Incident endometriosis diagnosis and AMH: how surgical staging and typology relate to serum AMH levels

In our sample of women undergoing gynecologic surgery, we observed that moderate to severe (Stage III-IV) endometriosis and ovarian endometriomas were associated with lower AMH levels compared to those without endometriosis. This association was greater among individuals who had not experienced infertility. We observed some degree of influence in our sensitivity analyses excluding concurrent PCOS, previous abdominal/pelvic surgery, and medications; however, the overall patterns of association remained consistent. Further, upon comparison to a post-operative diagnosis of a normal pelvis, incident endometriosis diagnosis and moderate to severe endometriosis staging were associated with statistically significant lower AMH levels. Additionally, we observed that endometriosis typologies were associated with lower AMH levels; however, this was likely driven by the inclusion of ovarian endometriomas across all subtypes. These findings align with prior studies that have evaluated the association between endometriosis and AMH levels. In a large, prospective cohort of women nurses in the United States, those with endometriosis were found to have 29.6% (95% CI −45.5, −9.2) lower AMH levels compared to those without endometriosis; of note, these lower AMH levels were ascertained several years after surgical diagnosis. Additionally, the study was unable to extrapolate differences by endometriosis location and severity and unable to determine whether endometriosis or endometriosis surgery influenced AMH levels [ 41 ]. Other studies have showed that surgical intervention for endometriosis can damage ovarian reserve [ 17 , 22 , 23 ]. In a retrospective study, those who had surgery for endometriosis—regardless of previous ovarian surgery—had lower average AMH levels (2.5 vs. 3.6 ng/mL) compared to those with male factor infertility or no history of infertility [ 17 ]. Additionally, those with endometriosis were more likely to have AMH levels <1ng/mL (odds ratio (OR) 2.4 and 2.7, respectively) [ 17 ]. However, they did not observe that increasing stage of endometriosis was associated with AMH levels [ 17 ]. AMH levels have been shown to remain lower at one month [ 22 ], three months [ 23 ], and six months [ 23 ] after unilateral and bilateral laparoscopic surgery. Our findings, however, remained consistent upon excluding those with previous abdominal/pelvic surgery ( Supplemental Table 7 ) suggesting that endometriosis lesions themselves regardless of surgical intervention may reduce ovarian reserve. Few studies evaluated AMH levels prior to surgical intervention as performed in our study. In a case-control study of 65 women undergoing their first laparoscopy for incident endometriosis and 130 controls undergoing routine gynecologic treatment, individuals with stage III-IV endometriosis had statistically significantly lower mean preoperative serum AMH levels compared to controls, prior to surgical intervention (1.0 vs. 1.7 ng/mL) [ 25 ]. In a separate prospective cohort study of individuals seeking fertility care, mild endometriosis (Stage I-II) was associated with lower pre-operative AMH levels compared to those without a diagnosis of endometriosis (1.3 vs. 2.0 mg/mL) [ 19 ]. These studies suggest a pattern similar to our observation, that individuals with endometriosis have lower AMH levels even before surgical intervention. In our sample, ovarian endometriomas was associated with 54.3% lower AMH levels compared to those without endometriosis and 60.8% lower compared to those with a post-operative diagnosis of normal pelvis. In a prospective study of women with ovarian endometriomas diagnosed via imaging, the median percent decline in serum AMH levels measured over 6 months was higher among those with ovarian endometriomas compared to controls (26.4% vs. 7.4%) [ 24 ]. Given our mutually inclusive definition of typology in which individuals with ovarian endometriomas are included in both superficial and deep subtypes, we hypothesize that ovarian endometriomas are a primary driver in the association between typology and serum AMH levels. It has been suggested in prior research, that more than half of women with endometriosis experience infertility and/or a shortened reproductive window [ 7 , 10 ]. Our findings highlight a potential pathway in which endometriosis lesions may influence ovarian follicles. Based on previous research demonstrating the negative effect of both inflammation and anatomic distortions on AMH levels, we hypothesize that endometriosis associated inflammation and/or anatomic distortions may lead to a decrease in ovarian follicles, measured via AMH, that can in turn influence both fertility and timing to menopause [ 