A Pilot Study on the Co-existence of Diabetes and Endometriosis in Reproductive-Age Women: Potential for Endometriosis Progression

Endometriosis (ENDO) is a chronic estrogen-dependent gynecological condition that affects reproductive-age women, causing pelvic pain, infertility, and increased risk for ovarian cancer. Diabetes mellitus (DM) is a metabolic disease with significant morbidity and mortality and rising incidence worldwide. The occurrence of DM among ENDO patients remains understudied, despite commonalities in these conditions’ immune, inflammatory, and metabolic dysfunctions. This pilot study evaluated whether a subset of women with ENDO manifests DM co-morbidity and if so, whether DM promotes ENDO status. Archived ectopic lesions obtained at ENDO surgery from non-diabetic (ENDO-N; n = 11) and diabetic (ENDO-DM; n = 15) patients were identified by a search of an electronic health database. Retrieved samples were analyzed by immunohis- tochemistry for markers of proliferation (Ki67, PTEN), steroid receptor signaling (ESR, PGR) and macrophage infiltration (CD68). Immunostaining data were expressed as percentages of immune-positive cells in lesion stroma and epithelium. In lesion stroma, the percentages of nuclear immune-positive cells were higher for ESR2 and lower for PGR-T, in ENDO-DM than ENDO-N patients. The percentages of nuclear immune-positive cells for ESR1 and PTEN tended to be higher and lower, respectively, in ENDO-DM than ENDO-N groups. In lesion glandular epithelium, the percentages of nuclear immune-positive cells were higher for ESR1 and ESR2, in ENDO-DM than ENDO-N groups. ENDO-N lesions had lower percentages of stromal CD68 immune-positive cells than ENDO-DM Type 1 lesions. Findings demonstrate DM in a subset of women with ENDO, which was associated with significant changes in lesion stromal and epithelial nuclear steroid hormone receptor levels, suggestive of disease progression.

Endometriosis · Diabetes · Estrogen receptor · Progesterone receptor · Co-morbidity Abbreviations BMI Body mass index DM Diabetes mellitus ENDO Endometriosis ESR1 Estrogen receptor α ESR2 Estrogen receptor β FFPE Formalin-fixed paraffin embedded GE Glandular epithelial PTEN Phosphate and tensin homolog PGR-T Progesterone receptor-total PGR-B Progesterone receptor-isoform B ST Stromal, T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus

Endometriosis (ENDO) is a condition characterized by the presence of uterine endometrial-like epithelium and stroma in extra-uterine sites, causing debilitating pelvic pain, dysmenorrhea, and infertility in 50% of afflicted women, and with an annual economic burden of ~ 50B in * Rosalia C. M. Simmen [email protected] Iad Alhallak [email protected] Charles M. Quick [email protected] Garrett L. Graham [email protected] 1 Department of Physiology & Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA 3 The Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2430 Reproductive Sciences (2023) 30:2429–2438 1 3 the USA alone [1 ]. While ENDO is considered benign in its initial stage, a history of ovarian/tubal ENDO is an independent risk factor in two subtypes of ovarian cancer, namely, clear-cell and endometrioid [2 , 3]. The patho- genesis of ENDO-associated ovarian carcinoma remains unclear and is an area of intense investigations [ 4, 5]. Nevertheless, recent findings have shown that endome - triotic lesions without concurrent cancers contain cancer- associated somatic mutations including for KRAS, PTEN, ARID1, and PIK3CA [ 6, 7]. The latter suggests that ovar - ian endometriotic lesions can progress to malignancy, given the proper signals and context. Diabetes is a progressive disease affecting over 400 mil- lion people worldwide [8]. Type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are characterized by significant genetic predispositions and shared dysfunctions in glucose homeostasis, resulting in elevated blood glucose levels in affected individuals. These conditions have distinct pathogenesis: T1DM results from the autoimmune destruc- tion of pancreatic β-cells and hence, early progressive loss of insulin production while T2DM, a condition increasing with age, stems from the loss of insulin sensitivity of tar - get cells, leading to defects in glucose clearance [9 , 10]. T1DM appears first in children and adolescents, and while its adult-onset is increasingly diagnosed, the basis for the delayed manifestation is not well-understood [11]. T2DM is significantly affected by lifestyle factors (diet, physical activity, alcohol, and tobacco use), is preventable, and is generally manageable by lifestyle modifications [12]. Simi- lar to ENDO [13, 14], T1DM and T2DM manifest significant inflammatory, immune, and metabolic dysfunctions and are associated with increased risk and progression of ovarian cancer [15–17]. There is no known predominance of ENDO in women with either T1DM or T2DM in the general population; how- ever, women (and young girls upon initiation of menses) may suffer unknowingly from co-morbid conditions throughout their reproductive years since ENDO is difficult to diagnose, identify, and treat [1]. Importantly, because ENDO and DM are chronic conditions, women with co-morbidities are antic- ipated to have lower quality of life, may develop drug-drug interactions leading to reduction in treatment efficacies for each condition, and may face advancement in ENDO status. The present investigation constitutes a pilot study to eval- uate whether a subset of women with ENDO may suffer DM co-morbidity and if so, whether DM may progress ENDO. Using archived FFPE ectopic (ovarian/tubal) lesions from women with ENDO alone and with co-incidence of DM (T1DM or T2DM), we report herein that DM and ENDO may co-exist in reproductive-age women and that DM asso- ciation in women with ENDO confers significant changes in lesion nuclear steroid hormone receptor levels, suggestive of increased estrogen dependency and heightened progesterone resistance, both of which constitute markers of endometrio- sis progression.

