{"paper_id":"8d670509-d3f0-400a-ae10-9500e9780b52","body_text":"Vol.:(0123456789)1 3\nArchives of Gynecology and Obstetrics (2023) 308:1327–1340 \nhttps://doi.org/10.1007/s00404-023-07110-9\nGENERAL GYNECOLOGY\nMechanisms of peripheral sensitization in endometriosis patients \nwith peritoneal lesions and acyclical pain\nRenata Voltolini Velho1  · Jalid Sehouli1 · Sylvia Mechsner1 \nReceived: 20 December 2022 / Accepted: 13 June 2023 / Published online: 5 July 2023 \n© The Author(s) 2023\nAbstract\nPurpose Endometriosis (EM) is one of the most frequent differential diagnoses concerning chronic pelvic pain. Women \nunder hormonal therapy (HT) often benefit from it but sometimes suffer a setback and develop acyclical pelvic pain. Due \nto the assumption that mechanisms of neurogenic inflammation are involved in the generation of chronic pelvic pain, we \naimed to investigate the expression of sensory nerve markers in EM-associated nerve fibers of patients with/without HT.\nMethods Laparoscopically excised peritoneal samples from 45 EM and 10 control women were immunohistochemically \nstained for: PGP9.5, Substance P (SP), NK1R, NGFp75, TRPV-1, and TrkA. Demographics and severity of pain were \ndocumented.\nResults EM patients showed a higher nerve fiber density (PGP9.5 and SP) and increased expression of NGFp75, TRPV1, \nTrkA, and NK1R in blood vessels and immune cells compared with controls. Patients with HT have cycle-dependent pelvic \npain but suffer from acyclical pelvic pain. Interestingly, reducing NK1R expression in blood vessels under HT was observed. \nA correlation between dyspareunia severity and nerve fibers density and between NGFRp75 expression in blood vessels and \ncycle-dependent pelvic pain severity was observed.\nConclusion Patients under HT have no ovulation and no (menstrual) bleeding, which correlate with inflammation and \ncyclical pain. However, acyclical pain seems to be due to peripheral sensitization once it is present under treatment. Neu-\nrotransmitters, like SP and their receptors, are involved in mechanisms of neurogenic inflammation, which are relevant for \npain initiation. These findings indicate that in both groups (EM with/without HT), neurogenic inflammation is present and \nresponsible for acyclical pain.\nKeywords Endometriosis · Nociceptive markers · Nociceptive receptors · Hormonal treatment · Pelvic pain\nWhat does this study add to the clinical work \nEndometriosis (EM) is one of the most frequent dif-\nferential diagnoses concerning chronic pelvic pain. \nOur findings indicate that in EM patients with and \nwithout hormonal therapy, neurogenic inflammation \nis present and responsible for acyclical pain.\nIntroduction\nCharacterized by the ectopic deposition and growth of \nendometrial-like tissues, endometriosis (EM) is an estrogen-\ndependent and inflammatory disorder [1]. EM lesions infiltrate \nadjacent organs (e.g., genitals, bladder, intestine, abdomen), \nresulting in inflammation, formation of scar tissue, and func-\ntional impairments of affected organs [1, 2]. The symptoms \noften affect patients’ psychological and social well-being and \nimpose a substantial economic burden on society. For this rea-\nson, EM is considered a disabling condition that may signifi-\ncantly compromise social relationships, sexuality, and mental \nhealth [3–5]. Approximately 10% of women of reproductive \nages are affected, i.e., 2 million women in Germany and 270 \nmillion worldwide, and 30–50% of them suffer from infertility \n[6]. Despite its negative impact on the quality of a patient’s \nlife, many issues related to EM remain unclear.\n * Sylvia Mechsner \n sylvia.mechsner@charite.de\n1 Department of Gynecology Charité with Center \nof Oncological Surgery, Endometriosis Research Center \nCharité, Campus Virchow-Klinikum, Berlin, Germany\n\n1328 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\nDue to the duration of pain and dissemination of endometri-\notic lesions, the associated symptoms showed a wide variation \nincluding cyclic and acyclic lower abdominal pain, dysmenor-\nrhoea, dyspareunia, dyschezia, dysuria and sub- or infertility. \nThe pathogenesis of pain generation is very complex [1 , 7, \n8]. Recent evidence demonstrates that the peripheral nervous \nsystem plays an important role in the pathophysiology of this \ndisease. Our group focus on understanding pain generation due \nto peritoneal lesions and already demonstrated wide changes \nin the innervation in the EM-affected peritoneum [9 ]. The \nhigher density of sensory nerve fibers, the lower density of \nsympathetic nerve fibers, the release of proinflammatory neu-\nrotransmitters like SP and CGRP as well as periendometriotic \ninflammation suggested neurogenic inflammatory reaction in \nthis tissue. Peripheral sensitization of EM-associated nerve \nfibers might be one key player in the modulation and severity \nof pain [10].