{"paper_id":"06013c62-5bfc-4693-b154-dad866386e2d","body_text":"Vol.:(0123456789)1 3\nReproductive Sciences (2023) 30:2429–2438 \nhttps://doi.org/10.1007/s43032-023-01190-3\nENDOMETRIOSIS: ORIGINAL ARTICLE\nA Pilot Study on the Co‑existence of Diabetes and Endometriosis \nin Reproductive‑Age Women: Potential for Endometriosis Progression\nIad Alhallak1 · Charles M. Quick2 · Garrett L. Graham1 · Rosalia C. M. Simmen1,3 \nReceived: 16 May 2022 / Accepted: 2 February 2023 / Published online: 14 February 2023 \n© The Author(s) 2023\nAbstract\nEndometriosis (ENDO) is a chronic estrogen-dependent gynecological condition that affects reproductive-age women, \ncausing pelvic pain, infertility, and increased risk for ovarian cancer. Diabetes mellitus (DM) is a metabolic disease with \nsignificant morbidity and mortality and rising incidence worldwide. The occurrence of DM among ENDO patients remains \nunderstudied, despite commonalities in these conditions’ immune, inflammatory, and metabolic dysfunctions. This pilot \nstudy evaluated whether a subset of women with ENDO manifests DM co-morbidity and if so, whether DM promotes ENDO \nstatus. Archived ectopic lesions obtained at ENDO surgery from non-diabetic (ENDO-N; n = 11) and diabetic (ENDO-DM; \nn = 15) patients were identified by a search of an electronic health database. Retrieved samples were analyzed by immunohis-\ntochemistry for markers of proliferation (Ki67, PTEN), steroid receptor signaling (ESR, PGR) and macrophage infiltration \n(CD68). Immunostaining data were expressed as percentages of immune-positive cells in lesion stroma and epithelium. In \nlesion stroma, the percentages of nuclear immune-positive cells were higher for ESR2 and lower for PGR-T, in ENDO-DM \nthan ENDO-N patients. The percentages of nuclear immune-positive cells for ESR1 and PTEN tended to be higher and lower, \nrespectively, in ENDO-DM than ENDO-N groups. In lesion glandular epithelium, the percentages of nuclear immune-positive \ncells were higher for ESR1 and ESR2, in ENDO-DM than ENDO-N groups. ENDO-N lesions had lower percentages of \nstromal CD68 immune-positive cells than ENDO-DM Type 1 lesions. Findings demonstrate DM in a subset of women with \nENDO, which was associated with significant changes in lesion stromal and epithelial nuclear steroid hormone receptor \nlevels, suggestive of disease progression.\nKeywords Endometriosis · Diabetes · Estrogen receptor · Progesterone receptor · Co-morbidity\nAbbreviations\nBMI  Body mass index\nDM  Diabetes mellitus\nENDO  Endometriosis\nESR1  Estrogen receptor α\nESR2  Estrogen receptor β\nFFPE  Formalin-fixed paraffin embedded\nGE  Glandular epithelial\nPTEN  Phosphate and tensin homolog\nPGR-T  Progesterone receptor-total\nPGR-B  Progesterone receptor-isoform B\nST  Stromal,\nT1DM  Type 1 diabetes mellitus\nT2DM  Type 2 diabetes mellitus\nIntroduction\nEndometriosis (ENDO) is a condition characterized by \nthe presence of uterine endometrial-like epithelium and \nstroma in extra-uterine sites, causing debilitating pelvic \npain, dysmenorrhea, and infertility in 50% of afflicted \nwomen, and with an annual economic burden of ~ 50B in \n * Rosalia C. M. Simmen \n simmenrosalia@uams.edu\n Iad Alhallak \n IAlhallak@uams.edu\n Charles M. Quick \n QuickCharlesM@uams.edu\n Garrett L. Graham \n GLGraham@uams.edu\n1 Department of Physiology & Cell Biology, University \nof Arkansas for Medical Sciences, Little Rock, AR, USA\n2 Department of Pathology, University of Arkansas for Medical \nSciences, Little Rock, AR, USA\n3 The Winthrop P Rockefeller Cancer Institute, University \nof Arkansas for Medical Sciences, Little Rock, AR, USA\n\n2430 Reproductive Sciences (2023) 30:2429–2438\n1 3\nthe USA alone [1 ]. While ENDO is considered benign \nin its initial stage, a history of ovarian/tubal ENDO is an \nindependent risk factor in two subtypes of ovarian cancer, \nnamely, clear-cell and endometrioid [2 , 3]. The patho-\ngenesis of ENDO-associated ovarian carcinoma remains \nunclear and is an area of intense investigations [ 4, 5]. \nNevertheless, recent findings have shown that endome -\ntriotic lesions without concurrent cancers contain cancer-\nassociated somatic mutations including for KRAS, PTEN, \nARID1, and PIK3CA [ 6, 7]. The latter suggests that ovar -\nian endometriotic lesions can progress to malignancy, \ngiven the proper signals and context.