42 , 43 ]. Further research is needed to evaluate the longitudinal effect of endometriosis on ovarian reserve levels and how various treatment regiments affect this association. Additionally, recent research suggests that AMH may play a role in the pathology of endometriosis. Both AMH and the AMHII Receptor (AMHRII) have been shown to be differentially expressed in peritoneal fluid of endometriosis patients [ 44 ]. This presence may indicate a role in the growth and progression of peritoneal endometriosis. Additionally, AMH and AMHRII expressions have also been found in the endometrium and endometriosis lesions in endometriosis patients, further suggesting a possible involvement of AMH in the pathology of endometriosis [ 45 ]. Further research should further evaluate the role of AMH in endometriosis pathology. The primary strength of this study is our ability to evaluate AMH levels concurrent with the incident endometriosis diagnosis, prior to surgical diagnosis/therapy. Additionally, endometriosis diagnosis, staging, and typology were ascertained using the gold standard method of surgical and histological confirmation. While standardization of endometriosis classification is still in development, we were able to expand our analysis beyond incident diagnosis and evaluate how typology may influence AMH levels. Our method for characterizing endometriosis typology is in line with suggestions from the International Working Group Terminology for Endometriosis [ 46 ]. However, there is a potential for misclassification of typology based on the definition chosen, however we expect this misclassification to be non-differential with respect to AMH and thus attenuate any associations. Further, our subgroup analyses had limited statistical power and we were unable to disentangle the influence of ovarian endometriomas on the association between deep and superficial endometriosis and AMH levels, as well as endometriosis stage. Given that our study sample consisted of women undergoing surgery for any indication, our findings cannot be generalized to all reproductive age women. Serum AMH was stored for 15-17 years prior to being sent for AMH testing. However, research has shown that AMH remains stable when stored at −80°C, as was conducted with our samples [ 47 ]. While this is an important potential limitation, if there were any measurement error for AMH, we would expect it to be non-differential with respect to our exposure and therefore attenuate any observed associations. Our analysis was restricted to only one marker of functional ovarian reserve and did not include other markers, such as follicle-stimulating hormone, estradiol, and antral follicle count (AFC). Moderate to severe (Stage III-IV) and ovarian endometriomas were associated with statistically significantly lower AMH levels compared to those without endometriosis. Compared to those with a post-operative diagnosis of normal pelvis, incident endometriosis and moderate to severe stages (Stage III-IV) were associated with statistically significantly lower AMH levels. Additionally, typology (deep, ovarian, and superficial) was associated with statistically significant lower AMH level, however, this association was likely driven by the presence of ovarian endometriomas across all sub-types. These findings are consistent with previous studies and demonstrate that endometriosis lesions themselves, independent of surgical intervention, influence AMH levels.

The average age at enrollment among those with and without endometriosis was 31.7 (SD 6.8) and 33.0 (SD 7.3), respectively ( Table 1 ). Most of the participants were non-Hispanic, white (77.9%). Those with a diagnosis of endometriosis had a lower mean BMI (kg/m 2 ) compared to those without endometriosis (26.7 vs. 29.6). Those without endometriosis reported a lower proportion of infertility (18.8%) compared to those with endometriosis (41.03%). Additionally, the non-endometriosis group were more likely to be gravid (78.7% vs 57.7%) and subsequently parous (73.0% vs. 48.1%). Those with endometriosis had a higher proportion of individuals that reported a diagnosis of PCOS (9.0% vs. 6.3%). Mean serum cotinine values were higher among the non-endometriosis group (34.0 ng/mL) than the endometriosis group (19.5 ng/mL). In our fully adjusted multivariable model, overall endometriosis diagnosis was associated with modestly lower AMH levels; however, these findings did not reach statistical significance (−19.8% 95% CI −37.0, 1.0) ( Figure 1 ; Supplementary Table 1 ). Surgical staging did not reach statistical significance for stage I-II endometriosis compared to those without endometriosis (−10.4 95% CI −31.6, 17.4). However, those with stage III-IV endometriosis had 40.1% lower AMH levels (95% CI −58.9, −12.7) compared to individuals