The effect of GnRH-a on the angiogenesis of endometriosis

Endometriosis is a chronic benign gynecological disease that is characterized by the presence of endometriotic tissue outside the uterine cavity [ 1]. It is an estrogen-dependent Apostolos Kaponis [email protected]; [email protected] 1 Dept. of Obstetrics & Gynecology, Patras University School of Medicine, Patras, Greece 2 Laboratory of Forensic Medicine and Toxicology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece 3 Dept. of Obstetrics & Gynecology, Tottori University Faculty of Medicine, Yonago, Japan

Purpose Neoangiogenesis is necessary for adhesion and invasiveness of endometriotic lesions in women affected by endo- metriosis. Vascular endothelial growth factor (VEGF) is one of the main components of angiogenesis and is part of the major pathway tissue factor (TF)-protease activated receptor-2 (PAR-2)-VEGF that leads to neoangiogenesis. Specificity protein 1 (SP1) is a transcriptional factor that has recently been studied for its crucial role in angiogenesis via a specific pathway. We hypothesize that by blocking angiogenetic pathways we can suppress endometriotic lesions. Gonadotrophin-releasing hormone-agonists (GnRH-a) are routinely used, especially preoperatively, in endometriosis. It would be of great interest to clarify which angiogenetic pathways are affected and, thereby, pave the way for further research into antiangiogenetic effects on endometriosis.

We used quantitative real-time polymerase chain reaction (qRT-PCR) to study mRNA expression levels of TF, PAR-2, VEGF, and SP1 in endometriotic tissues of women who underwent surgery for endometriosis and received GnRH-a (leuprolide acetate) preoperatively.

VEGF, TF, and PAR-2 expression is significantly lower in patients who received treatment (p < 0,001) compared to those who did not, whereas SP1 expression is not altered (p = 0.779).

GnRH-a administration does affect some pathways of angiogenesis in endometriotic lesions, but not all of them. Therefore, supplementary treatments that affect the SP1 pathway of angiogenesis should be developed to enhance the antiangiogenetic effect of GnRH-a in patients with endometriosis. Trial registration Clinicaltrial.gov ID: NCT06106932.

Endometriosis · Angiogenesis · GnRH-a · VEGF · SP1 Received: 20 November 2023 / Accepted: 3 April 2024 / Published online: 19 April 2024 © The Author(s) 2024 The effect of GnRH-a on the angiogenesis of endometriosis Theodoros Filindris1 · Efthymia Papakonstantinou1 · Maria Keramida1 · Eleftherios Panteris2 · Sotiris Kalogeropoulos1 · Neoklis Georgopoulos1 · Fuminori Taniguchi3 · George Adonakis1 · Tasuku Harada3 · Apostolos Kaponis1 1 3 Hormones (2024) 23:509–515 lesions [5–7]. Many angiogenic growth factors have been shown to be overexpressed in endometriotic lesions and in the peritoneal fluid of women with endometriosis [ 8, 9]. Τhere is increasing evidence that the vascular endothelial growth factor (VEGF) family is involved in the etiology and maintenance of peritoneal endometriosis [ 9]. 17β-estradiol (E2) up regulates VEGF expression in human endometrial stromal cells [10, 11]. Tissue factor (TF) is also known to be involved in angio- genesis via intracellular signaling that utilizes protease acti- vated receptor-2 (PAR-2), as indicated in multiple studies [12, 13]. Specificity protein 1 (SP1) is thought to regulate VEGF expression in several carcinomas, such as pancreatic adenocarcinoma and ovarian cancer [14, 15]. Gonadotropin-releasing hormone agonists (GnRH-a) have long been used for the management of endometrio - sis. The administration of long-lasting GnRH agonists has a central effect, causing pituitary down-regulation and a reduction in gonadotropin release, thus exerting an impact on endometriotic lesions [ 16]. According to the latest ESHRE guideline, it is strongly recommended that GnRH-a be prescribed in patients to reduce endometriosis-associated pain [17]. Considering the significance of angiogenesis in endometriotic lesions, it has been hypothesized that GnRH- a might influence angiogenic mechanisms in endometrial cell growth [ 16]. According to a published study, GnRH-a (leuprolide acetate, LA) has a direct effect on endometriotic tissue, partly by interfering in inhibiting angiogenesis [ 18]. There is, hence, great interest in utilizing this knowledge since it may pave the way to further investigation regarding the effect of GnRH analogs on angiogenesis in endometri - otic lesions. In the current study, we explore the effect of the long- term administration of a GnRH-a on angiogenesis factors which promote neoangiogenesis via two different and inde- pendent pathways, namely, the TF-PAR-2-VEGF pathway and the SP-1-VEGF pathway.