\nBecause the pathogenesis of EM is still unresolved, no \ncausal treatment options are available. The primary treat-\nment goals are to relieve pain and eliminate fertility issues in \nwomen who wish to conceive [11]. Hormonal therapy is the \nfirst line of treatment for women with EM [12]. This treatment \ndecreases the production of the estrogen-induced release of \nprostaglandins and consequently inflammation [13]. With con-\ntinuous hormonal treatment, dysmenorrhea may be reduced \ncompared to cyclic use, but the incidence of erratic bleeding \nmay increase, and safety issues have not been fully studied \n[12]. Also, the development of acyclical pain under hormonal \ntreatment is possible and described. We observed a high grade \nof inflammation especially in peritoneal lesions of sympto-\nmatic patients under hormonal treatment [7]. Taking together, \nwe aimed to understand this pathway of pain generation in \nmore detail and investigate the expression of sensory nerve \nmarkers in the periphery of peritoneal EM under the influ-\nence of hormonal therapy. The characteristics of tissue were \nalso analyzed concerning the acyclic pelvic pain experience \nof EM patients.\nMethods\nPatients\nThis prospective study enrolled 55 women. Forty-five EM \npatients, who underwent laparoscopy due to symptomatic \nEM with excision of endometriotic lesions, were included. \nThe diagnosed EM was staged according to the revised \nclassification of the American Society of Reproductive \nMedicine (rASRM) as I: minimal, II: mild, III: moder -\nate, and IV: severe. In the analysis, two stages had been \nconsidered: mild (rASRM I and II) and severe (rASRM III \nand IV). Ten control samples were collected from women \nwithout EM, who had undergone laparoscopy for benign \ngynaecological presentations such as nonendometriosis \nassociated with ovarian cysts, uterine fibroids, hydrosal-\npinx, pelvic pain, peritonealized tissue or the unfulfilled \nwish to have children.\nPatients were selected based on clinical intraoperative \nand subsequent histopathologic findings. All patients had \nbeen given a complete gynaecological examination. The \nseverity of pain was documented using a standardized \nquestionnaire with a visual analog scale (VAS). The pain \nintensity was determined with the help of a visual numeri -\ncal analog scale (0 = no pain, 10 = strongest imaginable \npain). The women were divided into two groups based on \nthe pain scale: moderate pain (0–5 on the scale) and severe \npain (6–10 on the scale).\nThe study was approved by the Institutional Review \nBoard of the Charité University Medical Centre (Ethic \nvote EA4/036/12). All patients gave their consent.\nSample collection and immunohistochemistry \nof peritoneal endometriotic lesions processing\nAll the surgically excised lesions (EM patients) and \nhealthy peritoneum (control samples) were immediately \nfixed in buffered formalin 4% for 12 h and thereafter \nembedded in paraffin. Two μm thickness sections were \nimmunohistochemically stained with antibodies (Sup-\nplemental Table I) against the nerve fibers markers: pro -\ntein gene product 9.5 (PGP9.5), Substance P (SP); and \nnociceptive receptors: Neurokinin-1 Receptor (NK1R), \nNerve Growth Factor Receptor p75 (NGFp75), Transient \nReceptor Potential Vanilloid 1 (TRPV-1), and Tropomyo-\nsin Receptor Kinase A (TrkA).\nNegative control sections were processed by omitting \nthe specific primary antibody. A skin incision and a tissue \nsection of peritoneal EM with large nerve incisions were \nused as the positive control. Staining was detected using \nan axiophot (Carl Zeiss, Göttingen, Germany) microscope. \nPhotomicrographs were taken at different magnifications \n(100 and 400) and were further processed using Adobe \nPhotoshop (Adobe Systems, Unterschleissheim, Germany).\nDetermination of nerve fiber density\nThe density of PGP9.5 and SP-positive nerve fibers was \nassessed by counting the number of immunostained nerves \nproximal to the endometriotic lesions (epithelial, stromal, \nand smooth muscle cells) and in the distal area at 1  mm2.\nThe “hotspot” method [14] was used to determine the \nnerve fiber density of the control tissue. The immunostained \nsection was scanned at low magnification (10 ×), and the \n\n1329Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3\ntissue area with the greatest number of nerves (“hotspot”) \nwas selected. Five hotspots were evaluated and averaged \nfor each control. The density was measured by the sequen-\ntial assessment of two investigators. In cases of discrepant \nresults, both observers repeated the analysis together and \nreached a consensus.\nStatistical analysis\nStatistical analysis was performed using Graphpad Prism \n9 and non-parametric (Mann–Whitney-U , Wilcoxon, \nKruskal–Wallis or Spearman correlation test). χ 2 and Fish-\ner’s exact tests were used for the qualitative variable. Statisti-\ncally significance was assumed for p < 0.05.