Endometriosis (EM) is a chronic, inflammatory gynaecological disorder defined by the presence of endometrial-like tissue outside the uterine cavity, most frequently affecting the ovaries, peritoneum, and uterosacral ligaments. Despite its prevalence and the significant impact on life quality, EM is often underdiagnosed, with an average delay of about nine years, particularly affecting adolescents and young women. The complex aetiology involves genetic, environmental, and immune factors, with whole-exome sequencing (WES) emerging as a potential tool for identifying relevant genetic variants. Research indicates that innate immune dysfunction, mechanotransduction, and epithelial-to-mesenchymal transition promote endometrial cell migration and lesion formation, processes regulated by nuclear envelope integrity and cytoskeletal dynamics. The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, specifically Nesprin-1 and Nesprin-2, encoded by SYNE1 and SYNE2, is crucial for these processes. Genome-wide studies have linked SYNE genes to EM risk, showing downregulation in affected patients, and rare variants in these genes have been identified, though their functional implications are still unclear. To this purpose, WES was performed on 204 EM patients to identify rare (MAF <0.1%), damaging variants in SYNE1/2. Primary endometriotic cells (EMCs) were isolated from ovarian lesions of variant carriers (n=4) and wild-type (WT) non-carrier controls (n=4). Functional characterization included somatic WES, RT-qPCR, Western blot, confocal immunofluorescence, and Transwell migration assays. WES identified 11 rare, likely damaging SYNE1/2 variants in 12 patients. Immunofluorescence revealed a distinct protein mislocalization, WT EMCs displayed physiological Nesprin-2 confinement at the nuclear envelope, whereas variant carriers exhibited a diffuse cytoplasmic distribution polarized along actin stress fibres. We demonstrated that SYNE1/2 mutated EMCs had a markedly higher migratory capacity compared to WT controls. Here, in vitro experiments demonstrated, for the first time, the involvement of Nesprin-2 in endometrial cell migration, supporting a mechanistic link between nuclear–cytoskeletal disruption and the invasive phenotype of endometriotic cells (EMCs). These findings provide new insights into EM pathogenesis and highlight SYNE2 as a promising molecular marker for improved diagnosis and disease management.

Introduction:The reproductive microbiome plays a key role in disease progression and fertility in women with endometriosis. Vaginal and endometrial dysbiosis has been increasingly linked to inflammation, impaired reproductive outcomes, and symptom severity. Although estro-progestins, progestins, and GnRH agonists are widely used, their impact on microbial communities remains poorly understood, highlighting the need to clarify microbiome-therapy interactions. This systematic review aims to comprehensively synthesize current evidence on how hormonal therapies influence the reproductive microbial environment and to offer insights for optimizing clinical management of endometriosis. Methods:Literature screening and data extraction followed PRISMA guidelines using PubMed, Scopus, and Google Scholar. The search combined terms on endometriosis, hormonal therapy, and reproductive microbiome. Non-English studies, reviews, and those without original data were excluded. Risk of bias was assessed with ROBINS-I-V2, and microbial composition and diversity were analyzed and synthesized qualitatively. Results:The literature search retrieved 577 publications, of which 6 met eligibility criteria and were analyzed. The evidence collected through sequencing or culture-based methods suggested that the use of hormonal therapies to treat endometriosis may impact both vaginal and endometrial microbiome, favoring the colonization of bacterial species associated with infertility. GnRHa resulted to foster the dominance of potentially pathogenic bacteria, as Gardnerella and Streptococcaceae, in the endometrium, and supporting bacterial vaginosis by increasing intermediate flora (Nugent score 4-6). A similar effect on the vaginal environment has been reported upon the use of oral contraceptive pills, which was shown to prompt the increase of Prevotella, Ureaplasma, Streptococcus anginosus and Streptococcus agalactiae, among other pathogenic microbes, and to enhance the Bacillota/Bacteroidota ratio. Discussion:Despite affected by several limitations and heterogeneity of included studies, this review provides a preliminary overview of the possible pejorative effect of hormonal therapy on the reproductive microbiome of endometriosis patients. While further investigations are required to consolidate these findings, the observations raised offer a valuable basis for opening a discussion about improving management strategies for affected women. By highlighting confounding factors overlooked in the selected papers, the present work will also be functional to optimize the design of future studies. Systematic review registration:https://www.crd.york.ac.uk/PROSPERO/view/CRD420251042858, identifier PROSPERO (CRD420251042858).