\nDiabetes is a progressive disease affecting over 400 mil-\nlion people worldwide [8]. Type 1 diabetes mellitus (T1DM) \nand type 2 diabetes mellitus (T2DM) are characterized by \nsignificant genetic predispositions and shared dysfunctions \nin glucose homeostasis, resulting in elevated blood glucose \nlevels in affected individuals. These conditions have distinct \npathogenesis: T1DM results from the autoimmune destruc-\ntion of pancreatic β-cells and hence, early progressive loss \nof insulin production while T2DM, a condition increasing \nwith age, stems from the loss of insulin sensitivity of tar -\nget cells, leading to defects in glucose clearance [9 , 10]. \nT1DM appears first in children and adolescents, and while \nits adult-onset is increasingly diagnosed, the basis for the \ndelayed manifestation is not well-understood [11]. T2DM \nis significantly affected by lifestyle factors (diet, physical \nactivity, alcohol, and tobacco use), is preventable, and is \ngenerally manageable by lifestyle modifications [12]. Simi-\nlar to ENDO [13, 14], T1DM and T2DM manifest significant \ninflammatory, immune, and metabolic dysfunctions and are \nassociated with increased risk and progression of ovarian \ncancer [15–17].\nThere is no known predominance of ENDO in women \nwith either T1DM or T2DM in the general population; how-\never, women (and young girls upon initiation of menses) may \nsuffer unknowingly from co-morbid conditions throughout \ntheir reproductive years since ENDO is difficult to diagnose, \nidentify, and treat [1]. Importantly, because ENDO and DM \nare chronic conditions, women with co-morbidities are antic-\nipated to have lower quality of life, may develop drug-drug \ninteractions leading to reduction in treatment efficacies for \neach condition, and may face advancement in ENDO status.\nThe present investigation constitutes a pilot study to eval-\nuate whether a subset of women with ENDO may suffer DM \nco-morbidity and if so, whether DM may progress ENDO. \nUsing archived FFPE ectopic (ovarian/tubal) lesions from \nwomen with ENDO alone and with co-incidence of DM \n(T1DM or T2DM), we report herein that DM and ENDO \nmay co-exist in reproductive-age women and that DM asso-\nciation in women with ENDO confers significant changes in \nlesion nuclear steroid hormone receptor levels, suggestive of \nincreased estrogen dependency and heightened progesterone \nresistance, both of which constitute markers of endometrio-\nsis progression.\nMaterials and Methods\nSample Tissue Collection\nThe study was approved by the Institutional Review Board \nof the University of Arkansas for Medical Sciences (UAMS \nIRB#205,177). The Arkansas Clinical Data Repository, \nwhich is affiliated with the TriNetX health research platform, \nwas used to identify female patients (20–60 years old inclu-\nsive) diagnosed from 2015 to 2019, using the search words \n“Endometriosis with no diabetes (ENDO-N),” “Endometrio-\nsis with Type 1 diabetes (ENDO-T1DM),” and “Endome-\ntriosis with Type 2 diabetes (ENDO-T2DM).” De-identified \nreferral numbers for patients meeting the specific criteria \nwere sent to the UAMS Department of Pathology and corre-\nsponding FFPE sections in the storage inventory, when avail-\nable, were retrieved by our team pathologist (CMQ). The \npatient data (age and BMI at ENDO surgery, race/ethnicity, \npresence or absence of ovarian mass, use of progesterone \nfor ENDO management) were subsequently obtained for all \nanalyzed samples.\nImmunohistochemistry\nFFPE samples were sectioned (5 μm) and processed by \nheat-induced epitope retrieval (citrate buffer) and subse-\nquent incubation with designated antibodies as previously \ndescribed [18]. Table  1 provides the list of the primary \nantibodies with their unique Research Resource Identifier \n(RRID), used at working dilutions following the RRID infor-\nmation (antibody registry.org) at incubation conditions of \n4 °C for 16–24 h. Immunoreactivity was detected using the \nVectastain Elite ABC kit (Vector Laboratories) and bioti-\nnylated anti-rabbit secondary antibodies (Vector Laborato-\nries), and slides were counterstained with hematoxylin. The \nstained slides were digitized using the Leica Digital Pathol-\nogy Whole Slide Scanner (Aperio Image Scope). Cells were \nscored as non-staining (i.e., only background staining) based \non sections that were processed in parallel with the omission \nof the primary antibody. For each antibody-treated tissue \nsection, a total of ~ 100 cells for each glandular epithelial and \nstromal compartment in 3–4 random fields were counted for \nnumbers of nuclear-staining and non-staining cells. Analy -\nses for CD68-immune-staining followed the same procedure \nexcept that stromal non-nuclear (cytoplasmic/membrane) \nstaining cells were counted. Data are expressed as the per -\ncentages of nuclear-stained (Ki67, PTEN, ESR1, ESR2, \nPGR-T, PGR-B) or cytoplasmic/membrane-stained (CD68) \ncells, relative to the total number of cells counted.