\nResults\nPatients’ characteristics\nDemographic and clinical variables for the 55 women \nrecruited for this study are summarized in Table 1. Our case \ngroup comprised 45 EM patients, 20 (44.44%) presented \nminimal to mild EM (rASRM I and II) and 25 (55.56%) \nmoderate to severe (rASRM III and IV). Twenty-three \n(23/45) of them were under hormonal therapy at the time \nof the surgery. Of these 23, 8 women received progestin-\nonly therapy, 10 a combined progestin–estrogen, and in 5 \nEM patients, the preparation taken could no longer be deter-\nmined. The mean age of the EM patients was 31.1 (19–53) \nyears. EM patients who took hormones were on average \nyounger (28.1) than women who did not take hormonal \npreparations (34.3; p = 0.0037).\nThe control group was a compost of ten patients, four of \nthem received hormonal therapy. In one case, it was a pure \nprogestin therapy, in two others, a combined progestin–estro-\ngen therapy and in one other case, the product ingested could \nno longer be determined. Women in the control group were \non average 32.1 (20–46) years old. No significant difference \nin age between the non-EM patients who took hormones \n(32.5) and the ones who did not take (31.8) was observed.\nPain characterization\nForty-two EM patients (95.5%) reported pelvic pain. One \npatient under hormonal treatment reported no pelvic pain. \nIn two cases, no statement was made, these women were \nnot taking any hormones. In the control group, six patients \n(54.5%, 3 under hormonal therapy) stated to suffer from \npelvic pain. Three (27.3%) denied suffering from this pain \n(one under hormonal therapy), and in two cases (18.2%), no \ninformation was given. All pain and patient characterization \nare summarized in Table  1.\nCycle‑dependent pelvic pain (CDPP)\nOf the 42 EM patients reporting pelvic pain, 15 (33.3%) \ncommunicate suffering from CDPP. Four of them were on \nhormonal therapy and eleven were not. A statistical differ -\nence (p  = 0.0096) could be seen in the hormonal therapy \nand this pain. No CDPP was reported in seven EM patients \n(one without hormonal therapy). For 23 patients with EM \n(13 positives for hormone therapy), this information was \nmissing. In the control group, three (27.3%) women reported \nCDPP. Two (18.2%) of them were on hormonal therapy. In \nseven cases (70%), this statement was missing.\nRegarding the strength of the CDPP, EM patients (data \nfrom 13 women—10 negatives for hormonal therapy) suf-\nfered on average from a pain level of 5.5 (2–9). No difference \nin the average pain severity was found between EM patients \nwho were on hormonal treatment (6.7; 4–7) and those who \nwere not (5.1; 2–8). In the control group (data from three \npatients), the severity of the CDPP was an average of 6 (4–9) \nand did not differ from the group of EM patients.\nCycle‑independent abdominal pain (CIAP)\nOnly ten (22.2%) EM patients describe suffering from CIAP \nwith an average severity of 5.7 (2–8). Five EM patients \nunder hormonal therapy report on average a CIAP of 5.2 \n(4–7) and for the five patients without hormonal therapy, a \npain average of 6.3 (2–8). Two (20%) women under hormo-\nnal therapy from the control group also describe feeling this \npain in severity 2 and 3.\nDysmenorrhea\nDysmenorrhea or painful bleeding in cyclical modus of \ncombined pills (withdrawal bleeding) was a symptom com-\nmunicated from 39 (86.7%) EM patients, with 21 under hor-\nmonal therapy. Two patients (4.4%—1 receiving hormonal \ntreatment), said do not suffer from this and the other four \n(8.9%) did not answer. In the control group, dysmenorrhea \nwas expressed as a symptom for four women (40%), two \nof these being on hormonal therapy. Two patients (20%, 1 \ntaking hormones) affirmed no painful bleeding in the cycli-\ncal modus of combined pills. This information was missing \nfor four patients (40%) in the control group, two under the \nhormonal treatment.\nThe severity of the dysmenorrhea or painful bleeding was \ninformed for 21 EM patients (46.7%), 9 of them were under \n\n1330 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\nTable 1  Characteristics of the study population\nGroup Nr Age rASRM Hormonal therapy Pain characterization (pain intensity)\nPelvic pain Cycle-dependent \npelvic pain\nCycle-independ-\nent pelvic pain\nDysmenorrhea Dyspareunia Dyschezia Dysuria\nEM patients H− 1 35 Severe – Yes NA NA Yes (NA) NA NA NA\n2 33 Mild – Yes Yes (3) No Yes (3) Yes (2) No No\n3 32 Severe – Yes Yes (NA) NA Yes (3) Yes (10) No No\n4 39 Mild – Yes Yes (2) NA Yes (3) Yes (NA) Yes (NA) NA\n5 43 Severe – Yes NA NA Yes NA NA NA\n6 25 Severe – Yes Yes (8) Yes (6) Yes (7) Yes (8) No No\n7 29 Severe – Yes Yes (5) No Yes (7) Yes (1) No No\n8 36 Severe – Yes Yes (5,5) Yes (7,5) Yes (5,5) Yes (4,5) No No\n9 53 Severe – Yes NA NA Yes (NA) NA Yes (NA) NA\n10 27 Severe – Yes Yes (3) No Yes (3) Yes (3) Yes (3) No\n11 32 Severe – Yes Yes (5) No Yes (7) Yes (2) Yes (6) No\n12 42 Severe – Yes NA NA NA No Yes (NA) No\n13 26 Severe – Yes NA NA Yes (NA) Yes (NA) Yes (NA) Yes (NA)\n14 30 Severe – NA NA NA NA NA NA NA\n15 38 Severe – Yes Yes (5) Yes (2) Yes (4) Yes (2) Yes (4) No\n16 29 Mild – Yes NA NA Yes (NA) Yes (NA) No Yes (NA)\n17 28 Mild – Yes Yes (6) Yes (8) Yes (8) Yes (3) No Yes (5)\n18 22 Severe – Yes Yes (9) Yes (8) Yes (9) Yes (9,5) No Yes (6)\n19 42 Severe – Yes NA NA Yes (NA) NA NA NA\n20 38 Mild – Yes No No Yes (3) Yes (2) Yes (5) No\n21 27 Mild – Yes NA NA NA NA NA NA\n22 