Does the homeostatic molecule Multimerin-2 (MMRN2) undergo alterations in preeclampsia (PE) and oocyte donation (OD) pregnancies, potentially contributing to the increased PE risk in OD? Multimerin-2, is profoundly remodeled in pregnancy and decreases in PE and in OD placentas, suggesting a potential pathogenetic role. Assisted reproductive techniques are associated with a higher risk of developing PE, with a two-fold rise in homologous (HOM) and a four-fold rise in heterologous (OD) settings. PE is a systemic vascular disorder characterized by endothelial cells dysfunction, yet its precise mechanisms remain unclear. A key element granting vascular homeostasis is MMRN2, an extracellular molecule specifically expressed by endothelial cells. While its function in pregnancy is not well understood, its regulatory role in vessel efficiency suggests a potential involvement in PE as well. Investigating MMRN2 alterations in PE and OD pregnancies may provide novel insights into disease pathogenesis. This is an observational pilot study conducted over 12 months. Six patient groups were included: 1) OD pregnancies, not diagnosed with PE (OD, n = 10); 2) homologous ART pregnancies, not diagnosed with PE (HOM, n = 7); 3) spontaneous pregnancies diagnosed with PE (PE, n = 7); 4) uneventful spontaneous pregnancies (SP, n = 8); 5) women occurring physiological pregnancies who chose to undergo voluntary abortion within the first gestational trimester (IVG, n = 6); 6) non-pregnant women (CTRL, n = 20). Placental samples were collected after delivery in groups 1-4 and after IVG in group 5, and MMRN2 deposition analyzed through qPCR and immunohistochemistry. Serum samples have been obtained from CTRL women, as reference, and from PE and SP patients at two time points: within the first (11-13th week) and third (34-38th week) trimesters. The concentration of MMRN2 fragments have been evaluated by ELISA. Statistical comparisons were made using ANOVA and student t-test when appropriate. Immunohistochemistry analysis indicated that MMRN2 is deposited within the placental capillary wall during the first trimester and remains stable in SP and HOM pregnancies. By contrast, PE and OD samples showed a consistent reduction of MMRN2. qPCR analyses confirmed this trend, showing a comparable decrease in MMRN2 expression in PE and OD groups compared to SP and HOM patients, respectively, uncovering novel shared molecular features between the two conditions. Additionally, we observed a marked increase in MMRN2 fragments, products of proteolytic cleavage, in first- and third-trimester sera from SP women compared to non-pregnant controls, indicating MMRN2 undergoes remodeling during pregnancy. ELISA analysis of PE samples revealed MMRN2 fragment levels comparable to those from SP in the first trimester, but significantly decreased by the third trimester, suggesting a dysregulated MMRN2 remodeling in PE pregnancies. Given MMRN2 essential role for vessel homeostasis, its absence in the placenta may contribute to vascular dysfunction. Moreover, altered MMRN2 deposition in OD patients suggests a link between the loss of the molecule and the higher risk of developing the disease. Collectively, our findings propose MMRN2 as a promising biomarker for PE and a potential tool for understanding common pathways in PE and OD pregnancies. The small sample size of this pilot study highlights the need for larger cohorts to confirm the observed trends and enhance the reliability of these findings. Additional molecular investigations are required to better characterize the impact of MMRN2 loss in PE and OD in terms of vessel functionality and efficiency. This study highlights excessive MMRN2 remodeling as a potential contributor to vascular dysfunction in PE and provides insight into how OD pregnancies carry a higher risk of developing the disease, supporting the evaluation of MMRN2 as a biomarker and target to improve pregnancy outcomes. No

