Ovarian reserve markers of women with superficial endometriosis

Endometriosis affects up to 10% of reproductive age women and is associated with pelvic pain and subfertility. While previous studies have shown an association between deep and ovarian endometriosis to reduced ovarian reserve, there is no data on the effect of superficial endometriosis on ovarian reserve markers. Hence, we aimed to compare ovarian reserve markers of women with superficial endometriosis to that of women without endometriosis.


| INTRODUC TI ON
[6] Diminished ovarian reserve, as reflected by low levels of anti-Mullerian hormone (AMH), has previously been reported in women with endometriomas, even when they had not been subjected to surgery. 7,8Similarly, a significant decrease in serum AMH levels was observed also in women with deep endometriosis, as compared to the general population. 9ritoneal endometriosis, the most common endometriosis type, is characterized by superficial lesions in the peritoneum and in most cases correlates to minimal to mild disease as per the revised American Society for Reproductive Medicine (rASRM) endometriosis score. 10It is well established that despite normal pelvic anatomy, superficial endometriosis may still negatively impact fertility in women. 11,12However, to date, it is unknown if superficial endometriosis is associated with diminished ovarian reserve.
In recent years, endometriosis awareness is increasing. 13,14As such, women with all types of endometriosis often express concern on the implications of their disease on future fertility. 15With delayed childbearing and the growing awareness of ovarian reserve, fertility preservation, which could be carried out with oocyte and/or embryo cryopreservation, is often considered as an option for women at risk for reduced ovarian reserve. 16,17Information is needed on whether superficial endometriosis affects ovarian reserve and whether fertility preservation is warranted.
Hence, we sought to assess the association of surgically proven superficial endometriosis with ovarian reserve markers.

| Study design
We conducted a matched case control prospective study at a tertiary university-affiliated hospital.The study was approved by the institutional review board (0331-21-HMO July 2021) and all participants gave written informed consent.

| Patient selection
To recruit patients, our medical records were reviewed for all endometriosis surgeries between the years 2019-2021.All relevant files were retrieved and searched for patients aged 18-40 who underwent surgery for investigation of pelvic pain and/or subfertility with no preoperative evidence of deep or ovarian endometriosis (as per physical examination and/or endometriosis dedicated imaging).We then reviewed all surgical and histopathology reports and included only patients who had surgical evidence of superficial endometriosis, confirmed by histopathology, with no ovarian or deep lesions.
All surgical notes were reviewed, and stage of disease was assessed according to the American Society for Reproductive Medicine classification (ASRM) score.
Each case was matched to a control by age groups (18-40), body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) groups (BMI ≤ 25 and BMI > 25) and parity (0, 1, 2+).
The control group was comprised of medical staff or women presenting to a routine gynecologic check-up.All candidates for the control group completed a prestudy questionnaire with questions on pain symptoms including dysmenorrhea, dyspareunia and chronic pelvic pain on a scale of 0-3 (0 = none, 1 = mild, 2 = moderate and 3 = severe).Women with more than mild pain in any of the pain symptoms or with more than one scoring of mild pain, were excluded from the control group.Women with risk factors for ovarian failure and with other gynecological disorders were excluded from the study.

| Intervention
Each participant completed a questionnaire that included demographic, medical and gynecologic data.Then, each patient underwent AMH testing and an ultrasound to assess their antral follicular count (AFC).Venous blood samples for AMH testing and an ultrasound scan for AFC was performed on the same day by three of the authors (NL, AG and UD).Serum AMH levels were measured by a fully automated AMH assay (AMH Plus kit) on the Cobas e601 device.This method is sensitive to values in the range of 0.01-23 ng/ mL with an accuracy level of 0.03 ng/mL and was found to be highly accurate and reliable. 18C testing was carried out via a transvaginal ultrasound.For uniform and proper count of antral follicles, the ovary occupied at least 50% of the screen along its largest axis and the ovary was scanned from one ovarian margin to the other.All ovarian follicular structures 2-10 mm in diameter that were identified were counted. 19

| Outcomes
The primary outcome was percentage of patients with endometriosis under the AMH tenth percentile as per Han et al., 20 as compared to the control group.Secondary outcomes were: (1, 2)   mean AMH levels and mean number of antral follicles of women with superficial endometriosis, as compared to the control group; and (3) percent of patients with endometriosis under a wide range of standardized percentiles of AMH, as compared to the control group.