without endometriosis. Participants with ovarian endometriomas had 54.4% lower AMH levels (95% CI −69.4, −31.8) compared to those without endometriosis, a statistically significant difference. Deep endometriosis and superficial endometriosis had a trend towards lower levels of AMH compared to those without a diagnosis (Deep: −24.1% 95% CI −48.2, 11.0; Superficial: −15.5% 95% CI −34.6, 9.3), however the findings were not statistically significant. In analyses restricting our comparison group to only those with a post-operative diagnosis of normal pelvis, we observed statistically significant findings for incident endometriosis diagnosis, stage III-IV, deep, ovarian, and superficial endometriosis. Incident diagnosis of endometriosis was associated with 26.8% lower AMH levels (95% CI −44.6, −3.4) compared to those with normal pelvis ( Figure 1 ; Supplemental Table 2 ). We saw an increase in the effect size between stage III-IV and ovarian endometriomas compared to a normal pelvis (Stage III-IV: −47.8% 95% CI −65.8, −23.2; Ovarian: −60.8% 95% CI −74.4, −39.9). We also noted an increase in the effect size for both deep and superficial endometriosis (Deep: −34.3% 95% −56.2, −1.4 Superficial: −24.8% 95% CI −43.9, −0.8). In our sensitivity analysis excluding individuals diagnosed with PCOS and those with an AMH ≥10 ng/mL, we saw an attenuation on the effect size for all exposures ( Supplemental Table 3 ). We observed an overall increase in the effect size across our exposures when expanding our sample to include outliers ( Supplemental Table 4 ). Similarly, in our sensitivity analysis excluding hormonal contraceptive use, we saw an attenuation in the effect size for stage and ovarian endometriomas ( Supplemental Table 5 ). Upon exclusion of additional medications that may impact AMH, we observed a modest increase in the effect size for stage of endometriosis and ovarian endometriomas ( Supplemental Table 6 ). Upon the exclusion of individuals that had reported a previous pelvic surgery we noted an overall increase in the effect sizes across all exposures ( Supplemental Table 7 ). In our sensitivity analysis including MRI diagnosed ovarian endometriomas, we noted a mild attenuation in the effect size ( Supplemental Table 8 ). There was no statistically significant effect modification by infertility on endometriosis diagnosis, stage, or deep and superficial locations; however, we observed a statistically significant effect modification by infertility on the association between ovarian endometriomas and AMH levels (p=0.01) ( Supplemental Table 9 ). Among individuals without infertility, those with ovarian endometriomas had 72.6% lower AMH levels compared to those without endometriosis (95% CI −85.4, −48.5). Among those with infertility, ovarian endometriomas was associated with 39.2% lower AMH compared to those without endometriosis (95% CI −67.3, 12.9).

The ENDO Study is a matched exposure cohort study that enrolled participants between 2007 and 2009 [ 26 ]. The study consisted of an operative and population cohort. The operative cohort included women ages 18-44 years that were scheduled for diagnostic and/or therapeutic laparoscopy or laparotomy at one of the participating surgical centers in Utah and San Francisco [ 26 ]. The population cohort was matched to the operative cohort by age and residence. Both cohorts were restricted to English or Spanish speakers. Premenopausal women without a previous surgical confirmation of endometriosis and who did not have a previous diagnosis of cancer except for non-melanoma skin cancer were eligible to participate in both the population and operative cohorts [ 26 ]. Inclusion was further restricted to those that had not breastfed for ≥6 months or had used injectable hormonal treatment within the last two years [ 26 ]. All human subjects approval was obtained for the study and participants provided informed consent prior to data collection [ 26 ]. The analyses were approved by the University of Arizona institutional review board (STUDY00004218). The ENDO Study consisted of 600 participants ( Figure 1 ). Only participants from the Utah center with complete survey information were included as these samples were sent for AMH testing (n=476). Individuals with missing AMH values due to insufficient biospecimen availability (n=51). To prevent misclassification of the endometriosis diagnosis and to evaluate surgical staging and typology, individuals from the population cohort were excluded (n=69). Those who had a previous unilateral oophorectomy (n=8) were excluded from the analysis. The final sample consisted of 348 participants. Incident endometriosis diagnosis, American Society for Reproductive Medicine (ASRM) staging and typology was ascertained via surgical and histological confirmation