& methods The subjects in this study were women of reproductive age. From January 2016 to December 2022, 60 women with known endometriosis (stages 2 and 3) were recruited. The staging of endometriosis was based on the rASRM classi - fication system [19]. Stage 2 includes women with ovarian endometrioma and superficial ovarian endometriosis, peri - toneal filmy adhesions, or deep peritoneal endometriosis. Stage 3 includes women with ovarian endometrioma, deep peritoneal endometriosis with dense adhesions, and partial obliteration of the cul de sac. Their mean age was 38 years. They were nulliparous and had a mean body mass index (BMI) of 27 kg/m2. The ovarian endometrioma present in all the participants was diagnosed using ultrasonography and/or magnetic resonance imaging. Women with peri - menopausal symptoms such as hot flashes, night sweats, and/or irregular menstrual period were excluded from the current study. The inclusion and exclusion criteria are pre - sented in Table 1. This was a randomized follow-up study with analysis of ovarian samples derived from GnRH-a-treated and non- GnRH-a-treated women before surgery. The randomiza - tion was performed by accessing a central internet-based randomization program MinimRan [ 20]. The random allo - cation sequence and the assignment of the participants to interventions were made by two of the authors (A.K. and S.K). After enrollment, the women were randomized into two groups (Table 2). Group A (GnRHa+) consisted of 30 women with a mean age of 35.5 years and a mean BMI of 27 kg/m2. Seventeen of them had stage 2 and 13 had stage 3 endometriosis. They received GnRH-a (LA) for a period of 3 months prior to surgery and had not received any hor - monal treatment within the 12 months before the surgical procedure. Group B (GnRHa-) consisted of 30 women with a mean age of 38 years and a mean BMI of 27 kg/m2. Six- teen of them had stage 2 and 14 had stage 3 endometrio - sis. They did not receive GnRH-a treatment before surgery. In addition, no treatment with oral contraceptives or other hormonal therapy had been administered within 12 months prior to surgery. Table 1 Inclusion and exclusion criteria of women Inclusion criteria Exclusion criteria Reproductive age Perimenopausal symptoms (hot flashes, night sweats, irregular menstrual bleeding) Personal history of infertility FSH > 12 IU/dl* Endometriosis stage II or greater Obesity** BMI 5 cm *Measured on 2nd day of menstrual period. **BMI >35 kg/m2. Table 2 Demographic parameters of the study population and controls. Stage of endometriosis No. of participants 30 30 - Group A Group B - Age [median, 95% Cis] 35.5 [29–40] 38.00 [34–46] 0.388 BMI [median, 95% Cis] 27.00 27.00 0.910 Duration of Treatment [months] 3 - - Stage 2 endometriosis 17 16 0.924 Stage 3 endometriosis 13 14 0.956 Menstrual cycle phase Amenorrhea Proliferative - 1 3 510 Hormones (2024) 23:509–515 During laparoscopy, biopsy specimens of the ovarian endometrioma were collected. In group B, surgery was performed during the proliferative phase of the menstrual cycle. All biopsy specimens were collected in accordance with the guidelines of the Declaration of Helsinki and with the approval of the ethical committee of the General Uni - versity Hospital of Patras. Informed consent was obtained from all women. qRT-PCR Quantitative real-time polymerase chain reaction (qRT- PCR) is used to study the expression of genes in various tissues. This method is one of the most common tools, enabling relative quantification of target gene expression by comparison with the expression of a “reference” or “house- keeping” gene. A “housekeeping” gene is defined as being constitutively expressed in the tissue under study [ 21]. The

gene should have stable expression under all experimental conditions (i.e., patients and controls) and be expressed appropriately in the tissue studied, otherwise