49 Severe – NA NA NA No NA NA NA\nH + 23 22 Mild E, POP No NA NA NA NA NA NA\n24 26 Mild COC Yes No Yes (6) Yes (0) No No No\n25 19 Mild COC Yes NA NA Yes (NA) Yes (NA) Yes (NA) NA\n26 35 Mild COC Yes Yes (7) Yes (7) Yes (8) Yes (7) Yes (5) No\n27 28 Severe NA Yes NA NA Yes (NA) Yes (NA) No No\n\n1331Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3Table 1  (continued)\nGroup Nr Age rASRM Hormonal therapy Pain characterization (pain intensity)\nPelvic pain Cycle-dependent \npelvic pain\nCycle-independ-\nent pelvic pain\nDysmenorrhea Dyspareunia Dyschezia Dysuria\n28 35 Mild POP Yes NA NA Yes (NA) NA NA NA\n29 26 Mild NA Yes NA NA Yes (NA) No NA Yes (NA)\n30 23 Mild POP Yes NA NA Yes (NA) Yes (NA) NA NA\n31 30 Severe POP Yes Yes (5) No Yes (7) Yes (3) Yes (7) No\n32 22 Severe POP Yes No Yes (4) Yes (6) Yes (7) Yes (8) Yes (5)\n33 39 Mild COC Yes NA NA Yes (NA) Yes (NA) No NA\n34 32 Mild POP Yes No NA Yes (6) Yes (NA) No No\n35 36 Mild COC Yes NA NA Yes (NA) Yes (NA) Yes (NA) No\n36 27 Mild NA Yes NA NA Yes (NA) NA NA NA\n37 29 Severe POP Yes No NA Yes (0) Yes (5) No No\n38 23 Severe COC Yes No Yes (4) Yes (10) Yes (6) Yes (8) No\n39 25 Severe COC Yes NA NA Yes (NA) NA No No\n40 24 Severe NA Yes Yes (NA) Yes (5) No Yes (NA) No No\n41 27 Mild NA Yes NA NA Yes (NA) NA Yes (NA) NA\n42 30 Severe COC Yes NA NA Yes (NA) NA NA Yes (NA)\n43 24 Mild POP Yes No No Yes (4) No Yes (3) No\n44 35 Mild POP Yes NA NA Yes (NA) NA NA NA\n45 30 Severe COC Yes Yes (8) NA Yes (8) Yes (NA) No Yes (NA)\nControl H− C1 26 – – Yes NA NA No NA NA NA\nC2 38 – – NA NA NA NA NA NA NA\nC3 45 – – NA NA NA NA NA NA NA\nC4 27 – – Yes Yes (9) No Yes (3) Yes (9) No No\nC5 28 – – No NA NA Yes (NA) Yes (NA) NA NA\nC6 27 – – Yes NA NA NA NA NA NA\nH + C7 46 – NA No NA NA No NA NA NA\nC8 43 – POP Yes NA NA NA NA NA NA\nC9 20 – COC Yes Yes (5) Yes (2) Yes (NA) No No NA\nC10 21 – COC Yes Yes (4) Yes (3) Yes (4) Yes (4) Yes (4) Yes (2)\nH+  hormonal therapy, H− no hormonal therapy, mild rASRM I and II, severe rASRM III and IV, E estrogen, POP progestogen, COC combined pills, NA no information\n\n1332 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\nhormonal therapy. No statistical difference was found in the \nseverity of the dysmenorrhea between EM patients who were \nunder hormonal therapy (5.4; 0–10) and those who were not \n(5.2; 3–9). In the control group, two (20%) women reported \nthe severity of this symptom. One patient who was taking \nhormones reported a pain level of 4 while the other, who was \nnot taking hormones, reported a pain level of 3.\nDyspareunia\nTwenty-seven (60%, 13 on hormonal therapy) EM patients \nreported dyspareunia. Four women (8.9%, 3 under hormonal \ntreatment) stated no pain during sexual intercourse. This \ninformation was not given for 14 EM patients (31.1%, 7 \nunder hormonal treatment). No information was given for \nsix women (60%, two received hormones, four did not) from \nthe control group. Three patients (27.3%, one taking hor -\nmones) suffered from dyspareunia. Only one control patient \n(10%), who was under hormonal therapy, affirmed not to \nhave this pain.\nEM patients reported an average degree of dyspareunia of \n4.7 (1–10). Five patients, who were under hormonal therapy, \nreported a mean pain score of 5.6 (3–7). The other 11, who \nwere not taking hormones, reported an average pain of 4.3 \n(1–10). Two patients from the control group (one patient in \nhormonal therapy) reported a pain score of 4 and 9.\nDyschezia\nDyschezia was indicated as a symptom in 16 (35.5%) \npatients with EM, 8 of them on hormonal therapy. Another \n16 women (35.5%) stated that they did not have dyschezia. \nOf these, eight women took hormones. Thirteen EM patients \n(28.9%) did not answer this question. In the control group, \none woman who received hormones suffered from dysche-\nzia severity 4. Two women, one of them taking hormones, \ndenied suffering from this condition (20%). In seven cases \n(70%, two did take hormones), the corresponding informa-\ntion was lacking in the questionnaire.\nA mean pain severity of 5.4 (3–8) concerning dyschezia \nwas given in 16 EM patients (35.6%). Eight patients who \nwere on hormone therapy estimated pain intensity to be a \nmean of 6.2 (3–8). However, the other eight women who did \nnot receive hormones reported an average pain of 4.5 (3–6).\nDysuria\nEight (17.8%) EM patients suffered from dysuria, four of \nthem were on hormone therapy. In this group (only three \npatients answered this question), the average severity of \nthe dysuria was given as 5.3 (5–6). In 21 cases (46.7%, 11 \non therapy), the dysuric pain was denied and in 16 cases \n(35.6%), the information was missing. One control patient \naffirmed suffering from dysuria with a severity of 2, another \ndenied it. Both were under hormonal treatment. In eight \ncases (80%), this information was missing.\nImmunohistochemistry results\nAll the results are presented as median, 25–75% percen-\ntile. Figure  1 shows the immunohistochemistry results as \nan example.