Sample Tissue Collection The study was approved by the Institutional Review Board of the University of Arkansas for Medical Sciences (UAMS IRB#205,177). The Arkansas Clinical Data Repository, which is affiliated with the TriNetX health research platform, was used to identify female patients (20–60 years old inclu- sive) diagnosed from 2015 to 2019, using the search words “Endometriosis with no diabetes (ENDO-N),” “Endometrio- sis with Type 1 diabetes (ENDO-T1DM),” and “Endome- triosis with Type 2 diabetes (ENDO-T2DM).” De-identified referral numbers for patients meeting the specific criteria were sent to the UAMS Department of Pathology and corre- sponding FFPE sections in the storage inventory, when avail- able, were retrieved by our team pathologist (CMQ). The patient data (age and BMI at ENDO surgery, race/ethnicity, presence or absence of ovarian mass, use of progesterone for ENDO management) were subsequently obtained for all analyzed samples. Immunohistochemistry FFPE samples were sectioned (5 μm) and processed by heat-induced epitope retrieval (citrate buffer) and subse- quent incubation with designated antibodies as previously described [18]. Table  1 provides the list of the primary antibodies with their unique Research Resource Identifier (RRID), used at working dilutions following the RRID infor- mation (antibody registry.org) at incubation conditions of 4 °C for 16–24 h. Immunoreactivity was detected using the Vectastain Elite ABC kit (Vector Laboratories) and bioti- nylated anti-rabbit secondary antibodies (Vector Laborato- ries), and slides were counterstained with hematoxylin. The stained slides were digitized using the Leica Digital Pathol- ogy Whole Slide Scanner (Aperio Image Scope). Cells were scored as non-staining (i.e., only background staining) based on sections that were processed in parallel with the omission of the primary antibody. For each antibody-treated tissue section, a total of ~ 100 cells for each glandular epithelial and stromal compartment in 3–4 random fields were counted for numbers of nuclear-staining and non-staining cells. Analy - ses for CD68-immune-staining followed the same procedure except that stromal non-nuclear (cytoplasmic/membrane) staining cells were counted. Data are expressed as the per - centages of nuclear-stained (Ki67, PTEN, ESR1, ESR2, PGR-T, PGR-B) or cytoplasmic/membrane-stained (CD68) cells, relative to the total number of cells counted. 2431Reproductive Sciences (2023) 30:2429–2438 1 3 Data Analysis Data were analyzed by the Shapiro–Wilk’s test for normal- ity and compared for statistical significance of difference between experimental groups using the Mann–Whitney U test. The statistical tests were performed using the GraphPad Prism software (version 6). Data are presented as box plots indicating the upper and lower quartiles, range, and median (middle line) with whiskers indicating the maximum and minimum points. A p value ≤ 0.05 was considered to be sig- nificant. Principal component, multivariate regression, and binary logistic regression analyses were used to analyze the associations between the patient variables (age at surgery, race, progesterone usage, and BMI) with the protein bio- markers in glandular epithelial and stromal compartments.