\n\n2431Reproductive Sciences (2023) 30:2429–2438 \n1 3\nData Analysis\nData were analyzed by the Shapiro–Wilk’s test for normal-\nity and compared for statistical significance of difference \nbetween experimental groups using the Mann–Whitney U \ntest. The statistical tests were performed using the GraphPad \nPrism software (version 6). Data are presented as box plots \nindicating the upper and lower quartiles, range, and median \n(middle line) with whiskers indicating the maximum and \nminimum points. A p value ≤ 0.05 was considered to be sig-\nnificant. Principal component, multivariate regression, and \nbinary logistic regression analyses were used to analyze the \nassociations between the patient variables (age at surgery, \nrace, progesterone usage, and BMI) with the protein bio-\nmarkers in glandular epithelial and stromal compartments.\nResults\nPatients’ Demographic Information\nFigure  1a provides a schematic summary of sample retrieval \nand subsequent analyses of tissue sections by immunohis-\ntochemistry (IHC). FFPE-processed blocks from women \nwith ENDO without DM (n  = 11), with TIDM (n  = 8), and \nwith T2DM (n = 7) were identified from surgical pathology \nreports of the Arkansas Clinical Data Repository based on \na web-based search of the TriNetX Health Research Plat-\nform for the period covering 2015–2019. Medical records \nwere de-identified and available FFPE sections at the UAMS \nDepartment of Pathology storage inventory were retrieved \nby our team pathologist (CMQ). Lesions were largely tubal \nand ovarian (with an exception of one omental) and classi-\nfied as stages 3–4, based on the American Society of Repro-\nductive Medicine guidelines [19]. Because of the small sam-\nple sizes of the ENDO-T1DM and ENDO-T2DM groups, \nthese sets were combined and designated as ENDO-DM \n(Table  2). The mean age (years, y) of women undergoing \nsurgery for removal of ENDO lesions was higher (p < 0.001) \nfor patients without diabetes (ENDO-N; 41.9 ± 0.8 y) when \ncompared to those with diabetes (ENDO-DM; 31.6 ± 1.8 \ny). Mean BMI (kg/m 2) was higher at surgery (p  = 0.007) \nfor ENDO patients with DM (45.0 ± 3.4) than without DM \n(ENDO-N; 31.4 ± 2.7). The duration of progestin use for \neach group could not be determined due to lack of docu -\nmentation. However, the numbers of patients within each \ngroup using progestin for ENDO treatment were compara-\nble ( p = 0.86). Similarly, there was no race/ethnic dispar -\nity in the patient population between the ENDO-N and the \nENDO-DM groups (p = 0.19). Corresponding ovaries for all \npatients did not contain ovarian carcinoma, as reported from \npathology records and a review of the surgical pathology \nslides (Table  2).\nImmunohistochemistry\nIn a previous report [18], we showed that Ki67, PGR-T, \nESR1, and ESR2 constitute valid biomarkers for endome-\ntriosis progression since their levels and patterns of nuclear \nimmunopositivity in ectopic lesions differed significantly \nfrom those of corresponding eutopic endometria or non-dis-\neased endometria. PTEN protein levels in lesions were addi-\ntionally evaluated in the present study, given the protein’s \nanti-proliferative/pro-apoptotic actions and the reported \nPTEN mutations in ovarian endometriosis lesions [20]. Sim-\nilar analyses for PGR-B were performed since disruption of \nPGR-B expression has been demonstrated in many uterine \ndisorders including endometriosis [21– 23]. A composite of \nrepresentative immunostaining of lesions from women with \nENDO-N, using specific antibodies to each protein, is shown \nin Fig.  1b. Nuclear staining for all proteins was demonstrated \nin both stromal and epithelial compartments.\nStromal Immunoreactivity in ENDO Lesions \nwith Diabetes Status\nStromal cells of ENDO lesions from women without DM \ndisplayed varying levels of nuclear-localized immunore-\nactivities (expressed as percent of nuclear-positive cells) \nfor the evaluated ENDO biomarkers (Fig.  2). In ENDO-N \nTable 1  Antibodies used for \nIHC\n1 Research Resource Identifier (antibodyregistry.org)\nProtein Vendor/catalog number RRID1 Working dilution\nEstrogen receptor α Santa Cruz/sc542 AB_63140 1:250\nEstrogen receptor β Millipore Sigma/05–824 AB_310195 1:200\nKi67 Abcam/Ab16667 AB_302459 1:200\nProgesterone Receptor (total) Santa Cruz/sc7208 AB_2164331 1:200\nProgesterone Receptor-B Cell Signaling/3157S AB_2252606 1:400\nPhosphatase and Tensin homologue Cell Signaling/138G6 AB_823618 1:200\nMacrophage CD68 Thermo Fisher Scientific/\nMA5-13,324\nAB_10987212 1:200\n\n2432 Reproductive Sciences (2023) 30:2429–2438\n1 3\nlesions, the highest percent immunoreactivities were \nseen for PTEN, PGR-T, and PGR-B, with Ki67 and ESR2 \nimmunoreactivities displaying the lowest levels. The co-\noccurrence of DM in women with ENDO increased and \ndecreased, respectively, nuclear-localized stromal immu-\nnoreactivities for ESR2 (p  = 0.04) and PGR-T (p  = 0.05), \nrelative to those of women without DM. Stromal cells of \nENDO lesions with DM tended to show reduced (p  = 0.07) \nand increased (p = 0.06) nuclear PTEN and ESR1 immuno-\nreactivity levels, respectively, relative to those of women \nwithout DM. Levels of nuclear immunoreactivities for \nKi67 and PGR-B were not affected by DM status (Fig.  2).