\nFig. 1  Expression of PGP9.5 (A), Substance P (B), NK1R-positive \nblood vessels (C), immune cells (D), NGFRp75 (E), TRPV1 (F), and \nTrkA (G) (red arrow) surrounding the endometriotic lesion (black \nmarked) was counted. All pictures are in 400 × magnification, except \nD, which is in 1000 × magnification\n\n1333Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3\nNerve fibers density in endometriotic lesions\nUsing anti-PGP9.5 and anti-SP, nerve fibers were detected \nin peritoneal specimens from women with EM and healthy \nperitoneum from women without EM. PGP9.5 nerve fib -\ners density was significantly increased in endometriotic \nlesions (0.57, 0.0–2.0) compared to healthy peritoneum \n(0.0, 0.0–0.072; p  = 0.0079) both in the view of nerve fib-\ners per  mm2 and in the hotspot image (EM: 2.0, 1.0–6.0; \nControl: 0.0, 0.0–0.25; p  = 0.0017) (Fig.  2A and C). EM \npatients showed significantly more SP-positive nerve fibers \nin the hotspot view (EM: 1.0, 0.0–2.0; control: 0.0, 0.0–2.0; \np = 0.0393) but no difference in the density of SP-positive \nnerve fibers per  mm2 (EM: 0.0, 0.0–0.47; control: 0.0, \n0.0–0.86; p = 0.0874) (Fig. 2E and G).\nWhen the hormonal therapy is taken into consideration, \nstatistical significance is observed in the EM group with \n(EM H+) and without hormonal intake (EM H−) compared \nwith the control without treatment (Ctr H−) for PGP9.5 \nboth in the nerve fibers per  mm2 (EM H + : 0.51, 0.0–1.7; \np = 0.0123; EM H−: 1.0, 0.4–3.0; p  = 0.0001; Ctr H−: 0.0, \n0.0–0.0) and in the hotspot image view (EM H + : 1.0, \n0.7–5.0; p = 0.0028; EM H−: 4.0, 1.0–6.0; p  < 0.0001; Ctr \nH−: 0.0, 0.0–0.0) (Fig.  2B and D).\nNo correlation could be seen between the nerve density \nand the rASRM stages. The PGP9.5 (hotspot view, r = 0.728; \np = 0.0029) and SP (hotspot and nerven/mm 2, r = 0.5741; \np = 0.0278 and r = 0.7118; p = 0.004, respectively) nerve fib-\ners density correlated with dyspareunia pain levels (Table 2).\nIncreased expression of NK1R in blood vessels and immune \ncells of EM patients\nEM patients presented more NK1R-positive stained ves-\nsels than the control group (EM: 16.0, 2.0–32.0; Ctr: 4.5, \n0.75–7.75; p = 0.0302) (Fig.  3A). A statistical difference \ncould also be found when the treatment was taken into \naccount. EM patients under hormonal therapy showed \nfewer NK1R-positive stained vessels compared with EM \npatients without treatment (EM H + : 11.0, 0.0–24.0; \nFig. 2  Nerve fibers density in endometriotic lesions and healthy peri-\ntoneum. PGP9.5-positive nerve fibers per  mm2 (A–B) and hotspot \n(C–D). Substance P (SP)-positive nerve fibers per  mm2 (D–E) and \nhotspot (F–G). EM endometriosis patients, Crt control, H +  under \nhormonal treatment, H− without hormonal treatment; all the results \nare presented as median, 25–75% percentile. Mann–Whitney test and \nKruskal–Wallis with Dunn’s multiple comparison tests. *p < 0.05; \n**p < 0.01; ***p < 0.001\n\n1334 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\nTable 2  Correlation analysis\nP value\nHormonal therapy in EMa Cycle-dependent pelvic pain 0.0096*\nCycle-independent lower pain 0.377\nDysmenorrhea 0.9153\nDyspareunia 0.3159\nDyschezia >0.9999\nDysuria 0.9087\nPain level in EM and Controla Cycle-dependent pelvic pain 0.6866\nCycle-independent lower pain0 .356\nDysmenorrhea 0.2105\nDyspareunia> 0.9999\nDyschezia> 0.9999\nDysuria >0.9999\nPain level and hormonal therapya Cycle-dependent pelvic pain 0.5846\nCycle-independent lower pain 0.2308\nDysmenorrhea 0.1009\nDyspareunia 0.551\nDyschezia 0.2745\nDysuria >0.9999\nPain level and rARSMa Cycle-dependent pelvic pain 0.703\nCycle-independent lower pain0 .076\nDysmenorrhea 0.628\nDyspareunia0 .404\nDyschezia0 .106\nDysuria 0.022*\nCycle-dependent pelvic pain and nerve fibres density/ nerve fibres receptorsb Hotspot 0.7461\nNerve fibres/ mm2 0.8456\nNGFR\nBlood vessels/ mm2 0.0334*\nBlood vessels/ mm2 0.2129NK1R\nImmune cells/ mm2 0.6101\nHotspot 0.8502PGP9.5\nNerve fibres/ mm2 0.5730\nHotspot 0.3114SP\nNerve fibres/ mm2 0.7346\nHotspot 0.8476\nNerve fibres/ mm2 0.9604\nTrkA\nBlood vessels/ mm2 0.6203\nHotspot 0.3552\nNerve fibres/ mm2 0.7353\nTRPV1\nBlood vessels/ mm2 0.2547\nCycle-independent lower pain and nerve fibres density/ nerve fibres receptorsb Hotspot 0.7905\nNerve fibres/ mm2 0.6111\nNGFR\nBlood vessels/ mm2 0.6299\nBlood vessels/ mm2 0.2234NK1R\nImmune cells/ mm2 0.2682\nHotspot 0.4417PGP9.5\nNerve fibres/ mm2 0.3664\nHotspot 0.7659SP\nNerve fibres/ mm2 0.9865\nHotspot 0.6990\nNerve fibres/ mm2 0.7947\nTrkA\nBlood vessels/ mm2 0.5098\nHotspot 0.5463\nNerve fibres/ mm2 0.4317\nTRPV1\nBlood vessels/ mm2 0.5088\nDysmenorrhea and nerve fibres density/ nerve fibres receptorsb Hotspot 0.9351\nNerve fibres/ mm2 0.4078\nNGFR\nBlood vessels/ mm2 0.7560\nBlood vessels/ mm2 0.3581NK1R\nImmune cells/ mm2 0.3963\nHotspot 0.2640PGP9.5\nNerve fibres/ mm2 0.4078\nHotspot 0.9358SP\nNerve fibres/ mm2 0.1580\nHotspot 0.3239\nNerve fibres/ mm2 0.3997\nTrkA\nBlood vessels/ mm2 0.1797\nHotspot 0.9879\nNerve fibres/ mm2 0.2799\nTRPV1\nBlood vessels/ mm2 0.9986\n\n1335Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3\nTable 2  (continued)\nDyspareunia and nerve fibres density/ nerve fibres receptorsb Hotspot 0.0991\nNerve fibres/ mm2 0.1937\nNGFR\nBlood vessels/ mm2 0.6278\nBlood vessels/ mm2 0.5823NK1R\nImmune cells/ mm2 0.8075\nHotspot 0.0029**PGP9.5\nNerve fibres/ mm2 0.0659\nHotspot 0.004**SP\nNerve fibres/ mm2 0.0029**\nHotspot 0.4392\nNerve fibres/ mm2 0.1720\nTrkA\nBlood vessels/ mm2 0.2187\nHotspot 0.8811\nNerve fibres/ mm2 0.8315\nTRPV1\nBlood vessels/ mm2 0.9921\nDyschezia and nerve fibres density/ nerve fibres receptorsb Hotspot 0.2829\nNerve fibres/ mm2 0.1263\nNGFR\nBlood vessels/ mm2 0.6422\nBlood vessels/ mm2 0.6975NK1R\nImmune cells/ mm2 0.9926\nHotspot 0.6153PGP9.5\nNerve fibres/ mm2 0.3081\nHotspot 0.0542SP\nNerve fibres/ mm2 0.1275\nHotspot 0.3313\nNerve fibres/ mm2 0.2546\nTrkA\nBlood vessels/ mm2 0.9908\nHotspot 0.0826\nNerve fibres/ mm2 0.7677\nTRPV1\nBlood vessels/ mm2 0.4181\nDysuria and nerve fibres density/ nerve fibres receptorsb Hotspot 0.6985\nNerve fibres/ mm2 0.6103\nNGFR\nBlood vessels/ mm2 0.2279\nBlood vessels/ mm2 0.1544NK1R\nImmune cells/ mm2 0.1397\nHotspot 0.8971PGP9.5\nNerve fibres/ mm2 0.9706\nHotspot 0.6985SP\nNerve fibres/ mm2 0.6324\nHotspot >0.9999\nNerve fibres/ mm2 0.6691\nTrkA\nBlood vessels/ mm2 0.2868\nHotspot >0.9999\nNerve fibres/ mm2 >0.9999\nTRPV1\nBlood vessels/ mm2 >0.9999\nrARSM and nerve fibres density/ nerve fibres receptorsb Hotspot 0.3202\nNerve fibres/ mm2 0.4664\nNGFR\nBlood vessels/ mm2 0.4483\nBlood vessels/ mm2 0.8568NK1R\nImmune cells/ mm2 0.7104\nHotspot 0.2244PGP9.5\nNerve fibres/ mm2 0.4243\nHotspot 0.3644SP\nNerve fibres/ mm2 0.7765\nHotspot 0.1818\nNerve fibres/ mm2 0.2018\nTrkA\nBlood vessels/ mm2 0.9271\nHotspot 0.7402\nNerve fibres/ mm2 0.5557\nTRPV1\nBlood vessels/ mm2 0.1895\nBold values highlight the significant correlations\nAnalyses were made with aχ2 or Fisher and bSpearman correlation\n*p < 0.05; **p < 0.005\n\n1336 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\n\n\n1337Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3\nEM H−: 21.0, 11.0–41.5; p  = 0.0465) (Fig.  3B). Also, \nthe EM group of patients without treatment differ from \nthe control group (p  = 0.0025), a difference that was not \nmaintained when the EM patients with hormonal therapy \nwere evaluated (p  = 0.3312) (Fig.  3B). Regarding immune \ncells NK1R-positive, EM patients also have an increased \namount of positive cells compared with the controls (EM: \n10.0, 0.0–24.0; Ctr: 2.0, 0.0–5.5; p  = 0.0415) (Fig.  3C). \nThis statistical difference was maintained when EM with-\nout treatment was compared with controls also without \nhormonal therapy (EM H−: 13.0, 3.0–41.0; Ctr H−: 1.0, \n0.0–5.5; p = 0.0184) (Fig.  3D).\nNo correlation could be seen between the NK1R expres-\nsion and the rASRM stages. The NK1R-positive stained ves-\nsels and immune cells also did not correlate with the pain \nlevels (Table  2).\nEM patients showed increased expression of nociceptive \nmarkers\nNGFRp75 staining showed a significant difference between \nEM patients and the control group when looking at the \nhotspot (EM: 4.0, 3.0–6.0; Ctr: 0.0, 0.0–5.0, p  = 0.0064) \nand nerve fibers per  mm2 (EM: 1.5, 0.33–3.26; Ctr: 0.0, \n0.0–0.58; p = 0.0067) (Fig.  3E and G). EM patients under \nhormonal therapy and without also presented increased \nNGFRp75-positive nerve fibers in the hotspot (EM H + : \n4.0, 0.0–5.0; EM H−: 5.0, 4.0–8.5; Ctr H−: 0.0, 0.0–1.25) \nand per  mm2 view (EM H + : 1.31, 0.0–3.0; EM H−: 2.29, \n0.98–4.43; Ctr H−: 0.0, 0.0–0.36) when compared with con-\ntrols without treatment (hotspot p = 0.0429 and 0.0017;  mm2 \np = 0.0285 and 0.0011) (Fig.  3F and H). No difference was \nobserved in the NGFRp75 stained vessels (data not shown).\nWhen looking at TRPV1-positive colored nerves, EM \npatients showed more nerve fibers than the control group \nin the hotspot view (EM: 4.0, 3.0–6.0; Ctr: 0.0, 0.0–2.0; \np = 0.039) (Fig.  3E), but not when looking at positively \ncolored fibers per  mm2 (EM: 1.5, 0.33–3.26; Ctr: 0.0, \n0.0–0.58; p = 0.7520) (Fig. 3I and K) or taking into consid-\neration the hormonal treatment (Fig.  3J and L). The analysis \nof TRPV1-stained vessels also showed no significant differ-\nence between these two groups (data not shown).\nWomen with EM showed an increased amount of TrkA \ncolored nerve fibers compared to the control patients when \nlooking at the hotspot view (EM: 1.0, 0.0–3.0; Ctr: 0.0, \n0.0–0.25; p = 0.0242) (Fig.  3O), which was not the case \nwhen the nerve fibers per  mm2 ( p = 0.1079) (Fig.  3M) \nand the stained blood vessels (data not shown) were ana -\nlyzed. In addition, a statistical difference was only obtained \nbetween EM and control patients without hormonal treat -\nment for the hotspot (EM H−: 2.0, 0.0–3.0; Ctr H−: 0.0, \n0.0–0.25; p = 0.0313) and nerve fibers per  mm2 (EM H−: \n0.6, 0.0–1.04; Ctr H−: 0.0, 0.0–0.08, p  = 0.0234) (Fig.  3N \nand P).\nNo correlation could be seen between NGFRp75, TRPV1, \nand TrkA expression and the rASRM stages. However, a \ncorrelation between NGFRp75 stained blood vessels and the \nseverity of the cycle-dependent pelvic pain was observed \n(r = − 0.5398; p = 0.0334) (Table 2).