Patients’ Demographic Information Figure  1a provides a schematic summary of sample retrieval and subsequent analyses of tissue sections by immunohis- tochemistry (IHC). FFPE-processed blocks from women with ENDO without DM (n = 11), with TIDM (n = 8), and with T2DM (n = 7) were identified from surgical pathology reports of the Arkansas Clinical Data Repository based on a web-based search of the TriNetX Health Research Plat- form for the period covering 2015–2019. Medical records were de-identified and available FFPE sections at the UAMS Department of Pathology storage inventory were retrieved by our team pathologist (CMQ). Lesions were largely tubal and ovarian (with an exception of one omental) and classi- fied as stages 3–4, based on the American Society of Repro- ductive Medicine guidelines [19]. Because of the small sam- ple sizes of the ENDO-T1DM and ENDO-T2DM groups, these sets were combined and designated as ENDO-DM (Table  2). The mean age (years, y) of women undergoing surgery for removal of ENDO lesions was higher (p < 0.001) for patients without diabetes (ENDO-N; 41.9 ± 0.8 y) when compared to those with diabetes (ENDO-DM; 31.6 ± 1.8 y). Mean BMI (kg/m 2) was higher at surgery (p = 0.007) for ENDO patients with DM (45.0 ± 3.4) than without DM (ENDO-N; 31.4 ± 2.7). The duration of progestin use for each group could not be determined due to lack of docu - mentation. However, the numbers of patients within each group using progestin for ENDO treatment were compara- ble ( p = 0.86). Similarly, there was no race/ethnic dispar - ity in the patient population between the ENDO-N and the ENDO-DM groups (p = 0.19). Corresponding ovaries for all patients did not contain ovarian carcinoma, as reported from pathology records and a review of the surgical pathology slides (Table  2). Immunohistochemistry In a previous report [18], we showed that Ki67, PGR-T, ESR1, and ESR2 constitute valid biomarkers for endome- triosis progression since their levels and patterns of nuclear immunopositivity in ectopic lesions differed significantly from those of corresponding eutopic endometria or non-dis- eased endometria. PTEN protein levels in lesions were addi- tionally evaluated in the present study, given the protein’s anti-proliferative/pro-apoptotic actions and the reported PTEN mutations in ovarian endometriosis lesions [20]. Sim- ilar analyses for PGR-B were performed since disruption of PGR-B expression has been demonstrated in many uterine disorders including endometriosis [21– 23]. A composite of representative immunostaining of lesions from women with ENDO-N, using specific antibodies to each protein, is shown in Fig.  1b. Nuclear staining for all proteins was demonstrated in both stromal and epithelial compartments. Stromal Immunoreactivity in ENDO Lesions with Diabetes Status Stromal cells of ENDO lesions from women without DM displayed varying levels of nuclear-localized immunore- activities (expressed as percent of nuclear-positive cells) for the evaluated ENDO biomarkers (Fig.  2). In ENDO-N Table 1 Antibodies used for IHC 1 Research Resource Identifier (antibodyregistry.org) Protein Vendor/catalog number RRID1 Working dilution Estrogen receptor α Santa Cruz/sc542 AB_63140 1:250 Estrogen receptor β Millipore Sigma/05–824 AB_310195 1:200 Ki67 Abcam/Ab16667 AB_302459 1:200 Progesterone Receptor (total) Santa Cruz/sc7208 AB_2164331 1:200 Progesterone Receptor-B Cell Signaling/3157S AB_2252606 1:400 Phosphatase and Tensin homologue Cell Signaling/138G6 AB_823618 1:200 Macrophage CD68 Thermo Fisher Scientific/ MA5-13,324 AB_10987212 1:200 2432 Reproductive Sciences (2023) 30:2429–2438 1 3 lesions, the highest percent immunoreactivities were seen for PTEN, PGR-T, and PGR-B, with Ki67 and ESR2 immunoreactivities displaying the lowest levels. The co- occurrence of DM in women with ENDO increased and decreased, respectively, nuclear-localized stromal immu- noreactivities for ESR2 (p = 0.04) and PGR-T (p = 0.05), relative to those of women without DM. Stromal cells of ENDO lesions with DM tended to show reduced (p = 0.07) and increased (p = 0.06) nuclear PTEN and ESR1 immuno- reactivity levels, respectively, relative to those of women without DM. Levels of nuclear immunoreactivities for Ki67 and PGR-B were not affected by DM status (Fig.  2). a) Experimental Design Ovarian Lesions (FFPE) a) TRINET –X Platform b) UAMS Pathology Department Non-DM (n=11) T1DM (n=8) T2DM (n=7) IHC Ki67 PTEN ESR1 ESR2 PGR-T PGR-B b) Representative IHC C Ki67 50μm ESR1 PTEN 50μm Ab 50μm 50μm 50μm 50μm ESR2 50μm 50μm PGR-T 50μm 50μm PGR-B 50μm 50μm C Ab C Ab C