\na) Experimental Design\nOvarian Lesions (FFPE)\na) TRINET –X Platform\nb) UAMS Pathology Department\nNon-DM\n(n=11)\nT1DM\n(n=8)\nT2DM\n(n=7)\nIHC\nKi67\nPTEN\nESR1\nESR2\nPGR-T\nPGR-B\nb) Representative IHC\nC\nKi67\n50μm\nESR1\nPTEN\n50μm\nAb\n50μm 50μm\n50μm 50μm\nESR2\n50μm 50μm\nPGR-T\n50μm 50μm\nPGR-B\n50μm 50μm\nC Ab\nC Ab C Ab\nC Ab\nC Ab\nFig. 1  Analyses of endometriotic lesions. a. Schematic of tissue \nretrieval and analyses. Tissue samples for the study were identi-\nfied from search of the Arkansas Clinical Data Repository, using \nthe TriNetX health research platform, and retrieved from the UAMS \nDepartment of Pathology repository. Formalin-fixed paraffin-embed-\nded sections were subjected to immunohistochemistry using the \nlisted antibodies (Table  1). b Representative H&E-stained sections \nof lesions from non-diabetic women with ENDO. For each section \nstained with the indicated antibody (anti-Ki67, anti-PTEN, anti-\nESR1, anti-ESR2, anti-PGR-T, anti-PGR-B), a corresponding section \nwas processed in parallel in the absence of antibody to serve as con-\ntrol (labeled C)\n\n2433Reproductive Sciences (2023) 30:2429–2438 \n1 3\nGlandular Epithelial Immunoreactivity in ENDO \nLesions with Diabetes Status\nGlandular epithelial cells of ENDO lesions from women \nwith DM displayed nuclear-localized immunoreactivities \nfor ESR1 (p = 0.001) and ESR2 (p = 0.005) that were higher \nthan those from women without DM (Fig.  3). By contrast, \nnuclear immunoreactivities for Ki67, PTEN, PGR-T, and \nPGR-B in these cells were not affected by DM status.\nMacrophage Biomarker CD68 Immunoreactivity \nin ENDO Lesions\nMacrophage infiltration of ovarian endometriomas has been \npreviously reported [24]. Furthermore, we have shown in a \nmouse model of endometriosis that progression of ENDO in \nectopic lesions with high fat diet was associated with increased \nlocalization of macrophages in stromal cells as measured by \nF/480 immunostaning [25]. Here, we used the human mac -\nrophage/monocyte selective biomarker CD68 to evaluate \npotential changes in macrophage infiltration of ENDO-N \nrelative to ENDO-T1DM lesions. The limited availability of \nENDO-T2DM lesions precluded parallel analyses of these \nsamples. ENDO lesions stained positive for CD68 preferen-\ntially in cytoplasmic/membrane-associated compartments of \nlesion stromal cells (Fig. 4a). Immunostaining levels in stromal \ncells were higher for ENDO-T1DM than ENDO-N (Fig. 4b).\nDiscussion\nEndometriosis (ENDO) and diabetes (DM) individually \naffect women of reproductive age, yet the occurrence of \nENDO and DM co-morbidity and its potential contribution \nTable 2  Patient demographics\n* t-test (p ≤ 0.05, compared to ENDO-N)\nRace/Ethnicity Age at ENDO Surgery BMI at ENDO Surgery Ovarian Mass Progesterone\nENDO-N\n  Black/African American 43 25.0 No No\n  Black/African American 43 46.2 No Yes\n  White/Caucasian 38 39.5 No Yes\n  Black/African American 39 21.6 No Yes\n  Black/African American 44 22.5 No Yes\n  Black/African American 45 29.9 No No\n  Black/African American 43 29.5 No Yes\n  Black/African American 43 18.2 No Yes\n  Black/African American 45 36.9 No No\n  Black/African American 39 38.3 No Yes\n  Black/African American 39 38.3 No Yes\nMean ± SEM 41.9 ± 0.8 31.4 ± 2.7\nENDO-DM\n  White/Caucasian 24 33.2 No Yes\n  White/Caucasian 25 39.7 No Yes\n  Black/African American 34 36.5 No Yes\n  White/Caucasian 24 33.1 No Yes\n  Black/African American 28 52.8 No No\n  Black/African American 29 61.1 No No\n  Black/African American 31 52.4 No No\n  White/Caucasian 26 21.6 No No\n  Black/African American 40 52.9 No No\n  Black/African American 29 61.8 No Yes\n  Black/African American 36 24.2 No No\n  Black/African American 44 46.2 No Yes\n  Black/African American 26 61.8 No Yes\n  Black/African American 34 49.8 No No\n  Hispanic/Latino 44 48.2 No Yes\nMean ± SEM 31.6 ± 1.8* 45.0 ± 3.4*\n\n2434 Reproductive Sciences (2023) 30:2429–2438\n1 3\nto ENDO progression remain unexplored. In this pilot study, \nwe provide clinical data to show the co-existence of ENDO \nin a subset of women with DM (T1DM and T2DM). Further, \nwe demonstrate that DM status in ENDO women confers \nsignificant changes in steroid hormone receptor levels in \nlesion stromal and glandular epithelial compartments, rela-\ntive to those of ENDO women without DM. Progesterone \nresistance, estrogen-dependency, and immune activation are \nhallmarks of ENDO development and progression. Reduc-\ntions in levels of stromal ligand-bound PGR exacerbate \nENDO status [18, 26, 27] and are associated with resistance \nto progestin therapy in women with ENDO [28]. Moreover, \nenhanced estrogen signaling leads to heightened cell inflam-\nmation mediated by ESR2 [29] and promotes cell prolifera-\ntion mediated by ESR1 [30]. Here, we show that nuclear lev-\nels of PGR-T (stroma) were decreased while those of ESR1 \n(epithelia) and ESR2 (epithelia, stroma) were