may be biased. For primer design The gene sequences of the exons and introns used for the design of the specific primers were obtained from the ensembl database (EMBL-EBI) (Online Resource 1). Primer design, purchased from Thermo Fisher Scientific, using the gene sequences was performed with the NCBI tool “Primer- Blast” according to the instructions of the manufacturer. The following criteria were considered in the development of the primers [22]: 1. Length of 18–24 bases 2. 40–60% G/C content 3. Starts and ends with 1–2 G/C pairs 4. Melting temperature (TM) of 50–65 °C 5. The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield 6. Primer pairs should not have complementary regions 7. The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 bp and for standard PCR it is near 500 bp (Online Resource 2; Online Resource 3) Fresh tissue samples were cut < 0.5 cm and immersed in 5–10 volumes of RNAlater Stabilization Solution (Invi - trogen, Cat. No. AM7020), stored at 4 °C overnight, and then moved to − 80 °C until RNA extraction for long-term storage. Tissue lysis and RNA extraction Prior to RNA isolation, the samples were lysed and homog- enized. The frozen tissue was placed on ice and 0.5 mL of TRIzol Reagent (TRIzol Reagent, Cat. No: 15,596,026, Invitrogene) was added at optimal sample size (50–70 mg) and homogenized at 25 Hz for 3 min. 0.1 mL of chloro - form was added to 0.5 mL of Trizol reagent, shaken vigor - ously by hand for 15 s, and incubated at room temperature for 3 min. The samples were centrifuged at 11.600 x g for 15 min at 4 °C. An equal volume of ice-cold 75% ethanol was added to the upper phase and transferred to a High Pure Filter Tube of the High Pure RNA Isolation Kit (Cat. No. 11 828 665 001, Roche) [ 23]. RNA isolation was performed according to the isolation kit protocol [ 24]. The concentra- tion and purity of RNA was determined by measuring the absorbance at 260 nm and 280 nm in a spectrophotometer. The yield of total RNA was 0.5–0.8 µg/mg. DNA (cDNA) synthesis was performed with a mixture of anchored-oligo (dT) primers and 1 µg of total RNA, according to the manufacturer’s instructions (Transcriptor First Strand cDNA Synthesis Kit, Cat. No. 04897030001; Roche Applied Science). Real-time PCR was carried out in the LightCycler 2 Instrument (Roche) using the FastStart Universal SYBR Green Master (Roche Hellas). Four independent experiments were analyzed in dupli - cates for all data shown. GAPDH was used as a reference gene for normalization. To analyze qPCR data, REST-MCS beta software version 2 was used. Statistical methods Power analysis was performed using GPower 3.1.9.6 for the comparison of patients with and without GnRH-a for a power level of = 0.8 with effect size = 0.7. Effect size was deemed to be large (0.7), as a large difference is expected between RNA expression of the main regulators of angio - genesis between patients with and without GnRH-a [ 25]. A sample size of 28 per group was indicated for the specified power level; thus, 60 patients were recruited to allow for dropouts/analysis issues. The data were analyzed using nonparametric methods via SPSS (Statistical Package for the Social Sciences) v. 26 (SPSS, Inc. Chicago, IL, USA) to generate graphs and anal- yses. As parameters do not follow the normal distribution as per the Shapiro-Wilk normality test, the Mann-Whitney U test was used for multiple variables with a significance level of 0.05. Median and 95% confidence intervals (95% CIs) were recorded for all continues variables. 1 3 511 Hormones (2024) 23:509–515

In normal endometrial stromal cells, VEGF is highly expressed, its levels depending on the effect of estrogen and progesterone [26–28]. It is widely accepted that in women with endometriosis, VEGF is highly expressed in perito - neal fluid as well as in ectopic endometrial tissue [9, 26, 29, 30]. Estrogen is a proangiogenic hormone whose effects on neovascularization and angiogenesis in the uterus and endo- metrium through proliferation and migration of endothelial cells are widely studied [26]. Previous studies have reached the conclusion that GnRH-a (LA) administration in women with endometriosis or uterine fibroids downregulated VEGF expression and affected the vascular pattern via decrease of microvessel density in the endometria studied [31–33]. The above observations enable us to formulate the hypothesis that reduction in the size of endometriomas after treatment