Ab C Ab C Ab Fig. 1 Analyses of endometriotic lesions. a. Schematic of tissue retrieval and analyses. Tissue samples for the study were identi- fied from search of the Arkansas Clinical Data Repository, using the TriNetX health research platform, and retrieved from the UAMS Department of Pathology repository. Formalin-fixed paraffin-embed- ded sections were subjected to immunohistochemistry using the listed antibodies (Table  1). b Representative H&E-stained sections of lesions from non-diabetic women with ENDO. For each section stained with the indicated antibody (anti-Ki67, anti-PTEN, anti- ESR1, anti-ESR2, anti-PGR-T, anti-PGR-B), a corresponding section was processed in parallel in the absence of antibody to serve as con- trol (labeled C) 2433Reproductive Sciences (2023) 30:2429–2438 1 3 Glandular Epithelial Immunoreactivity in ENDO Lesions with Diabetes Status Glandular epithelial cells of ENDO lesions from women with DM displayed nuclear-localized immunoreactivities for ESR1 (p = 0.001) and ESR2 (p = 0.005) that were higher than those from women without DM (Fig.  3). By contrast, nuclear immunoreactivities for Ki67, PTEN, PGR-T, and PGR-B in these cells were not affected by DM status. Macrophage Biomarker CD68 Immunoreactivity in ENDO Lesions Macrophage infiltration of ovarian endometriomas has been previously reported [24]. Furthermore, we have shown in a mouse model of endometriosis that progression of ENDO in ectopic lesions with high fat diet was associated with increased localization of macrophages in stromal cells as measured by F/480 immunostaning [25]. Here, we used the human mac - rophage/monocyte selective biomarker CD68 to evaluate potential changes in macrophage infiltration of ENDO-N relative to ENDO-T1DM lesions. The limited availability of ENDO-T2DM lesions precluded parallel analyses of these samples. ENDO lesions stained positive for CD68 preferen- tially in cytoplasmic/membrane-associated compartments of lesion stromal cells (Fig. 4a). Immunostaining levels in stromal cells were higher for ENDO-T1DM than ENDO-N (Fig. 4b).

Endometriosis (ENDO) and diabetes (DM) individually affect women of reproductive age, yet the occurrence of ENDO and DM co-morbidity and its potential contribution Table 2 Patient demographics * t-test (p ≤ 0.05, compared to ENDO-N) Race/Ethnicity Age at ENDO Surgery BMI at ENDO Surgery Ovarian Mass Progesterone ENDO-N   Black/African American 43 25.0 No No   Black/African American 43 46.2 No Yes   White/Caucasian 38 39.5 No Yes   Black/African American 39 21.6 No Yes   Black/African American 44 22.5 No Yes   Black/African American 45 29.9 No No   Black/African American 43 29.5 No Yes   Black/African American 43 18.2 No Yes   Black/African American 45 36.9 No No   Black/African American 39 38.3 No Yes   Black/African American 39 38.3 No Yes Mean ± SEM 41.9 ± 0.8 31.4 ± 2.7 ENDO-DM   White/Caucasian 24 33.2 No Yes   White/Caucasian 25 39.7 No Yes   Black/African American 34 36.5 No Yes   White/Caucasian 24 33.1 No Yes   Black/African American 28 52.8 No No   Black/African American 29 61.1 No No   Black/African American 31 52.4 No No   White/Caucasian 26 21.6 No No   Black/African American 40 52.9 No No   Black/African American 29 61.8 No Yes   Black/African American 36 24.2 No No   Black/African American 44 46.2 No Yes   Black/African American 26 61.8 No Yes   Black/African American 34 49.8 No No   Hispanic/Latino 44 48.2 No Yes Mean ± SEM 31.6 ± 1.8* 45.0 ± 3.4* 2434 Reproductive Sciences (2023) 30:2429–2438 1 3 to ENDO progression remain unexplored. In this pilot study, we provide clinical data to show the co-existence of ENDO in a subset of women with DM (T1DM and T2DM). Further, we demonstrate that DM status in ENDO women confers significant changes in steroid hormone receptor levels in lesion stromal and glandular epithelial compartments, rela- tive to those of ENDO women without DM. Progesterone resistance, estrogen-dependency, and immune activation are hallmarks of ENDO development and progression. Reduc- tions in levels of stromal ligand-bound PGR exacerbate ENDO status [18, 26, 27] and are associated with resistance to progestin therapy in women with ENDO [28]. Moreover, enhanced estrogen signaling leads to heightened cell inflam- mation mediated by ESR2 [29] and promotes cell prolifera- tion mediated by ESR1 [30]. Here, we show that nuclear lev- els of PGR-T (stroma) were decreased while those of ESR1 (epithelia) and ESR2 (epithelia, stroma) were increased, in ENDO-DM relative to ENDO-N lesions. Furthermore, we found increased macrophage localization (CD68 immu- noreactivity) indicative