by operating surgeons [ 26 ]. Endometriosis diagnosis was categorized as a binary variable (yes vs. no). Surgical staging was determined using the revised American Society for Reproductive Medicine (rASRM) classification of endometriosis categorized into stage I-II and stage III-IV [ 5 , 26 ]. Endometriosis typology was ascertained using the rASRM standardized form among participants that had information on lesion depth (superficial or deep) and size (<1cm, 1–3 cm, and <3cm) for the ovary, peritoneum, and posterior cul de sac obliteration. We used expert clinical guidance and The International Glossary on Infertility and Fertility Care to characterize endometriosis [ 27 ] [ 28 ]. Per the International Glossary on Infertility and Care, “endometriosis can be located superficially on the peritoneum (peritoneal endometriosis), can extend 5mm or more beneath the peritoneum (deep endometriosis) or can be present as an ovarian endometriotic cyst (endometrioma) [ 27 , 28 ]”. Using the complete rASRM standardized form, we characterized lesions into three exclusive categories: a) superficial endometriosis which consisted of superficial lesions of the ovary or on any peritoneal surface; b) deep endometriosis which consisted of any deep lesions on the peritoneal surface OR posterior cul-de-sac obliteration (partial or complete); and c) ovarian endometrioma which consisted of deep endometriosis of the ovary [ 29 ] Blood samples were collected prior to surgery (2009-2011) and serum samples were aliquoted and stored at −80°C in a continuously monitored/alarmed storage facility with no history of temperature variance. In 2024, banked serum samples were thawed and aliquoted into ARUP standard transport tube, refrozen, and delivered over dry ice to the Associated Regional and University Pathologists Inc. (ARUP) Laboratories © (Salt Late City, Utah, USA) within 24 hours of processing for AMH testing. AMH was ascertained using the quantitative enzyme-linked immunosorbent assay. For the statistical analysis, values below the limit of detection (LOD) (0.003ng/mL) were substituted using the LOD/√2 (n=3). Covariates were collected at the time of enrollment via a self-reported questionnaire. Which covariates were added to the statistical model was determined a priori from the literature and included variables that are associated with endometriosis and/or serum AMH. Because of the strong association between age and AMH, both age (continuous) and age (squared) were utilized in the analysis [ 11 ]. Other covariates that were adjusted for in the statistical model included body mass index (BMI, continuous) [ 30 , 31 ], age at menarche (continuous) [ 31 , 32 ], current cigarette smoke exposure [ 33 ] ascertained via serum cotinine values (ng/mL) (continuous) and reported current exogenous hormonal contraceptive use [ 34 ]. Current exogenous hormonal contraceptive use ( “Currently, which of the following is your primary method of birth control?”) included the pill, patch, NuvaRing, and injectables. Outliers were determined based on a threshold of >1.25 times the third quartile value (6.52 ng/mL) + 1.5*Interquartile Range (5.01). Based on this threshold, AMH values >14.0 ng/mL were excluded (n=20). The analytic sample included 333 participants. Serum AMH levels were log-transformed to account for non-normal distribution. Multivariable linear regression models were conducted to evaluate the association between each exposure (endometriosis diagnosis, stage, and location) and the log-transformed AMH values. The percentage difference in AMH (exp(β)-1) *100) and the 95% confidence intervals (CI) were reported for each model. The first model adjusted for age (continuous and squared) and BMI (continuous). The second model adjusted for age (continuous and squared), BMI (continuous), age at menarche (continuous) and exogenous hormonal use (yes/no). Lastly, the third model additionally adjusted for serum cotinine (continuous). A secondary analysis was conducted restricting our comparison group to include only those with a post-operative diagnosis of “normal pelvis.” Gynecologic conditions that were excluded from the comparison group were uterine fibroids, pelvic adhesions, benign ovarian cysts, neoplasm, and congenital mullerian anomalies. The multivariable model was adjusted for age (continuous and squared), BMI (continuous), age at menarche (continuous) and exogenous hormonal use (yes/no), serum cotinine (continuous). Six sensitivity analyses were conducted related to study inclusion/exclusion criteria. First, because PCOS is highly associated with AMH levels and endometriosis diagnosis [ 35 , 36 ], individuals with a self-reported diagnosis of polycystic ovary syndrome (PCOS) and/or an AMH value ≥10 ng/mL were excluded (n=63) [ 37 ]. Conversely, we conducted