P-332 Decoding autophagy dynamics in adenomyosis: insights of a murine model

citation ID: deae108.695 P-331 Unlocking metformin’s potential: exploring its impact on inflammation and oxidative pathways in an endometriosis mouse model A. C. Neto Cerqueira 1,2, M. Botelho1, A. Rodrigues1,2, S. Lamas4, B. Arau´j o5, J. T. Guimar ~aes6,7, A. Gouveia1,2, H. Almeida1,2, D. Neves1,2 1University of Porto Faculty of Medicine, Biomedicine - Experimental Biology Unit, Porto, Portugal 2i3S - Institute for Research and Innovation in Health, Ageing and Stress, Porto, Portugal 3University of Porto- Faculty of Nutrition and Food Science, n.a., Porto, Portugal 4i3S - Institute for Research and Innovation in Health, Animal Facility, Porto, Portugal 5Hospital de Braga, Patologia Clinica, Braga, Portugal 6University of Porto Faculty of Medicine, Biomedicine - Biochemistry Unit, Porto, Portugal 7Centro Hospitalar de S. Jo ~ao EPE, Patologia Clinica, Porto, Portugal Study question: Could endometriosis-associated pro-oxidative and pro-in- flammatory condition be mitigated by metformin? Summary answer: Metformin treatment tends to reduce fibrosis in ectopic tissues, while up-regulating anti-oxidative response enzymes. What is known already: Endometriosis is a pro-inflammatory and pro-oxi- dative disease, which causes pain and infertility. Hormonal treatments used to control the disease must be stopped when a woman attempts to conceive, meaning new therapies are necessary. We had shown that endometriosis reduces fertility in a mouse model, which metformin treatment led to its reversal. Metformin, in addition to reducing blood glucose, reduces inflammation and systemic oxidation. It has also been shown to mitigate the growth of en- dometrial implants in the animal model. The objective of this work is to evaluate the intervention of metformin in the inflammatory and oxidative re- sponse pathways associated with endometriosis. Study design, size, duration: Female mice were randomly divided into groups: 1-Endometriosis (n ¼ 24); 2-Sham (n ¼ 12); 3-Endometriosis with metformin (n ¼ 21); 4-Sham with metformin (n ¼ 16). Endometriosis was sur- gically induced by heterologous transplantation of endometrium from a donor into recipients of the same strain. Metformin (0,5mg/mL) was administered orally for 3 months. Half of the mice in each group were mated for the fertil- ity study. Tissues analysed are described with “Eu” for eutopic and “Ec” for ectopic tissues, respectively. Participants/materials, setting, methods: The implants were confirmed and monitored by ultrasound and serum glucose, estradiol and lipoprotein values were quantified by enzymatic-colorimetric methods. Fibrosis in eutopic and ectopic tissues was quantified after computer-assisted analysis in Masson’s Trichrome images. Dual-labelling immunofluorescence was per- formed for the proteins F4/80 and Galectin-3 (gal-3) and Western Blot (WB) was performed to quantify their expression and also NFkB, HO-1 and GPX- 1, both in eutopic and ectopic tissues. Main results and the role of chance: No differences were found in serum analysis between groups, apart from a decrease in total cholesterol and an in- crease in estradiol in pregnant compared to non-mated mice. Masson’s Trichrome stained sections revealed increased stromal fibrosis in both eutopic and ectopic tissues of non-mated Endometriosis groups com- pared to Sham groups(EuEnd vs. EuSham p ¼ 0.018; EcEnd vs. EuSham p ¼ 0.022; EuEnd vs. EuShamMet p ¼ 0.0008; EcEnd vs EuShamMet p ¼ 0,001). Metformin treatment showed a decreasing trend in fibrosis both in non-mated and pregnant, particularly in ectopic tissue, although statistical significance was not reached. Both eutopic and ectopic endometrium presented positive cells for f4/80 and gal-3, with some co-localization, in all groups. Metformin treatment led to the increase of the inflammation markers in the tissues of mice with ENDO: F4/80 was increased in eutopic tissue(p ¼ 0.0002), while gal-3 (p ¼ 0.004) and NF-kB (p ¼ 0.03) augmented in ectopic tissue of non-mated. Pregnancy condition, on the other hand had a tendency for the decrease of these