of immune activation known to be associated with ENDO progression [1, 25], in lesion stromal cells of ENDO women with Type I DM, relative to those of ENDO-N women. The tending decrease in lesion stromal PTEN immunoreactivity with DM status is consistent with earlier reports that subtle reductions of PTEN expression level are sufficient to promote cell proliferation and hence, cancer susceptibility [ 31]. Unexpectedly, there was a lack of coincident increases in the levels of Ki67 immunoreac- tivities in both lesion stromal and epithelial cells with DM status. We suggest that this may reflect in part the relatively advanced endometriotic stage of the lesions analyzed in the present study (largely stages 3–4) and the previously docu- mented inability of Ki67 dynamics to accurately capture cellular proliferation index [32]. Multivariate analysis (data not shown) showed no significant association of patient vari- ables of progesterone usage, race, BMI, and age of ENDO surgery with the evaluated protein biomarkers in lesion epi- thelial and stromal compartments, suggesting diabetes status as the major driver in the noted differences in these proteins’ expression levels. In this study, the tissue samples were retrieved from patients in the age range of 20–60 years because ENDO symptoms (i.e., pelvic pain, heavy menses) are pronounced around age 20 (although the condition may initiate earlier) and recurrence in affected women may extend beyond the menopausal period. Interestingly, the study participants in the ENDO-N group were notably older than those of the ENDO/DM group. The significance of this finding is unclear since the data were obtained at ENDO surgery and not dur- ing initial diagnoses. However, we speculate that the earlier age of ENDO surgery for ENDO-DM patients may reflect their greater degree of pain/discomfort. The relationship between pain severity in ENDO women and DM status mer- its further investigation. The linkage of adiposity (measured by BMI) and endometriosis is complex and may be depend- ent on disease severity [33]. The higher BMIs shown for ENDO patients with DM align with the positive association of BMI and diabetes mellitus [34, 35]. By contrast, epide- miological studies indicate a negative association for obe- sity and ENDO progression [ 36, 37]. Nevertheless, since obesity does not protect against endometriosis [38] and in mouse models of the disease, high-fat diet induced obesity and inflammation increased endometriosis development [25, 39], there is a need for further evaluation of this relationship. Despite the significant health and economic challenges imposed individually by DM and ENDO in female patients, the prospects of their co-incidence in a subset of women have not been assessed. Indeed, no work to date has clini- cally addressed whether DM status promotes ENDO and if ENDO treatments may complicate glycemic control in women with DM [40]. In a recent prospective study using data from the Nurses’ Health Study, Missmer and colleagues [41] reported no overall increased risk of T2DM for women with ENDO. However, the reverse relationship, whether DM promotes ENDO, has not been studied. Our pilot study, 0.0 0.2 0.4 0.6 0.8 1.0 Percent Nuclear Positive Cells * * # # ESR2 PGR-TKi67 ESR1PTEN PGR-B Stromal Cells N DM N DM N DM N DM N DM N DM Fig. 2 Stromal immunoreactivities of ENDO markers in ectopic lesions of women with and without diabetes. Tissue sections from non-diabetic women with ENDO (N) and from women with ENDO and diabetes (DM) were processed for immunohistochemistry, using specific antibodies as described under “Materials and Methods.” The percentages of nuclear-localized Ki67, PTEN, ESR1, ESR2, PGR-T, and PGR-B in lesion stromal cells were determined by counting the number of immunopositive-staining nuclei over the total number of cells counted × 100. For each tissue section, 3–4 random visual fields representing a total of ~ 100 cells were assessed. Data represent the percentages of nuclear immunopositive cells from 6 to 10 individual samples per group and are presented as box plots indicating the upper and lower quartiles, range, and median (middle line) with whisk - ers specifying the maximum and minimum points. *P ≤ 0.05; #0. 