A sample of 60 women, 30 treated with GnRH-a in the amenorrhea phase and 30 controls without GnRH-a treat - ment in the proliferative menstrual cycle phase participated in this study. The stage of endometriosis was similar in both groups (p = 0.956). There were no demographic differences among the patients as age and BMI were similar in the two groups, with a median age of 38 years old (34–46, 95%CIs) for the control group, who did not receive GnRH-a antago - nists, and a median age of 35.5 years old (30–41, 95%CIs) (p = 0.388) for the experimental group, who did. The BMI was also identical between the two groups ( p = 0.910). Table 2 displays the demographics. Median expressions of the relevant mRNAs were sta - tistically differentiated between the two groups. In detail, TF mRNA median expression was 3.2 (3.1–3.6, 95%CIs) in control vs. 0.7 (0.7–0.9, 95%CIs) in the experimental group. Similarly, P AR-2 mRNA median expression was 7.65 (7.5–8.2, 95%CIs) vs. 2.1 (1.9–2.7, 95%CIs) in the control versus the experimental group and VEGF mRNA median expression was also significantly differentiated with 1.52 (1.4–1.8 95%CIs) vs. 0.3 (0.3–0.4, 95%CIs) in the control versus the experimental group, respectively. In contrast, SP1 mRNA did not show any differentiation between the two groups, with SP1 median expression being 1.57 (1.43– 1.69, 95%CIs) vs. 1.51 (1.36–1.69, 95%CIs) in the control versus the experimental group, respectively. Table 3; Fig. 1 presents the latter specifics in detail. Table 3 Gene expression between women with endometriosis who received a GnRH-a (Group A) and those who did not receive GnRH-a (Group B) Group A Group B Mann -Whit- ney U test Median 95.0% CΙs Median 95.0% CΙs P value TF 0.7 0.70–0.90 3.2 3.10–3.60 < 0.001 PAR-2 2.1 1.90–2.70 7.65 7.50–8.20 < 0.001 VEGF 0.3 0.30–0.40 1.52 1.40–1.80 < 0.001 SP1 1.51 1.36–1.69 1.57 1.43–1.69 0.779 Fig. 1 Gene expression of PAR-2, TF, VEGF, and SP-1 in endometriotic tissues of women with and without GnRH-a administration 1 3 512 Hormones (2024) 23:509–515 Hormonal treatments, such as GnRH-a, are not suitable for women desiring to preserve their fertility and act only for symptomatic relief and not for actual improvement of fertility [ 42]. Bevacizumab, an anti-VEGF, non-hormonal factor has been studied for possible treatment of endome - triosis; however, it carries serious, not easily tolerated side effects (i.e., gastrointestinal perforation, thrombosis, severe bleeding, impaired kidney function, and wound healing) [42]. In addition, statins work in a dose-dependent way, either promoting angiogenesis (at low doses) or blocking angiogenesis (at higher doses). However, their long-term side effects, such as myopathy and rhabdomyolysis, as well as their controversial effectiveness remain a deterrent factor to their widespread use [42]. Cabergoline, a dopamine ago- nist, has also been studied for the treatment of endometrio - sis and, in some studies, it has been found to downregulate VEGF receptors in endometriotic implants [ 42, 43]. Future research is essential to highlight the role of other treatments, hormonal or non-hormonal, in downregulating both the TF- PAR2-VEGF and SP1 pathways of angiogenesis, resulting in ultimately diminishing endometrioma size and not only relieving the symptoms. Recent studies on the treatment of endometriosis have focused on the development of antiangiogenic drugs, such as anti-VEGF antibodies, VEGFR tyrosine kinase, COX-2 inhibitors, and dopamine agonists [ 44]. This is why the present study is of