Abstract Background About 50% of cutaneous melanoma (CM) harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently demonstrated that knockout of Spry1 (Spry1KO) in BRAFV600-mutant CM led to cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Despite these findings, however, the precise molecular mechanism linking Spry1 to BRAFV600-mutant CM remains to be elucidated. Materials and methods Immunoprecipitation coupled to mass spectrometry was employed to gain insight into Spry1 interactome. Spry1 gene was knocked-out using the CRISPR strategy in the BRAF-mutant cell lines. Transmission electron microscopy was used to assess the relationship between Spry1 expression and mitochondrial morphology. By using in vitro and in vivo models, the effects of Spry1KO were investigated through RNA-sequencing, quantitative real-time PCR, Western blot, and immunofluorescence analyses. The Seahorse XF24 assay allowed real-time measurement of cellular metabolism in our model. Angiogenic potential was assessed through in vitro tube formation assays and in vivo CD31 staining. Results Spry1 was mainly located in mitochondria in BRAFV600-mutant CM cells where it interacted with key molecules involved in mitochondrial homeostasis. Spry1 loss resulted in mitochondrial shape alterations and dysfunction, which associated with increased reactive oxygen species production. In agreement, we found that nuclear hypoxia-inducible factor-1 alpha (HIF1α) protein levels were reduced in Spry1KO clones both in vitro and in vivo along with the expression of its glycolysis related genes. Accordingly, Ingenuity Pathway Analysis identified “HIF1α Signaling” as the most significant molecular and cellular function affected by Spry1 silencing, whereas the glycolytic function was significantly impaired in Spry1 depleted BRAFV600-mutant CM cells. In addition, our results indicated that the expression of the vascular endothelial growth factor A was down-regulated following Spry1KO, possibly as a result of mitochondrial dysfunction. Consistently, we observed a substantial impairment of angiogenesis, as assessed by the tube formation assay in vitro and the immunofluorescence staining of CD31 in vivo. Conclusions Altogether, these findings identify Spry1 as a potential regulator of mitochondrial homeostasis, and uncover a previously unrecognized role for Spry1 in regulating nuclear HIF1α expression and angiogenesis in BRAFV600-mutant CM. Significance Spry1KO profoundly impacts on mitochondria homeostasis, while concomitantly impairing HIF1α-dependent glycolysis and reducing angiogenesis in BRAF-mutant CM cells, thus providing a potential therapeutic target to improve BRAFV600-mutant CM treatment.

[This retracts the article DOI: 10.1016/j.mbplus.2020.100029.].

The extracellular matrix is a complex network of macromolecules that support the growth and homeostatic development of organisms. By conveying multiple signaling cascades, it impacts on several biological processes and influences the behaviour of numerous cell types. During the endometrial cycle and the key events necessary for a correct embryo implantation and placentation, this bioactive meshwork is substantially modified to favour endometrial receptivity and vascular adaptation, trophoblast cell migration, and immune activation as well. A correct extracellular remodeling is fundamental for the establishment of a physiological pregnancy; indeed, the occurrence of altered matrix modifications associates with gestational disorders such as preeclampsia. In the present review, we will critically evaluate the role of pivotal matrix constituents in regulating the key steps of embryo implantation and placentation, provide up-to-date information concerning their primary mechanisms of action and discuss on their potential as a novel source of biomarkers and therapeutic targets.

BRCA1/2 mutations are classically associated with hereditary breast and ovarian cancer, yet growing evidence indicates that even heterozygous BRCA1 status may alter ovarian physiology before malignant transformation. Carriers of BRCA1 mutations display reduced ovarian reserve and accelerated reproductive ageing, but the cellular and molecular mechanisms behind these changes remain unclear. Given the fundamental role of angiogenesis in follicle survival and stromal homeostasis, we investigated whether BRCA1 haploinsufficiency disrupts the ovarian microvascular environment, predisposing to both impaired ovarian function and a pro-tumorigenic niche. We isolated ovarian endothelial cells (OVECs) from biopsies of healthy women, carrying and non-carrying the BRCA1 mutation. Our observations indicated distinct growth behaviours in vitro , particularly in terms of morphology and replication rate. Transcriptomic analysis revealed a distinct gene expression profile in mut compared to WT OVECs. Mut cells exhibited an enrichment of gene signatures associated with vascular remodelling, such as migration, proliferation, and sensitivity to endothelial-to-mesenchymal transition. Functionally, mut OVECs showed increased angiogenic behaviour and a shift toward mesenchymal traits. Histological analysis of ovarian tissues confirmed aberrant vascular architecture and increased microvessel density in BRCA1-mut ovaries, consistent with endothelial activation and remodelling. In conclusion, phenotypic and functional differences between wild-type and mut OVECs were proved, demonstrating that heterozygous mutations in BRCA1 can induce a tissue-specific endothelial dysfunction.