10 ≤ P ≤ 0.05 between N and DM 2435Reproductive Sciences (2023) 30:2429–2438 1 3 despite small sample sizes, provides support for further con- sideration of this possibility. We acknowledge several limitations in the present study — these include small sample numbers, lack of ethnic diver- sity which does not allow for generalizability of results in the population, missing information on onset of DM status and on initial ENDO diagnoses, and lack of consideration on the possibility of other pre-existing/underlying disease in patients. Due to the small sample sizes, ENDO patients with T1DM or T2DM were analyzed as one group relative to ENDO patients with no DM. Given the distinct pathogen- esis of T1DM and T2DM, future studies should consider the individual impact of T1DM vs T2DM on ENDO progression to inform screening or preventive interventions. A recent study showed that serum glucose levels were lower in ovar- ian ENDO patients than in healthy controls and that glucose together with those of inflammatory cytokine tumor necro- sis factor-α, interleukin-6, and monocyte chemoattractant protein-1 may be useful as diagnostic serum biomarkers for staging of ENDO [42]. Since the participants in the reported study were not diabetic, the significance of the results in the context of our study is not clear. Nonetheless, these collec- tive findings suggest that metabolic status may constitute a significant contributor to ENDO progression, consistent with the metabolic underpinnings of ENDO as suggested by us [25] and others [43]. Further research is merited to under - stand whether a potential feed-forward relationship between ENDO and DM exists and with relevance to glycemic con- trol and other metabolic features in the patient population with co-morbidities. In summary, while endometriosis is considered a largely benign disorder from a clinical perspective, co-morbidity with DM may lead to a progressive condition. Given that epithelial cells from endometriomas with no associated car- cinoma express numerous cancer-associated mutations [7 ], effective management of ENDO and DM may constitute a promising strategy against the development of ovarian can- cer. Moreover, a mechanistic understanding of a causal rela- tionship between ENDO and DM may have implications for the treatment of ENDO in a subset of women with DM and 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Percent Nuclear Positive Cells ESR2 PGR-TKi67 ESR1 ** ** PTEN PGR-B Epithelial Cells N DM N DM N DM N DM N DM N DM Fig. 3 Glandular epithelial immunoreactivities of ENDO markers in ectopic lesions of women with and without diabetes. Tissue sec- tions from non-diabetic women with ENDO (N) and from women with ENDO and diabetes (DM) were processed for immunohisto- chemistry, using specific antibodies as described under “Materials and Methods.” The percentages of nuclear-localized Ki67, PTEN, ESR1, ESR2, PGR-T, and PGR-B in lesion glandular epithelial cells were determined by counting the number of immunopositive-staining nuclei over the total number of cells counted × 100. For each tissue section, 3–4 random visual fields representing a total of ~ 100 cells were assessed. Data represent the percentages of nuclear immuno- positive cells from 6 to 10 individual samples per group and are pre- sented as box plots indicating the upper and lower quartiles, range, and median (middle line) with whiskers specifying the maximum and minimum points. **P ≤ 0.005 between N and DM 2436 Reproductive Sciences (2023) 30:2429–2438 1 3 for long-term glycemic control in patients with co-morbid DM and ENDO.

The authors are grateful to Dr. Frank A Simmen for critical feedback during the course of this study and for the techni- cal assistance provided by the UAMS Translational Research Institute (Kim Gates, Shaymaa Al-Shukri) and the UAMS Experimental Pathol- ogy Core (Jennifer James). Funding This work was supported in part by the National Institutes of Health (NIH)-NICHD (RO1 HD21961), NIH-National Center for Advancing Translational Sciences (NIH-UL1 TR003107), and the Uni- versity of Arkansas for Medical Sciences Sturgis Diabetes Endowment Funds. Declarations Conflict of Interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

1. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers. 2018;4(1):9. 2. Hermens M, van Altena AM, Nieboer TE, Schoot BC, van Vliet HAAM, Siebers AG, et al. Incidence of endometrioid and clear- cell ovarian cancer in histological proven endometriosis: the ENOCA population-based cohort study. Am J Obstet Gynecol. 2020;223(1):107. 3. Kvaskoff M, Mahamat-Saleh Y, Farland LV, Shigesi N, Terry KL, Harris HR, et  al. Endometriosis and cancer: a systematic review and meta-analysis. Hum Reprod Update. 2021;27(2):393–420. 4. Bulun SE, Wan Y, Matei D. Epithelial mutations in endometriosis: link to ovarian cancer. Endocrinol. 