increased, \nin ENDO-DM relative to ENDO-N lesions. Furthermore, \nwe found increased macrophage localization (CD68 immu-\nnoreactivity) indicative of immune activation known to be \nassociated with ENDO progression [1, 25], in lesion stromal \ncells of ENDO women with Type I DM, relative to those of \nENDO-N women. The tending decrease in lesion stromal \nPTEN immunoreactivity with DM status is consistent with \nearlier reports that subtle reductions of PTEN expression \nlevel are sufficient to promote cell proliferation and hence, \ncancer susceptibility [ 31]. Unexpectedly, there was a lack \nof coincident increases in the levels of Ki67 immunoreac-\ntivities in both lesion stromal and epithelial cells with DM \nstatus. We suggest that this may reflect in part the relatively \nadvanced endometriotic stage of the lesions analyzed in the \npresent study (largely stages 3–4) and the previously docu-\nmented inability of Ki67 dynamics to accurately capture \ncellular proliferation index [32]. Multivariate analysis (data \nnot shown) showed no significant association of patient vari-\nables of progesterone usage, race, BMI, and age of ENDO \nsurgery with the evaluated protein biomarkers in lesion epi-\nthelial and stromal compartments, suggesting diabetes status \nas the major driver in the noted differences in these proteins’ \nexpression levels.\nIn this study, the tissue samples were retrieved from \npatients in the age range of 20–60 years because ENDO \nsymptoms (i.e., pelvic pain, heavy menses) are pronounced \naround age 20 (although the condition may initiate earlier) \nand recurrence in affected women may extend beyond the \nmenopausal period. Interestingly, the study participants in \nthe ENDO-N group were notably older than those of the \nENDO/DM group. The significance of this finding is unclear \nsince the data were obtained at ENDO surgery and not dur-\ning initial diagnoses. However, we speculate that the earlier \nage of ENDO surgery for ENDO-DM patients may reflect \ntheir greater degree of pain/discomfort. The relationship \nbetween pain severity in ENDO women and DM status mer-\nits further investigation. The linkage of adiposity (measured \nby BMI) and endometriosis is complex and may be depend-\nent on disease severity [33]. The higher BMIs shown for \nENDO patients with DM align with the positive association \nof BMI and diabetes mellitus [34, 35]. By contrast, epide-\nmiological studies indicate a negative association for obe-\nsity and ENDO progression [ 36, 37]. Nevertheless, since \nobesity does not protect against endometriosis [38] and in \nmouse models of the disease, high-fat diet induced obesity \nand inflammation increased endometriosis development [25, \n39], there is a need for further evaluation of this relationship.\nDespite the significant health and economic challenges \nimposed individually by DM and ENDO in female patients, \nthe prospects of their co-incidence in a subset of women \nhave not been assessed. Indeed, no work to date has clini-\ncally addressed whether DM status promotes ENDO and \nif ENDO treatments may complicate glycemic control in \nwomen with DM [40]. In a recent prospective study using \ndata from the Nurses’ Health Study, Missmer and colleagues \n[41] reported no overall increased risk of T2DM for women \nwith ENDO. However, the reverse relationship, whether \nDM promotes ENDO, has not been studied. Our pilot study, \n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nPercent Nuclear Positive Cells\n*\n*\n#\n#\nESR2 PGR-TKi67 ESR1PTEN PGR-B\nStromal Cells\nN DM N DM N DM N DM N DM N DM\nFig. 2  Stromal immunoreactivities of ENDO markers in ectopic \nlesions of women with and without diabetes. Tissue sections from \nnon-diabetic women with ENDO (N) and from women with ENDO \nand diabetes (DM) were processed for immunohistochemistry, using \nspecific antibodies as described under “Materials and Methods.” The \npercentages of nuclear-localized Ki67, PTEN, ESR1, ESR2, PGR-T, \nand PGR-B in lesion stromal cells were determined by counting the \nnumber of immunopositive-staining nuclei over the total number of \ncells counted × 100. For each tissue section, 3–4 random visual fields \nrepresenting a total of ~ 100 cells were assessed. Data represent the \npercentages of nuclear immunopositive cells from 6 to 10 individual \nsamples per group and are presented as box plots indicating the upper \nand lower quartiles, range, and median (middle line) with whisk -\ners specifying the maximum and minimum points. *P ≤ 0.05; #0. \n10 ≤ P ≤ 0.05 between N and DM\n\n2435Reproductive Sciences (2023) 30:2429–2438 \n1 3\ndespite small sample sizes, provides support for further con-\nsideration of this possibility.