Abstract Study question Do stromal alterations occur during placentation upon heterologous assisted reproductive techniques (ART) and are they associated with a higher risk of developing preeclampsia? Summary answer The remodelling of extracellular matrix molecules, key for vascularization and immunity, is altered in pregnancies from oocyte donation and resembles the miss-regulations observed in preeclampsia. What is known already Preeclampsia represents one of the main causes of maternal and perinatal morbidity, affecting 2-8% of spontaneous pregnancies. Its incidence increases two times upon homologous ART and up to four times upon oocyte donation, however the underlying factors are still unknown. Preeclampsia is characterized by high blood pressure and endothelial cell dysfunction, but its pathogenesis is multifactorial relying on vascular, metabolic and immune alterations. All these elements are tightly influenced by the extracellular matrix (ECM) that, during an uneventful pregnancy, is remodelled to support an efficient placentation and immunotolerance. Despite its key functions, little is known regarding ECM remodelling in preeclampsia. Study design, size, duration This is an observational pilot study conducted over a 12 months period. Four groups were included: 1) pregnant women that underwent homologous ART (n = 7; HOM); 2) pregnant women that underwent oocyte donation (n = 10; OD); 3) women that conceived spontaneously affected by PE (n = 7); and 4) uneventful spontaneous pregnancies (n = 8; control group CTRL). Participants/materials, setting, methods For each patient placental samples have been collected upon delivery and processed to perform molecular and proteomic analysis by real time PCR and immunohistochemistry The statistical differences among the groups have been evaluated by the ANOVA and Mann-Whitney tests. Main results and the role of chance We focused our study on specific ECM elements known to modulate angiogenesis and immunity, two key players in preeclampsia occurrence. Our data indicated first that collagen I, collagen IV and Multimerin-2, important components of the vessel wall that contribute to vessel stabilization and efficiency, are significantly decreased in OD compared to HOM group (coll I P = 0.04; coll IV P = 0.043; Multimerin-2 P = 0.036). Next, we analyzed EMILIN-2 and versican, two ECM components exerting not only angiogenic but also immunomodulatory functions. We found that EMILIN-2 is decreased in OD compared to CTRL placentas (P = 0.043), whereas versican undergoes an opposite regulation, being slightly increased in the OD placentas (P = 0.045). We observed an analogous mis-regulation when comparing PE to the CTRL group, suggesting that these stromal alterations may take part in the establishment of a microenvironment more prone to preeclampsia onset and associate with the higher risk related to OD pregnancies. We also found that some features of ECM remodelling seem to be strongly associated with OD. Precisely, our data denoted that the glycoprotein tenascin-C decreased specifically in OD patients, while was comparable between HOM, PE and CTRL groups (P = 0.042). Limitations, reasons for caution The main limitation of this pilot study is the restricted number of patients. Further investigations on a larger sample and prospective cohort of patients might provide more robust data. Wider implications of the findings Our data highlighted for the first time that the placental stroma undergoes an altered remodelling in OD and PE pregnancies. We envision that a deep characterization of ECM will help better understanding of the mechanisms behind the pathogenesis of preeclampsia with possible implications for prediction and prevention. Trial registration number N/A

Abstract Background: Cutaneous melanoma (CM) is a very aggressive malignancy that still represents the deadliest form of skin cancer. About 50% of CM harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently reported that Spry1 knockdown (Spry1KO) reduced the expression of several markers of epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Loss of E-cadherin is a hallmark of EMT in CM, and the presence of aberrant E-cadherin expression in CM is usually associated with a significantly worse overall survival. Based on these premises, in this study we wondered to explore whether Spry1 influences EMT through the modulation of E-cadherin expression. Material and Methods: SPRY1 gene was knocked-out using the CRISPR strategy in BRAF wild-type (wt) and in BRAF-mutant CM cell lines. By using in vitro and in vivo models, the effects of Spry1KO on E-cadherin expression and localization was investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass spectrometry (IP-MS) was performed. Results and Discussion: E-cadherin mRNA and protein levels were significantly increased both in vitro and in vivo in Spry1KO clones. In addition, immunofluorescence analysis revealed a sustained redistribution of E-cadherin to the plasma membrane following Spry1 loss, thus confirming that Spry1 plays a role in EMT in CM. E-cadherin requires p120-catenin (p120) to maintain cell adhesion and functional adherens junctions. Indeed, p120 dysregulation results in the rapid turnover of E-cadherin complexes. Intriguingly, IP-MS analyses identified several Spry1 protein partners, including p120. Although a number of functional studies are still ongoing, preliminary western blot analysis of cytosolic and mitochondrial proteins indicated that Spry1 and p120 were mostly located in mitochondria. Conclusion: Spry1 reduced E-cadherin expression in vitro and in vivo. Starting from preliminary data, we hypothesized that Spry1-p120 interaction might influence E-cadherin distribution over the plasma membrane. Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula Anneli Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 targeting enhances E-cadherin expression in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 202.