2019;160(3):626–38. Fig. 4 Monocyte/macrophage CD68 protein localization in ectopic lesions of women with and without Type 1 diabetes. Tissue sections from non- diabetic women with ENDO (ENDO-N) and from women with ENDO and type 1 diabetes (ENDO-T1DM) were processed for immunohistochemistry, using anti-CD68 antibody as described under “Materials and Methods.” a Representative images of anti-CD68 immu- nostaining for ENDO-N (middle panel) and for ENDO-T1DM (right panel) sections are shown. Left panel is an ENDO-N section processed in parallel in the absence of antibody. b For each tissue section, 3–4 random visual fields representing a total of ~ 100 stromal cells were assessed. Data represent the percentages of immunopositive stromal cells from 5 individual samples per group and are pre- sented as box plots indicating the upper and lower quartiles, range, and median (middle line) with whiskers specifying the maximum and minimum points. *P ≤ 0.05 between ENDO-N and ENDO-T1DM 0.00 0.05 0.10 0.15 0.20 0.25 a) Representative IHC b) Stromal Cells ENDO ENDO ENDO/T1DMA bAbControl 50μm 100μm 50μm Percent Positive Nuclear Cells * ENDO-N ENDO/ T1DM 2437Reproductive Sciences (2023) 30:2429–2438 1 3 5. Teasley HE, Beesley A, Kim TH, Risinger J, Young SL, Jeong JW, et al. Differential expression of KRAS and SIRT1 in ovar - ian cancers with and without endometriosis. Reprod Sci. 2020;27(1):145–51. 6. Anglesio MS, Papadopoulos N, Ayhan A, Nazeran TM, Noë M, Horlings HM, et al. Cancer-associated mutations in endometriosis without cancer. N Engl J Med. 2017;376(19):1835–48. 7. Suda K, Nakaoka H, Yoshihara K, Ishiguro T, Tamura R, Mori Y, et al. Clonal expansion and diversification of cancer-associated mutations in endometriosis and normal endometrium. Cell Rep. 2018;24(7):1777–89. 8. World Health Organization. Global Reports on Diabetes. Cam- bridge University Press; 2016. 9. Kwon BC, Anand V, Achenbach P, Dunne JL, Hagopian W, Hu J, et al. Progression of type 1 diabetes from latency to sympto- matic disease is predicted by distinct autoimmune trajectories. Nat Commun. 2022;13(1):1514. 10. Reed J, Bain S, Kanamarlapudi V. A review of current trends with type 2 diabetes epidemiology, aetiology, pathogenesis, treatments and future perspectives. Diabetes Metab Syndr Obes. 2021;14:3567–602. 11. Harding JL, Wander PL, Zhang X, Li X, Karuranga S, Chen H, et al. The incidence of adult-onset type 1 diabetes: a sys- tematic review from 32 countries and regions. Diabetes Care. 2022;45(4):994–1006. 12. Brown MR, Matveyenko AV. It’s what and when you eat: an overview of transcriptional and epigenetic responses to dietary perturbations in pancreatic islets. Front Endocrinol. 2022;13:842603. 13. Vallvé-Juanico J, George AF, Sen S, Thomas R, Shin MG, Kushnoor D, et al. Deep immunophenotyping reveals endome- triosis is marked by dysregulation of the mononuclear phago- cytic system in endometrium and peripheral blood. BMC Med. 2022;20(1):158. 14. Zolbin MM, Mamillapalli R, Nematian SE, Goetz TG, Taylor HS. Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte meta- bolic gene expression. Reprod Biol Endocrinol. 2019;17(1):36. 15. Swerdlow AJ, Laing SP, Qiao Z, Slater SD, Burden AC, Botha JL, et al. Cancer incidence and mortality in patients with insulin-treated diabetes: a UK cohort study. Br J Cancer. 2005;92(11):2070–5. 16. Yang J, Stack MS. Lipid regulatory proteins as potential thera- peutic targets for ovarian cancer in obese women. Cancers (Basel). 2020;12(11):3469. 17. Wang L, Zhong L, Xu B, Chen M, Huang H. Diabetes mel- litus and the risk of ovarian cancer: a systematic review and meta-analysis of cohort and case-control studies. BMJ Open. 2020;10(12):e040137. 18. Brown DM, Lee HC, Liu S, Quick CM, Fernandes LM, Simmen FA, et al. Notch-1 signaling activation and progesterone recep- tor expression in ectopic lesions of women with endometriosis. J Endocr Soc. 2018;2(7):765–78. 19. American Society for Reproductive Medicine. Revised Ameri- can Society for Reproductive Medicine classification of endo- metriosis. Fertil Steril. 1997;67(5):817–21. 20. Sato N, Tsunoda H, Nishida M, Morishita Y, Takimoto Y, Kubo T, Noguchi M. Loss of heterozygosity on 10q23.3 and mutation of the tumor suppressor gene PTEN in benign endometrial cyst of the ovary: possible sequence progression from benign endo- metrial cyst to endometrioid carcinoma and clear cell carcinoma of the ovary. Cancer Res. 2000;60(24):7052–6. 21. Pabona JM, Simmen FA, Nikiforov MA, Zhuang D, Shankar K, Velarde MC, et al. Krüppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis. J Clin Endocrinol Metab. 2012;97(3):E376–92. 