\nWe acknowledge several limitations in the present study \n— these include small sample numbers, lack of ethnic diver-\nsity which does not allow for generalizability of results in \nthe population, missing information on onset of DM status \nand on initial ENDO diagnoses, and lack of consideration \non the possibility of other pre-existing/underlying disease \nin patients. Due to the small sample sizes, ENDO patients \nwith T1DM or T2DM were analyzed as one group relative \nto ENDO patients with no DM. Given the distinct pathogen-\nesis of T1DM and T2DM, future studies should consider the \nindividual impact of T1DM vs T2DM on ENDO progression \nto inform screening or preventive interventions. A recent \nstudy showed that serum glucose levels were lower in ovar-\nian ENDO patients than in healthy controls and that glucose \ntogether with those of inflammatory cytokine tumor necro-\nsis factor-α, interleukin-6, and monocyte chemoattractant \nprotein-1 may be useful as diagnostic serum biomarkers for \nstaging of ENDO [42]. Since the participants in the reported \nstudy were not diabetic, the significance of the results in the \ncontext of our study is not clear. Nonetheless, these collec-\ntive findings suggest that metabolic status may constitute a \nsignificant contributor to ENDO progression, consistent with \nthe metabolic underpinnings of ENDO as suggested by us \n[25] and others [43]. Further research is merited to under -\nstand whether a potential feed-forward relationship between \nENDO and DM exists and with relevance to glycemic con-\ntrol and other metabolic features in the patient population \nwith co-morbidities.\nIn summary, while endometriosis is considered a largely \nbenign disorder from a clinical perspective, co-morbidity \nwith DM may lead to a progressive condition. Given that \nepithelial cells from endometriomas with no associated car-\ncinoma express numerous cancer-associated mutations [7 ], \neffective management of ENDO and DM may constitute a \npromising strategy against the development of ovarian can-\ncer. Moreover, a mechanistic understanding of a causal rela-\ntionship between ENDO and DM may have implications for \nthe treatment of ENDO in a subset of women with DM and \n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\nPercent Nuclear Positive Cells\nESR2 PGR-TKi67 ESR1\n**\n**\nPTEN PGR-B\nEpithelial Cells\nN DM N DM N DM N DM N DM N DM\nFig. 3  Glandular epithelial immunoreactivities of ENDO markers \nin ectopic lesions of women with and without diabetes. Tissue sec-\ntions from non-diabetic women with ENDO (N) and from women \nwith ENDO and diabetes (DM) were processed for immunohisto-\nchemistry, using specific antibodies as described under “Materials \nand Methods.” The percentages of nuclear-localized Ki67, PTEN, \nESR1, ESR2, PGR-T, and PGR-B in lesion glandular epithelial cells \nwere determined by counting the number of immunopositive-staining \nnuclei over the total number of cells counted × 100. For each tissue \nsection, 3–4 random visual fields representing a total of ~ 100 cells \nwere assessed. Data represent the percentages of nuclear immuno-\npositive cells from 6 to 10 individual samples per group and are pre-\nsented as box plots indicating the upper and lower quartiles, range, \nand median (middle line) with whiskers specifying the maximum and \nminimum points. **P ≤ 0.005 between N and DM\n\n2436 Reproductive Sciences (2023) 30:2429–2438\n1 3\nfor long-term glycemic control in patients with co-morbid \nDM and ENDO.\nAcknowledgements The authors are grateful to Dr. Frank A Simmen \nfor critical feedback during the course of this study and for the techni-\ncal assistance provided by the UAMS Translational Research Institute \n(Kim Gates, Shaymaa Al-Shukri) and the UAMS Experimental Pathol-\nogy Core (Jennifer James).\nFunding This work was supported in part by the National Institutes \nof Health (NIH)-NICHD (RO1 HD21961), NIH-National Center for \nAdvancing Translational Sciences (NIH-UL1 TR003107), and the Uni-\nversity of Arkansas for Medical Sciences Sturgis Diabetes Endowment \nFunds.\nDeclarations \nConflict of Interest The authors declare no competing interests.\nOpen Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article’s Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nReferences\n 1. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, \nViganò P. Endometriosis. Nat Rev Dis Primers. 2018;4(1):9.\n 2. Hermens M, van Altena AM, Nieboer TE, Schoot BC, van Vliet \nHAAM, Siebers AG, et al. Incidence of endometrioid and clear-\ncell ovarian cancer in histological proven endometriosis: the \nENOCA population-based cohort study. Am J Obstet Gynecol. \n2020;223(1):107.\n 3. Kvaskoff M, Mahamat-Saleh Y, Farland LV, Shigesi N, \nTerry KL, Harris HR, et  al. Endometriosis and cancer: a \nsystematic review and meta-analysis. Hum Reprod Update. \n2021;27(2):393–420.\n 4. Bulun SE, Wan Y, Matei D. Epithelial mutations in endometriosis: \nlink to ovarian cancer. Endocrinol. 2019;160(3):626–38.