| Statistical analysis
Based a power level of 80%, a two-tailed significance level of 0.05 and on the assumption that a 20% absolute difference between groups would be considered clinically significant (i.e., to promote change of practice and advocate for ovarian reserve testing in patients with superficial endometriosis), in order to perform a fully powered study -the required sample size of each group was calculated to be 62.Hence, we aimed to recruit a total of 124 eligible patients.Statistical analysis was performed using IBM SPSS Statistics (version 25, IBM Corporation, Armonk, NY, USA).Patient demographics and characteristics were reported by means ± standard deviation or percent, as appropriate.We then compared percentages of women with AMH levels under respective AMH percentiles as per Han et al. 20 who reported the 10th, 50th and 90th percentiles and as per Anckaert et al. 21who reported the 2.5th, 5th, 50th, 95th and 97.5th percentiles.Of note, Anckaert et al. 21published a table of measured AMH values in healthy women with regular menstrual cycle aged 20-50 years, divided according to the percentiles as above.Their results were used as the reference values of the Cobas e601 device by the manufacturer.
We further compared mean AMH and AFC levels between women with endometriosis and the control group using unpaired t-test and chi-square test, as appropriate.We then carried out a multivariable logistic regression to account for potential residual confounding including variables that were not used as matching variables.A P value of less than 0.05 was considered statistically significant for all analyses.

| RE SULTS
A total of 124 patients were included in the study.Of these, 62 had surgically and histopathology proven superficial endometriosis and 62 women comprised the control group.Baseline demographic and clinical characteristics of study participants are presented in Table 1.
The matching variables (age, BMI and parity) did not differ between the two groups.While none of the women in the control group reported a past or current history of subfertility, 16.1% (N = 10) of the women in the endometriosis group reported past or current subfertility (P = 0.001).Of the women in the endometriosis group, the mean ± SD of the ASRM score was 6.5 ± 3.0 with a maximal score of 15, reflecting stage I-II/minimal-mild disease.
Four of the candidates to the control group reported moderate or severe dysmenorrhea, dyspareunia or chronic pelvic pain and were not included in the analysis.Eight women from the control group indicated mild dysmenorrhea and three indicated mild dyspareunia and were included in the study.None of the women indicated any level of chronic pelvic pain.No statistically significant differences were observed between the study and control groups in mean ± SD serum AMH levels (3.0 ± 2.8 ng/L vs 2.8 ± 1.8 ng/mL for the study and control groups, respectively, P = 0.71) and in number of antral follicles (12.0 ± 6.6 vs 10.2 ± 5.0 for the study and control group, respectively, P = 0.15).Of note, AFC was not tested in 16 women with endometriosis and in 14 women from the control group due to their preference not to undergo a vaginal ultrasound.
An AFC of below seven was demonstrated in 13.0% (n = 6/46) of women in the superficial endometriosis group, as compared to 22.9% (n = 11/48) women in the control group (P = 0.29).An AMH level of 1 ng/mL or below was found in 19.4% (n = 12/62) of women in the superficial endometriosis group, as compared to 14.5% (n = 9/62) of women in the control group (P = 0.63).percentiles). 21The proportion of women with endometriosis under the Anckaert 50th percentile was significantly higher than controls.
No other significant differences in rates of women under the above percentiles were observed between women with endometriosis and controls.

