{"paper_id":"b928a2d4-df71-4e56-88d5-a2c8044d3e5d","body_text":".........................................\n...................................................\n...........\n.\n.....................................\n...............................\nAbstract citation ID: deae108.695\nP-331 Unlocking metformin’s potential: exploring its impact on\ninflammation and oxidative pathways in an endometriosis mouse\nmodel\nA. C. Neto Cerqueira 1,2, M. Botelho1, A. Rodrigues1,2, S. Lamas4,\nB. Arau´j o5, J. T. Guimar ~aes6,7, A. Gouveia1,2, H. Almeida1,2,\nD. Neves1,2\n1University of Porto Faculty of Medicine, Biomedicine - Experimental Biology Unit,\nPorto, Portugal\n2i3S - Institute for Research and Innovation in Health, Ageing and Stress, Porto,\nPortugal\n3University of Porto- Faculty of Nutrition and Food Science, n.a., Porto, Portugal\n4i3S - Institute for Research and Innovation in Health, Animal Facility, Porto,\nPortugal\n5Hospital de Braga, Patologia Clinica, Braga, Portugal\n6University of Porto Faculty of Medicine, Biomedicine - Biochemistry Unit, Porto,\nPortugal\n7Centro Hospitalar de S. Jo ~ao EPE, Patologia Clinica, Porto, Portugal\nStudy question: Could endometriosis-associated pro-oxidative and pro-in-\nflammatory condition be mitigated by metformin?\nSummary answer: Metformin treatment tends to reduce fibrosis in ectopic\ntissues, while up-regulating anti-oxidative response enzymes.\nWhat is known already: Endometriosis is a pro-inflammatory and pro-oxi-\ndative disease, which causes pain and infertility. Hormonal treatments used to\ncontrol the disease must be stopped when a woman attempts to conceive,\nmeaning new therapies are necessary. We had shown that endometriosis\nreduces fertility in a mouse model, which metformin treatment led to its\nreversal.\nMetformin, in addition to reducing blood glucose, reduces inflammation\nand systemic oxidation. It has also been shown to mitigate the growth of en-\ndometrial implants in the animal model. The objective of this work is to\nevaluate the intervention of metformin in the inflammatory and oxidative re-\nsponse pathways associated with endometriosis.\nStudy design, size, duration: Female mice were randomly divided into\ngroups: 1-Endometriosis (n ¼ 24); 2-Sham (n ¼ 12); 3-Endometriosis with\nmetformin (n ¼ 21); 4-Sham with metformin (n ¼ 16). Endometriosis was sur-\ngically induced by heterologous transplantation of endometrium from a donor\ninto recipients of the same strain. Metformin (0,5mg/mL) was administered\norally for 3 months. Half of the mice in each group were mated for the fertil-\nity study. Tissues analysed are described with “Eu” for eutopic and “Ec” for\nectopic tissues, respectively.\nParticipants/materials, setting, methods: The implants were confirmed\nand monitored by ultrasound and serum glucose, estradiol and lipoprotein\nvalues were quantified by enzymatic-colorimetric methods. Fibrosis in eutopic\nand ectopic tissues was quantified after computer-assisted analysis in\nMasson’s Trichrome images. Dual-labelling immunofluorescence was per-\nformed for the proteins F4/80 and Galectin-3 (gal-3) and Western Blot (WB)\nwas performed to quantify their expression and also NFkB, HO-1 and GPX-\n1, both in eutopic and ectopic tissues.\nMain results and the role of chance: No differences were found in serum\nanalysis between groups, apart from a decrease in total cholesterol and an in-\ncrease in estradiol in pregnant compared to non-mated mice.\nMasson’s Trichrome stained sections revealed increased stromal fibrosis in\nboth eutopic and ectopic tissues of non-mated Endometriosis groups com-\npared to Sham groups(EuEnd vs. EuSham p ¼ 0.018; EcEnd vs. EuSham\np ¼ 0.022; EuEnd vs. EuShamMet p ¼ 0.0008; EcEnd vs EuShamMet\np ¼ 0,001). Metformin treatment showed a decreasing trend in fibrosis both\nin non-mated and pregnant, particularly in ectopic tissue, although statistical\nsignificance was not reached.\nBoth eutopic and ectopic endometrium presented positive cells for f4/80\nand gal-3, with some co-localization, in all groups. Metformin treatment led to\nthe increase of the inflammation markers in the tissues of mice with ENDO:\nF4/80 was increased in eutopic tissue(p ¼ 0.0002), while gal-3 (p ¼ 0.004)\nand NF-kB (p ¼ 0.03) augmented in ectopic tissue of non-mated. Pregnancy\ncondition, on the other hand had a tendency for the decrease of these pro-\nteins. Metformin increased the expression of antioxidant defense enzymes\nHO-1 (p ¼ 0,005) and GPX-1 (p ¼ 0,0001) in the ectopic endometrium of\nnon-mated mice.