\nFig. 4  Monocyte/macrophage \nCD68 protein localization in \nectopic lesions of women with \nand without Type 1 diabetes. \nTissue sections from non-\ndiabetic women with ENDO \n(ENDO-N) and from women \nwith ENDO and type 1 diabetes \n(ENDO-T1DM) were processed \nfor immunohistochemistry, \nusing anti-CD68 antibody as \ndescribed under “Materials and \nMethods.” a Representative \nimages of anti-CD68 immu-\nnostaining for ENDO-N (middle \npanel) and for ENDO-T1DM \n(right panel) sections are shown. \nLeft panel is an ENDO-N \nsection processed in parallel in \nthe absence of antibody. b For \neach tissue section, 3–4 random \nvisual fields representing a \ntotal of ~ 100 stromal cells were \nassessed. Data represent the \npercentages of immunopositive \nstromal cells from 5 individual \nsamples per group and are pre-\nsented as box plots indicating \nthe upper and lower quartiles, \nrange, and median (middle line) \nwith whiskers specifying the \nmaximum and minimum points. \n*P ≤ 0.05 between ENDO-N \nand ENDO-T1DM\n0.00\n0.05\n0.10\n0.15\n0.20\n0.25\na) Representative IHC\nb) Stromal Cells\nENDO ENDO ENDO/T1DMA bAbControl\n50μm 100μm 50μm\nPercent Positive Nuclear Cells\n*\nENDO-N ENDO/\nT1DM\n\n2437Reproductive Sciences (2023) 30:2429–2438 \n1 3\n 5. Teasley HE, Beesley A, Kim TH, Risinger J, Young SL, Jeong \nJW, et al. Differential expression of KRAS and SIRT1 in ovar -\nian cancers with and without endometriosis. Reprod Sci. \n2020;27(1):145–51.\n 6. Anglesio MS, Papadopoulos N, Ayhan A, Nazeran TM, Noë M, \nHorlings HM, et al. Cancer-associated mutations in endometriosis \nwithout cancer. N Engl J Med. 2017;376(19):1835–48.\n 7. Suda K, Nakaoka H, Yoshihara K, Ishiguro T, Tamura R, Mori \nY, et al. Clonal expansion and diversification of cancer-associated \nmutations in endometriosis and normal endometrium. Cell Rep. \n2018;24(7):1777–89.\n 8. World Health Organization. Global Reports on Diabetes. Cam-\nbridge University Press; 2016.\n 9. Kwon BC, Anand V, Achenbach P, Dunne JL, Hagopian W, Hu \nJ, et al. Progression of type 1 diabetes from latency to sympto-\nmatic disease is predicted by distinct autoimmune trajectories. \nNat Commun. 2022;13(1):1514.\n 10. Reed J, Bain S, Kanamarlapudi V. A review of current trends \nwith type 2 diabetes epidemiology, aetiology, pathogenesis, \ntreatments and future perspectives. Diabetes Metab Syndr Obes. \n2021;14:3567–602.\n 11. Harding JL, Wander PL, Zhang X, Li X, Karuranga S, Chen \nH, et al. The incidence of adult-onset type 1 diabetes: a sys-\ntematic review from 32 countries and regions. Diabetes Care. \n2022;45(4):994–1006.\n 12. Brown MR, Matveyenko AV. It’s what and when you eat: \nan overview of transcriptional and epigenetic responses to \ndietary perturbations in pancreatic islets. Front Endocrinol. \n2022;13:842603.\n 13. Vallvé-Juanico J, George AF, Sen S, Thomas R, Shin MG, \nKushnoor D, et al. Deep immunophenotyping reveals endome-\ntriosis is marked by dysregulation of the mononuclear phago-\ncytic system in endometrium and peripheral blood. BMC Med. \n2022;20(1):158.\n 14. Zolbin MM, Mamillapalli R, Nematian SE, Goetz TG, Taylor \nHS. Adipocyte alterations in endometriosis: reduced numbers of \nstem cells and microRNA induced alterations in adipocyte meta-\nbolic gene expression. Reprod Biol Endocrinol. 2019;17(1):36.\n 15. Swerdlow AJ, Laing SP, Qiao Z, Slater SD, Burden AC, \nBotha JL, et al. Cancer incidence and mortality in patients \nwith insulin-treated diabetes: a UK cohort study. Br J Cancer. \n2005;92(11):2070–5.\n 16. Yang J, Stack MS. Lipid regulatory proteins as potential thera-\npeutic targets for ovarian cancer in obese women. Cancers \n(Basel). 2020;12(11):3469.\n 17. Wang L, Zhong L, Xu B, Chen M, Huang H. Diabetes mel-\nlitus and the risk of ovarian cancer: a systematic review and \nmeta-analysis of cohort and case-control studies. BMJ Open. \n2020;10(12):e040137.\n 18. Brown DM, Lee HC, Liu S, Quick CM, Fernandes LM, Simmen \nFA, et al. Notch-1 signaling activation and progesterone recep-\ntor expression in ectopic lesions of women with endometriosis. \nJ Endocr Soc. 2018;2(7):765–78.\n 19. American Society for Reproductive Medicine. Revised Ameri-\ncan Society for Reproductive Medicine classification of endo-\nmetriosis. Fertil Steril. 1997;67(5):817–21.\n 20. Sato N, Tsunoda H, Nishida M, Morishita Y, Takimoto Y, Kubo \nT, Noguchi M. Loss of heterozygosity on 10q23.3 and mutation \nof the tumor suppressor gene PTEN in benign endometrial cyst \nof the ovary: possible sequence progression from benign endo-\nmetrial cyst to endometrioid carcinoma and clear cell carcinoma \nof the ovary. Cancer Res. 2000;60(24):7052–6.\n 21. Pabona JM, Simmen FA, Nikiforov MA, Zhuang D, Shankar \nK, Velarde MC, et al. Krüppel-like factor 9 and progesterone \nreceptor coregulation of decidualizing endometrial stromal \ncells: implications for the pathogenesis of endometriosis. J Clin \nEndocrinol Metab. 2012;97(3):E376–92.