\nDiscussion\nThe majority of research into the mechanisms underlying \npain in EM has focused on the endometriotic lesions with the \nestrogen-dependent cyclical release of pain mediators as the \nprimary source of EM-associated pain [10, 15]. This reflects \nthe typical nociceptive pain, which disappears with the end \nof the menstrual bleeding and is the reason why hormonal \ntreatment and nonsteroidal anti-inflammatory substances \n(NSAP) work, especially at the beginning of the disease. \nHowever, not seldom do patients experience a shift from \ncyclical to more acyclical pain or develop acyclical pain \nunder hormonal treatment. This suggests the involvement \nof additional complex mechanisms.\nAlthough lesion-specific pain is undoubtedly essential \nfor the induction of EM-associated pain, lesion removal \ndoes not provide pain relief in all cases [7 ]. Furthermore, \nonly a marginal association exists between lesion size or \ndisease stage and the severity of pelvic symptoms [ 16]. A \nmore recent understanding of the mechanisms underlying \nthe development of a chronic pain state in EM implicates \ncyclical bleeding from lesions and subsequent inflammation \nat both lesion sites and in the peritoneal cavity. These proin-\nflammatory responses then result in sensory nerve activation \nand altered activation of nociceptive pathways The complex-\nity of peripheral and central sensitization makes research in \nthis field very difficult.\nIn this study, we focused on peripheral sensitization of \nEM-associated nerve fibers: we investigated (i) the nerve \nfiber density of sensory nerve fibers in symptomatic EM \npatients, (ii) analyzed the expression of the SP and their \nreceptor NK1R and (iii) the expression of nociceptive recep-\ntors and compared the findings between EM patients and \nFig. 3  Endometriosis patients showed increased expression of noci-\nceptive markers. NK1R-positive nerve fibers per  mm2 (A–B) and \nhotspot (C–D); NGFp75-positive nerve fibers per  mm2 (E–F) and \nhotspot (G–H); TRPV1-positive nerve fibers per  mm2 (I–J) and \nhotspot (K–L); and TrkA-positive nerve fibers per  mm2 (M–N) and \nhotspot (O–P). EM endometriosis patients, Crt control, H +  under \nhormonal treatment, H− without hormonal treatment; all the results \nare presented as median, 25–75% percentile. Mann–Whitney test and \nKruskal–Wallis with Dunn’s multiple comparison tests. *p < 0.05; \n**p < 0.01; ***p < 0.001\n◂\n\n1338 Archives of Gynecology and Obstetrics (2023) 308:1327–1340\n1 3\ncontrols and between patients using or not using hormonal \ntreatment.\nWe demonstrated the presence of sensory nerve fib-\ners in peritoneal endometriotic lesions in 45 women with \nconfirmed symptomatic EM. The density of nerve fibers in \nperitoneal endometriotic lesions was much greater than in \nnormal peritoneum in women with no EM, both in nerve fib-\ners per  mm2 and in hotspot image. EM patients also showed \nsignificantly more SP-positive nerve fibers in the hotspot \nview. Taken together, our data confirm the high innervation \nof the endometriotic lesion already seen by different groups \n[17–20]. These sensitive nerve fibers typically function as \nnociceptors, implicating them strongly in the generation of \nEM-associated pelvic pain [21, 22]. This supports the cor -\nrelation between dyspareunia and the density of nerve fibers \nusing anti-PGP9.5 and anti-SP seen in this study. The higher \nnerve fiber density goes in line with higher sensitivity in the \ncase of mechanically stretching of the tissue during inter -\ncourse. There was no difference in the nerve fiber density \nbetween patients with and without hormonal treatment.\nNeurogenic inflammation is caused by releasing the \nneurotransmitters from sensitive nerve endings, through \ninteraction with immune cells [15] and might be the \nmain source for a shift from cyclical to acyclical pain, \nor a reason for the development of acyclical pain under \nhormonal treatment. The expression of NK1R (recep-\ntor for SP) is reported to be upregulated by estrogen and \nTNF-α [19]. As local production of TNF-α and estrogen \nis increased in endometriotic lesions, NK1R expression \nwould be and has been reported to be elevated [23]. In our \nstudy, NK1R could be detected in blood vessels but also \nimmune cells at higher levels when compared with control \npatients. NK1R activation is involved in ERK1/2 protein \n(MAPK), p38 MAPK, NF-κB, PI3K, Akt, Src, EGFR and \nRho/Rock signaling pathways in different cell types [24]. \nImportantly, all these proteins have been implicated in the \ndevelopment of EM [19]. To the best of our knowledge, \nthis is the first study demonstrating this finding in perito-\nneal endometriotic lesions and gives the strong hint for \nevidence of neurogenic inflammation due to EM-associ-\nated nerve fibers.