pro- teins. Metformin increased the expression of antioxidant defense enzymes HO-1 (p ¼ 0,005) and GPX-1 (p ¼ 0,0001) in the ectopic endometrium of non-mated mice. Metformin has shown to act in the up-regulation of anti-oxidative defenses that intervene in the control of endometriosis progression, but this effect was not evident during pregnancy. Limitations, reasons for caution: Caution is necessary when extrapolating animal model data to humans due to species-specific differences in endome- triosis patterns. Further studies, assessing pro-oxidative species, total antioxidant capacity, and potential anti-inflammatory roles of studied mole- cules, are essential to clarify the unexpected results in inflammatory pathways. Wider implications of the findings: These findings highlight metformin, in a subtherapeutic dose, as a novel and safe approach to alleviate oxidative stress in endometriosis, offering a promising pharmacological solution for im- proving endometriosis-associated infertility, once we also had shown the reversal of infertility associated with endometriosis through metformin treatment. Trial registration number: not applicable

citation ID: deae108.696 P-332 Decoding autophagy dynamics in adenomyosis: insights of a murine model J. Vervier1, M. Squatrito2, M. Nisolle1, C. Munaut2 1CHU de Lie`ge, Obstetrics and Gynecology, Lie`ge, Belgium 2University of Lie`ge, Laboratory of Tumor and Development Biology- Giga-Cancer, Lie`ge, Belgium Study question: This study aimed to highlight the role of autophagy in the pathogenesis of adenomyosis using a murine model of medically induced adenomyosis. Summary answer: Autophagy seems to play a role in the early stages of the disease and is likely not the primary mechanism driving the progression of adenomyosis. What is known already: Autophagy is an intracellular mechanism involved in the recycling of aging organelles and non-functional proteins. Initially described as a phenomenon activated under stress or fasting conditions (non- selective autophagy), it is now accepted that a basal level of autophagy is es- sential for several cellular functions to maintain cellular homeostasis (selective autophagy). Autophagy is essential for cell survival and tissue growth, and an alteration of the autophagic pathway has been described in endometrial pa- thologies such as endometrial hyperplasia and carcinoma, infertility, and endometriosis. The contribution of autophagy to adenomyosis is still unclear due to conflicting findings in the existing literature. Study design, size, duration: This experimental study was conducted from October 2022 until January 2024. A total of 170 mice pups were treated (90 treated with tamoxifen – 80 with vehicle only). Participants/materials, setting, methods: Female neonatal CD1 mice re- ceived oral doses of 2.7 mmol/kg tamoxifen from days 2 to 5 after birth. Control mice received only the vehicle. After synchronizing their estrus cycle, the mice were euthanized at 1 month or 3 months old. The diagnostic and grading of adenomyosis were performed by histological examination (hema- toxylin-eosin and immuno-fluorescence). RT-PCR and Western Blot were used to study the modulation of autophagy pathways between the uterus of the two groups. Main results and the role of chance: Histological analysis of 3-month-old tamoxifen-treated group showed that 97% of the mice developed the disease. The grading method was defined by assessing the depth of lesions. After sen- sitivity enhancement through immunofluorescence staining (alpha-SMA, EpCAM and DAPI), grade 3 adenomyosis was diagnosed in up to 91% of our samples (vs 84% when analyzed by H&E only). Neonatal treatment of CD1 mice with tamoxifen reliably produces severe adenomyotic lesions. Immunofluorescence enhances sensitivity in histological examinations of myo- metrial lesions, combining alpha-SMA staining for smooth muscle detection with EpCAM for identifying epithelial cells. 