\n 22. Rocha CV Jr, Da Broi MG, Miranda-Furtado CL, Navarro PA, \nFerriani RA, Meola J. Progesterone receptor B (PGR-B) is par -\ntially methylated in eutopic endometrium from infertile women \nwith endometriosis. Reprod Sci. 2019;26(12):1568–74.\n 23. MacLean JA 2nd, Hayashi K. Progesterone actions and resist-\nance in gynecological disorders. Cells. 2022;11(4):647.\n 24. Wang L, Li L, Li Y, Huang C, Lian R, Wu T, et al. History \nof endometriosis is associated with decreased peripheral NK \ncytotoxicity and increased infiltration of uterine  CD68+ mac-\nrophages. Front Immunol. 2021;12:711231.\n 25. Heard ME, Melnyk SB, Simmen FA, Yang Y, Pabona JM, Sim-\nmen RC. High-fat diet promotion of endometriosis in an immu -\nnocompetent mouse model is associated with altered peripheral \nand ectopic lesion redox and inflammatory status. Endocrinol. \n2016;157(7):2870–82.\n 26. Li Y, Adur MK, Kannan A, Davila J, Zhao Y, Nowak RA, et al. \nProgesterone alleviates endometriosis via inhibition of uterine \ncell proliferation, inflammation and angiogenesis in an immu-\nnocompetent mouse model. PLoS ONE. 2016;11(10):e0165347.\n 27. Heard ME, Simmons CD, Simmen FA, Simmen RC. Krüppel-\nlike factor 9 deficiency in uterine endometrial cells promotes \nectopic lesion establishment associated with activated notch and \nhedgehog signaling in a mouse model of endometriosis. Endo-\ncrinol. 2014;155(4):1532–46.\n 28. Flores VA, Vanhie A, Dang T, Taylor HS. Progesterone receptor \nstatus predicts response to progestin therapy in endometriosis. \nJ Clin Endocrinol Metab. 2018;103(12):4561–8.\n 29. Han SJ, Jung SY, Wu SP, Hawkins SM, Park MJ, Kyo S, et al. \nEstrogen receptor β modulates apoptosis complexes and the \ninflammasome to drive the pathogenesis of endometriosis. Cell. \n2015;163(4):960–74.\n 30. Yilmaz BD, Sison CAM, Yildiz S, Miyazaki K, Coon VJ, et al. \nGenome-wide estrogen receptor-α binding and action in human \nendometrial stromal cells. F S Sci. 2020;1(1):59–66.\n 31 Carracedo A, Alimonti A, Pandolfi PP. PTEN level in \ntumor suppression: how much is too little? Cancer Res. \n2011;71(3):629–33.\n 32. Miller I, Min M, Yang C, Tian C, Gookin S, Carter D, Spencer \nSL. Ki67 is a graded rather than a binary marker of proliferation \nversus quiescence. Cell Rep. 2018;24(5):1105–12.\n 33. Byun J, Peterson CM, Backonja U, Taylor RN, Stanford JB, \nAllen-Brady KL, et al. Adiposity and endometriosis severity \nand typology. J Minim Invasive Gynecol. 2020;27(7):1516–23.\n 34 Ganz ML, Wintfeld N, Li Q, Alas V, Langer J, Hammer M. The \nassociation of body mass index with the risk of type 2 diabetes: \na case–control study nested in an electronic health records sys-\ntem in the United States. Diabetol Metab Syndr. 2014;6:50.\n 35. Waddell T, Bagur A, Cunha D, Thomaides-Brears H, Banerjee \nR, Cuthbertson DJ, et al. Greater ectopic fat deposition and liver \nfibroinflammation, and lower skeletal muscle mass in people \nwith type 2 diabetes. Obesity (Silver Spring). 2022;30:1231.\n 36. Shah DK, Correia KF, Vitonis AF, Missmer SA. Body size \nand endometriosis: results from 20 years of follow-up within \nthe Nurses’ Health Study II prospective cohort. Hum Reprod. \n2013;28(7):1783–92.\n 37. Lafay Pillet MC, Schneider A, Borghese B, Santulli P, Souza \nC, Streuli I, et al. Deep infiltrating endometriosis is associated \nwith markedly lower body mass index: a 476 case-control study. \nHum Reprod. 2012;27(1):265–72.\n 38. Holsworth-Carson SJ, Dior UP, Colgrave EM, Healey M, \nMontgomery GW, Rogers PAW, Girling JE. The association \nof body mass index with endometriosis and disease severity \nin women with pain. J Endometriosis Pelvic Pin Disorders. \n2018;10(2):79–87.\n\n2438 Reproductive Sciences (2023) 30:2429–2438\n1 3\n 39. Kim TH, Bae N, Kim T, Hsu AL, Hunter MI, Shin JH, Jeong \nJW. Leptin stimulates endometriosis development in mouse \nmodels. Biomedicines. 2022;10(9):2160. https:// doi. org/ 10.  \n3390/ biome dicin es100 92160.\n 40. Simmen RCM, Brown DM, Quick CM, Alhallak I, Rose TK, \nLiu S, Kelley AS. Co-morbidity of type 1 diabetes and endo -\nmetriosis: bringing a new paradigm into focus. J Endocrinol. \n2019;243:R47–57.\n 41. Farland LV, Degnan WJ, Harris HR, Tobias DK, Missmer SA. A \nprospective study of endometriosis and risk of type 2 diabetes. \nDiabetologia. 2021;64(3):552–60.\n 42. Tang T, Lai H, Huang X, Gu L, Shi H. Application of serum \nmarkers in diagnosis and staging of ovarian endometriosis. J \nObstet Gynaecol Res. 2021;47(4):1441–50.\n 43. Lu J, Ling X, Liu L, Jiang A, Ren C, Lu C, Yu Z. Emerging \nhallmarks of endometriosis metabolism: a promising target for \nthe treatment of endometriosis. Biochim Biophys Acta Mol Cell \nRes. 2022;1870(1):119381. https:// doi. org/ 10. 1016/j. bbamcr.  \n2022. 119381.\nPublisher's Note Springer Nature remains neutral with regard to \njurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}