| DISCUSS ION
The main finding of the present study was that serum AMH and AFC levels of women with superficial endometriosis are similar to those of the general population, suggesting that ovarian reserve of women with superficial endometriosis is not negatively affected by their disease.
Several studies have demonstrated a negative effect of deep and ovarian endometriosis on ovarian reserve markers.In a prospective cohort study, Uncu et al. 22 reported that women with endometriomas had lower ovarian reserve as demonstrated by lower AMH and AFC levels, as compared to healthy women.A recent study by the same group has further shown that women with ovarian endometriosis experience a faster progressive decline in serum AMH levels, as compared to healthy controls. 235][26] Pacchiarotti et al. 9 compared AMH levels between women with rASRM stage III-IV endometriosis and fertile healthy women.In their study women with rASRM stage III-IV endometriosis had significantly lower AMH levels, suggesting a negative effect of deep lesion on the ovarian reserve.Shebl et al. 26 have also shown a negative association between endometriosis severity and serum AMH levels.Importantly, in their study women with minimal to mild endometriosis had similar AMH levels to controls.A possible explanation for the negative effect of deep or severe endometriosis on ovarian reserve is significant pelvic inflammation that may impair ovarian function. 27previous prospective study of 17 patients has reported decreased serum AMH levels in patients with minimal to mild endometriosis, as compared to controls.28 However, in addition to the relatively small sample size, their results were not adjusted to parity or hormonal treatment.Similar to our findings and lending support TA B L E 2 Percentage of women with anti-Mullerian hormone levels under standardized anti-Mullerian hormone percentiles.to our main finding of no difference in ovarian reserve markers between women with without superficial endometriosis, they have not demonstrated any significant difference in AFC levels between women with minimal-mild endometriosis and controls.
The association of all endometriosis types and subfertility is well established. 29[32] Our study suggests that as opposed to deep and ovarian endometriosis, ovarian reserve does not play a role in the pathogenesis of subfertility of patients with superficial endometriosis.
4][35] These may advocate for active assessment of ovarian reserve in women with these types of endometriosis and consideration of fertility preservation when required.Our findings of no effect of superficial endometriosis on ovarian reserve markers may be reassuring and may obviate the need for routing ovarian reserve assessment for this large group of patients.
To our knowledge, this is the first case control study analyzing AMH levels and AFC values of women with surgically and histopathology-proven superficial endometriosis, as compared to controls.The matching carried out between the study and control groups reduced the baseline diversity between groups.Previous studies, most of which were limited in size, have shown a possible low rate of endometriosis even in asymptomatic women; 36 as such, it is possible that some of the women in the control group also had endometriosis and we recognize this as a limitation of the study.However, the ultrasound scan performed for all women in the control group as well as the prestudy questionnaire, ruling out pain symptoms and infertility in the control group, significantly decreased the chances of the presence of undiagnosed endometriosis.
We did not have information on the precise duration of endometriosis within the study group and hence were not able to assess the correlation between length of diagnosis and ovarian reserve.
While AMH levels remain consistent throughout the menstrual cycle and can therefore be measured at any time, 37 the effect of the cycle stage on AFC levels is uncertain.Some studies reported variations in AFC across the menstrual cycle with possible higher correlation to ovarian reserve at the late follicular phase. 38,39In our study, not all women underwent AFC testing at the same time of their cycle which possibly represents a limitation of the study.However, a recent study has demonstrated that random AFC testing has the same predictive value of AMH. 40though a sample size calculation was carried out before our study commenced, studies with higher numbers are warranted to confirm our findings.
This was a single center study, hence limiting its generalizability and repeating our study at other centers may be of benefit.

| CON CLUS ION
In our cohort, ovarian reserve markers of patients with superficial endometriosis were similar to those of patients with no suspected endometriosis.While further and larger studies are needed, to date, it is unjustified to assess ovarian reserve for patients with superficial endometriosis.

Figures 1 and 2
Figures1 and 2show box plots representing the median and interquartile range of AMH and AFC levels of both groups (Median (IQR) AMH levels: 2.01 (1.27-3.52)and 2.57 (1.52-3.9)for the

F I G U R E 1 F I G U R E 2
Comparison of anti-Mullerian hormone (AMH) levels between women with and without superficial endometriosis.Serum anti-Mullerian hormone (AMH) (ng/mL) comparison between women with surgically proven superficial endometriosis and controls.The box represents the interquartile range that contains 50% of values.The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers.The line across the box indicates the median.Median (IQR) AMH levels: 2.01 (1.27-3.52)and 2.57 (1.52-3.9)for the study and control groups, respectively.Comparison of antral follicular count (AFC) between women with and without superficial endometriosis.Antral follicular count (AFC) comparison between women with surgically proven superficial endometriosis controls.The box represents the interquartile range that contains 50% of values.The whiskers are lines that extend from the box to the highest and lowest values, excluding outliers.The line across the box indicates the median.Median (IQR) AFC levels: 9.0 (8.0-16.0)and 8.0 (6.75-12.0)for the study and control groups, respectively.

Table 2
shows a comparison between the study and control group of percentages of women with AMH levels under the respective AMH percentiles as per Han et al. (10th, 50th and 90th TA B L E 1 Baseline characteristics of study participants (n = 124).

Table 3
20esents a multivariable logistic regression AMH under the 10th percentile as per Han et al.20as the dependent vari- able and hormonal treatment (yes/no), comorbidities (yes/no) and presence or assumed absence of superficial endometriosis as the independent variables.None of the included variables were found to be significantly associated with AMH levels under the 10th percentile.