high originality, given the fact that it is the first time, to our knowledge, that a study has explored the impact of GnRH-a treatment preoperatively on angio - genetic pathways in women with endometriosis. One dis - advantage of the present study is that we studied only the mRNA expression of TF, PAR2, VEGF, and SP1 and not their protein expression using Western blot or enzyme- linked immunosorbent assay (ELISA). While GnRH-a can inhibit neoangiogenesis in endometriotic lesions, it cannot completely block all the angiogenetic pathways, since no alterations in expression of the SP1 pathway of angiogen - esis have been found. Further research should be conducted to discover new, more efficient treatments of endometrio - sis. Since endometriosis concerns many reproductive-aged women, discovering ways to affect its angiogenesis is very promising to moderate the role of angiogenesis in its patho- genesis and will give hope and new perspectives to patients with endometriosis. Abbreviations BMI Body mass index ELISA Enzyme-linked immunosorbent assay E2 17β-estradiol GnRH-a Gonadotropin-releasing hormone agonist IL-1β Interleukin 1β MVD Microvessel density with GnRH-a might be caused by reduction of angiogenesis in the pathologic lesions. Many other components apart from VEGF are involved in neoangiogenesis and thus play an equally significant role in this pathway. Angiogenesis has been widely stud - ied in neoplastic tissues given that angiogenesis is of great importance for tumor viability and progression [34, 35]. TF is a cell membrane-bound glycoprotein that binds to circu - lating factor VIIa to mediate the activation of both factors IX and X and, thus, has a crucial role in hemostasis [ 35]. According to a number of studies, TF-PAR2 signaling con- tributes to angiogenesis. When TF is exposed to the blood - stream, FVIIa binds to it on the cell surface, an event that promotes hemostasis. Furthermore, the binding of FVIIa to TF cleaves PAR-2, a cleavage that results in phosphoryla - tion of the TF cytoplasmic domain and inhibits the negative effect of PAR-2-mediated signaling, promoting angiogen - esis [35]. Several mitogen-activated protein kinase (MAPK) pathways are then activated, which leads to expression of several genes, one of them being the VEGF gene. High expression of VEGF has been reported after exposure of TF-expressing cells to FVIIa (via PAR-2 activation). Our observation that mRNA of TF and PAR-2 is downregulated in women receiving GnRH-a is significant as regards insight into the role of GnRH-a in angiogenesis, since it blocks one of the most important pathways, thereby causing endome - triosis regression. Transcription factor SP1 promotes tumor angiogenesis and invasion by activating VEGF expression in several tumors, such as ovarian and pancreatic cancer, follow - ing a different and independent pathway from that of TF- PAR2-VEGF [14, 35]. According to a recent study, SP1 can activate the transforming growth factor-β1/Sma and Mad proteins 2 (TGF-β1/SMAD2) pathway and promote VEGF secretion through TGF-β1, promoting angiogenesis in preosteoblasts [ 37]. In gastric cancer as well, transcrip - tion factor SP1 is an independent prognostic factor since it has been observed that the higher the expression of SP1, the higher the microvessel density (MVD) of the tumor [38]. In a recent study, SP1 mRNA and protein levels were found to be increased in ectopic and eutopic endometrium of women with stage III/IV endometriosis [39]. We therefore included SP1 transcription factor in our study and found that GnRH-a does not affect its expression in endometriomas. The non- involvement of SP1 could be due to its known post-transla- tional modification capacity, which regulates its expression, which action could override potential disruptors. SP1 is a unique transcription factor as it both initiates transcription and can regulate the activation and repression processes: it is thus a key component that must not be affected by exter - nal disruptors [40, 41]. 