\nMetformin has shown to act in the up-regulation of anti-oxidative defenses\nthat intervene in the control of endometriosis progression, but this effect was\nnot evident during pregnancy.\nLimitations, reasons for caution: Caution is necessary when extrapolating\nanimal model data to humans due to species-specific differences in endome-\ntriosis patterns. Further studies, assessing pro-oxidative species, total\nantioxidant capacity, and potential anti-inflammatory roles of studied mole-\ncules, are essential to clarify the unexpected results in inflammatory\npathways.\nWider implications of the findings: These findings highlight metformin, in\na subtherapeutic dose, as a novel and safe approach to alleviate oxidative\nstress in endometriosis, offering a promising pharmacological solution for im-\nproving endometriosis-associated infertility, once we also had shown the\nreversal of infertility associated with endometriosis through metformin\ntreatment.\nTrial registration number: not applicable\nAbstract citation ID: deae108.696\nP-332 Decoding autophagy dynamics in adenomyosis: insights of\na murine model\nJ. Vervier1, M. Squatrito2, M. Nisolle1, C. Munaut2\n1CHU de Lie`ge, Obstetrics and Gynecology, Lie`ge, Belgium\n2University of Lie`ge, Laboratory of Tumor and Development Biology- Giga-Cancer,\nLie`ge, Belgium\nStudy question: This study aimed to highlight the role of autophagy in the\npathogenesis of adenomyosis using a murine model of medically induced\nadenomyosis.\nSummary answer: Autophagy seems to play a role in the early stages of\nthe disease and is likely not the primary mechanism driving the progression of\nadenomyosis.\nWhat is known already: Autophagy is an intracellular mechanism involved\nin the recycling of aging organelles and non-functional proteins. Initially\ndescribed as a phenomenon activated under stress or fasting conditions (non-\nselective autophagy), it is now accepted that a basal level of autophagy is es-\nsential for several cellular functions to maintain cellular homeostasis (selective\nautophagy). Autophagy is essential for cell survival and tissue growth, and an\nalteration of the autophagic pathway has been described in endometrial pa-\nthologies such as endometrial hyperplasia and carcinoma, infertility, and\nendometriosis. The contribution of autophagy to adenomyosis is still unclear\ndue to conflicting findings in the existing literature.\nStudy design, size, duration: This experimental study was conducted from\nOctober 2022 until January 2024. A total of 170 mice pups were treated (90\ntreated with tamoxifen – 80 with vehicle only).\nParticipants/materials, setting, methods: Female neonatal CD1 mice re-\nceived oral doses of 2.7 mmol/kg tamoxifen from days 2 to 5 after birth.\nControl mice received only the vehicle. After synchronizing their estrus cycle,\nthe mice were euthanized at 1 month or 3 months old. The diagnostic and\ngrading of adenomyosis were performed by histological examination (hema-\ntoxylin-eosin and immuno-fluorescence). RT-PCR and Western Blot were\nused to study the modulation of autophagy pathways between the uterus of\nthe two groups.\nMain results and the role of chance: Histological analysis of 3-month-old\ntamoxifen-treated group showed that 97% of the mice developed the disease.\nThe grading method was defined by assessing the depth of lesions. After sen-\nsitivity enhancement through immunofluorescence staining (alpha-SMA,\nEpCAM and DAPI), grade 3 adenomyosis was diagnosed in up to 91% of our\nsamples (vs 84% when analyzed by H&E only). Neonatal treatment of CD1\nmice with tamoxifen reliably produces severe adenomyotic lesions.\nImmunofluorescence enhances sensitivity in histological examinations of myo-\nmetrial lesions, combining alpha-SMA staining for smooth muscle detection\nwith EpCAM for identifying epithelial cells.\n40th Hybrid Annual Meeting of the ESHRE, Amsterdam - The Netherlands, 7-10 July 2024 i351\n\n.........................................