40th Hybrid Annual Meeting of the ESHRE, Amsterdam - The Netherlands, 7-10 July 2024 i351 ......................................... ................................................... ........... . ..................................... ............................... RTqPCR analysis of lc3b, akt, bcl2, bclxl, ulk2, ulk1, cxcr4, tnfs10, dapk1, igf1, atg9, pik3c3, and bid showed an up-regulation of autophagy in one- month-old adenomyosis mice. This was also indicated by a decrease in akt and bcl2, which are inhibitors of the pathway, along with an increase in lc3b, a marker for phagophore formation. However, the conflicting decrease in ulk2, a component of the induction complex, raises questions. Importantly, these changes in autophagy are not observed in 3-month-old mice, suggesting that autophagy may play a role in the early stages of disease development. There were no statistical differences in Protein expression of pakt/akt, lc3b, and bcl2 between the groups. Limitations, reasons for caution: The analysis used protein or mRNA extracted from the whole uterus. Variations in results may arise if the endo- metrial and myometrial components are analyzed separately. Techniques like laser capture microdissection could prove valuable in this context. Continued research focusing on investigating the role of apoptosis in the study is essential. Wider implications of the findings: Given the frequent association of adenomyosis with endometriosis and/or uterine fibroids, using an experimen- tal model offers the advantage of eliminating bias from associated comorbidities. This approach presents a unique opportunity to unravel the molecular basis of adenomyosis, while enabling a translational analysis when juxtaposed with clinical studies. Trial registration number: not applicable

citation ID: deae108.697 P-333 Combining deep shotgun metagenomic sequencing and culturomics for in-depth characterization of a potential endometrial microbiome I. Rahou 1, R. Vanstokstraeten2, C. Nina Allonsius 3, I. De Boeck 3, S. Wittouck3, T. Van Rillaer 3, T. Demuyser4, S. Mackens5, S. Lebeer3 1University of Antwerp- Department of Bioscience Engineering- Antwerp- Belgium, Department of Bioscience Engineering, Antwerp, Belgium 2Vrije Universiteit Brussel VUB, Department of Microbiology and Infection Control, Brussel, Belgium 3University of Antwerp, Department of Bioscience Engineering, Antwerp, Belgium 4University of Antwerp, Department of Microbiology, Antwerp, Belgium 5Vrije Universiteit Brussel, GRAD Genetics Reproduction and Development, Brussel, Belgium Study question: Does the endometrium host a microbiome, and if so, are these taxa distinct from the vaginal/cervical microbiome? Summary answer: Multifaceted analysis of endometrial tissue reveals no distinct microbial taxa compared to the vaginal/cervical samples, questioning whether detected microbiota are endometrium-related or result from cross- contamination. What is known already: Recent studies suggest a role for the endometrial microbiota in reproductive health, yet the existence of an endometrial micro- biome and its specific bacterial taxa remain enigmatic. The female reproductive tract, with a bacterial abundance gradient, is predominantly Lactobacillus-dominated in the lower region. Inconsistent findings on Lactobacillus dominance in endometrial samples are often ascribed to cross- contamination and methodological constraints, highlighting the need for pre- cise methodologies in studying the low-biomass endometrial environment. Study design, size, duration: Fifteen women, undergoing a diagnostic hys- teroscopy and endometrial biopsy, as part of the subfertility work-up, were included in this study. Microbiota profiles of endometrial biopsies were com- pared to paired vaginal and cervical swabs from nine patients using 16S amplicon sequencing. Furthermore, the microbial composition of six additional endometrium biopsies was characterized using both a culturomics-based ap- proach and deep metagenomic sequencing. Participants/materials, setting, methods: Endometrial biopsies (n ¼ 15) were taken with a sterile Pipelle de Cornier and snap-frozen in liquid nitrogen, followed by -80 /C14 C storage and 16S amplicon sequencing (n ¼ 9) targeting the V4-region or deep shotgun metagenomic sequencing (n ¼ 6) (Prebiomics, Italy). Simultaneously, in nine women, paired