\nIncreased levels of neurotrophins such as NGF and their \nreceptors NGFRp75 and TrkA are also seen in endometrial \nbiopsies of women with EM [17, 25]. The greatly increased \nexpression of NGFRp75 and TrkA by endometriotic lesions \nmay also play a role in inducing the ingrowth of nerve fib-\ners into endometriotic tissue and may play a primary role in \nsetting up the mechanisms for the generation of pain [18, \nFig. 4  Summary of the cyclical and acyclical pain expression of noci-\nceptive markers. In the cyclical pain (left), only the prostaglandin \n(Pg) and their receptor (PgR—prostaglandin receptor) are involved in \nthe pain, in the nociceptive pain. In the acyclic pain (right), we have \nmore and activated nerve fibers, increased expression of TRPV-1, \nTrKA, NGFp75 in the nerves, increased release of substance P (SP) \nand increased expression of NK1R in immune cells as well as in \nblood vessels\n\n1339Archives of Gynecology and Obstetrics (2023) 308:1327–1340 \n1 3\n26, 27]. These effects are exacerbated by increased levels of \ncirculating estrogen in EM patients, as estrogen can enhance \nNGF activation of NGFRp75 and TrkA [28]. This is impor-\ntant as its downstream target is the well-known nociceptive \ncation channel TRPV1. The TRPV1 receptor is the most \nimportant activator of silent C-fibers. It was found to be \nupregulated in endometriomas and ectopic endometrial \ncells [29, 30], as well as in our EM samples suggesting the \nperipheral sensitization of the nerve fibers. This was, in all \nsymptomatic patients, upregulated, independent from the \nuse of hormonal treatment. An increase in the density of \nnerve endings throughout lesions and enhanced excitabil-\nity of nerves provide the basis for increased nociception at \nlesion sites [10].\nPain severity was assessed to determine if there could \nexist a correlation between nerve fiber density, receptors \nexpression and hormonal therapy. EM is associated with \nsexual pain, specifically, pain with deep penetration (dys -\npareunia). The etiology of dyspareunia in EM seems to be \nmultifactorial [31], but a higher density of nerve fiber bun-\ndles around the endometriotic lesion, compared to patients \nwithout dyspareunia, was already confirmed [32]. Now, we \nshow a correlation between this pain severity and the den-\nsity of the nerve fibers. Along these lines, we indicate the \ncorrelation between NGFRp75 expression in blood vessels \nand the cycle-dependent pelvic pain severity. As commented \nabove, this effect is aggravated by increased levels of estro-\ngen in EM patients, as estrogen can enhance NGFRp75 acti-\nvation [28].\nIt has been shown that traditional hormone therapies that \nalleviate EM-associated pain, including progestogens and \noral contraceptives, significantly reduced nerve fiber den-\nsity in ectopic endometrium [33]. What we observed was a \nreduction of the cycle-dependent pelvic pain in EM patients \nto the treatment and the marginally reduced expression \nof NK1R in the blood vessels of these patients compared \nto those that did not receive the hormonal therapy. Since \npatients under hormonal therapy do not ovulate or bleed, \nmakes sense that they also have no or less cyclic pain. As \nNK1R is related to inflammation (vasodilatation and inter -\nleukins release induction) [34], this makes us hypothesize \nthat the hormonal intake is efficient only against the inflam-\nmation due to the EM but not to the pain itself as the other \nmarkers did not decrease with the treatment and the patients \nstill suffer from acyclical pain (Fig. 4). This shows that neu-\nrogenic inflammation is present, and therefore causes the \nperipheral sensitization of the sensory nerve fibers.\nPatients under hormonal therapy have no ovulation and \nno (menstrual) bleeding, which are typically associated \nwith inflammation and cyclical pain. However, acyclical \npain seems to be due to peripheral sensitization once it is \npresent under the treatment. Neurotransmitters, like SP and \ntheir receptors, are involved in mechanisms of neurogenic \ninflammation, which are relevant for pain initiation in \nwomen affected by this chronic disease. Taking together, \nthese findings seem to indicate that in both groups (EM with/\nwithout hormonal treatment), neurogenic inflammation is \npresent and responsible for acyclical painful symptoms.\nSupplementary Information The online version contains supplemen-\ntary material available at https:// doi. org/ 10. 1007/ s00404- 023- 07110-9.\nAuthor contributions Conceptualization, RVV and SM; experiments, \nRVV and SM; writing—original draft preparation, RVV; writing—\nreview and editing, RVV, SM and JS.\nFunding Open Access funding enabled and organized by Projekt \nDEAL. We acknowledge support from the German Research Founda-\ntion (DFG) and the OpenAccess Publication Fund of Charité—Uni-\nversitätsmedizin Berlin.\nData availability Data available on reasonable request.\nDeclarations \nConflict of interest The authors declare no conflict of interest.\nEthical approval The study was approved by the Institutional \nReview Board of the Charité University Medical Centre (Ethic vote \nEA4/036/12). All subjects provided written informed consent and the \nstudies were conducted in accordance with the Declaration of Helsinki.\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. 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