\n...................................................\n...........\n.\n.....................................\n...............................\nRTqPCR analysis of lc3b, akt, bcl2, bclxl, ulk2, ulk1, cxcr4, tnfs10, dapk1,\nigf1, atg9, pik3c3, and bid showed an up-regulation of autophagy in one-\nmonth-old adenomyosis mice. This was also indicated by a decrease in akt\nand bcl2, which are inhibitors of the pathway, along with an increase in lc3b,\na marker for phagophore formation. However, the conflicting decrease in\nulk2, a component of the induction complex, raises questions. Importantly,\nthese changes in autophagy are not observed in 3-month-old mice, suggesting\nthat autophagy may play a role in the early stages of disease development.\nThere were no statistical differences in Protein expression of pakt/akt, lc3b,\nand bcl2 between the groups.\nLimitations, reasons for caution: The analysis used protein or mRNA\nextracted from the whole uterus. Variations in results may arise if the endo-\nmetrial and myometrial components are analyzed separately. Techniques like\nlaser capture microdissection could prove valuable in this context. Continued\nresearch focusing on investigating the role of apoptosis in the study is\nessential.\nWider implications of the findings: Given the frequent association of\nadenomyosis with endometriosis and/or uterine fibroids, using an experimen-\ntal model offers the advantage of eliminating bias from associated\ncomorbidities. This approach presents a unique opportunity to unravel the\nmolecular basis of adenomyosis, while enabling a translational analysis when\njuxtaposed with clinical studies.\nTrial registration number: not applicable\nAbstract citation ID: deae108.697\nP-333 Combining deep shotgun metagenomic sequencing and\nculturomics for in-depth characterization of a potential\nendometrial microbiome\nI. Rahou\n1, R. Vanstokstraeten2, C. Nina Allonsius 3, I. De Boeck 3,\nS. Wittouck3, T. Van Rillaer 3, T. Demuyser4, S. Mackens5,\nS. Lebeer3\n1University of Antwerp- Department of Bioscience Engineering- Antwerp- Belgium,\nDepartment of Bioscience Engineering, Antwerp, Belgium\n2Vrije Universiteit Brussel VUB, Department of Microbiology and Infection Control,\nBrussel, Belgium\n3University of Antwerp, Department of Bioscience Engineering, Antwerp, Belgium\n4University of Antwerp, Department of Microbiology, Antwerp, Belgium\n5Vrije Universiteit Brussel, GRAD Genetics Reproduction and Development, Brussel,\nBelgium\nStudy question: Does the endometrium host a microbiome, and if so, are\nthese taxa distinct from the vaginal/cervical microbiome?\nSummary answer: Multifaceted analysis of endometrial tissue reveals no\ndistinct microbial taxa compared to the vaginal/cervical samples, questioning\nwhether detected microbiota are endometrium-related or result from cross-\ncontamination.\nWhat is known already: Recent studies suggest a role for the endometrial\nmicrobiota in reproductive health, yet the existence of an endometrial micro-\nbiome and its specific bacterial taxa remain enigmatic. The female\nreproductive tract, with a bacterial abundance gradient, is predominantly\nLactobacillus-dominated in the lower region. Inconsistent findings on\nLactobacillus dominance in endometrial samples are often ascribed to cross-\ncontamination and methodological constraints, highlighting the need for pre-\ncise methodologies in studying the low-biomass endometrial environment.\nStudy design, size, duration: Fifteen women, undergoing a diagnostic hys-\nteroscopy and endometrial biopsy, as part of the subfertility work-up, were\nincluded in this study. Microbiota profiles of endometrial biopsies were com-\npared to paired vaginal and cervical swabs from nine patients using 16S\namplicon sequencing. Furthermore, the microbial composition of six additional\nendometrium biopsies was characterized using both a culturomics-based ap-\nproach and deep metagenomic sequencing.\nParticipants/materials, setting, methods: Endometrial biopsies (n ¼ 15)\nwere taken with a sterile Pipelle de Cornier and snap-frozen in liquid nitrogen,\nfollowed by -80\n/C14 C storage and 16S amplicon sequencing (n ¼ 9) targeting the\nV4-region or deep shotgun metagenomic sequencing (n ¼ 6) (Prebiomics,\nItaly). Simultaneously, in nine women, paired cervical and vaginal swabs\n(Copan Diagnostics, Italy) were taken and preserved in eNAT medium. In six\nparticipants, an additional endometrial biopsy was collected and stored in\nAmies medium for immediate culturomics.