22. Rocha CV Jr, Da Broi MG, Miranda-Furtado CL, Navarro PA, Ferriani RA, Meola J. Progesterone receptor B (PGR-B) is par - tially methylated in eutopic endometrium from infertile women with endometriosis. Reprod Sci. 2019;26(12):1568–74. 23. MacLean JA 2nd, Hayashi K. Progesterone actions and resist- ance in gynecological disorders. Cells. 2022;11(4):647. 24. Wang L, Li L, Li Y, Huang C, Lian R, Wu T, et al. History of endometriosis is associated with decreased peripheral NK cytotoxicity and increased infiltration of uterine CD68+ mac- rophages. Front Immunol. 2021;12:711231. 25. Heard ME, Melnyk SB, Simmen FA, Yang Y, Pabona JM, Sim- men RC. High-fat diet promotion of endometriosis in an immu - nocompetent mouse model is associated with altered peripheral and ectopic lesion redox and inflammatory status. Endocrinol. 2016;157(7):2870–82. 26. Li Y, Adur MK, Kannan A, Davila J, Zhao Y, Nowak RA, et al. Progesterone alleviates endometriosis via inhibition of uterine cell proliferation, inflammation and angiogenesis in an immu- nocompetent mouse model. PLoS ONE. 2016;11(10):e0165347. 27. Heard ME, Simmons CD, Simmen FA, Simmen RC. Krüppel- like factor 9 deficiency in uterine endometrial cells promotes ectopic lesion establishment associated with activated notch and hedgehog signaling in a mouse model of endometriosis. Endo- crinol. 2014;155(4):1532–46. 28. Flores VA, Vanhie A, Dang T, Taylor HS. Progesterone receptor status predicts response to progestin therapy in endometriosis. J Clin Endocrinol Metab. 2018;103(12):4561–8. 29. Han SJ, Jung SY, Wu SP, Hawkins SM, Park MJ, Kyo S, et al. Estrogen receptor β modulates apoptosis complexes and the inflammasome to drive the pathogenesis of endometriosis. Cell. 2015;163(4):960–74. 30. Yilmaz BD, Sison CAM, Yildiz S, Miyazaki K, Coon VJ, et al. Genome-wide estrogen receptor-α binding and action in human endometrial stromal cells. F S Sci. 2020;1(1):59–66. 31 Carracedo A, Alimonti A, Pandolfi PP. PTEN level in tumor suppression: how much is too little? Cancer Res. 2011;71(3):629–33. 32. Miller I, Min M, Yang C, Tian C, Gookin S, Carter D, Spencer SL. Ki67 is a graded rather than a binary marker of proliferation versus quiescence. Cell Rep. 2018;24(5):1105–12. 33. Byun J, Peterson CM, Backonja U, Taylor RN, Stanford JB, Allen-Brady KL, et al. Adiposity and endometriosis severity and typology. J Minim Invasive Gynecol. 2020;27(7):1516–23. 34 Ganz ML, Wintfeld N, Li Q, Alas V, Langer J, Hammer M. The association of body mass index with the risk of type 2 diabetes: a case–control study nested in an electronic health records sys- tem in the United States. Diabetol Metab Syndr. 2014;6:50. 35. Waddell T, Bagur A, Cunha D, Thomaides-Brears H, Banerjee R, Cuthbertson DJ, et al. Greater ectopic fat deposition and liver fibroinflammation, and lower skeletal muscle mass in people with type 2 diabetes. Obesity (Silver Spring). 2022;30:1231. 36. Shah DK, Correia KF, Vitonis AF, Missmer SA. Body size and endometriosis: results from 20 years of follow-up within the Nurses’ Health Study II prospective cohort. Hum Reprod. 2013;28(7):1783–92. 37. Lafay Pillet MC, Schneider A, Borghese B, Santulli P, Souza C, Streuli I, et al. Deep infiltrating endometriosis is associated with markedly lower body mass index: a 476 case-control study. Hum Reprod. 2012;27(1):265–72. 38. Holsworth-Carson SJ, Dior UP, Colgrave EM, Healey M, Montgomery GW, Rogers PAW, Girling JE. The association of body mass index with endometriosis and disease severity in women with pain. J Endometriosis Pelvic Pin Disorders. 2018;10(2):79–87. 2438 Reproductive Sciences (2023) 30:2429–2438 1 3 39. Kim TH, Bae N, Kim T, Hsu AL, Hunter MI, Shin JH, Jeong JW. Leptin stimulates endometriosis development in mouse models. Biomedicines. 2022;10(9):2160. https:// doi. org/ 10. 3390/ biome dicin es100 92160. 40. Simmen RCM, Brown DM, Quick CM, Alhallak I, Rose TK, Liu S, Kelley AS. Co-morbidity of type 1 diabetes and endo - metriosis: bringing a new paradigm into focus. J Endocrinol. 2019;243:R47–57. 41. Farland LV, Degnan WJ, Harris HR, Tobias DK, Missmer SA. A prospective study of endometriosis and risk of type 2 diabetes. Diabetologia. 2021;64(3):552–60. 42. Tang T, Lai H, Huang X, Gu L, Shi H. Application of serum markers in diagnosis and staging of ovarian endometriosis. J Obstet Gynaecol Res. 2021;47(4):1441–50. 43. Lu J, Ling X, Liu L, Jiang A, Ren C, Lu C, Yu Z. Emerging hallmarks of endometriosis metabolism: a promising target for the treatment of endometriosis. Biochim Biophys Acta Mol Cell Res. 2022;1870(1):119381. https:// doi. org/ 10. 1016/j. bbamcr. 2022. 119381. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.