1 3 513 Hormones (2024) 23:509–515 4. Groothuis PG, Endometriosis (2012) Sci Pract 2013:190–199. https://doi.org/10.1002/9781444398519.ch19 5. Lebovic DI, Bentzien F, Chao V A et al (2000) Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1β. Mol Hum Reprod 6:269–275. https://doi. org/10.1093/molehr/6.3.269 6. Arici A (2002) Local cytokines in endometrial tissue: the role of interleukin-8 in the pathogenesis of endometriosis. Ann N Y Acad Sci 955:396–406. https://doi.org/10.1111/j.1749-6632.2002. tb02770.x 7. Cohen T, Nahari D, Cerem LW et al (1996) Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 271:736–741. https://doi.org/10.1074/jbc.271.2.736 8. Taylor RN, Lebovic D, Mueller MD (2002) Angiogenic factors in endometriosis. Ann N Y Acad Sci 955:89–100. https://doi. org/10.1111/j.1749-6632.2002.tb02769.x 9. McLaren J (2000) Vascular endothelial growth factor and endo - metriotic angiogenesis. Hum Reprod Update 6:45–55. https://doi. org/10.1093/humupd/6.1.45 10. Liu S, Fan W, Gao X et al (2019) Estrogen receptor alpha regu - lates the Wnt/β-catenin signaling pathway in colon cancer by tar- geting the NOD-like receptors. Cell Signal 61:86–92. https://doi. org/10.1016/j.cellsig.2019.05.009 11. Zhang L, Xiong W, Xiong Y et al (2016) 17 β-Estradiol promotes vascular endothelial growth factor expression via the Wnt/β- catenin pathway during the pathogenesis of endometriosis. Mol Hum Reprod 22:526–535. https://doi.org/10.1093/molehr/ gaw025 12. Krikun G (2012) Endometriosis, angiogenesis and tis - sue factor. Scientifica (Cairo) 2012(306830). https://doi. org/10.6064/2012/306830 13. van den Hengel LG, Versteeg HH (2011) Tissue factor signal - ing: a multi-faceted function in biological processes. Front Biosci (Schol Ed) 3:1500–1510. https://doi.org/10.2741/240 14. Su F, Geng J, Li X et al (2017) SP1 promotes tumor angiogen - esis and invasion by activating VEGF expression in an acquired trastuzumabresistant ovarian cancer model. Oncol Rep 38:2677– 2684. https://doi.org/10.3892/or.2017.5998 15. Wang L, Guan X, Zhang J et al (2008) Targeted inhibition of Sp1- mediated transcription for antiangiogenic therapy of metastatic human gastric cancer in orthotopic nude mouse models. Int J Oncol 33:161–167 16. Tesone M, Bilotas M, Barañao RI, Meresman G (2008) The role of GnRH analogues in endometriosis-associated apoptosis and angiogenesis. Gynecol Obstet Invest 66 Suppl 110–18. https:// doi.org/10.1159/000148026 17. Christian M, Becker A, Bokor O, Heikinheimo et al (2022) ESHRE guideline: endometriosis. Hum Reprod Open. https://doi. org/10.1093/hropen/hoac009 18. Meresman GF, Bilotas MA, Lombardi E et al (2003) Effect of GnRH analogues on apoptosis and release of interleukin-1β and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 18:1767–1771. https://doi.org/10.1093/humrep/deg356 19. Rock JA (1995) The revised American Fertility Society classifi - cation of endometriosis: reproducibility of scoring. ZOLADEX endometriosis Study Group. Fertil Steril 63:1108–1110 20. Xiao L, Huang Q, Yank V , Ma J (2013) An easily accessible web- based minimization Random Allocation System for clinical trials. J Med Internet Res 15:e139. https://doi.org/10.2196/jmir.2392 21. Joshi CJ, Ke W, Drangowska-Way A et al (2022) What are house- keeping genes? PLoS Comput Biol 18:e1010295. https://doi. org/10.1371/journal.pcbi.1010295 22. Bustin S, Huggett J (2017) qPCR primer design revisited. Biomol Detect Quantif 14:19–28. https://doi.org/10.1016/j. bdq.2017.11.001 MAPK Mitogen activated protein kinase PAR-2 Protease activated receptor 2 qRT-PCR Quantitative real-time polymerase chain reaction SP1 Specificity protein 1 SMAD2 Sma and Mad proteins from Caenorhabditis elegans and Drosophilla, respectively TF Tissue factor TGF-β1 Transforming growth factor β1 VEGF Vascular endothelial growth factor Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s42000- 024-00559-6.