a sensitivity analysis expanding our inclusion criteria to include outliers and increase our sample size (n=348). Because of the potential effect of exogenous hormonal contraception on serum AMH levels [ 38 ], a sensitivity analysis was performed excluding current exogenous hormonal contraceptive users (n=49). An additional sensitivity analysis was conducted that further excluded self-reported medications that have been suggested to affect AMH levels including any type of exogenous hormone (n=83) [ 34 ], selective serotonin reuptake inhibitors (SSRI)(n=47) [ 39 ], vitamin D (n=2) [ 34 ], and Metformin (13) [ 34 ]. Individuals that had reported having previous pelvic surgery, including appendectomy, laparoscopy, or laparotomy, (n=85) were excluded as pelvic surgery has been associated with AMH levels [ 6 ]. Because ovarian endometriomas can be detected with a high level of sensitivity using magnetic resonance imaging (MRI) [ 40 ], an additional sensitivity analysis was conducted including additional individuals from the population cohort (n=63) to assess the effect of ovarian endometriomas diagnosed via surgical confirmation (N=33) and MRI (N=38) on serum AMH levels in our sample. To evaluate effect modification by infertility, a sensitivity analysis was conducted stratifying by infertility, defined as categorized as having reported trying to conceive for ≥12 months without conception. A likelihood ratio test was performed to determine whether there were statistically significant differences in the influence of endometriosis on AMH by infertility history.

Approximately 10% of reproductive age women have been diagnosed with endometriosis [ 1 ], a chronic, inflammatory condition in which tissue similar to the lining of the uterus implants throughout the body [ 2 ]. However, the true prevalence of endometriosis is likely higher than given the lack of a non-invasive, highly sensitive diagnostic measure [ 3 , 4 ]. Endometriosis is typically characterized by surgical staging (I-IV) and/or lesion depth and location [ 2 , 5 ]Symptoms of endometriosis include pelvic pain, painful menstruation, and infertility [ 1 , 2 , 6 ] Indeed, the association between endometriosis and infertility is well established [ 7 ]. However, the exact mechanism by which endometriosis contributes to infertility is not well understood [ 7 ]. Potential hypothesized mechanisms include increased inflammation and a reduction in ovarian reserve and ovarian function [ 7 ]. Potential hypothesized mechanisms include, but are not limited to, increased inflammation, anatomic distortion, implantation abnormalities, and a reduction in ovarian reserve and ovarian function [ 7 – 9 ]. Premature loss of ovarian function may lead to a shortened reproductive window. Previous research has observed that women with endometriosis have a 50% greater risk of early natural menopause compared to those without endometriosis, which could result from a reduction in ovarian reserve and function [ 10 ]. Anti-müllerian hormone (AMH), a member of the transforming growth factor-β family, is secreted in the granulosa cells of the ovary [ 11 , 12 ]. Serum AMH levels are considered a reliable proxy for the quantity and quality of primordial follicles in the ovary [ 11 ]. Serum AMH levels reflect a woman’s reproductive age more accurately than chronological age and can better predict transition to menopause compared to other classic ovarian markers such as follicle-stimulating hormone (FSH) and antral follicle count (AFC) [ 11 , 13 , 14 ]. Clinically, AMH is used as an important biomarker in fertility treatments [ 15 ]. Previous literature show a negative association between endometriosis and AMH [ 6 , 16 – 25 ]. Some of these studies were restricted to infertile populations, limiting the generalizability of the findings [ 16 , 17 ]. A significant portion of the literature focused on ovarian damage iatrogenic to surgery and was limited by small sample sizes and short follow-up times [ 17 , 22 , 23 ]. Additionally, few studies have been able to disentangle the effect endometriosis typology on AMH levels [ 16 , 18 , 19 , 24 , 25 ]. Our study overcomes these limitations by evaluating pre-surgical serum AMH levels in women with and without endometriosis regardless of infertility history from the Eunice Kennedy Shriver Natural Institute of Child Health and Human Development (NICHD) funded Endometriosis: Natural History, Diagnosis, and Outcomes (ENDO) Study [ 26 ]. Further, we were able to assesses whether AMH levels differ by endometriosis surgical staging and typology. This approach enables us to more precisely evaluate whether endometriosis is associated with diminished ovarian reserve prior to any surgical intervention and how this association may differ by specific disease characteristics.