cervical and vaginal swabs (Copan Diagnostics, Italy) were taken and preserved in eNAT medium. In six participants, an additional endometrial biopsy was collected and stored in Amies medium for immediate culturomics. Main results and the role of chance: We observed a high similarity in the microbial composition of endometrial biopsies, cervical, and vaginal samples using 16 amplicon sequencing, but also some taxa were only found in the en- dometrial samples. Notably, the presence of Gemmobacter, a waterborne genus, was only found in endometrial biopsies and could be a source of con- tamination from water used during hysteroscopy to flush the uterus. Most endometrial microbiome profiles were Lactobacillus dominated, with excep- tions observed in three patients. No significant differences for alpha diversity measures (richness and evenness) of the microbiota composition were ob- served between the different niches. For the first time we applied both culturomics and deep shotgun metage- nomic sequencing for the characterization of the microbial composition in the same endometrial biopsies. Commonly identified genera using both techni- ques include Lactobacillus, Prevotella, Bifidobacterium, Anaerococcus, and Cuticubacterium. Remarkably, at the species level, the two approaches revealed almost no overlapping species within each biopsy, underscoring the importance of combining multiple methodologies in examining low-biomass niches like the endometrium. Limitations, reasons for caution: A limitation of this study is the low-bio- mass nature of endometrial biopsies which are prone to cross-contamination with the lower female reproductive tract. This challenges downstream analy- sis, as it becomes difficult to distinguish between a relevant microbiological signal and contaminating noise. Wider implications of the findings: This study emphasizes the need to consider a multifaceted microbiome approach when exploring low-biomass environments, like the endometrium. The critical consideration of potential cross-contamination by the lower female reproductive tract and its implica- tions for downstream analysis is paramount in clinical microbiology research and demands meticulous handling. Trial registration number: not applicable

citation ID: deae108.698 P-334 Hysterosalpingo-Contrast-Sonography: a key step in the female infertility workup? R. Y. Chaala 1, M. Chanegriha2, F. Yendjah1, A. Atmani1, B. Bellala1, N. Rabhi1, M. Atir1, S. Hassani1, S. Mouhoub1 1Mother-child hospital of army, assisted reproductive technologies department, Algiers, Algeria 2Mother-child hospital of army, medical department, Algiers, Algeria Study question: The main objective of this study is to estimate the possibil- ity of offering Hystero-Salpingo-Contrast-Sonography (HyCoSy) as a first-line diagnosis test in the female infertility exploration Summary answer: HyCoSy represents a key step in decision-making re- garding subsequent steps in infertility diagnosis and treatment What is known already: In this age of rising rates of infertility; the evalua- tion of the fallopian tubes and the endometrial cavity in an essential part of the infertility investigation. Traditionally hysterosalpingography has been used to evaluate tubal pa- tency; but it has many disadvantages; laparoscopy and hysteroscopy are at present considered as the gold standard techniques for tubal and endometrial cavity assessment, respectively. Hysterosalpingo- Contrast –Sonography or HyCoSy was introduced over 20 years ago since 1981; it allows to gynecolo- gist to evaluate the fallopian tubes the uterus and the ovaries with only one ultrasound in the office. Study design, size, duration: During two years, one hundred and sixty- seven patients with a context of female infertility with normal spermogram were included in our prospective and descriptive study, Participants/materials, setting, methods: Our patients underwent: a Hysterosalpingography (HSG), a Hysteroscopy, a Laparoscopy and as part of our study: a Hystero-Salpingo-Contrast-Sonography (HyCoSy), a comparison i352 40th Hybrid Annual Meeting of the ESHRE, Amsterdam - The Netherlands, 7-10 July 2024