [21]. According to the VHI, atrophy is defined as a score lower than 15 [21]. The VHI does not include clear examination instructions and is, therefore, dependent on examiner's

interpretation, thus lacking uniformity. Like other measures used, it is, at least partially, an estrogen-dependent index, as pH level comprises one fifth of its value.

VMI is another tool that represents vaginal estrogen influence on epithelial cytology, through calculating the relative percentage of superficial (mature) cells compared to intermediate and para-basal epithelial cells [3,8].

The main strength of our study is the introduction of a new measurement, which is easy to use and interpret. To our knowledge, previous studies that aimed to test the amount of vaginal discharge included usage of swabbing the entire vagina during an exam and weighing the swab [22], weighing tampons after inserting them for 8 h [23], by aspiration of vaginal fluid [24], or by pad weighing [25]. Nevertheless, these modalities are either effortand time-consuming or lack precision, as the weighed fluid is not necessarily comprised of vaginal discharge alone and may also contain sweat or urine. Other drawbacks include patient discomfort and functionality. In addition, none of these measures were studied in relation to vaginal atrophy.

Furthermore, the current tool may allow an objective and direct evaluation of dryness symptoms when there is a discrepancy between symptoms and findings. For example, in patients who complain of dryness but have an apparently normal examination, this tool may allow confirmation of a normal amount of discharge, suggesting a sensory issue or vulvodynia and directing further evaluation or suitable treatment.

Our study is limited for several reasons, including the small number of participants, the lack of measures among asymptomatic menopausal women, and the lack of measurement comparison before and after treatment. The "modified Schirmer test" should be further evaluated in larger scale studies, which will generate more accurate and specific ranges of measurements, representing and distinguishing between menopause-associated-vaginalatrophy, menopause without atrophy, and an estrogenized state. Furthermore, larger sample sizes may allow the division of measurement ranges into subranges, such as premenopausal women using hormonal contraceptives with and without vaginal dryness, compared to age-matched women who are not using hormonal contraceptives.

The main advantage of the "modified Schirmer test" is its potential to serve as an objective test in assessing vaginal dryness/moisture level in relation to non-estrogenic treatments for vaginal dryness. Therefore, the validity of this test should be further studied by comparison of measurements before and after treatments aimed to relieve vaginal atrophy.