\nMain results and the role of chance: We observed a high similarity in the\nmicrobial composition of endometrial biopsies, cervical, and vaginal samples\nusing 16 amplicon sequencing, but also some taxa were only found in the en-\ndometrial samples. Notably, the presence of Gemmobacter, a waterborne\ngenus, was only found in endometrial biopsies and could be a source of con-\ntamination from water used during hysteroscopy to flush the uterus. Most\nendometrial microbiome profiles were Lactobacillus dominated, with excep-\ntions observed in three patients. No significant differences for alpha diversity\nmeasures (richness and evenness) of the microbiota composition were ob-\nserved between the different niches.\nFor the first time we applied both culturomics and deep shotgun metage-\nnomic sequencing for the characterization of the microbial composition in the\nsame endometrial biopsies. Commonly identified genera using both techni-\nques include Lactobacillus, Prevotella, Bifidobacterium, Anaerococcus, and\nCuticubacterium. Remarkably, at the species level, the two approaches\nrevealed almost no overlapping species within each biopsy, underscoring the\nimportance of combining multiple methodologies in examining low-biomass\nniches like the endometrium.\nLimitations, reasons for caution: A limitation of this study is the low-bio-\nmass nature of endometrial biopsies which are prone to cross-contamination\nwith the lower female reproductive tract. This challenges downstream analy-\nsis, as it becomes difficult to distinguish between a relevant microbiological\nsignal and contaminating noise.\nWider implications of the findings: This study emphasizes the need to\nconsider a multifaceted microbiome approach when exploring low-biomass\nenvironments, like the endometrium. The critical consideration of potential\ncross-contamination by the lower female reproductive tract and its implica-\ntions for downstream analysis is paramount in clinical microbiology research\nand demands meticulous handling.\nTrial registration number: not applicable\nAbstract citation ID: deae108.698\nP-334 Hysterosalpingo-Contrast-Sonography: a key step in the\nfemale infertility workup?\nR. Y. Chaala\n1, M. Chanegriha2, F. Yendjah1, A. Atmani1, B. Bellala1,\nN. Rabhi1, M. Atir1, S. Hassani1, S. Mouhoub1\n1Mother-child hospital of army, assisted reproductive technologies department,\nAlgiers, Algeria\n2Mother-child hospital of army, medical department, Algiers, Algeria\nStudy question: The main objective of this study is to estimate the possibil-\nity of offering Hystero-Salpingo-Contrast-Sonography (HyCoSy) as a first-line\ndiagnosis test in the female infertility exploration\nSummary answer: HyCoSy represents a key step in decision-making re-\ngarding subsequent steps in infertility diagnosis and treatment\nWhat is known already: In this age of rising rates of infertility; the evalua-\ntion of the fallopian tubes and the endometrial cavity in an essential part of\nthe infertility investigation.\nTraditionally hysterosalpingography has been used to evaluate tubal pa-\ntency; but it has many disadvantages; laparoscopy and hysteroscopy are at\npresent considered as the gold standard techniques for tubal and endometrial\ncavity assessment, respectively. Hysterosalpingo- Contrast –Sonography or\nHyCoSy was introduced over 20 years ago since 1981; it allows to gynecolo-\ngist to evaluate the fallopian tubes the uterus and the ovaries with only one\nultrasound in the office.\nStudy design, size, duration: During two years, one hundred and sixty-\nseven patients with a context of female infertility with normal spermogram\nwere included in our prospective and descriptive study,\nParticipants/materials, setting, methods: Our patients underwent: a\nHysterosalpingography (HSG), a Hysteroscopy, a Laparoscopy and as part of\nour study: a Hystero-Salpingo-Contrast-Sonography (HyCoSy), a comparison\ni352 40th Hybrid Annual Meeting of the ESHRE, Amsterdam - The Netherlands, 7-10 July 2024","source_license":"CC0","license_restricted":false}