None Funding Open access funding provided by HEAL-Link Greece. Declarations Disclosure The authors declare no conflict of interest for this article. Human rights statement and informed consent All procedures fol - lowed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the 1964 Declaration of Helsinki and its later amendments. The study conformed to the Greek Federal Policy for the Protection of Hu- man Subjects. The appropriate ethical review committee approval was received on 11-05-2015/83. Informed consent was obtained from all patients for inclusion in the study. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, 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://creativecommons. org/licenses/by/4.0/.

1. Bulun SE (2009) Endometriosis. N Engl J Med 360:268–279. https://doi.org/10.1056/NEJMra0804690 2. Gazvani R, Templeton A (2002) Peritoneal environment, cyto - kines and angiogenesis in the pathophysiology of endome - triosis. Reproduction 123:217–226. https://doi.org/10.1530/ rep.0.1230217 3. Körbel C, Gerstner MD, Menger MD, Laschke MW (2018) Notch signaling controls sprouting angiogenesis of endometri - otic lesions. Angiogenesis 21:37–46. https://doi.org/10.1007/ s10456-017-9580-7 1 3 514 Hormones (2024) 23:509–515 adenomyosis and uterine myoma after GnRH agonist therapy. Hum Reprod 25:642–653. https://doi.org/10.1093/humrep/dep437 34. Ruf W, Yokota N, Schaffner F (2010) Tissue factor in cancer pro- gression and angiogenesis. Thromb Res 125:S36–S38. https:// doi.org/10.1016/S0049-3848(10)70010-4 35. Bluff JE, Brown NJ, Reed MW, Staton CA (2008) Tissue factor, angiogenesis and tumour progression. Breast Cancer Res 10:204. https://doi.org/10.1186/bcr1871 36. Wang L, Guan X, Zhang J, Jia Z, Wei D, Li Q, Yao J, Xie K (2008) Targeted inhibition of Sp1-mediated transcription for anti- angiogenic therapy of metastatic human gastric cancer in ortho - topic nude mouse models. Int J Oncol 33:161–167 37. Ding A, Bian Y-Y , Zhang Z-H (2020) SP1/TGFβ1/SMAD2 path- way is involved in angiogenesis during osteogenesis. Mol Med Rep 21:1581–1589. https://doi.org/10.3892/mmr.2020.10965 38. Wang L, Guan X, Gong W et al (2005) Altered expression of transcription factor Sp1 critically impacts the angiogenic pheno - type of human gastric Cancer. Clin Exp Metastasis 22:205–213. https://doi.org/10.1007/s10585-005-5684-3 39. Licong S, Xiaxia H, Yang L et al (2020) The miR-25-3p/ Sp1 pathway is dysregulated in ovarian endometrio - sis. J Int Med Res 48:300060520918437. https://doi. org/10.1177/0300060520918437 40. Tan NY , Khachigian LM (2009) Sp1 phosphorylation and its regulation of gene transcription. Mol Cell Biol 29:2483–2488. https://doi.org/10.1128/MCB.01828-08 41. Suske G (1999) The Sp-family of transcription factors. Gene 238:291–300. https://doi.org/10.1016/S0378-1119(99)00357-1 42. Thanatsis N, Filindris T, Siampalis A, Papageorgiou E, Panago - dimou E, Adonakis G, Kaponis A (2021) The Effect of Novel Medical Nonhormonal treatments on the angiogenesis of endo - metriotic lesions. Obstet Gynecol Surv 76:281–291. https://doi. org/10.1097/OGX.0000000000000888 43. Novella-Maestre E, Carda C, Ruiz-Sauri A, Garcia-Velasco JA, Simon C, Pellicer A (2010) Identification and quantification of dopamine receptor 2 in human eutopic and ectopic endometrium: a novel molecular target for endometriosis therapy. Biol Reprod 83:866–873. https://doi.org/10.1095/biolreprod.110.084392 44. Bo C, Wang YF (2024) Angiogenesis signaling in endometriosis: molecules, diagnosis and treatment. Mol Med Rep 29:43. https:// doi.org/10.3892/mmr.2024.13167 Publisher’s Note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional affiliations. 23. Roy D, Tomo S, Modi A et al (2020) Optimizing total RNA qual- ity and quantity by phenol-chloroform extraction method from human visceral adipose tissue: a standardisation study. MethodsX 7:101113. https://doi.org/10.1016/j.mex.2020.101113 24. Hummon AB, Lim SR, Difilippantonio MJ, Ried T (2007) Isolation and solubilization of proteins after TRI zol ® extraction of RNA and DNA from patient material follow - ing prolonged storage. Biotechniques 42:467–472. https://doi. org/10.2144/000112401 25. Thanatsis N, Filindris T, Siampalis A et al (2021) The Effect of Novel Medical Nonhormonal treatments on the angiogenesis of endometriotic lesions. Obstet Gynecol Surv 76:281–291. https:// doi.org/10.1097/OGX.0000000000000888 26. Chung MS, Han SJ (2022) Endometriosis-Associated Angiogen- esis and anti-angiogenic therapy for endometriosis. Front Glob Womens Health 3. https://doi.org/10.3389/fgwh.2022.856316 27. Shifren JL, Tseng JF, Zaloudek CJ et al (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 81:3112–3118. https://doi.org/10.1210/jcem.81.8.8768883 28. Lebovic DI, Shifren JL, Ryan IP et al (2000) Ovarian steroid and cytokine modulation of human endometrial angiogenesis. Hum Reprod 15:67–77. https://doi.org/10.1093/humrep/15.suppl_3.67 29. Bourlev V , V olkov N, Pavlovitch S et al (2006) The relationship between microvessel density, proliferative activity and expres - sion of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 132:501–509. https://doi.org/10.1530/rep.1.01110 30. Donnez J, Smoes P, Gillerot S et al (1998) Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 13:1686– 1690. https://doi.org/10.1093/humrep/13.6.1686 31. Meresman GF (2003) Effect of GnRH analogues on apoptosis and release of interleukin-1 and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 18:1767–1771. https://doi.org/10.1093/humrep/deg356 32. Di Lieto A, De Falco M, Mansueto G, De Rosa G, Pollio F, Staibano S (2005) Preoperative administration of GnRH-a plus tibolone to premenopausal women with uterine fibroids: evalu - ation of the clinical response, the immunohistochemical expres - sion of PDGF, bFGF and VEGF and the vascular pattern. Steroids 70:95–102. https://doi.org/10.1016/j.steroids.2004.10.008 33. Khan KN, Kitajima M, Hiraki K et al (2010) Changes in tis - sue inflammation, angiogenesis and apoptosis in endometriosis, 1 3 515